Foot and Mouth Disease Virus: Current Research and Emerging Trends [1 ed.] 9781910190524, 9781910190517

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Foot-and-mouth Disease Virus

Current Research and Emerging Trends

Edited by

Francisco Sobrino and Esteban Domingo

Caister Academic Press

Foot-and-mouth Disease Virus Current Research and Emerging Trends

Edited by Francisco Sobrino and Esteban Domingo Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Madrid Spain

Caister Academic Press

Copyright © 2017 Caister Academic Press Norfolk, UK www.caister.com British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-1-910190-51-7 (paperback) ISBN: 978-1-910190-52-4 (ebook) Description or mention of instrumentation, software, or other products in this book does not imply endorsement by the author or publisher. The author and publisher do not assume responsibility for the validity of any products or procedures mentioned or described in this book or for the consequences of their use. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publisher. No claim to original U.S. Government works. Cover design adapted from Figures 4.1(a) and 9.1. Ebooks Ebooks supplied to individuals are single-user only and must not be reproduced, copied, stored in a retrieval system, or distributed by any means, electronic, mechanical, photocopying, email, internet or otherwise. Ebooks supplied to academic libraries, corporations, government organizations, public libraries, and school libraries are subject to the terms and conditions specified by the supplier.

Contents

Contributorsv Prefacexi 1

Introduction: Foot-and-mouth Disease – Much Progress But Still a Lot to Learn David J. Rowlands

2

1

Genome Organization, Translation and Replication of Foot-and-mouth Disease Virus RNA

13

3

Foot-and-mouth Disease Virus Proteinases and Polyprotein Processing

43

4

The Foot-and-mouth Disease Virion: Structure and Function

61

5

Foot-and-mouth Disease Virus Receptors: Multiple Gateways to Initiate Infection

107

6

The RNA-dependent RNA Polymerase 3D: Structure and Fidelity

137

7

Quasispecies Dynamics Taught by Natural and Experimental Evolution of Foot-and-mouth Disease Virus

147

8

Clinical Signs and Pathology of Foot-and-mouth Disease

171

9

Natural Habitats in which Foot-and-mouth Disease Viruses are Maintained

179

Innate to Adaptive: Immune Defence Handling of Foot-and-mouth Disease Virus

211

Encarnación Martinez-Salas and Graham J. Belsham Fiona Tulloch, Garry A. Luke and Martin D. Ryan Mauricio G. Mateu

Paul Lawrence and Elizabeth Rieder

Cristina Ferrer-Orta and Nuria Verdaguer

Esteban Domingo, Ignacio de la Higuera, Elena Moreno, Ana I. de Ávila, Rubén Agudo, Armando Arias and Celia Perales

10

Charles Nfon, Oliver Lung, Carissa Embury-Hyatt and Soren Alexandersen Wilna Vosloo and Gavin R. Thomson

Kenneth C. McCullough, Margarita Sáiz and Artur Summerfield

iv  | Contents

11

Laboratory Diagnostic Methods to Support the Surveillance and Control of Foot-and-mouth Disease Anna Ludi, Valerie Mioulet, Nick J. Knowles and Donald P. King

12

275

Quality Attributes of Current Inactivated Foot-and-mouth Disease Vaccines and their Effects on the Success of Vaccination Programmes287 Eliana N. Smitsaart and Ingrid E. Bergmann

13

Peptide Vaccines Against Foot-and-mouth Disease

317

14

Control of Foot-and-mouth Disease by Using Replication-defective Human Adenoviruses to Deliver Vaccines and Biotherapeutics

333

15

Antiviral Therapies for Foot-and-mouth Disease

357

16

Mathematical Models of the Epidemiology and Control of Foot-and-mouth Disease

385

The Role of International Organizations in the Control of Foot-and-mouth Disease

409

Overview of Foot-and-mouth Disease and its Impact as a Re-emergent Viral Infection

417

Esther Blanco, David Andreu and Francisco Sobrino

Fayna Diaz-San Segundo, Gisselle N. Medina, Marvin J. Grubman and Teresa de los Santos Annebel R. De Vleeschauwer, David J. Lefebvre and Kris De Clercq

Michael J. Tildesley, William J.M. Probert and Mark E.J. Woolhouse

17

Bernard Vallat, Joseph Domenech and Alejandro A. Schudel

18

Brian W.J. Mahy and Graham J. Belsham

Index427

Contributors

Rubén Agudo Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Consejo Superior de Investigaciones Científicas (CSIC) Campus de Cantoblanco Madrid Spain

Ana I. de Ávila Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Consejo Superior de Investigaciones Científicas (CSIC) Campus de Cantoblanco Madrid Spain

[email protected]

[email protected]

Soren Alexandersen Deakin University Geelong-Geelong Centre for Emerging Infectious Diseases Health Education and Research Building University Hospital Geelong Geelong, VIC Australia

Graham J. Belsham DTU National Veterinary Institute Technical University of Denmark Lindholm, Kalvehave Denmark

[email protected]

Ingrid E. Bergmann Centro de Virología Animal (CEVAN) Instituto de Ciencia y Tecnología Dr. César Milstein CONICET Buenos Aires Argentina

David Andreu Department of Experimental and Health Sciences Pompeu Fabra University Barcelona Spain [email protected] Armando Arias Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Consejo Superior de Investigaciones Científicas (CSIC) Campus de Cantoblanco Madrid Spain; and Section for Virology National Veterinary Institute Technical University of Denmark Frederiksberg C Denmark [email protected]

[email protected]

[email protected] Esther Blanco Animal Health Research Center (CIS-INIA) Madrid Spain [email protected] Kris De Clercq Unit of Vesicular and Exotic Diseases Operational Direction Viral Diseases CODA-CERVA Veterinary and Agrochemical Research Centre Brussels Belgium [email protected]

vi  | Contributors

Annebel R. De Vleeschauwer Unit of Vesicular and Exotic Diseases Operational Direction Viral Diseases CODA-CERVA Veterinary and Agrochemical Research Centre Brussels Belgium [email protected] Fayna Diaz-San Segundo Plum Island Animal Disease Center (PIADC) ARS USDA Greenport, NY; and Department of Pathobiology and Veterinary Science University of Connecticut Storrs, CT USA [email protected] Joseph Domenech World Organisation for Animal Health Paris France [email protected] Esteban Domingo Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Consejo Superior de Investigaciones Científicas (CSIC) Campus de Cantoblanco Madrid; and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) Barcelona Spain [email protected] Carissa Embury-Hyatt National Centre for Foreign Animal Disease Canadian Food Inspection Agency Winnipeg, MB Canada [email protected]

Cristina Ferrer-Orta Structural Biology Unit Molecular Biology Institute of Barcelona Spanish Research Council (CSIC) Barcelona Spain [email protected] Marvin J. Grubman Plum Island Animal Disease Center (PIADC) ARS USDA Greenport, NY USA [email protected] Ignacio de la Higuera Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Consejo Superior de Investigaciones Científicas (CSIC) Campus de Cantoblanco Madrid Spain [email protected] Donald P. King World Reference Laboratory for Foot-and-mouth Disease The Pirbright Institute Pirbright UK [email protected] Nick J. Knowles World Reference Laboratory for Foot-and-mouth Disease The Pirbright Institute Pirbright UK [email protected] Paul Lawrence Agricultural Research Service US Department of Agriculture Plum Island Animal Disease Center Greenport, NY USA [email protected]

Contributors |  vii

David J. Lefebvre Unit of Vesicular and Exotic Diseases Operational Direction Viral Diseases CODA-CERVA Veterinary and Agrochemical Research Centre Brussels Belgium

Mauricio G. Mateu Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM); and Department of Molecular Biology Universidad Autónoma de Madrid Madrid Spain

[email protected]

[email protected]

Anna Ludi World Reference Laboratory for Foot-and-mouth Disease The Pirbright Institute Pirbright UK

Kenneth C. McCullough Immunology Department Institute of Virology and Immunology; and University of Bern Mittelhäusern and Bern Switzerland

[email protected]

[email protected]

Garry A. Luke Biomedical Sciences Research Complex School of Biology University of St. Andrews St. Andrews UK

Gisselle N. Medina Plum Island Animal Disease Center (PIADC) ARS USDA Greenport, NY USA

[email protected]

[email protected]

Oliver Lung National Centre for Foreign Animal Disease Canadian Food Inspection Agency Winnipeg, MB Canada

Valerie Mioulet World Reference Laboratory for Foot-and-mouth Disease The Pirbright Institute Pirbright UK

[email protected] Brian W.J. Mahy Wolfson College University of Cambridge Cambridge UK [email protected] Encarnación Martinez-Salas Centro de Biología Molecular ‘Severo Ochoa’ (CSICUAM) Madrid Spain [email protected]

[email protected] Elena Moreno Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Consejo Superior de Investigaciones Científicas (CSIC) Campus de Cantoblanco Madrid Spain [email protected] Charles Nfon National Centre for Foreign Animal Disease Canadian Food Inspection Agency Winnipeg, MB Canada [email protected]

viii  | Contributors

Celia Perales Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Consejo Superior de Investigaciones Científicas (CSIC) Campus de Cantoblanco Madrid; and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd); and Liver Unit Internal Medicine Laboratory of Malalties Hepàtiques Vall d’Hebron Institut de Recerca-Hospital Universitari Vall d’Hebron (VHIR-HUVH) Universitat Autònoma de Barcelona Barcelona Spain [email protected] William J.M. Probert Warwick Infectious Disease Epidemiology Research (WIDER) Group School of Life Sciences and Mathematics Institute University of Warwick Coventry UK [email protected] Elizabeth Rieder Agricultural Research Service US Department of Agriculture Plum Island Animal Disease Center Greenport, NY USA [email protected] David J. Rowlands School of Molecular and Cellular Biology, and The Astbury Centre for Structural Molecular Biology Faculty of Biological Sciences University of Leeds Leeds UK [email protected]

Martin D. Ryan Biomedical Sciences Research Complex School of Biology University of St. Andrews St. Andrews UK [email protected] Margarita Sáiz Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Universidad Autónoma de Madrid Madrid Spain [email protected] Teresa de los Santos Plum Island Animal Disease Center (PIADC) ARS USDA Greenport, NY USA [email protected] Alejandro A. Schudel Animal Health and Food Safety (PROSAIA) Foundation Buenos Aires Argentina [email protected] Eliana N. Smitsaart Biogénesis Bagó S.A. Buenos Aires Argentina [email protected] Francisco Sobrino Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) Madrid Spain [email protected] Artur Summerfield Immunology Department Institute of Virology and Immunology; and University of Bern Mittelhäusern and Bern Switzerland [email protected]

Contributors |  ix

Gavin R. Thomson TADScientific Pretoria South Africa

Bernard Vallat World Organisation for Animal Health (OIE) Paris France

[email protected]

[email protected]

Michael J. Tildesley Warwick Infectious Disease Epidemiology Research (WIDER) Group School of Life Sciences and Mathematics Institute University of Warwick Coventry UK

Nuria Verdaguer Structural Biology Unit Molecular Biology Institute of Barcelona Spanish Research Council (CSIC) Barcelona Spain

[email protected] Fiona Tulloch Biomedical Sciences Research Complex School of Biology University of St. Andrews St. Andrews UK [email protected]

[email protected] Wilna Vosloo CSIRO-Australian Animal Health Laboratory Geelong, VIC Australia [email protected] Mark E.J. Woolhouse Centre for Immunity, Infection and Evolution The University of Edinburgh Edinburgh UK [email protected]

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Preface

Foot-and-mouth disease virus (FMDV) maintains a continuing fascination not only because of its worldwide implications for economic development, but also because it makes us relive the events of an outbreak of disease upon unsuspecting areas of our planet. On top of this, many fundamental questions about its replication, transmission, detection, spread and persistence do not yet have an answer, since the virus displays unique features even when compared with its closest picornavirus relatives. As with other small viruses, FMDV is endowed with complex biological behaviour for an apparently simple pathogen. An invitation to produce a book similar in content to the 2004 book is a clear sign that not only the first edition was well received by the scientific community, but also that many problems and questions remain, and that the unsolved issues have a very relevant scientific and economic impact in our increasingly global world. Unanswered questions are, for example, the limited knowledge about host range determinants, or the lack of cost-effective vaccines, as alternatives to the chemically inactivated conventional vaccines. The limited amount of funding devoted to FMDV research in the EU is surprising considering the potential economic impact of a disease outbreak within the EU or in neighbouring countries. A very important change regarding the social perception of the disease has taken place since 2004. Perhaps as a result of the terrible images of mass animal slaughtering during the 2001 European epidemic, witnessed on television by the public at large, there is a growing trend to consider alternative means to deal with the disease. In particular, the non-vaccination policy and the possibility of new types of antiviral interventions are gaining

impetus, and gradually diminishing the traditional support for a slaughter-based control strategy. This is reflected in renewed effort on vaccine designs and the consideration of antiviral agents to control or prevent the infection, either by administering the agents alone or as a complement to vaccination or other immunization-based interventions. This book reflects this trend by including a chapter on antiviral therapies that was not even considered in the 2004 version where small molecule inhibitors or RNA interference or silencing (to name just two points) were not even mentioned. In planning the new volume, we have done our best as authors to invite those experts that in our view have contributed either recently or historically to construct the body of current knowledge on FMDV. Of course, they are not the only ones in this endeavour, and we apologize for any omissions of experts that could have been invited as authors. Many names are listed in a remarkable number of references that should serve as further reading to complement the core information gained by reading the 18 chapters. This book is not a reprint or even an updated version of the 2004 book. While many topics have been retained, each chapter has been written afresh, so as to include recent progress as evidenced by the large number of references to publications of the last decade. It is our hope that the present book will provide an updated overview of several interconnected aspects of FMDV and its disease, including the structure of the viral particle and encoded proteins, expression of the genetic material, natural habitats of the virus, diagnostic procedures, epidemiological spread, and control measures. As in the 2004 book, great attention has been paid to what is known, and what is not, regarding the

xii  | Preface

innate and acquired immune responses elicited by the virus and their implications for classical vaccines improvement and the development of new immunization strategies. We thank all authors for their timely contributions, and for reflecting recent developments as well

as historical developments by many devoted scientists some of whom, unfortunately, are no longer among us. Finally, our appreciation goes also to Caister Academic Press, and in particular to Hugh Griffin, for his friendly and constructive work in the planning and production of this book. Francisco Sobrino and Esteban Domingo Centro de Biología Molecular ‘Severo Ochoa’ Cantoblanco

Introduction: Foot-and-mouth Disease – Much Progress But Still a Lot to Learn David J. Rowlands

Abstract Several massive outbreaks of foot-and-mouth disease (FMD) around the turn of the century, and in particular the 2001 outbreak in the UK, provided the incentive for assembling and publishing the first edition of Foot-and-mouth Disease: Current Perspectives in 2004. It is now more than a decade since the first edition was published, and the editors deemed that it is appropriate to publish this second edition, which summarizes some of the advances in control and prevention of the disease and in fundamental understanding of virus replication that have occurred during that time. Much progress has been made, as evidenced in the following chapters, but FMD remains as one of the most important threats to the agriculture industry and more needs to be done. Similarly, our fundamental understanding of the structure and function of the virus and its genome has advanced significantly but there remain a number of intriguing unanswered questions, many of which appear to be specific to this fascinating virus. Introduction In 2001, the UK experienced the worst outbreak of foot-and-mouth disease (FMD) in its history. More than 10 million cattle sheep and pigs were slaughtered and burned on huge funeral pyres. These scenes were broadcast daily on television and engendered a feeling of revulsion in the general public. The cost in compensation to farmers who had lost their livestock was approximately £1.1 billion, but the associated effects on trade in the agriculture industry were also costly and longlasting. The scale of the slaughter of animals and their transportation and disposal was so great that

1

the army was called in to organize and manage the operation. In addition, restrictions on movement in large areas of the country had a massive effect on the tourism and leisure industry since much of the countryside was ‘closed for business’ for the best part of a year. The overall cost to the nation was estimated to have been £8 billion, but the human cost associated with loss of stock or rural businesses cannot be estimated. The trauma and severity of this outbreak brought the threat of FMD sharply into focus, at least in Europe, and was a powerful wake-up call after the complacency engendered by many disease free years. Other notable outbreaks of FMD around the world around the turn of the century include Taiwan in 1997 (Yang et al., 1999), when 40% of the country’s pig population was lost, and in Argentina, which suffered a massive outbreak in 2000–2001 (Mattion et al., 2004) just a few years after the country had been declared FMD free. The renewed interest in the disease and its causative agent, foot-and-mouth disease virus (FMDV), that was engendered by these outbreaks stimulated the publication of three volumes of review articles; Foot-and-mouth Disease (ed. D.J. Rowlands, 2003), Foot-and-mouth Disease: Current Perspectives (ed. F. Sobrino and E. Domingo, 2004) and Foot-andmouth Disease Virus (ed. B. W. J. Mahy, 2005). These books provided broad, authoritative and in-depth discussions of all aspects of the disease and the virus. The current volume of Foot-andmouth Disease: Current Research and Emerging Trends is intended to review the developments in our understanding of FMDV in the 11 years since the publication of the first issue. The chapters contained within the book cover most aspects of the disease spanning from control to the structure and

2  | Rowlands

molecular biology of the viral genome. Although there has been significant progress in all/most areas, FMD is still a continuing threat to the global agricultural economy and there remains a pressing need for better control measures, including diagnostics and improved vaccines. At the molecular end of the spectrum there are still many aspects of FMDV replication that are poorly understood. The trauma of the 2001 outbreak triggered a number of investigations into the state of FMDV research, its control and the response capability of the UK. These included a government enquiry (Foot-and-mouth Disease 2001: Lessons to be Learned), an enquiry sponsored and conducted by the Royal Society (Royal Society Infectious Disease in Livestock Inquiry Follow-Up Review) and a policy commission report on the future of farming and food (Foot-and-mouth disease: Applying the Lessons). The general conclusions drawn from these enquiries were that there was considerable scope for improvement in virtually all aspects of FMD surveillance and control. At about the same time (2002) the BBSRC, the UK research council responsible for maintenance and operation of the UK high security laboratory at Pirbright, commissioned a review of its status (Review of the Institute for Animal Health – the Pirbright Laboratory). One of the concerns expressed in this report was that the Pirbright laboratory, one of the oldest and most respected research institutions working with FMDV, had fallen into disrepair and required considerable investment to modernize its facilities. As a consequence of all these reviews into the shortcomings of the support for veterinary viral diseases in general, and FMD in particular, extra funds were allocated for a site redevelopment programme at Pirbright. However, it was not until thirteen years later these recommendations were realized with the opening of the BBSRC National Virology Centre: the Plowright Building, which is now one of the most sophisticated high security research facilities in the world. There have also been significant developments in the management and construction of high containment facilities for the handling of FMD elsewhere in the world. For example, new and improved laboratories were opened at the National Veterinary Institute at Lindholm in Denmark. Also, after much debate in the USA, the first steps in the construction of a new high containment laboratory

complex in Kansas to replace the ageing facilities at Plum Island, New York, have been taken. In addition to these developments in the infrastructure necessary to conduct work on FMD safely and effectively, there have been significant movements to improve international coordination of FMD on the global scale. For example, the Global Foot-and-Mouth Disease Research Alliance (GFRA) is a consortium of partners from FMD free and endemic countries which plays a valuable role in bringing together scientists who are trying to tackle FMD from very different perspectives. The role of international organizations in the co-ordination of approaches to deal with the global problems relating to FMD are discussed in Chapter 17. Progress and challenges in the management of FMD The disastrous epidemic in the UK in 2001 served to emphasize a number of specific issues of FMDV outbreak management that required improvement or reappraisal. These include surveillance, diagnostics, persistence, outbreak control, improved vaccines, improved facilities and improved security at national borders. Although the importance of all of these factors relating to FMD control was highlighted by the UK outbreak they are of continued relevance to the global efforts to control the disease. In the last decade or so advances have been made on all of these areas relating to FMD control, some of which are briefly discussed below: Surveillance and diagnostics Effective surveillance is to a large extent dependent on the availability of rapid, accurate and sensitive diagnostic tools. Cost too is an important factor in many parts of the world. Initial diagnosis is based on observation of the clinical presentations typical of FMD and in classic infection in cattle the symptoms are usually obvious. However, in other species, sheep for example, the symptoms may not be as simple to detect and it is vitally important that livestock owners and veterinarians are appropriately trained to observe infected animals. Expansion of international training and education is of great importance in the local and global efforts to control the spread of FMD. International schemes to improve awareness of FMD and the importance of rapid confirmation of the disease have expanded

FMDV: Advances and Challenges |  3

in recent years with the involvement of several international organizations [e.g. European Commission for the Control of Foot-and-Mouth Disease (EUFMD), the Food and Agriculture Organization (FAO) of the United States, World Organisation for Animal Health (OIE)] with the advent of GFRA playing an important role Diagnosis of current infections has traditionally required that virus in clinical samples is identified by serological techniques such as ELISA, often requiring the virus to be grown in tissue culture to provide sufficient antigen to perform the test. This can require multiple blind passages and take several days. Diagnostic approaches that rely on these techniques have many drawbacks including the time required to provide a conclusive result, the problems attendant on maintaining the sample in a sufficiently well preserved state to recover viable virus and the occasional difficulty/ inability to grow field strains on conventional tissue culture cells. Among attempts to overcome these restrictions is the development of cells modified to express the most effective FMDV (Yang et al., 1999) receptors (King et al., 2011). Along similar lines there has been progress in the development of universal capture methods for ELISA by replacing an immobilized antiviral capture antibody with recombinantly expressed integrin receptor (Ferris et al., 2011). This has the advantage of potentially capturing all FMDVs without the serotype restriction inherent in the use of antibody capture. The development of sensitive and reliable pen-side tests is another important research objective. There have been significant advances in the development of diagnostic procedures that rely on detection of the viral RNA irrespective of the ability to grow the virus. Nucleic acid based methods have a number of advantages over the older more traditional diagnostic procedures including speed and sensitivity. In addition, full genome, deep sequencing methods (Wright et al., 2013) are providing highly detailed information that is invaluable for the study of viral transmission during the course of epidemics and can inform predictive mathematic modelling of the patterns of spread of infection (Cottam et al., 2006). Persistence It has long been known that a significant proportion of infected animals, especially cattle, continue

to shed virus (or at least virus/antibody complexes) long after clinical evidence of disease has gone (Sutmoller et al., 1967). In addition, vaccinated animals can be infected to become carriers if their immune status is sufficient to prevent clinical symptoms but not to prevent all virus replication (Anderson et al., 1974). Interestingly, the acquisition of carrier status is somewhat species dependent, being usually observed in bovines or ovines but not in porcines but the reasons for these differences are not known. In Africa the indigenous African buffalo is particularly prone to persistent infection, often with multiple serotypes simultaneously (Ayebazibwe et al., 2010). The phenomenon of persistence of FMDV in symptomless animals has long been a thorn in the side of regulatory authorities and has strongly influenced decisions on the use of vaccination as an emergency procedure to control epidemics of FMD. There are three important questions relating to the problem of persistence of FMDV: how to detect carrier animals, what is the relevance of persistence to disease prevention and control and finally what are the viral and host factors involved in the maintenance of a persistently infected state? How to detect potential carrier animals? In regions where the virus is endemic and control is by vaccination it has long been perceived that diagnostic procedures that could distinguish between animals that had seroconverted following vaccination from those that may have been infected, and are therefore potential carriers, would be of great value. The earliest attempt to provide such a discriminatory test relied on detecting the presence of antibodies to the so-called VIA (virus infection associated) antigen (Cowan and Graves, 1966). This was subsequently shown to be 3D, the viral RNA dependent RNA polymerase which is surprisingly immunogenic in FMDV infections. It is also antigenically cross-reactive over the serotype spectrum. A problem associated with the detection of anti-VIA antibodies as an aid to discrimination between vaccinated and infected animals is that 3D, the VIA antigen, is frequently present in FMD vaccines, unless these have been scrupulously purified, and so vaccinated animals would also develop anti-VIA antibodies. There has been a lot of work to try to identify other non-structural protein marker assays that can successfully distinguish infected

4  | Rowlands

from vaccinated animals and several of these appear to work well and are being used in screening procedures (Brocchi et al., 2006; Sorensen et al., 1998). An alternative approach has been to develop strains of vaccine virus (‘differentiating infected from vaccinated animals’ or DIVA vaccines) that have been manipulated to assist distinction between the immune response to the vaccine or to infection with wild type virus. Removal of an epitope from the vaccine virus without compromising its protective ability has been used for this purpose (Fowler et al., 2014; Uddowla et al., 2012). Presence of antibody to the deleted epitope would therefore imply that the animals had been infected with wild-type virus. What is the relevance of persistence to disease prevention and control? This is a major question for regulatory and control authorities. It is well established that infectious virus can be isolated from persistently infected animals in the laboratory but how efficiently such virus, normally present in immune complexes, can transmit infection to naive animals under field conditions is not certain. Although there is considerable epidemiological evidence accrued over many years to suggest that such transmission can occur in the field numerous attempts to replicate this under controlled laboratory conditions have failed. However, it is impossible to discount the possibility of transmission from a carrier and there is great reluctance to risk the presence of clinically normal persistently infected animals as a possible source of disease through transmission to uninfected animals. The virus shed from persistently infected animals is mostly in the form of immune complexes and so effectively neutralized. However, infectious virus can be isolated directly from samples collected from the oropharynx of carrier animals by probang sampling, although the infectivity titre of such samples can be increased 10- to 100-fold by extraction with organic solvents to remove inhibitory materials such as antibodies (Alexandersen et al., 2002). In addition, it is know that FMDV/ antibody complexes can be taken into and infect cells bearing fc receptors (Baxt and Mason, 1995; Mason et al., 1993). Consequently it is theoretically possible that, should an immune complexed virus particle encounter an fc receptor bearing cell,

such as a macrophage, an infection might ensue. The incredible infectiousness of FMDV makes this scenario unacceptable, no matter how unlikely it might be. What are the viral and host factors involved in the maintenance of a persistently infected state? Although the phenomenon of long-term persistent shedding of FMDV from convalescent bovines has been known for many years, there has been considerable uncertainty regarding the mechanism of persistence and the precise location of the tissue in which it is supported. Persistent virus can be isolated from probang sampling from the oropharynx and there is evidence to suggest that it is mostly derived from the mucosal epithelia of the oropharynx (Alexandersen et al., 2002). It has been proposed that basal cells in these areas can support non-lytic viral replication possibly in conjunction with down regulation of genes controlling innate immunity. Alternatively, it has been reported that FMDV particles can be retained in intact form at the surfaces of dendritic cells within germinal centres of regional lymph nodes ( Juleff et al., 2008, 2012). Presumably there must be a continuous reseeding of infectious virus particles held at dendritic cell surfaces from a source of replication since infectivity of FMDV is lost quite rapidly at body temperature. Although cell cultures persistently infected by FMDV can be selected in vitro (de la Torre et al., 1985), the relevance of these observations to the mechanisms underlying persistent infection in vivo is unclear. In the cell culture persistence studies a very few residual surviving cells present after high level acute infection of BHK cell cultures were found to be infected with the virus. As has been observed with persistently infected cell cultures with other viruses (e.g. VSV) there is a continuous genetic battle between the host cell and the virus to maintain a quasi-stable equilibrium (Martin Hernandez et al., 1994) Interestingly, in long-term passage studies the ratios of the different forms of viral RNA in the cells changed dramatically (Herrera et al., 2008). In a normal lytic infection positive-strand RNA is in large excess over negative strand RNA but as passage of the persistently infected cells progressed so this changed to reach a condition in which both strand are present at

FMDV: Advances and Challenges |  5

similar levels, resulting in a high proportion of double-stranded RNA. Intriguingly, a similar low level viral persistence has been implied in examples of chronic heart failure related to Coxsackievirus infection (Archard et al., 1991). As interesting as these studies are there is little evidence to suggest that the observations made in the artificial in vitro situations are comparable to the mechanisms underlying persistent virus infection and shedding in vivo. However, given the implication of some studies that the site of persistent infection resides in basal cells of the mucosal epithelia it is possible that continual mutational response of the cells to infection, as seen in tissue culture, may play a role in the maintenance of infection. Persistent infection is still an important and unresolved problem. The biological mechanisms that enable the virus to survive and continue replicating are far from clear as is the means of transmission from carrier animals to naive stock. Given that this can occur, albeit rarely and by undefined mechanisms, can we contemplate methods of clearing persistently maintained viral infection – maybe with antiviral drugs? Control in epidemic situations The basic principles of control of FMD epidemics have remained unchanged for many decades. In non-endemic countries, such as the UK, control of the spread of the disease following introduction from an external source is achieved by strict animal movement control, slaughter of stock that have been infected or are ‘at risk’ of infection together with general decontamination and quarantine measures. In endemic regions vaccination is an additional tool for disease control. However there are numerous and complex issues underlying these simplistic descriptions of the availability and application of disease control measures. In 2001 the UK adopted the standard procedures for stamping out occasional outbreaks that have been successful in the past and allowed the country to maintain a disease-free status, which is of great importance for international trading purposes. However a number of factors relating to that outbreak provided the ‘perfect storm’ situation in which the seeds of the epidemic had become widespread throughout the country before its full distribution had become apparent. As a consequence the scale of the control measures that were applied was so

great that they became almost unsustainable and engendered much public distress. Not only was the number of outbreaks occurring at the peak of the epidemic exceedingly high but the ‘tail end’ of the outbreak continued for many months after the initial introduction of the disease (Chis Ster et al., 2009). Interestingly at about the same time as the UK outbreak FMD occurred in regions of South America, such as Argentina, that had remained free of disease for several years. In contrast to the European situation the control measures adopted in South America were to revert to the practice of mass vaccination that had been used to eliminate FMD prior to its reintroduction. This was highly successful, resulting in a somewhat more rapid resolution of the epidemic than was achieved in the UK with ‘stamping out’ measures (Perez et al., 2004). These and other global experiences of the effects of FMD and its control have stimulated a great deal of research and debate concerning the best ways to handle the disease in future. Improving our abilities to understand and predict the scale and course of epidemic spread is of particular importance for improved management following introduction of FMD into non-endemic regions. Retrospective analyses of outbreaks such as that in 2001 have been of great value for determining the mechanisms of spread between premises. For example, the role of meteorological conditions; it has been known for many years that transmission via wind-borne droplets can occur under favourable conditions (Donaldson et al., 1970). In addition, the varying roles played by different host species (cattle, sheep and pigs) in maintaining and spreading infection could be analysed (Donaldson et al., 2001). The massive increases in recent years in the power of genomic sequencing have enabled the fine definition of transmission routes to be determined with unprecedented precision (Cottam et al., 2006). Furthermore, data from all of these sources have enabled the development of increasingly sophisticated mathematical modelling approaches for the prediction of epidemic progression (see Chapter 16). Important improvements in the speed, accuracy, sensitivity and cost of diagnostic methods and devices have been made in recent years. Rapid and easily applied diagnostic tests are of special importance to the control of FMD in the face of its extraordinary rate of spread. Recent advances in the

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methods of detection and control are discussed in Chapter 11. Improved vaccines for global protection Of course, vaccination has been and remains of major importance for the prevention and control of FMD. The importance of vaccination and vaccine development is reflected in the fact that more than one chapter in this book is devoted to the subject. Although current vaccines are very effective if used appropriately (in fact they are the biggest single component responsible for the eradication of FMD from many regions of the globe), there are many ways in which they might be improved. These include potency, thermostability during distribution to point of use, duration of protection induced, breadth of cover against variant strains of virus and safety of production procedures and facilities. In addition to improvements in conventional vaccine technologies there is a lot of interest and progress into novel approaches for vaccine development. These fall into several categories including; the delivery of FMDV antigens by expression from a DNA virus vector, improvement of the stability of seed viruses used to make conventional vaccines; recombinant expression of stabilized virus-like particles (VLPs); in vitro assembly of E. coli expressed capsid proteins; the construction of live attenuated viruses and the reassessment of peptide vaccines. These approaches are discussed in detail in other chapters in this book. Security of facilities handling FMDV The serious consequences of escape of FMDV into the environment necessitate that work with the virus, be it diagnostic surveillance, vaccine production or research, is carried out under conditions of maximum biosecurity. Consequently there is a limited number of high security facilities suitable for FMDV work and the maintenance of their biosecurity is of paramount importance. Breaches of biosecurity at a few institutions in recent years have been the justifiable cause for public alarm. For example, in 1978 virus was found to have escaped from the high containment area at Plum Island. Fortunately, the virus did not escape the confines of Plum Island to reach the mainland (http://www. homelandsecuritynewswire.com/governmenta d m i t s - a c c i d e n t s - p l u m - i s l a n d - b i o l a b) .

However, this was technically a serious breach of containment and other ‘within facility’ unwitting transmissions have been reported. In the UK, in 2007, virus escaped from the high-level security site at Pirbright (https://www.gov.uk/government/ uploads/system/uploads/attachment_data/ file/250363/0312.pdf). The site incorporates both research facilities and a commercial vaccine production plant and these share some common containment infrastructure such as waste disposal drains. Although the exact means by which the virus escaped from the site to infect cattle on nearby farmland will never be known for sure, deficiencies in maintenance of the infrastructure were almost certainly involved. Incidents such as these serve to highlight the importance of ensuring the continual safe maintenance and operation of high containment facilities devoted to work with FMDV and other high risk pathogens. Emergency steps were taken to improve biosecurity as a result of these unfortunate occurrences and, as mentioned above, these aged facilities have been (in the case of Pirbright) or are being (in the case of the USA facility) replaced with new ‘state of the art’ laboratories. Progress and challenges in understanding the molecular biology of FMDV In addition to the issues discussed above, which are highly pertinent to the control of FMD, there are many intriguing aspects of the genome structure and function that are still not well understood, although significant progress has been made in the past 11 years. Some of these features are peculiar to or unique to FMDV and must contain the molecular explanations of the specific pathogenic characteristics of the virus. The following is an (incomplete) list of some of the aspects of FMDV structure, function and replication that are still poorly understood despite progress in recent years: The FMDV particle The atomic structure of the FMDV particle was solved in 1989 and it provided molecular insight into some of its biological properties (Acharya et al., 1989). For example, the nature of the receptor binding domain was revealed as loop-like projection from the virus surface; quite unlike

FMDV: Advances and Challenges |  7

the groove or ‘canyon’ seen in the enteroviruses. This provided an explanation of the extraordinary success of experimental simple peptide vaccines in inducing protective immunity in laboratory animals compared with peptide vaccines against other viruses (Bittle et al., 1982). In addition, the location of specific amino acid residues, especially histidines, at the interfaces of the pentameric subunits of the particle provided a rational explanation of the extraordinary acid sensitivity of FMDV. The structural insights into properties of the virion, such as acid lability, provided the background for directed mutagenesis of the particle to modify these characteristics for the purposes of novel vaccine development. In particular, modifying the pentameric interfaces by introducing cysteine residues to facilitate the formation of disulphide bonds to strengthen subunit interactions (Porta et al., 2013). Other mutations have been described in different serotypes of the virus which also have stabilizing consequences (Kotecha et al., 2015). Recombinant expression of the structural proteins containing stabilizing mutations at the pentamer interfaces, together with the viral protease, has produced empty capsids (virus-like particles or VLPs) with greatly increased resistance to heat or acid mediated disruption (Porta et al., 2013). This work has interesting potential for the development of safe, virus free vaccine production, as discussed elsewhere in this book. Knowledge of the structure of the FMDV particle has also proved useful in other vaccine related studies. For example, modification of selected residues at subunit interfaces has resulted in viruses which maintain their infectivity but have increased thermal stability, a useful potential advance for the production of conventional vaccines that are better preserved in the field. Knowledge of the virus structure has also been exploited for the production of marker vaccines in which specific epitopes have been removed or replaced (Fowler et al., 2011). It is intended that the immune responses to such modified viruses will help to distinguish vaccinated from infected animals. Finally, it has been shown that epitope tags can be inserted at positions that do not interfere with the immunogenic potential of the virus but can facilitate immuno-affinity-based procedure for facile purification and concentration of the particles (Seago et al., 2012). Despite the potential for modification of the

virus for vaccine development purposes, there are still important aspects of the biological functioning of the particle about which we know little. In common with many other viruses we do not understand the assembly process. Genome RNA is preferentially/exclusively packaged within virions and the basis for this specificity is unclear. For other picornaviruses assembly involves the packaging of nascent RNA and the same is likely true for FMDV. For poliovirus there is genetic evidence for the involvement of the putative helicase, P2C (Liu et al., 2010), but whether the same is true for FMDV is not known. In addition, there is enormous condensation required to enclose the 8500 nucleotide genome into the 30-nm-diameter capsid and how this is achieved is, as yet, a mystery. The function of a virus particle is to ensure safe delivery of the genome to a new host cell. For the enteroviruses there is increasing evidence for a mechanism by which the endocytosed virion undergoes receptor or acid mediated conformational changes resulting in the externalization of the internal protein, VP4, and the N terminal domain of VP1. This results in a breaching of the endosomal membrane, most likely by formation of a protective channel through which the genomic RNA is transported into the cytoplasm to initiate infection while the capsid remains intact as an empty particle (Levy et al., 2010). It has been known for many years that acidification of the endosome is necessary and sufficient to trigger FMDV uncoating and penetration into the host cell. However, the details of the process are not understood. Exposure of FMDV particles to pH levels found in endosomes, and necessary for uncoating, result in dissociation into subunits and RNA and it is difficult to envisage how the RNA could be protected from hydrolysis in the lumen of the endosome and how it might be transported across the membrane. There is evidence for FMDV and the related equine rhinovirus A virus (ERAV) for the transient production of empty particles during acidification but whether these play a comparable role to the intermediate and empty particles found during enterovirus cell entry is unclear (Tuthill et al., 2009). Hidden features of the genome sequence – GORS Detailed bioinformatic analysis of RNA virus genomes revealed the presence of genome-scale

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ordered RNA structure (GORS), a previously unrecognized feature inherent in viral genomes (Simmonds et al., 2004). Even more surprising was the comparative level of GORS in different virus groups, even within the same virus family. For example, the GORS score for most picornaviruses is rather modest but this is completely different for FMDVs, which have an extremely high GORS score, indicating a high level of inherent and conserved RNA structure. An interpretation of this phenomenon is that a viral genome with a high GORS score is likely to be degraded to produce small double stranded RNA products within a restricted size range and that these fragments have important interactions with, and evasion of, innate immune mechanisms with infected cells (Witteveldt et al., 2014). Other viruses characterized by a high GORS score, such as hepatitis C virus, are reported to be able to induce a persistent infected state and it has been proposed that this ‘hidden’ feature of the FMDV genome is related to the ability of the virus to induce chronic infection. Functions of the UTRs All picornaviruses have long 5′ untranslated regions (UTRs), largely due to the presence of a large and complicated structure, the internal ribosome entry site (IRES) which enables translation of the viral open reading frame in the absence of a 5′ CAP. However, the 5′UTR of FMDVs is extraordinarily long, comprising approximately 1,300 nucleotides and containing at least five apparently independent domains (Belsham, 2005). The roles of two of these; the IRES (~450 nt) and a stem/ loop involved in templating the uridilylation of VPg and termed the cre (McKnight and Lemon, 1998) or bus (Tiley et al., 2003) are known but the functions of the remaining three domains are still not understood even though their presence has been recognized for many decades. The most 5′ structure is the S-fragment (Sangar et al., 1980), which is a generally well-conserved sequence of up to ~ 400 nt which is predicted to be largely double stranded and comprises a single long stem/loop (Newton et al., 1985). Although usually 350 or more nt in length, some isolates with a significantly shorter S-fragment have been identified (Valdazo-Gonzalez et al., 2013). The function of this extraordinary structure is unknown but by

analogy with enteroviruses, which have a highly structured ‘clover leaf ’ at the 5′ end of the genome, it is predicted that the S-fragment is involved in genome circularization and control of the switch from translation to replication. There is some evidence to suggest that the S-fragment is involved in host species specificity but the structural and functional interactions of this distinctive feature with host and viral proteins and its role in viral replication are subjects of current research. Another striking feature is the poly(C) tract, a contiguous stretch of pyrimidine residues, almost entirely cytidines, that varies in length between different isolates and is in the range of 100–200 residues long. This feature was first recognized in the 1970s but its function is still obscure (Harris and Brown, 1976). Studies in the 1970s also suggested that it may have a role in attenuation but this is still unclear (Harris and Brown, 1977). However, in the cardioviruses, many of which also have a poly(C) tract, there is good evidence for a relationship between poly(C) tract length and pathogenicity in mice. Mengovirus is highly pathogenic in mice but a form from which the poly(C) tract had been deleted was non-pathogenic, even though it grew well in tissues culture (Duke et al., 1990). Clearly there is still a lot to learn about this ubiquitous FMDV feature. The final structural domain found in the 5′ UTR is a string of pseudoknots lying between the poly(C) tract and the cre element (Clarke et al., 1987). Variable numbers of these pseudoknots are found in different isolates of FMDV, each being 43 nt in length. The pseudoknot sequences are precisely deleted/inserted in different viruses (Escarmis et al., 1995), supporting the suggestion that they are genuine structures with functional roles. However, the role(s) of structures is unknown. The 3′ UTR is much shorter than the 5′UTR and terminates in a polyA tail. There are two conserved stem/loops within the heteropolymeric region which clearly have roles in replication of the genome. Interactions between these stem/ loops and sequences within the 5′ UTR have been described, suggesting that they are involved in genome circularization during replication (Serrano et al., 2006). However, the molecular ballet that must accompany the assembly and function of the replication complex and the role of circularization are still far from being understood.

FMDV: Advances and Challenges |  9

Two forms of L The L protease is the most N-terminal domain of the viral polyprotein and cleaves the host protein translation initiation factor eIF4G, so giving the viral RNA a translation advantage over cellular mRNAs. However, there are two forms of Lpro (Lab and La), each arising from a different initiation site and so differing by ~ 28 amino acids at their N-termini. Why this should be is unclear. Virus in which the L protein has been deleted is viable but the nucleic acid stretch between the two natural initiation sites appears to be essential although its sequence is not well conserved (Piccone et al., 1995). Role of 2A The C-terminal extension of VP1, which occupies the genome location of P2A in other picornaviruses, facilitates separation of the polyprotein by a non-proteolytic ‘ribosome skip’ mechanism (Donnelly et al., 2001). It has been suggested that the rationale for this unusual ‘cleavage’ mechanism is that it provides a means for altering the relative levels of translation of the structural proteins and non-structural proteins during infection, i.e. enabling higher levels of production of capsid proteins relative to non-structural proteins at later stages of the infection cycle. There is some evidence to support this for the cardiovirus, Theiler’s virus but whether this applies to FMDV has yet to be demonstrated. 2B and 2C – membrane interactions, helicase function The 2B and 2C proteins are involved in manipulation of the cellular environment to facilitate viral replication in ways that are different from that seen in the enteroviruses but the functional basis for these differences is not known. As with other picornaviruses 2C has an ATPase activity (Rodriguez and Carrasco, 1993) and is a putative helicase. It has been suspected for many years that 2C has an involvement in viral RNA replication as mutations that overcome the RNA replication inhibitory action of guanidine are located in this protein (Saunders and King, 1982). However, how 2C is structurally and functionally aligned with the other components that go to make up the replication complex is not understood for FMDV or any other picornavirus. Genetic evidence has implicated the 2C protein of poliovirus in the RNA encapsidation

process and it will be interesting to learn whether the same is true for FMDV. 3A – species specificity The role of the P3A protein in FMDV replication is not clear but it seems to differ significantly from the equivalent protein in other picornaviruses such as the enteroviruses. It can be found in infected cells alone or as a series of precursor proteins still fused to the downstream 3B, or VPg proteins. Whether these precursor proteins are the donors for uridilylation of the VPg proteins to facilitate their use as primers for initiation of RNA synthesis or whether they serve a separate function is not yet clear. Another poorly understood feature of 3A is its involvement in defining host specificity for the virus. Porcine-tropic strains of FMDV, first identified in Taiwan (Knowles et al., 2001) were found to have significant deletion in 3A. Subsequently it has been shown that deliberate deletion of portions of the 3A can favour growth in porcine cells and inhibit the ability to grow in bovine cells (Pacheco et al., 2003). 3Bs – why 3 (in all but one case so far)? All other known picornaviruses survive perfectly well with a single copy of the primer peptide 3B (VPg) whereas FMDV encodes three almost identical copies arrayed in tandem (Boothroyd et al., 1981; Forss et al., 1984). A small exception to this conserved feature has recently been discovered in a virus isolated from pigs in South Korea which only encodes two copies of VPg. Why does FMDV (almost) invariably encode three copies? All three are used equally in the initiation of viral RNA replication. Are their functions identical? There are suggestions that this might not be the case and VPg3 may have a special role in encapsidation (Arias et al., 2010). However, viruses from which one or even two copies of VPg have been deleted can still replicate in tissue culture cells. The (almost) invariable presence of three copies of VPg implies that there is strong election pressure to maintain this configuration in the genome but we are still some way from understanding why. 3C, 3CD and 3D and RNA replication The functions of 3C as the viral protease and 3D as the basic RNA polymerase enzyme are quite well

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understood. However, the precursor protein, 3CD, appears to improve uridilylation of VPg in in vitro assays, having some catalytic role in the process. There is also uncertainty about the role of specific precursor proteins in the donation of uridilylated VPg to initiate RNA synthesis. There is in vitro evidence for poliovirus suggesting that VPg is donated from the precursor, 3BCD (Pathak et al., 2008), which implies that a complex involving 3D, as the enzymatic workhorse, and 3BCD as the primer donor must form for each RNA replication initiation event. If this scenario holds true for FMDV it might partly explain the rapid growth kinetics of the virus as a single complex assembly process could accomplish three initiation events. An amusing speculation. FMDV replicons – a safer way to study replication Until recently research on the replication of FMDV was restricted to the relatively few facilities world-wide that are licensed to work with live virus. However, in 2009 the regulatory authorities in the UK agreed that FMDV replicons, in which the structural coding regions of the genome are removed and usually replaced with a convenient reporter gene, do not represent an infection hazard. This has paved the way for institutions, such as Universities, outwith the high security virus handling laboratories, to work on replication competent but non-infectious FMDV genomes. This simple regulatory decision should result in a significant expansion of fundamental studies on FMDV replication to the general benefit to the field. Summary FMD is of undiminished importance to global agriculture and is a continual financial threat or burden to countries world-wide, be they endemically infected or disease free. Progress towards controlling the disease more effectively is being made continuously but there is still a long way to go. In addition to the economic importance of FMDV it is a fascinating virus to study from a fundamental science perspective. It has numerous unusual features to its structure and function that distinguish it from most/all other picornaviruses and understanding these will be both intellectually satisfying and provide knowledge that can better

inform approaches to control and eliminate the disease. References Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D., and Brown, F. (1989). The three-dimensional structure of foot-and-mouth disease virus at 2.9 A resolution. Nature 337, 709–716. Alexandersen, S., Zhang, Z., and Donaldson, A.I. (2002). Aspects of the persistence of foot-and-mouth disease virus in animals – the carrier problem. Microbes. Infect. 4, 1099–1110. Anderson, E.C., Doughty, W.J., and Anderson, J. (1974). The effect of repeated vaccination in an enzootic foot-and-mouth disease area on the incidence of virus carrier cattle. J. Hyg. 73, 229–235. Archard, L.C., Bowles, N.E., Cunningham, L., Freeke, C.A., Olsen, E.G., Rose, M.L., Meany, B., Why, H.J., and Richardson, P.J. (1991). Molecular probes for detection of persisting enterovirus infection of human heart and their prognostic value. Eur. Heart J. 12 Suppl D, 56–59. Arias, A., Perales, C., Escarmís, C., and Domingo, E. (2010). Deletion mutants of VPg reveal new cytopathology determinants in a picornavirus. PLOS ONE 5, e10735. Ayebazibwe, C., Mwiine, F.N., Tjørnehøj, K., Balinda, S.N., Muwanika, V.B., Ademun Okurut, A.R., Belsham, G.J., Normann, P., Siegismund, H.R., and Alexandersen, S. (2010). The role of African buffalos (Syncerus caffer) in the maintenance of foot-and-mouth disease in Uganda. BMC. Vet. Res. 6, 54. Baxt, B., and Mason, P.W. (1995). Foot-and-mouth disease virus undergoes restricted replication in macrophage cell cultures following Fc receptor-mediated adsorption. Virology 207, 503–509. Belsham, G.J. (2005). Translation and replication of FMDV RNA. Curr. Top. Microbiol. Immunol. 288, 43–70. Bittle, J.L., Houghten, R.A., Alexander, H., Shinnick, T.M., Sutcliffe, J.G., Lerner, R.A., Rowlands, D.J., and Brown, F. (1982). Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature 298, 30–33. Boothroyd, J.C., Highfield, P.E., Cross, G.A., Rowlands, D.J., Lowe, P.A., Brown, F., and Harris, T.J. (1981). Molecular cloning of foot-and-mouth disease virus genome and nucleotide sequences in the structural protein genes. Nature 290, 800–802. Brocchi, E., Bergmann, I.E., Dekker, A., Paton, D.J., Sammin, D.J., Greiner, M., Grazioli, S., De Simone, F., Yadin, H., Haas, B., et al. (2006). Comparative evaluation of six ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus. Vaccine 24, 6966–6979. Chis Ster, I., Singh, B.K., and Ferguson, N.M. (2009). Epidemiological inference for partially observed epidemics: the example of the 2001 foot-and-mouth epidemic in Great Britain. Epidemics 1, 21–34. Clarke, B.E., Brown, A.L., Currey, K.M., Newton, S.E., Rowlands, D.J., and Carroll, A.R. (1987). Potential secondary and tertiary structure in the genomic RNA of foot-and-mouth disease virus. Nucleic Acids Res. 15, 7067–7079.

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Cottam, E.M., Haydon, D.T., Paton, D.J., Gloster, J., Wilesmith, J.W., Ferris, N.P., Hutchings, G.H., and King, D.P. (2006). Molecular epidemiology of the foot-and-mouth disease virus outbreak in the United Kingdom in 2001. J. Virol. 80, 11274–11282. Cowan, K.M., and Graves, J.H. (1966). A third antigenic component associated with foot-and-mouth disease infection. Virology 30, 528–540. de la Torre, J.C., Davila, M., Sobrino, F., Ortin, J., and Domingo, E. (1985). Establishment of cell lines persistently infected with foot-and-mouth disease virus. Virology 145, 24–35. Donaldson, A.I., Alexandersen, S., Sørensen, J.H., and Mikkelsen, T. (2001). Relative risks of the uncontrollable (airborne) spread of FMD by different species. Vet. Rec. 148, 602–604. Donaldson, A.I., Herniman, K.A., Parker, J., and Sellers, R.F. (1970). Further investigations on the airborne excretion of foot-and-mouth disease virus. J. Hyg. 68, 557–564. Donnelly, M.L., Luke, G., Mehrotra, A., Li, X., Hughes, L.E., Gani, D., and Ryan, M.D. (2001). Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. The J. Gen. Virol. 82, 1013–1025. Duke, G.M., Osorio, J.E., and Palmenberg, A.C. (1990). Attenuation of Mengo virus through genetic engineering of the 5’ non-coding poly(C) tract. Nature 343, 474–476. Escarmís, C., Dopazo, J., Dávila, M., Palma, E.L., and Domingo, E. (1995). Large deletions in the 5’-untranslated region of foot-and-mouth disease virus of serotype C. Virus Res. 35, 155–167. Ferris, N.P., Grazioli, S., Hutchings, G.H., and Brocchi, E. (2011). Validation of a recombinant integrin alphavbeta6/monoclonal antibody based antigen ELISA for the diagnosis of foot-and-mouth disease. J. Virol. Methods 175, 253–260. Forss, S., Strebel, K., Beck, E., and Schaller, H. (1984). Nucleotide sequence and genome organization of foot-and-mouth disease virus. Nucleic Acids Res. 12, 6587–6601. Fowler, V., Bashiruddin, J.B., Belsham, G.J., Stenfeldt, C., Bøtner, A., Knowles, N.J., Bankowski, B., Parida, S., and Barnett, P. (2014). Characteristics of a foot-and-mouth disease virus with a partial VP1 G-H loop deletion in experimentally infected cattle. Vet. Microbiol. 169, 58–66. Fowler, V.L., Bashiruddin, J.B., Maree, F.F., Mutowembwa, P., Bankowski, B., Gibson, D., Cox, S., Knowles, N., and Barnett, P.V. (2011). Foot-and-mouth disease marker vaccine: cattle protection with a partial VP1 G-H loop deleted virus antigen. Vaccine 29, 8405–8411. Harris, T.J., and Brown, F. (1976). The location of the ploy(C) tract in the RNA of foot-and-mouth disease virus. J. Gen. Virol. 33, 493–501. Harris, T.J., and Brown, F. (1977). Biochemical analysis of a virulent and an avirulent strain of foot-and-mouth disease virus. J. Gen. Virol. 34, 87–105. Herrera, M., Grande-Pérez, A., Perales, C., and Domingo, E. (2008). Persistence of foot-and-mouth disease virus in cell culture revisited: implications for contingency in evolution. J. Gen. Virol. 89, 232–244.

Juleff, N., Windsor, M., Reid, E., Seago, J., Zhang, Z., Monaghan, P., Morrison, I.W., and Charleston, B. (2008). Foot-and-mouth disease virus persists in the light zone of germinal centres. PLOS ONE 3, e3434. Juleff, N.D., Maree, F.F., Waters, R., Bengis, R.G., and Charleston, B. (2012). The importance of FMDV localisation in lymphoid tissue. Vet. Immunol. Immunopathol. 148, 145–148. King, D.P., Burman, A., Gold, S., Shaw, A.E., Jackson, T., and Ferris, N.P. (2011). Integrin sub-unit expression in cell cultures used for the diagnosis of foot-and-mouth disease. Vet. Immunol. Immunopathol. 140, 259–265. Knowles, N.J., Davies, P.R., Henry, T., O’Donnell, V., Pacheco, J.M., and Mason, P.W. (2001). Emergence in Asia of foot-and-mouth disease viruses with altered host range: characterization of alterations in the 3A protein. J. Virol. 75, 1551–1556. Kotecha, A., Seago, J., Scott, K., Burman, A., Loureiro, S., Ren, J., Porta, C., Ginn, H.M., Jackson, T., Perez-Martin, E., et al. (2015). Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design. Nat. Struct. Mol. Biol. 22, 788–794. Levy, H.C., Bostina, M., Filman, D.J., and Hogle, J.M. (2010). Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy. J. Virol. 84, 4426–4441. Liu, Y., Wang, C., Mueller, S., Paul, A.V., Wimmer, E., and Jiang, P. (2010). Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis. PLOS Pathog. 6, e1001066. Martín Hernández, A.M., Carrillo, E.C., Sevilla, N., and Domingo, E. (1994). Rapid cell variation can determine the establishment of a persistent viral infection. Proc. Natl. Acad. Sci. U.S.A. 91, 3705–3709. Mason, P.W., Baxt, B., Brown, F., Harber, J., Murdin, A., and Wimmer, E. (1993). Antibody-complexed foot-and-mouth disease virus, but not poliovirus, can infect normally insusceptible cells via the Fc receptor. Virology 192, 568–577. Mattion, N., König, G., Seki, C., Smitsaart, E., Maradei, E., Robiolo, B., Duffy, S., León, E., Piccone, M., Sadir, A., et al. (2004). Reintroduction of foot-and-mouth disease in Argentina: characterisation of the isolates and development of tools for the control and eradication of the disease. Vaccine 22, 4149–4162. McKnight, K.L., and Lemon, S.M. (1998). The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication. RNA 4, 1569–1584. Newton, S.E., Carroll, A.R., Campbell, R.O., Clarke, B.E., and Rowlands, D.J. (1985). The sequence of foot-and-mouth disease virus RNA to the 5’ side of the poly(C) tract. Gene 40, 331–336. Pacheco, J.M., Henry, T.M., O’Donnell, V.K., Gregory, J.B., and Mason, P.W. (2003). Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-and-mouth disease virus. J. Virol. 77, 13017–13027. Pathak, H.B., Oh, H.S., Goodfellow, I.G., Arnold, J.J., and Cameron, C.E. (2008). Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation. J. Biol. Chem. 283, 30677-30688.

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Perez, A.M., Ward, M.P., and Carpenter, T.E. (2004). Control of a foot-and-mouth disease epidemic in Argentina. Prev. Vet. Med. 65, 217–226. Piccone, M.E., Rieder, E., Mason, P.W., and Grubman, M.J. (1995). The foot-and-mouth disease virus leader proteinase gene is not required for viral replication. J. Virol. 69, 5376–5382. Porta, C., Kotecha, A., Burman, A., Jackson, T., Ren, J., Loureiro, S., Jones, I.M., Fry, E.E., Stuart, D.I., and Charleston, B. (2013). Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen. PLOS Pathog. 9, e1003255. Rodriguez, P.L., and Carrasco, L. (1993). Poliovirus protein 2C has ATPase and GTPase activities. J. Biol. Chem. 268, 8105–8110. Sangar, D.V., Black, D.N., Rowlands, D.J., Harris, T.J., and Brown, F. (1980). Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA. J. Virol. 33, 59–68. Saunders, K., and King, A.M. (1982). Guanidine-resistant mutants of aphthovirus induce the synthesis of an altered nonstructural polypeptide, P34. J. Virol. 42, 389–394. Seago, J., Jackson, T., Doel, C., Fry, E., Stuart, D., Harmsen, M.M., Charleston, B., and Juleff, N. (2012). Characterization of epitope-tagged foot-and-mouth disease virus. J. Gen. Virol. 93, 2371–2381. Serrano, P., Pulido, M.R., Sáiz, M., and Martínez-Salas, E. (2006). The 3’ end of the foot-and-mouth disease virus genome establishes two distinct long-range RNA–RNA interactions with the 5’ end region. J. Gen. Virol. 87, 3013–3022. Simmonds, P., Tuplin, A., and Evans, D.J. (2004). Detection of genome-scale ordered RNA structure (GORS) in genomes of positive-stranded RNA viruses: Implications for virus evolution and host persistence. RNA 10, 1337–1351. Sørensen, K.J., Madsen, K.G., Madsen, E.S., Salt, J.S., Nqindi, J., and Mackay, D.K. (1998). Differentiation of infection from vaccination in foot-and-mouth disease

by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus. Arch. Virol. 143, 1461–1476. Sutmoller, P., Cottral, G.E., and McVicar, J.W. (1967). A review of the carrier state in foot-and-mouth disease. Proc. Annu. Meet. U. S. Anim. Health. Assoc. 71, 386–395. Tiley, L., King, A.M., and Belsham, G.J. (2003). The foot-and-mouth disease virus cis-acting replication element (cre) can be complemented in trans within infected cells. J. Virol. 77, 2243–2246. Tuthill, T.J., Harlos, K., Walter, T.S., Knowles, N.J., Groppelli, E., Rowlands, D.J., Stuart, D.I., and Fry, E.E. (2009). Equine rhinitis A virus and its low pH empty particle: clues towards an aphthovirus entry mechanism? PLOS Pathog. 5, e1000620. Uddowla, S., Hollister, J., Pacheco, J.M., Rodriguez, L.L., and Rieder, E. (2012). A safe foot-and-mouth disease vaccine platform with two negative markers for differentiating infected from vaccinated animals. J. Virol. 86, 11675–11685. Valdazo-González, B., Timina, A., Scherbakov, A., Abdul-Hamid, N.F., Knowles, N.J., and King, D.P. (2013). Multiple introductions of serotype O foot-and-mouth disease viruses into East Asia in 2010– 2011. Vet. Res. 44, 76. Witteveldt, J., Blundell, R., Maarleveld, J.J., McFadden, N., Evans, D.J., and Simmonds, P. (2014). The influence of viral RNA secondary structure on interactions with innate host cell defences. Nucleic Acids Res. 42, 3314–3329. Wright, C.F., Knowles, N.J., Di Nardo, A., Paton, D.J., Haydon, D.T., and King, D.P. (2013). Reconstructing the origin and transmission dynamics of the 1967–68 foot-and-mouth disease epidemic in the United Kingdom. Infect. Genet. Evol. 20, 230–238. Yang, P.C., Chu, R.M., Chung, W.B., and Sung, H.T. (1999). Epidemiological characteristics and financial costs of the 1997 foot-and-mouth disease epidemic in Taiwan. Vet. Rec. 145, 731–734.

Genome Organization, Translation and Replication of Foot-and-mouth Disease Virus RNA

2

Encarnación Martinez-Salas and Graham J. Belsham

Abstract Foot-and-mouth disease virus (FMDV), a picornavirus, has a positive-sense RNA genome that encodes a large polyprotein. The intact polyprotein is never observed; it is co- and post-translationally processed, largely by virus-encoded proteases, to produce 15 different mature proteins plus a variety of precursors. The RNA genome also has to act as the template for RNA replication. This process occurs in two stages. Initially, synthesis of negative strands occurs using the positive strand template and then the production of many positive-sense infectious RNAs is achieved from the negative strands. The infectious RNAs are then packaged by the capsid proteins to produce new virus particles. Particular emphasis in this review is placed on the role of distinct RNA structures within the viral genome in the initiation of protein synthesis and in the initiation of RNA replication. Early on in FMDV-infected cells, the synthesis of host cell proteins is inhibited, due to modification of the cellular translation machinery that is induced by a virus-encoded protease. However, viral protein synthesis is maintained under these conditions. Replication of the viral RNA is achieved by the virus encoded RNA-dependent RNA polymerase within replication complexes assembled in the cytoplasm of the cell. Thus both viral RNA and viral protein production are dependent on the functions of virus-encoded proteins and conserved RNA structures. Introduction Foot-and-mouth disease virus (FMDV) is the causative agent of one of the most economically important diseases of farm animals. The virus is

classified within the Aphthovirus genus of the family Picornaviridae. Like all picornaviruses, FMDV has a single stranded positive-sense RNA genome. A single copy of the genome is contained within each virus particle. Each virus consists of a protein shell (capsid) comprising 60 copies of the four different structural proteins 1A (VP4, which is internal), 1B (VP2), 1C (VP3) and 1D (VP1) surrounding the genomic RNA. The FMDV genome is over 8300 nt in length and its structure has certain similarities to eukaryotic cellular mRNAs in that it contains a single, large, open reading frame (ORF) and has a poly(A) tail at its 3′ terminus (see Fig. 2.1). However, in contrast to cellular mRNAs, there is no cap structure (m7GpppN…) at the 5′ terminus of the genomic RNA but a virus-encoded peptide, termed VPg (or 3B), is covalently linked to the terminal nucleotide. The 5′-untranslated region (5′-UTR) of the FMDV RNA is about 1300 nt in length. This is much longer than the 5′UTRs of most cellular mRNAs. The large open reading frame within the RNA encodes a polyprotein. However, the complete polyprotein is never detected since protein processing, largely by virus-encoded proteases, commences during protein synthesis to generate the mature viral proteins required for RNA replication and virus assembly. A notable feature of the FMDV genome is the presence of two different sites on the RNA at which the initiation of protein synthesis occurs. This feature is conserved in all natural strains and leads to the production of two alternative forms of the N-terminal component of the viral polyprotein, the leader (L) protease (see Fig. 2.1), these are commonly termed Lab and Lb. A very important feature of picornavirus RNA is that it is infectious (see, for example, Belsham and Bostock, 1988). Thus, no viral proteins are

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Figure 2.1 Genome organization of FMDV. Important features of the FMDV RNA and its encoded proteins are indicated and described in detail within the text. The FMDV RNA encodes a single polyprotein that is processed mainly by the virus-encoded proteases (Lpro and 3Cpro; the cleavages generated by these proteases are indicated by black arrows) to 15 mature products plus multiple precursors (only some of which are shown). The break in the polyprotein that occurs at the 2A/2B junction is indicated by a green arrow. Abbreviations used are: S, S-fragment; CCC(n), poly(C) tract; PK, pseudoknot; cre, cis-acting replication element; IRES, internal ribosome entry site; L, Leader protein; VPg, virus protein genome-linked; VP1, VP2, VP3 and VP4 are the virus capsid proteins which assemble around a single copy of the RNA to form a virus particle.

required by the viral RNA to initiate the infection cycle. Hence, the virus particle essentially serves to protect the genome while it is outside of cells and to bring about its delivery to the cytoplasm of a cell where a new cycle of infection can be initiated. The entire replication cycle of the virus occurs in the cytoplasm of cells and, indeed, for certain picornaviruses it has been demonstrated that their replication is maintained in enucleated cells (Follett et al., 1975). The viral RNA contains all the information required to allow the virus to take over the cellular machinery. Most picornaviruses induce, within infected cells, the shut-down of nearly all of the host macromolecular synthesis and, hence, only the production of viral RNA and viral proteins is observed. FMDV RNA is replicated with high efficiency within susceptible cells and after a few hours of infection the amount of viral RNA (all genome length) within cells can reach a level similar to that of all the cellular cytoplasmic mRNAs (about 5% of total RNA). The viral RNA is synthesized by the virus encoded RNA-dependent RNA polymerase (3Dpol) (see Fig. 2.1). This process also requires

other viral proteins, e.g. VPg (3B); the latter acts as the primer for RNA synthesis, inside a membraneassociated replication complex that contains several other viral proteins. The infectivity of viral RNA demonstrates that the first phase of the infection cycle has to be translation of the viral RNA so that each of the viral proteins is made within the infected cell. These viral proteins are required for the formation of RNA replication complexes, to modify cellular activities and to generate new virus particles. At some point, it appears that there has to be a switch in the function of the input viral RNA so that translation is blocked and RNA replication can commence. This seems necessary since the process of translation of picornavirus RNA (in which ribosomes move along the RNA in a 5′–3′ direction) is apparently not compatible with the initial step in the replication of the viral RNA that is achieved by the movement of the RNA polymerase from the 3′ end to the 5′ terminus (see Gamarnik and Andino, 1998). It is clear that the distinct processes of protein synthesis, RNA replication and RNA packaging (to form virus particles) need to be regulated and

Translation and Replication of FMDV RNA |  15

possibly differentially localized within the cell. Separating the various activities to different sites within the cell could help to overcome potential incompatibilities between these functions that each require positive-sense RNA and hence may compete. The targets of any signals required for regulation of function must be contained within the sequence of the genome. This chapter will seek to examine the different features of the FMDV genome, which are responsible for directing the production of virus proteins and viral RNA within the infected cell. There is much that remains unknown about these processes and we will seek to identify these areas and point out where relevant information is available from other related viruses. However, it should always be borne in mind that the various members of the picornavirus family are distinct; hence, what is true for one virus may not always apply to others. Several unique features of FMDV are already known and undoubtedly more remain to be identified. The structure and function of the FMDV RNA 5′ UTR As indicated above, the 5′-UTR of FMDV RNA is about 1300nt long. This is significantly larger than most other picornavirus 5′-UTRs, e.g. the 5′-UTR of poliovirus (PV) RNA is about 740 nt in length while the encephalomyocarditis virus (EMCV) RNA 5′-UTR is about 830 nt long. The 5′-UTR of each picornavirus RNA includes sequences required for the initiation of protein synthesis on the viral RNA, for RNA replication and probably for other, currently unknown, functions. The FMDV RNA 5′-UTR can be considered in several distinct regions, including the S-fragment, a poly(C) tract, several pseudoknots and elements termed the cis-acting replication element (cre) and the internal ribosome entry site (IRES) (see Fig. 2.1). The IRES is about 450 nt in length and directs the initiation of protein synthesis on the viral RNA (see Belsham and Sonenberg, 1996; Belsham and Jackson, 2000; Martinez-Salas et al., 2001, 2015; Belsham, 2009), its properties will be discussed in detail below. The cre (about 55 nt in length) is required for FMDV RNA replication (Mason et al., 2002; Nayak et al., 2005, 2006) and will also be discussed further below.

RNA replication elements The S-fragment At the 5′-terminus of the FMDV genome is a region of about 360 nt, termed the S-fragment, which is predicted to form a large hairpin structure (Clarke et al., 1987; Escarmis et al., 1992; Witwer et al., 2001). The S-fragment is the name given to the smaller of the two RNA fragments that are generated when viral RNA is treated with oligo(dG) and RNaseH (Rowlands et al., 1978). This treatment cleaves the RNA within the poly(C) tract and generates the 5′-terminal small (S)-fragment plus the major portion of the genome (the large or L-fragment) that includes the rest of the 5′ UTR, the polyprotein coding region and the 3′ UTR. It is assumed that the S-fragment is required for RNA replication (since initiation of positive strand RNA synthesis will commence at the complement of this sequence on the negative strand). It has been reported that the cellular RNA helicase A (RHA) interacts with the S-fragment and depletion of RHA from cells inhibited FMDV replication (Lawrence and Rieder, 2009). Interestingly the RHA also co-immunoprecipates with FMDV 2C and 3A proteins, these also play a role in RNA replication (see below). Similarly, there is a considerable body of data indicating that both cellular and viral proteins interact with the ‘cloverleaf ’ structure that is located at the 5′ terminus of the PV genome (Gamarnik and Andino, 1997; Parsley et al., 1997). This element (only about 80nt in length) binds to a cellular RNA binding protein termed the poly(rC) binding protein 2 and to the PV 3CD protein (by interaction with sequences within 3C). It has been suggested that they may play a role in the switch from translation to replication (Gamarnik and Andino, 1998). Additionally, these interactions may serve to circularize the RNA (since the poly(A) binding protein can bind to both the 3′ terminal poly(A) tail and the poly(rC) binding protein) which may facilitate replication and/or translation (Herold and Andino, 2001). It is also possible that the presence of proteins at (or near) the RNA termini serves to protect the viral RNA from degradation (Murray et al., 2001; Kempf and Barton, 2008). Further information about the interaction between the 5′ and 3′-termini within FMDV RNA is included in ‘Implications of long-range 5′–3′ viral RNA interactions on IRES activity’.

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The poly(C) tract and pseudoknots On the 3′ side of the S-fragment within FMDV RNA there is a long, almost homopolymeric, stretch of C residues termed the poly(C) tract. This is a common feature of the genomes of FMDV and most cardioviruses (e.g. EMCV, but not Theiler’s murine encephalitis virus). There is considerable variation in the length of this poly(C) tract amongst different strains of FMDV, from about 80 nt to about 420 nt. In general, the shorter tracts are found in laboratory strains and typically field strains have a tract of about 200 nt (Brown et al., 1974; Harris and Brown, 1977). However, the largest poly(C) tract identified was found in a strain of FMDV (VR 100) recovered from persistently infected cells in culture (Escarmis et al., 1992) but the significance of the size of this homopolymeric sequence is not clear. The VR 100 strain has been described as hypervirulent in BHK-21 cells (de la Torre et al., 1988) but it is attenuated for growth in mice and cattle (Diez et al., 1990). However, since the VR100 genome is different from the parental virus at about 80 positions (1% of the genome), and these changes occur all across the genome including within the IRES region (Martinez-Salas et al., 1993), it is not clear which changes (including those within the long poly(C) tract) are responsible for the different phenotypes. There is a strong selection pressure for the presence of the poly(C) tract within the viral RNA. When virus was recovered from infectious RNA transcripts, derived from plasmids that initially contained as few as 6 C residues at this location, it was found that the length of the poly(C) tract in the replicated RNA had increased to about 80 nt or more (Rieder et al., 1993). In contrast, RNA transcripts containing just two C residues at the location of the poly(C) tract did not regenerate a poly(C) tract but these rescued viruses grew fairly poorly in tissue culture cells. However, each of the rescued viruses retained virulence in mice irrespective of the length of the poly(C) tract (Rieder et al., 1993). The instability in the length of this tract in FMDV RNA is in marked contrast to observations made using mengovirus RNA (Duke et al., 1989, 1990). Modified mengoviruses, with differently sized poly(C) tracts, were rescued in tissue culture and their virulence was determined in mice. The viruses with short poly(C) tracts were greatly attenuated but remarkably no selection for viruses with enlarged tracts occurred within these

animals (Duke et al., 1990). Surprisingly, the role of the poly(C) tract in EMCV, another cardioviruslike mengovirus, appears quite different (Hahn and Palmenberg, 1995). Mutant EMC viruses, containing very short poly(C) tracts, were about as pathogenic in animals as wt EMCV and replicated at very similar rates in cells as judged by single-step growth curves. Between the poly(C) tract and the IRES element in FMDV RNA there is another stretch of sequence, about 250 nt in length, that is believed to contain multiple pseudoknots. Different FMDV strains have been predicted to contain 2–4 pseudoknots (Clarke et al., 1987; Escarmis et al., 1995). Interestingly the cardiovirus RNAs are predicted to contain multiple pseudoknots located on the 5′ side of their poly(C) tract rather than on the 3′ side as in FMDV RNA (Martin and Palmenberg, 1996). It may be that the pseudoknots are involved, in some manner, in a joint function with the poly(C) tract. A cis-acting replication element (cre) In addition to sequences at the termini of the viral RNA, it has been shown that internal RNA sequences are required for RNA replication. One such feature has been termed a ‘cis-acting replication element’ (cre). The initial characterization of cre structures within picornavirus RNAs resulted from the observation that a region within the P1-coding sequence was required to achieve efficient replication of replicons based on human rhinovirus-14 (HRV-14) (McKnight and Lemon, 1996). This contrasts with studies on the closely related PV RNA that have demonstrated that the entire capsid coding sequence can be deleted without affecting RNA replication (Barclay et al., 1998). Further work (McKnight and Lemon, 1998) showed that HRV-14 RNA replication required a specific stem– loop structure located within the coding region for the capsid protein 1D (VP1). This structure was termed a ‘cre’ and subsequently, analogous elements have been identified in other picornavirus genomes (e.g. Lobert et al., 1999, Goodfellow et al., 2000; Gerber et al., 2001). The cre elements from HRV-14, HRV-2, cardioviruses and PV are within the coding regions for 1D, 2A, 1B and 2C respectively. However, these cre structures can be moved without blocking function. Interestingly, Mason et al. (2002) provided functional evidence for a cre, located within the 5′-UTR of FMDV, just upstream

Translation and Replication of FMDV RNA |  17

of the IRES (see Fig. 2.1). Each of the cre structures defined within the entero-, rhino-, cardio- and aphthovirus RNAs include a conserved sequence motif, AAACA, located within a loop at the top of a stable stem-structure (Rieder et al., 2000; Gerber et al., 2001; Mason et al., 2002). It has been demonstrated that insertion of the wt FMDV cre into the 3′ UTR could restore replication ability to an RNA transcript containing a mutated (and defective) cre within its 5′UTR (Mason et al., 2002). It was shown that the PV and HRV-2 cre sequences act as a template for the uridylylation of VPg (3B) by the viral RNA polymerase in vitro (Paul et al., 2000; Gerber et al., 2001) and similar observations have been reported for the analogous FMDV structure (Nayak et al., 2005, 2006). This reaction generates the products VPgpU and/or VPgpUpU that act as primers for viral RNA synthesis. Thus, it is believed that this process is an essential first step in RNA replication. Each of the three distinct FMDV VPg peptides can take part in this reaction in vitro (Nayak et al., 2005), consistent with the use of each form of VPg within infected cells (King et al., 1980). The reaction, in vitro, requires a number of components; in addition to the VPg and the 3Dpol, it is necessary to include an RNA template including the cre and the 3CD precursor protein (Nayak et al., 2005). The role of the 3CD can be achieved by 3C alone, albeit less efficiently, and modification of specific residues within 3C, that are involved in RNA binding, strongly inhibited VPg uridylylation activity in vitro and also blocked virus replication within cells (Nayak et al., 2006). When the cre structure in FMDV RNA was identified it provided an explanation for some results that had demonstrated the presence of a temperature-sensitive (ts) mutation within the 5′-UTR of FMDV RNA. This mutation, located within the stem–loop structure that constitutes the cre, destabilized the structure. A non-ts revertant virus has a compensating mutation within the cre that restores the stability of the stem (Tiley et al., 2003). An important feature of this ts mutant is that the defect in replication can be complemented in trans by other ts FMDVs (with defects elsewhere in the genome). This result seems compatible with the earlier observations that a pool of free VPgpUpU is generated within picornavirus-infected cells (Crawford and Baltimore, 1983). Thus, the

term ‘cre’ (cis-acting replication element) may not be entirely appropriate, at least for the FMDV element, and an alternative title of 3B-uridylyaltion site (bus) was proposed (Tiley et al., 2003) but the original terminology has persisted. The IRES element – structure and function Translation of FMDV RNA is initiated internally, under the control of a sequence known as the IRES (Belsham and Brangwyn, 1990; Kuhn et al., 1990; Martinez-Salas et al., 1993). The FMDV IRES consists of a highly structured region, located several hundred nucleotides away from the uncapped 5′ end of the genomic RNA (see Fig. 2.1). Initiation of translation mediated by IRES elements represents an alternative to the cap-dependent translation initiation mechanism used for most cellular mRNAs. The 5′ end of all cytoplasmic eukaryotic mRNAs has a cap structure (m7GpppN…) which plays a crucial role during translation initiation, it is recognized by the initiation factor eIF4F (Gingras et al., 1999; Hershey and Merrick, 2000; Sonenberg and Hinnebush, 2009). This heterotrimer comprises the translation initiation factors eIF4E (that binds to the cap), eIF4A (an RNA helicase) and eIF4G. The latter component has distinct binding sites not only for eIF4E and eIF4A but also for other proteins including eIF3 (located on the small ribosomal subunit), the poly(A) binding protein (PABP) and Mnk - 1 (an eIF4E kinase). It is generally believed that the small ribosomal subunit interacts with the eIF4F complex (bound at the 5′ cap) and then migrates with it along the 5′ UTR in a 5′ to 3′ direction. The presence of eIF4A is thought to facilitate the unwinding of RNA secondary structure (see Svitkin et al., 2001a). This migration is called scanning and continues until an AUG codon in an appropriate context (as defined by Kozak, 1989) is encountered. At this point, the ribosome pauses, the large ribosomal subunit joins and polypeptide synthesis can commence. The ability of the scanning process to recognize the correct initiation codon can be inhibited by complex RNA structure and by the presence of additional AUG codons. In contrast, internal initiation of translation mediated by an IRES involves the direct recruitment of the translational machinery to an internal position in the mRNA usually with the help of cellular

18  | Martinez-Salas and Belsham

trans-acting factors. This process may, or may not, be followed by ribosome scanning (see below). IRES elements were first identified within picornavirus RNAs (Pelletier and Sonenberg, 1988; Jang et al., 1988). Consistent with their role in picornavirus translation, IRES-dependent translation initiation can bypass stress conditions that are inhibitory for cap-dependent translation initiation (Hellen and Sarnow, 2001; Martinez-Salas et al., 2001, 2008). Such conditions occur following eIF4G cleavage (Gradi et al., 1998) that is induced during picornavirus infection as a consequence of the action of viral proteases Lb (FMDV) or 2A (poliovirus), or due to the dephosphorylation of the translational repressor 4E-BP1 in EMCVinfected cells (Gingras et al., 1996). The element in the viral RNA that constitutes the IRES is believed to adopt a three-dimensional structure that is essential for internal initiation of translation. However, IRES elements belonging to the picornavirus family do not show extensive primary sequence conservation. Two major groups of picornavirus IRES structures were initially described, from the entero-/rhinoviruses and from the cardio-/aphthoviruses (Belsham and Jackson, 2000). Neither of these types of IRES has any apparent similarity to that of hepatitis C virus (HCV) (Honda et al., 1999). However, it has become apparent that at least five different types of IRES element are present within the incessantly growing family of picornaviruses (see reviews by Belsham, 2009; Hellen and de Breyne, 2007; Martinez-Salas et al., 2015). One type, initially identified within the porcine teschoviruses (Kaku et al., 2002; Pisarev et al., 2004; Chard et al., 2006a) is closely related to the HCV IRES and has now been identified in multiple genera of picornaviruses (Chard et al., 2006b, Bakhshesh et al., 2008; Belsham, 2009; Hellen and de Breyne, 2007). The absence of apparent structural conservation between the different types of IRES element may reflect the diversity of strategies used by IRES elements to interact with the translational machinery. However, it is noteworthy that the IRES elements from FMDV and PV, albeit of different types, each interact directly with eIF4G (López de Quinto and Martinez-Salas, 2000; Stassinopoulos and Belsham, 2001; de Breyne et al., 2009). We discuss here the features of the RNA structure of the FMDV IRES that are known to be functionally relevant and

compare them to the structural features described for other IRES elements. Structure and function of the FMDV IRES element RNA virus genomes are reservoirs of structural elements that perform specific steps in the viral replication cycle, as illustrated by 3′ cap-independent translation enhancers (3′CITE), tRNA-like structures (TLS), internal ribosome entry site elements (IRES), cis-acting replication elements (cre), etc. (Paul and Wimmer, 2015; Simon, 2015). In particular, IRES elements adopt specialized threedimensional structures, which act in concert with host factors to recruit the translation machinery internally on the RNA using a mechanism that is independent of the 5′-terminus. However, IRESs belonging to different groups of RNA viruses lack conservation of primary sequence and secondary RNA structure; they also differ in their host factor requirements to recruit the translation machinery. The sequences of picornavirus IRESs are generally longer than other viral IRESs, and their secondary structure is more heterogeneous (Martinez-Salas et al., 2008). According to their secondary structures, picornavirus IRESs are classified into five different types (Sweeney et al., 2012). Furthermore, while some motifs are found in various picornavirus IRESs, others are exclusive for one type suggesting a specialized requirement for factors (MartinezSalas et al., 2015). In spite of the diversity of primary sequence, each type of picornavirus IRES harbours conserved sequence motifs and a common RNA structure core maintained by covariant substitutions. Evolutionary conserved motifs tend to preserve both RNA structures and RNA–protein interactions that determine the activity of the IRES element (Lozano and Martinez-Salas, 2015). Compelling evidence for the relevance of RNA structures for IRES function was initially provided by functional studies. Indeed, mutations disrupting certain stems impaired IRES activity and, conversely, compensatory mutations restored IRES function (Fernandez et al., 2011a; Haller and Semler, 1992; Haller et al., 1996; Hoffman and Palmenberg, 1996; MartinezSalas et al., 1993, 1996; Serrano et al., 2007, 2009; van der Velden et al., 1995; Witherell et al., 1995). The RNA structure of the enterovirus IRES is organized in modular domains (designated II to

Translation and Replication of FMDV RNA |  19

VI). These IRES elements span about 450 nt in which conserved pyrimidine tracts occur at the base of domain II, and in the central and basal part of domain V (Bailey and Tapprich, 2007). Domain IV harbours a C-rich loop and a GNRA motif (N stands for any nucleotide, and R for purine) (Du et al., 2004). Domain V provides the binding site for the polypyrimidine tract-binding protein (PTB), which partially overlaps with the binding sites for eIF4G and eIF4A during 48S complex assembly (Sweeney et al., 2014). The 3′-border of

the enterovirus IRES harbours the Yn-Xm-AUG motif in which Yn (a pyrimidine tract of 8–10 nt) is separated by a spacer (Xm, 10–20 nt) from an AUG triplet. This motif has been proposed to be the ribosome entry site, which in turn is separated from the functional initiator codon by a long non-conserved spacer, which is scanned by the initiation complex (Hellen et al., 1994). The RNA structure of the cardio- and aphthovirus IRES is arranged in modular domains (designated H to L, or 1 to 5) (see Fig. 2.2). Uniquely

Figure 2.2  Schematic representation of the RNA structure of the FMDV IRES with indication of the binding sites for host-factors. The secondary structure model was predicted by RNAstructure software imposing the values of SHAPE reactivity. Nucleotides are coloured according to their SHAPE reactivity (grey  0.5–1; dark red >1). The IRES domains (2 to 5, or H to L) are depicted at the bottom. Stem-loops (SL) and junctions (J) referred to in the text are shown in blue, whilst conserved RNA motifs are depicted in black letters. The sites on the IRES that interact with eIF4G, eIF4B, PTB, PCBP2 and Gemin5 are described in the text. The first functional AUG (AUG1) is boxed.

20  | Martinez-Salas and Belsham

for FMDV RNA, domain 1 of the IRES is located just downstream of the cre element (Mason et al., 2002). Domain 2 contains a conserved pyrimidine tract that provides a binding site for PTB ( Jang and Wimmer, 1990; Luz and Beck, 1991). Domain 3 is a self-folding cruciform structure (FernandezMiragall and Martinez-Salas, 2003) that harbours three conserved motifs (GNRA, RAAA, and the C-rich loop) (Fernandez-Miragall et al., 2009). The GNRA motif is essential for IRES activity in both FMDV and EMCV (Lopez de Quinto and Martinez-Salas, 1997; Robertson et al., 1999). Domain 4 consists of a Y-shape RNA structure (containing subdomains J and K) and provides the binding-site for eIF4G (Bassili et al., 2004; Kolupaeva et al., 1998; Lopez de Quinto and Martinez-Salas, 2000; Stassinopoulos and Belsham, 2001; Clark et al., 2003). Finally, domain 5 consists of a short hairpin with a conserved pyrimidine tract at its 3′ end that provides the binding site for eIF4B, PTB, and other RNA-binding proteins (Lopez de Quinto et al., 2001; Meyer et al., 1995; Pacheco et al., 2008). Overall, the data derived from functional analyses are in agreement with the conservation of structural motifs in highly variable viral genomes (Lozano and Martinez-Salas, 2015; Martinez-Salas, 2008). Likewise, the lack of accessibility towards RNases was in accordance with the stems defined by covariation data (Fernandez et al., 2011a). More recently, selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) reactivity data also supported a modular organization of the IRES element (Fig. 2.2), and reinforced the evidence for the localization of loops and internal bulges within the IRES structure in solution (Fernandez et al., 2011b; Lozano et al., 2014, 2016b). Incorporation of SHAPE reactivities as constraints to RNA structure software (Reuter and Mathews, 2010) generates RNA structure models, which are in agreement with the data for the RNA accessibility to DMS and RNases T1 and T2 (Fernandez-Miragall et al., 2009). Concerning the relevance of RNA structure for IRES function, the local RNA flexibility of specific IRES regions is sensitive to the concentration of Mg2+ ions in the RNA folding buffer (Lozano et al., 2014). Noteworthy, the apical loop of domain 3 is more flexible at a near physiological concentration (0.5 mM) of Mg2+ rather than at high concentration

(6 mM). Differences in RNA conformation are found in the hexaloop of SL3a, the loop of SL3b and the J1 junction that links SL1 with SL2 (see Fig. 2.2). Besides the higher flexibility of the apical region of domain 3 at low Mg2+ concentration, it is also worth noting that the interaction of the protein eIF4G with the FMDV IRES is impaired at high Mg2+ concentration (Lozano et al., 2014), in agreement with earlier reports showing that concentrations of Mg2+ above 5 mM inhibit IRESdriven protein synthesis (Shenvi et al., 2005). Thus, it appears that IRES activity is permissive at low Mg2+ concentration, coincident with a more flexible RNA conformation. Although the IRES of EMCV and FMDV differ in primary sequence (ca. 50% identity), they have a similar secondary structure. In both cases, domain 3 consists of two differentiated structural regions, located in the basal and the apical parts of this domain. In the FMDV IRES, the apical region of domain 3 harbours stem–loops designated SL1 (absent in EMCV IRES), SL2 and SL3abc. In addition to the previously mentioned conserved motifs GNRA and RAAA, the hexaloop of SL3a and the C-rich bulge (heptaloop) of SL2 are also conserved in both the EMCV and FMDV IRES elements (Lozano et al., 2016a). Mutational analysis carried out on SL1 and SL3b of the FMDV IRES showed that disruption of the stems reduced IRES activity whereas compensatory mutations restoring the secondary structure recovered translation (Fernandez et al., 2011a, 2013). Disruption of the SL3b stem abolishes IRES activity, and induces a reorganization of the entire apical region (Fernandez et al., 2011a). Based on the observations of inter- and intramolecular RNA–RNA interactions (Ramos and Martinez-Salas, 1999) it has been proposed that domain 3 plays a fundamental role in dictating the formation of a constrained structure, likely providing the correct orientation to recruit the ribosome subunits to the initiation site (Martinez-Salas, 2008). A notable combination of structural motifs is found in this domain. The GNRA tetraloop motif is frequently found in folded RNAs (Correll and Swinger, 2003); the loop–helix interactions combine base pairing and stacking to determine a tertiary conformation that stabilizes the global RNA folding. The GNRA motif adopts a tetraloop conformation in both FMDV and EMCV IRESs

Translation and Replication of FMDV RNA |  21

(Dupont and Snoussi, 2009; Fernandez-Miragall and Martinez-Salas, 2003; Phelan et al., 2004). It was proposed that the GNRA motif determines the folding of the apical region of domain 3, since its substitution by a stable UNCG tetraloop disrupts IRES function (Fernandez-Miragall and MartinezSalas, 2003). Additionally, structural analysis of GNRA substitution mutants (GUAA to GUAG, or to UCCG) showed a specific modification of the accessibility towards DMS and RNase T1 in the distant residue G240, together with a change in the local accessibility of the GNRA motif and a reorganization of SL3b. Conversely, substitution mutants affecting the G240 position induced reciprocal changes in the GNRA motif. These results led to the proposal of the existence of a potential GNRA receptor within the distant C-rich loop (Fernandez-Miragall et al., 2006). Computational modelling of domain 3 has been achieved by a divide-and-conquer approach using the secondary structure determined by mutational analysis and RNA probing to model RNA junction topology ( Jung and Schlick, 2013). Candidates for three-dimensional models generated using MC-Sym (Parisien and Major, 2008) subjected to molecular dynamics (MD) simulations identified energetically favourable and stable conformational states compatible with the GNRA tetraloop–receptor interactions (Fernandez-Miragall et al., 2006), in which the adenosines A180 and A181 form hydrogen bonds with the receptors C230/G242 and G231/ C241 base pair, respectively. A similar tertiary interaction was proposed to occur in the EMCV IRES using a synthetic 16mer harbouring the GCGA tetraloop and a 17mer corresponding to the C-rich heptaloop (Mohammed et al., 2014). The GCGA tetraloop of the 16mer folds into a standard GNRA conformation (Correll and Swinger, 2003), with the A residue being in the form of a G: A sheared base-pair. The C-rich loop of the 17-mer forms a compact tertiary loop motif, in which Mg2+ ions enhance RNA stability. Furthermore, stable RNA structures were generated using synthetic oligoribonucleotides that combine the GNRA tetraloop and the C-rich heptaloop of the EMCV IRES in 28-, 36- 44- and 48-mers as building blocks (Chan and Ramesh, 2012). In the dynamic simulations of the FMDV IRES, SL3a forms an L-shape configuration ( Jung and Schlick, 2014). Two four-way RNA junctions, J1

and J2, within the apical region of domain 3 connect the GNRA motif of SL3a and its potential receptor in SL2. Molecular dynamics simulation that explored the conformational variability in the J1 junction revealed transitions between parallel and antiparallel conformations via a perpendicular intermediate that maintains the coaxial stacks, providing a modular structural platform that can be adjusted by the binding of cofactors and ligands. Computational modelling of the entire domain 3 suggests that the basal region is set apart from the apical region ( Jung and Schlick, 2014). However, biochemical RNA probing of a transcript bearing solely this region showed a higher accessibility to residues 120–133 within an internal bulge (Fernandez-Miragall et al., 2006), suggesting that the stability and the global structure of the entire domain depends on the apical region. Host factors critical for picornavirus IRES activity Picornavirus IRES-dependent translation initiation bypasses stress situations that compromise cap-dependent translation initiation that occurs in FMDV-infected cells. Under normal situations, however, most eukaryotic mRNAs initiate translation by a mechanism that depends on the recognition of the m7GpppN residue (or cap) located at the 5′ end of most mRNAs (Sonenberg and Hinnebusch, 2009). Many cellular translation initiation factors (eIFs) play an essential role in IRES-dependent initiation promoted by the FMDV and EMCV IRES elements (reviewed by Belsham and Jackson, 2000; Martinez-Salas et al., 2001). The canonical initiation factors eIF4A, eIF4G, eIF2 and eIF3, are each required for 48S complex formation in a reconstituted 40S ribosome-binding assay with the EMCV or FMDV IRES elements (Pestova et al., 1996; Kolupaeva et al., 1998; Pilipenko et al., 2000). In particular, reconstitution assays have shown that assembly of 48S initiation complexes onto the FMDV IRES, extended through to the second AUG, requires eIF4A, eIF1, eIF3, and the C-terminal fragment of eIF4G resulting from the proteolytic cleavage induced by the L protease (Andreev et al., 2007). The region of eIF4G that interacts with the EMCV and FMDV IRES (residues 630–1560) contains the binding sites for eIF3 and eIF4A (Gingras et al., 1999). Accordingly, eIF4A stimulates binding of the central part

22  | Martinez-Salas and Belsham

of eIF4GI (amino acids 746–949) to the EMCV and FMDV IRES (Pilipenko et al., 2000; Lomakin et al., 2000). Consistent with the interaction of the eIF4G-Ct with these picornavirus IRES elements, internal initiation of translation promoted by these elements is highly efficient under conditions of eIF4G cleavage (Belsham and Brangwyn, 1990; Martinez-Salas et al., 1993; Ohlmann et al., 1996; Roberts et al., 1998; López de Quinto and Martinez-Salas, 2000). A transcript corresponding to FMDV domain 4 alone binds to eIF4G (see Fig. 2.2) as effectively as the full length IRES, strongly suggesting that no additional sites for recognition of this protein within the FMDV IRES are required. Furthermore, the carboxy-terminal end of the proteolytically processed form of eIF4G (eIF4G-Ct), present in cells transfected with the FMDV Lb protease, binds as efficiently to the FMDV IRES as the unprocessed protein (López de Quinto and Martinez-Salas, 2000). Fully consistent with this result, the use of RNA transcripts (ca. 150 nt) from the 3′ end of the FMDV IRES to deplete RRL extracts lead to a strong reduction of factors required for cap-dependent translation, including eIF4G (Stassinopoulos and Belsham, 2001). Disruption of the structural motif at the base of domain 4 is associated with the lack of binding of eIF4G to the FMDV IRES (López de Quinto and MartinezSalas, 2000). Consistent with this observation, eIF4G interacts with the related EMCV IRES in a similar position (Kolupaeva et al., 1998), and additional nucleotides within the J-domain have also been implicated in this interaction (Kolupaeva et al., 2003; Clark et al., 2003). The second species of eIF4G, termed eIF4GII, is also cleaved by the FMDV encoded Lb protease in vitro (López de Quinto et al., 2001) and within FMDV-infected cells (Gradi et al., 2004). The C-terminal fragment (Ct) also has a strong binding affinity for the FMDV IRES, suggesting its involvement in IRES activity. Interestingly, a fragment of the central region of eIF4GII interacts with the EMCV IRES through a non-canonical RRM region (Marcotrigiano et al., 2001). The strong correlation found between the eIF4G–IRES interaction and IRES activity in transfected cells demonstrates that eIF4G binding is an essential step in the recruitment of the translational machinery in vivo. Consistent with these results, it has been shown that dominant

negative mutants of eIF4A (which interfere with eIF4F function) block picornavirus IRES activity (Pause et al., 1994; Pestova et al., 1998; Svitkin et al., 2001a). The 3′ region of the FMDV IRES has been shown to interact with the eIF4B initiation factor (Meyer et al., 1995; López de Quinto and MartinezSalas, 2000; Stassinopoulos and Belsham, 2001). Sequence comparison of field isolates of FMDV showed that domain 5 contains a conserved hairpin (see Fernandez-Charmorro et al., 2016) that is also shared with the EMCV IRES and a high-affinity ligand for eIF4B that was selected in vitro (Methot et al., 1996; López de Quinto et al., 2001). However, substitution of conserved residues within the hairpin structure did not impair FMDV IRES activity to the same extent as the mutations introduced into the A-bulge of domain 4, or the GNRA loop of domain 3. In spite of the strong binding capacity of the FMDV IRES for eIF4B, mutants impaired in eIF4B-binding only reduce IRES activity two- to fourfold (López de Quinto et al., 2001). In agreement with this, the eIF4B initiation factor only stimulates 48S complex formation on the EMCV IRES about two-fold (Pestova et al., 1996). In the early studies on IRES elements, a number of proteins were found to associate with picornavirus IRES elements, e.g. PTB and La (reviewed in Belsham and Sonenberg, 1996, 2000). Identification of these proteins as non-canonical initiation factors was particularly interesting since it seemed possible they could contribute to IRES tropism in different cell types, and hence determine viral spread within an infected animal. Interestingly, a protein named unr (upstream of N-ras) is unique in its capacity to interact with the rhinovirus and PV IRES elements (Hunt et al., 1999; Boussadia et al., 2003) whereas PTB interacts with all IRES elements tested (Luz and Beck, 1991; Hunt and Jackson, 1999). PTB (also known as hnRNP I) was the first protein reported as an ITAF that stimulated the activity of cardio- and aphthovirus IRES elements ( Jang and Wimmer, 1990; Luz and Beck, 1991). This protein harbours four RNA recognition motifs (RRM) that recognize U/C-rich sequences (Conte et al., 2000). During early studies of the picornavirus IRES elements, it was noticed that a polypyrimidine tract was the only motif conserved between rhino-/enterovirus IRES elements and the cardio-/aphthovirus IRES elements (Meerovitch et

Translation and Replication of FMDV RNA |  23

al., 1991; Pilipenko, et al., 1992; Meerovitch and Sonenberg, 1993). Both, FMDV and EMCV have two polypyrimidine tracts located at each end of the IRES region. In agreement with the functional role of each pyrimidine tract on IRES activity it has been found that PTB constrains the EMCV IRES structure in a unique orientation by binding to the RNA with RRM1 – 2 contacting the 3′ end, and RRM3 contacting the 5′ end of the IRES (Kafasla et al., 2009). In contrast to the Theiler’s murine encephalitis virus (TMEV) and EMCV IRES elements, the FMDV IRES required binding to PTB and to the proliferation associated factor, ITAF45 (also known as Ebp1 or PA2G4), for 48S complex formation in vitro (Pilipenko et al., 2000). Therefore, even closely related IRES elements (i.e. from EMCV and FMDV) that share secondary structure and primary sequence in essential regions behave differently in terms of functional RNA–protein association. Other members of the hnRNP family that have been identified to associate with picornavirus IRESs are hnRNP K, PCBP1 (hnRNP E1) and PCBP2 (hnRNP E2) (Gamarnik et al., 2000; Lin et al., 2008; Sean et al., 2009). These proteins recognize poly-r(C) regions and share the KH RNA-binding domain. However, although PCBP2 binds to both EMCV and FMDV IRES, this factor only stimulates the first one (Walter et al., 1999), and is the only ITAF absolutely required to assemble 48S initiation complexes on enterovirus IRESs using purified components (Sweeney et al., 2014). Known IRES interacting proteins (unr, PTB, La, ITAF45 and PCBP2) contain several RNA binding motifs, and display multiple sites of interaction with the IRES molecule (Blyn et al., 1997; Hunt et al., 1999; Pilipenko et al., 2000; Conte et al., 2000; Kim et al., 2000). Hence, it has been suggested that these proteins act as RNA chaperones, directing or stabilizing the tertiary folding of the RNA. The EMCV and FMDV IRES elements seem to possess a modular organization, in which the different domains perform a precise function but none of them is active on its own. The 3′ region (domains J–K or 4–5) mediates the interaction with eIFs required for 48S complex formation in vitro (Kolupaeva et al., 1998; Pilipenko et al., 2000). This region establishes RNA–eIF4G interactions, which are essential for IRES activity in vivo (López

de Quinto and Martinez-Salas, 2000; López de Quinto et al., 2001, Stassinopoulos and Belsham, 2001). The 5′ and central regions (domains 1–2 and 3, or H and I) are probably involved in the organization of the IRES architecture, directing intramolecular RNA–RNA interactions (Ramos and Martinez-Salas, 1999). In agreement with this, the PTB protein that seems to act as an RNA chaperone has its main binding site near the 5′ end of the IRES but it also interacts with sequences near the 3′ end (Kolupaeva et al., 1996; Luz and Beck, 1991). In addition to eIFs, several RNA-binding proteins designated IRES-transacting factors (ITAFs), stimulate the assembly of 48S complexes in vitro on the cardio- and aphthovirus IRESs (Pilipenko et al., 2000; Pilipenko et al., 2001; Yu et al., 2011). ITAFs are RNA binding proteins identified previously as factors involved in transcription regulation, splicing, RNA transport, RNA stability, or translational control. Consistent with the fact that the entire picornavirus replication cycle occurs in the cell cytoplasm, many ITAFs are nuclear proteins that shuttle to the cytoplasm in infected cells. In this way, factors that normally participate in nuclear events associated with cellular RNA metabolism become relocalized and participate in translation modulation of viral RNAs lacking nuclear localization (Fitzgerald and Semler, 2011). Although early studies identified ITAFs as proteins stimulating IRES activity, various examples of IRES downregulators have also been found in later studies. For instance, the cellular mRNA decay protein AU-binding factor (AUF1) behaves as a negative regulator of EV71 and HRV infections (Cathcart et al., 2013). Another example of a repressor ITAF is Gemin5 (see Fig. 2.2), a cytoplasmic protein that binds directly to the FMDV IRES (Pineiro et al., 2013) and down-regulates translation (Pacheco et al., 2009). An additional feature of many ITAFs is their recognition as substrates of picornavirusencoded proteases. In some cases, this leads to the generation of truncated peptides with a different capacity to modulate translation than the uncleaved polypeptide. Accordingly, proteolysis of PTB in PV-infected cells results in truncated polypeptides that repress IRES activity (Back et al., 2002), and proteolysis of Gemin5 in FMDV-infected cells (Pineiro et al., 2012) leads to the appearance of C-terminal fragments. These fragments, which

24  | Martinez-Salas and Belsham

contain the IRES-repressor element on the most distal domain, harbour a noncanonical bipartite RNA-binding motif (RBS1–RBS2) (FernandezChamorro et al., 2014). In contrast, the FBP2 fragment lacking the C-terminal region behaves as an IRES stimulator (Chen et al., 2013). In summary, most ITAFs are proteins that shuttle between the nucleus and the cytoplasm, and in many cases are targets for viral proteases or undergo post-translational modifications leading to the reprogramming of gene expression within infected cells. Small molecules perturbing the local flexibility of domain 3 inhibit IRES activity Since RNA structure plays a critical role in the function of regulatory elements controlling expression of viral proteins, understanding the three-dimensional structure could help the development of antiviral drugs that inhibit virus multiplication. A variety of small molecules targeting specific RNA motifs essential for picornavirus multiplication have been explored as antiviral agents (de la Torre et al., 1987; Fajardo et al., 2012; Stone et al., 2008; Vagnozzi et al., 2007). Moreover, benzimidazole derivatives rearrange the SLIIa structural element of HCV-like IRESs, including those of picornaviruses (Boerneke et al., 2014). Similarly, a novel benzimidazole compound (IRAB) resulted in a negative effect on both the HCV and FMDV IRES activities (Lozano et al., 2015), suggesting some structural similarities between these distinct IRESs. Interestingly, following incubation of the FMDV IRES transcript with IRAB, SHAPE probing revealed changes in the local flexibility of SL3A. This involved residues of the hexaloop and the GNRA motif, in addition to SL3C and SL1, while residues located at the junction J1 of the C-rich loop were partially protected. Additionally, use of aminopurine-labelled oligoribonucleotides showed that the SL3A alone is enough to support binding to this compound (Lozano et al., 2015). However, fluorescence data in conjunction with SHAPE reactivity strongly support the hypothesis that the entire apical region of domain 3 may be important for IRAB binding, again supporting the idea that the entire apical region of domain 3 is a structural entity (Fernandez-Miragall and Martinez-Salas, 2003; Fernandez-Miragall et al., 2006). Therefore,

the RNA conformational changes induced by IRAB could result in a misfolded IRES structure leading to diminished internal initiation of translation. Implications of long-range 5′–3′ viral RNA interactions on IRES activity The 3′UTR of the picornavirus genome plays a critical role in virus multiplication (Duque and Palmenberg, 2001; Melchers et al., 1997; Saiz et al., 2001; Todd et al., 1997a). This region harbours a heterogeneous sequence, in addition to a polyA tail. An active role for interactions between the 5′ and 3′ ends of the viral RNA in controlling picornavirus translation was supported by the fact that the activity of the FMDV IRES was stimulated by its own 3′UTR (Garcia-Nunez et al., 2014; Lopez de Quinto et al., 2002), irrespective of the presence of a polyA tail and the coexpression of the L protease. In addition to RBPs, 5′–3′ end bridges involve direct long-distance RNA–RNA contacts (Serrano et al., 2006), presumably leading to a pseudocircularization of the viral RNA. It is worth noting that the 3′end region, which is essential for FMDV infectivity (Saiz et al., 2001), can mediate two different long-range interactions (Serrano et al., 2006), one with the IRES element and another with the S hairpin at the 5′end. These long-range interactions, in concert with the corresponding protein partners, provide a mechanistic basis for the stimulation of cap-independent translation of picornavirus RNAs resembling the synergistic stimulation of the cellular mRNA cap-dependent translation by the cap and polyA tail. The polyprotein coding region The major portion (ca. 7000 nt) of the viral genome comprises the coding sequence for a polyprotein of about 2330 amino acids. The length of the coding region can be increased to some extent; Seago et al. (2013) showed that insertions of up to 300nt within the coding region could be accommodated by the virus but inserts of 400 nt or more were unstable. The encoded polyprotein is never observed within infected cells or within in vitro translation systems (e.g. rabbit reticulocyte lysate, RRL) since this protein is rapidly processed by virus-encoded proteases that are present within the polyprotein sequence (see Chapter 3). A large variety of precursors can be generated by alternative cleavage pathways (see

Translation and Replication of FMDV RNA |  25

Fig. 2.1) and some of these precursors, as well as the mature products, may have distinct biological roles. The FMDV polyprotein yields 15 different mature proteins including the two forms of the Leader (L) protein and three different copies of VPg (3B1–3). The function of some of these products is the subject of other chapters within this volume and we will consequently give only a brief outline of these and thus will concentrate on other issues. Selection of the initiation site for protein synthesis The sequencing of FMDV RNA (Forss et al., 1984; Carroll et al., 1984) indicated that two in-frame AUG codons, 84 nt apart, were present in the virus genome that could potentially act as initiation codons. This feature is conserved across all seven serotypes of the virus (Sangar et al., 1987). Each initiation codon is preceded by a polypyrimidine tract; the arrangement of a polypyrimidine tract followed by an AUG codon is a general feature at the 3′ end of picornavirus IRES elements (Meerovitch and Sonenberg, 1993). On FMDV RNA both AUG codons are used as initiation sites for protein synthesis, generating two forms of the Leader (L) protein, termed Lab and Lb. Studies on EMCV RNA demonstrated that ribosomes initiated translation at AUG-11 on the EMCV-R strain RNA without encountering AUG-10 located just 8 nt upstream (Kaminski et al., 1990). The Lab start site on FMDV RNA is located at the same position relative to the IRES element as AUG-11. However, only a minor fraction of the ribosomes initiate protein synthesis at this point since Lb is made in excess over Lab in infected cells (Lopez de Quinto and Martinez-Salas, 1999). To determine the mechanism of recognition of the two start sites on FMDV RNA, Belsham (1992) analysed the usage of these start sites under a variety of conditions. RNA transcripts including the two FMDV initiation codons, either preceded by the FMDV IRES element or not, were expressed within cells and it was observed that both the Lab and Lb start sites were used on each RNA. The Lb start site was recognized preferentially in both cases and the presence of the IRES resulted in greater usage of the Lb site than occurred when the initiation codons were recognized by scanning ribosomes. When two additional AUG codons were introduced in-frame between the authentic initiation

codons, it was found that the 4 AUG codons were each recognized, independently of whether translation initiation occurred by an IRES-dependent or cap-dependent mechanism. Thus, the 84-nt region between the two natural initiation codons has unusual features. The most likely explanation given for the results was that the FMDV IRES directed ribosomes to bind initially to the RNA just upstream of the Lab start site, as for EMCV. The recognition of this first start site on FMDV RNA must be inefficient, however, so that many ribosomes can then scan downstream until the next initiation codon is encountered (Belsham, 1992). Subsequently, Cao et al. (1995) demonstrated that the Lab initiation codon was not required for virus viability but found that modification of the Lb initiation codon was lethal. A requirement for the Lb start site was also observed by Piccone et al. (1995a), who constructed viable mutants of FMDV that lacked the Lb protein coding region but found that transcripts lacking the Lb start site were noninfectious. To further analyze the initiation site, López de Quinto and Martinez-Salas (1999) modified the context of the Lab start site and showed that improving the context did increase the efficiency of translation initiation at this site. However, this had no apparent effect on the efficiency of initiation at the Lb start site. Hence, it was suggested that these two initiation codons could act independently. This view was supported by the observation that an antisense oligonucleotide, which annealed to the region of the Lab start site, blocked initiation at this site but had little effect on initiation at the Lb site. Thus, it may be that some ribosomes are directed by the FMDV IRES to bind to the RNA downstream of the Lab start site. However, this putative entry site must presumably be close to the Lab start site since the additional AUG codons introduced just 18 nt downstream of the Lab site were efficiently recognized during IRES-directed translation initiation (Belsham, 1992). Reconstitution studies using purified components have shown a differential requirement of eIF1 and eIF1A, depending on whether the 48S initiation complex is assembled at AUG1 or AUG2 using the FMDV IRES extended to the second AUG (Andreev et al., 2007), suggesting the existence of two distinct mechanisms operating at each of these codons. In summary, the FMDV IRES may direct ribosome attachment to the viral RNA either just

26  | Martinez-Salas and Belsham

upstream or just downstream of the Lab initiation site. Ribosomes landing upstream of the Lab site can initiate protein synthesis at this point but some may fail to do so and can then scan along the RNA until the Lb site is reached. However, ribosomes that land downstream of the Lab site can just migrate along the RNA to initiate translation at the Lb site. Different picornavirus IRES elements may direct ribosomes to the initiation codon with different degrees of precision (Ohlmann and Jackson, 1999). To analyse this process, chimeras (fused at the polypyrimidine tract) were made between the FMDV IRES and a region of the EMCV RNA around the initiation codon (AUG-11). It was shown that the FMDV IRES directed utilization of AUG-11 (the correct initiation site) and AUG-12 (12 bases downstream) with similar efficiency in vitro. In contrast, the EMCV IRES linked to its own RNA directed almost exclusive use of AUG-11. Furthermore, in the converse experiment, when the EMCV IRES was linked to the FMDV initiation codons, the Lab site was predominantly used, in contrast to the preferential use of the Lb site on natural FMDV RNA. The leader protease As indicated above, the use of two alternative initiation codons on the FMDV RNA results in the generation of two distinct forms of the Leader protein. Unusually amongst the picornaviruses, the Leader (L) protein of aphthoviruses, including FMDV and equine rhinitis A virus, is a protease (Strebel and Beck, 1986; Hinton et al., 2002). The L protease is a member of the papain-like cysteine proteases and is unrelated to other picornavirus protease sequences. The active site residues have been identified (Roberts and Belsham, 1995; Piccone et al., 1995b) and the 3D structure of FMDV Lb has been determined (Guarne et al., 1998). Both the FMDV Lab and Lb proteins have been shown to cleave the L/P1 junction within the polyprotein (Fig. 2.1), thus liberating the L protease from the rest of the molecule (Medina et al., 1993). The protease can work in trans and probably also in cis (see Glaser et al., 2001). Both forms of the L protease also induce the cleavage of the translation initiation factor eIF4G (Devaney et al., 1988; Medina et al., 1993), a component of the cap-binding complex eIF4F (see above).

Indeed both species of eIF4G, termed eIF4GI and eIF4GII (products of different genes), are cleaved in the presence of the L protease (see below). The consequence of this cleavage is the inhibition of cap-dependent translation initiation and hence the loss of nearly all host cell protein synthesis. The cleavage of eIF4G requires a very low level of the L protease and can be observed to occur within an hour of addition of virus to cells (Belsham et al., 2000) and hence well before any virus-encoded proteins can be detected directly. Indeed, replication-incompetent FMDV RNA transcripts that include the L coding sequence can still induce efficient eIF4G cleavage when they are introduced into cells by electroporation, even though protein can only be produced from the input RNA (Belsham et al., 2000). A third activity of the L protease is to stimulate the activity of certain picornavirus IRES elements (Borman et al., 1997; Roberts et al., 1998; Hinton et al., 2002). It is not known whether this effect is a direct consequence of the cleavage of eIF4G or whether the L protease also induces the cleavage of other proteins that modify IRES function [see Belsham and Jackson (2000) for review and below]. Recombinant Lb protease can cleave isolated eIF4GI directly in vitro. A cleavage site has been identified on the C-terminal side of residue 674 (Kirchweger et al., 1994), which is close to that determined for the in vitro cleavage of eIF4GI by the rhinovirus 2A protease (C-terminal side of residue 681; Lamphear et al. (1993). (The locations of these cleavage sites are numbered according to the system of Byrd et al. (2002) for the largest form of eIF4GI, termed eIF4GIa; multiple initiation codons are used resulting in multiple forms of eIF4GI.) However, in both cases, high levels of the proteases are required to achieve efficient eIF4G cleavage in vitro compared to the very low levels of the proteases that are required within cells. Hence, there are legitimate concerns about whether the cleavage of eIF4G induced by the FMDV L and rhinovirus 2A proteases within cells is a direct effect or is mediated through cellular protease(s) [see Belsham and Jackson (2000) for review]. It is noteworthy that the addition of eIF4E to eIF4G greatly enhances the efficiency of cleavage of eIF4G by the 2A protease in vitro; this may suggest that the conformation of the protein is important (Haghighat et al., 1996). Evidence has recently been presented

Translation and Replication of FMDV RNA |  27

that FMDV Lb only forms a stable complex with a fragment of eIF4GII in the presence of eIF4E (Aumayr et al., 2015) and the HRV2 2A protease also forms a similar trimeric complex. The identification of the cleavage site in eIF4G that is generated by low levels of the Leader protease within FMDV-infected cells remains to be achieved. Such analyses should help to resolve the issue of whether the FMDV L induced cleavage of eIF4G is achieved directly or not. The L protease is not essential for virus viability since the complete Lb coding sequence can be removed (Piccone et al., 1995a). This leaderless FMDV (lacking Lb) was attenuated in cattle (Brown et al., 1996); however, proof that the attenuation was reversed by re-introduction of the missing sequence was not obtained. In contrast to the deletion of Lb, it is not possible to delete the whole of the Lab coding sequence and retain infectivity. However, the 84 nt region between the two start sites (Belsham, 2013) can be deleted if the Lb coding region is still present. Thus all of the Lab coding sequence can be deleted but not all at the same time. The basis for this observation is not yet clear and is discussed further below. It may be that the primary advantage to the virus of inducing the inhibition of host cell protein synthesis lies in reducing the ability of the cell to mount an antiviral response. It has been shown that the mRNAs encoding the alpha/beta interferons are induced within FMDV-infected cells, presumably this is a response to the presence of dsRNA (Chinsangaram et al., 1999; Kaufman, 2000). The shut-off of protein synthesis induced by the L protease will inhibit synthesis of the alpha/ beta interferons within infected cells and hence reduce the cellular antiviral response (Chinsangaram et al., 1999). Interferons trigger a variety of responses within cells, these responses include an activation of the dsRNA-activated protein kinase (PKR, see review by Kaufman, 2000). This kinase phosphorylates the α-subunit of eIF2 and hence blocks the initiation step of both host and viral protein synthesis. Thus, this process will result in a reduction in virus production within infected cells. The inability to block this host response could explain, at least in part, the apparent attenuation of the leaderless FMDV. A comparison of gene expression within cells infected with wild type or leaderless FMDV found that 39 out of

about 22,000 transcripts tested were up-regulated by at least two-fold in the cells infected with the leaderless virus (Zhu et al., 2010) compared to the wt virus. Most of the apparently up-regulated genes were known interferon-inducible genes. However, a potential problem with such studies is that the leaderless viruses grow less well than the wt virus in some cells (Chinsangaram, et al., 1999; de los Santos et al., 2007; Belsham, 2013) and hence it is difficult to ensure that the cells infected with the different viruses are in the same stage of infection. This problem was well illustrated by the apparent difference in the degradation of the p65/relA subunit of NFκB in wt and leaderless virus infected cells (de los Santos et al., 2007); although less degradation was apparent, there was also much less expression of viral protein (VP1). Hence, the direct link between the L protease and loss of p65/relA in FMDV-infected cells was not clearly established. No cleavage products of p65/ relA have been reported within FMDV-infected cells. There is also a single report indicating that the FMDV L protease has an additional means of blocking the host interferon response by acting as a deubiquitinase (Wang et al., 2011). Several components of the interferon signalling pathway are ubiquitinated within cells and expression of the L protease appears to block this modification. However, it can be difficult to separate out some of the different effects of the L protease into direct and indirect effects (e.g. due to loss of host cell protein synthesis) and there is a need to isolate mutants of the protease which are clearly deficient in only individual activities of this protein. As indicated above, two forms of the L protein are generated within infected cells, precise deletion of Lb is tolerated by the virus (Piccone et al., 1995a) but deletion of the entire Lab coding sequence is not tolerated by the virus (see also Belsham, 2013). The function of the 84 nt between the two initiation sites is not entirely clear. Recent studies have shown that insertion of a 57 nt transposon or an epitope tag within this ‘spacer’ region can be tolerated but resulted in attenuation of the virus, whilst a 51 nt deletion did not adversely affect the growth of the virus in cell culture or the virulence of the virus in cattle (Piccone et al., 2010, 2011). In cell culture, a virus which retains this 84 nt sequence with both functional initiation codons but lacks the Lb

28  | Martinez-Salas and Belsham

region adapts rapidly during growth. The changes that occurred meant that initiation at the Lab start site does not add additional amino acid sequences onto the P1–2A precursor (Belsham, 2013). Precise deletion of the entire region between the two initiation sites can be tolerated when the Lb coding region is retained (Belsham, 2013). It may be that this region influences the efficiency of translation initiation under different conditions. When the virus initially infects the cell, the RNA has to function in competition with the host mRNAs using the intact cap-binding complex (eIF4F, including eIF4G, eIF4A and eIF4E). However, once the L protein is produced eIF4G is cleaved very rapidly (Belsham et al., 2000); then, the capped host cell mRNAs are no longer used by the translation machinery. Deletion of the Lb coding sequence from the viral genome means that the viral RNA has to continue to be translated under conditions similar to those encountered at the beginning of the infection cycle. Perhaps, under these conditions, there is a more efficient utilization of the RNA for translation if the sequence immediately downstream of the Lab initiation codon is present. Hence, loss of this ‘spacer’ sequence, under conditions of maximal competition with host mRNAs, may make the RNA sufficiently inefficient that it proves lethal (see Belsham, 2013). It has now been demonstrated that the L protease is able to induce the cleavage of the cellular protein Gemin5 (Pineiro et al., 2012). Binding of Gemin5 to the FMDV and the HCV IRES elements inhibits their activity (Pacheco et al., 2009). This protein, which was identified earlier as a component of the SMN complex, has the capacity to interact directly with the 3′end region of the FMDV IRES (Piñeiro et al., 2013) via a non-canonical RNA-binding site located in its C-terminal domain (FernandezChamorro et al., 2014). The proteolytic fragments of this protein, however, differ not only in their capacity to modulate IRES activity, but also in their stability within FMDV-infected cells. While the larger C-terminal fragment (p85) binds to the IRES and slightly stimulates translation, the short distal C-terminal fragment harbouring the RBS2 domain, is unstable in infected cells and harbours the repressor domain (Fernandez-Chamorro et al., 2104; Piñeiro et al., 2015). Thus the cleavage of the protein can be expected to enhance the IRES function.

The capsid precursor P1–2A The removal of the L protein from the N-terminus of the polyprotein reveals an N-terminal glycine residue on the P1–2A capsid precursor (see Fig. 2.1) that is present within the motif (GXXXS/T) required for recognition of proteins by the cellular myristoylation machinery. This modification is a common (but not universal) feature of the picornavirus capsid proteins. The addition of the C18 lipid to the N-terminus of 1A (VP4) is important for the assembly and/or stability of the FMDV and PV capsids (Chow et al., 1987; Abrams et al., 1995). The processing of the FMDV P1–2A precursor to 1AB (VP0), 1C (VP3) and 1D (VP1) is achieved by the 3C protease (see below) and these products can self-assemble into empty capsid particles (Abrams et al., 1995; Porta et al., 2013; Gullberg et al., 2013). The cleavage of 1AB to 1A and 1B normally occurs on encapsidation of the RNA genome but the precise mechanism of this cleavage is not known. There is some evidence that VP0 cleavage can occur within assembled empty capsid particles (Curry et al., 1995; Gullberg et al., 2013, 2014). The structure and properties of the FMDV capsid, including its ability to interact specifically with cellular receptors, are discussed elsewhere in this volume (see Chapter 4). The generation of the P1–2A precursor clearly requires the loss of any linkage to the 2B protein and this process is mediated by the 2A sequence. However, the 2A sequence in FMDV is very short, just 18 amino acids. It seems unlikely that such a short peptide could possess protease activity in its own right. This peptide sequence is closely related to the C-terminal region of the cardiovirus 2A protein (note that the cardiovirus 2A proteins are much larger but do not contain any motifs characteristic of known proteases either). It has been proposed that the FMDV 2A sequence may prevent the formation of the peptide bond at the 2A/2B junction rather than acting as a peptidase to break a bond that has been formed (see Chapter 3). Recent evidence indicates that the cleavage of the 1D/2A junction by the 3C protease is not essential for capsid assembly or virus infectivity (Gullberg et al., 2013, 2014). Thus infectious ‘2A-tagged’ virus particles containing VP1–2A can be produced. Cleavage of the VP1/2A junction in the P1–2A precursor is the last step in the capsid precursor processing (as indicated by the presence of

Translation and Replication of FMDV RNA |  29

VP1–2A when no other precursors remain; Ryan et al., 1989; Gullberg et al., 2013, 2014). Surprisingly, using in vitro assays with short peptide substrates, the cleavage of this site is the most efficient (Birtley et al., 2005). The P2 proteins The FMDV 2BC precursor is processed to 2B and 2C by the 3C protease (Fig. 2.1). Rather little is known about these P2 proteins. Within PV-infected cells, the P2 proteins are found within membraneassociated viral replication complexes (Bienz et al., 1987, 1990). However, their functions are also rather poorly understood. Different members of the picornavirus family have very different 2B proteins, the FMDV and EMCV 2B proteins show low levels of identity to enterovirus 2B proteins (Moffat et al., 2005; de Jong et al., 2008), hence it is not clear that they share functional activities. The enterovirus 2B protein can increase cell permeability, as judged by the susceptibility to the translation inhibitor hygromycin B (van Kuppeveld et al., 1997). However some mutants within 2B affect cell growth without affecting either of these functions, hence other activities for the 2B protein may still need to be defined (van Kuppeveld et al., 1997). The studies of de Jong et al. (2008) indicated that enterovirus 2B proteins affect calcium homeostasis and intracellular protein trafficking. It has been demonstrated that the FMDV 2BC precursor is able to inhibit trafficking of proteins to the cell surface (Moffat et al., 2005). This activity has been associated with the 3A protein of other picornaviruses (Doedens and Kirkegaard, 1995) but the 3A of FMDV does not show this activity. The function of FMDV 2BC was not obtained with the expression of either 2B or 2C alone but could be reproduced by the dual expression of both proteins (Moffat et al., 2007). It is believed that this effect on protein trafficking may block the presentation of viral proteins on the cell surface to the host immune system. The various picornavirus 2C protein sequences contain helicase and nucleotide binding motifs but until very recently no evidence for RNA helicase activity has ever been reported. However, Xia et al (2015) have now reported that the 2C protein of human EV71 has both RNA helicase and RNA chaperone activity; no doubt this will encourage

investigations of these activities in other picornaviruses. The 2C protein is the locus that determines the sensitivity of viral RNA replication to the inhibitor, guanidine (Saunders and King, 1982). Different strains of FMDV vary in their sensitivity to this agent. Single amino acid substitutions within the protein are sufficient to confer resistance to the inhibitor (Pariente et al., 2003; Belsham and Normann, 2008). The precise role of 2C in RNA replication is not established. Biochemical studies on the FMDV 2C protein, expressed in E. coli, have shown that the protein forms hexameric structures in vitro in the presence of ATP and RNA (Sweeney et al., 2010). These properties are characteristic of AAA + ATPases. The P3 proteins The FMDV P3 precursor (Fig. 2.1) is processed by the 3C protease to 3A, the three distinct copies of the 3B peptide (VPg1, VPg2 and VPg3), the 3C protease and the 3D RNA polymerase plus various intermediates (e.g. 3CD). The 3A protein has hydrophobic sequences that serve to anchor it to membranes and this may be the means by which RNA replication is localized to membrane vesicles. It is thought that the 3A protein also serves to deliver the 3B peptides to the sites of RNA replication. Various strains of FMDV have been isolated that contain in-frame deletions within the 3A coding sequence and these strains are attenuated in cattle (O’Donnell et al., 2001) but remain pathogenic in pigs. This presumably reflects some differences in the interaction of 3A with cellular factors between the two species. Expression of FMDV 3A alone disrupted the Golgi apparatus of keratinocytes. It is interesting to note that some picornaviruses that disrupt Golgi function, e.g. PV, are extremely sensitive to the effect of brefeldin A (Maynell et al., 1992). However, in contrast, it has been observed that FMDV is rather insensitive to this agent (O’Donnell et al., 2001), like EMCV (Iruzun et al., 1992). Brefeldin A modifies vesicle transport between the Golgi and the endoplasmic reticulum, and the different sensitivities of picornaviruses to this agent suggest that the different viruses recruit membranes to their replication complexes in different ways. The PV 3A protein has also been shown to block protein secretion (Doedens and Kirkegaard, 1995) whereas the 3A protein of FMDV does not have this activity (Moffat et al., 2005).

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Uniquely, FMDV RNA encodes three similar but distinguishable copies of the 3B (VPg) peptide. The coding sequences for these different peptides occur as a tandem array in the genome (see Fig. 2.1). Priming of RNA synthesis requires 3B (VPg) and this explains the linkage of this peptide to the 5′ terminus of both positive- and negative-sense RNA strands. As with the PV and HRV cre elements, the initial modification of VPg to VPgpU or VPgpUpU is achieved by the use of the ‘cre’ as a template (Paul et al., 2000; Gerber et al., 2001; Nayak et al., 2005). Each of the FMDV VPgs has been found attached to genomic RNA and thus each peptide is believed to be functionally equivalent (King et al., 1980) and indeed each functions in the in vitro uridylylation assay (Nayak et al., 2005). Even the closely related equine rhinitis A virus, the only member of the Aphthovirus genus other than FMDV, only has a single copy of this peptide. Using mutagenesis, deletion of one or more FMDV 3B sequences can be achieved but the mutant viruses replicate less efficiently than the wt virus (Falk et al., 1992). Indeed, deletion of 3B3 alone destroyed virus viability but this appeared to result from a defect in polyprotein processing rather than a direct effect on RNA replication. However, overall, it is not clear why the presence of three different VPg sequences within the FMDV RNA enhances its replication efficiency (Falk et al., 1992). The 3C protease The FMDV 3C protease is responsible for most of the cleavages within the polyprotein coding sequence. It functions alone and, in contrast to the PV 3C protease (see Ypma-Wong et al., 1988), it does not require 3D sequences for any of its processing activities. The key catalytic residues of the FMDV 3C have been identified (Grubman et al., 1995) and the protease is a member of the chymotrypsin-like family of serine proteases (except that the active serine is replaced by a cysteine; see Chapter 3). The 3D structure of the 3C protease has been determined (Birtley et al., 2005). In addition to cleaving the viral polyprotein it has been found that the FMDV 3C also modifies certain cellular proteins. The histone H3 was shown to be cleaved by this protease (Falk et al., 1990) and subsequently it has been shown that the 3C protease also cleaves the translation initiation factors eIF4A and eIF4GI within FMDV-infected cells (Belsham et al., 2000).

The precise location of the cleavage site generated by the 3C protease in eIF4AI has been identified (Li et al., 2001). The cleavage is specific for eIF4AI, since the closely related eIF4AII (92% identical) is not modified. It has been suggested that the cleavage of eIF4AI, which will inactivate the protein (Li et al., 2001), may contribute to the decrease in the level of viral protein synthesis during the later phase of virus infection (Belsham et al., 2000). As indicated above, the 3C protease also cleaves eIF4GI in some cells, e.g. BHK. This modification occurs on the C-terminal side of the site generated by the expression of the L protease (Belsham et al., 2000). This result is consistent with the loss of intact eIF4GI within BHK cells infected by the leaderless FMDV (Piccone et al., 1995a; Belsham et al., 2000). Sequential cleavage of eIF4GI within FMDVinfected BHK cells by the L protease and then of the C-terminal cleavage product by the 3C protease has been observed, both cleavages are complete by 3 hours post infection (Strong and Belsham, 2004). Thus at the time of peak viral protein synthesis the form of eIF4GI that supports IRES function is that generated by FMDV 3C. Later on during infection, further modification of eIF4GI, probably also generated by FMDV 3C, occurs and these cleavages may contribute to the decline in viral protein synthesis. It was found that human eIF4GI is not susceptible to cleavage by FMDV 3C (Strong and Belsham, 2004) and this observation facilitated the identification of the precise cleavage site for 3C within eIF4GI. It has been shown that a single amino acid substitution (Pro in human and Thr in BHK eIF4GI) at residue 713 is sufficient to modify the sensitivity of the host protein to 3C (Strong and Belsham, 2004). In addition to its proteolytic activity, FMDV 3C also has RNA binding activity. This probably accounts for the requirement of 3CD (or 3C itself) within the in vitro uridylylation assay (Nayak et al., 2006). Three specific basic residues involved in this interaction have been identified and modification of each of them individually either inhibits or blocks virus replication. These residues are located on the opposite face of the molecule from the catalytic site (Nayak et al., 2006). The 3D RNA polymerase The 3D protein is the RNA-dependent RNA polymerase. The replication of picornavirus RNA

Translation and Replication of FMDV RNA |  31

has two distinct aspects to it. To replicate the positive-sense genome, an antisense RNA has to be synthesized which then functions as the template for the production of new positive-sense infectious genomes (see Fig. 2.3). Within infected cells, a large excess of positive strands accumulates over negative strands. Presumably this reflects a differential recognition of the negative-sense template over the positive strand template by the RNA polymerase and/or differences in the stability of the transcripts. The 3′ terminus of the positive sense RNA (the poly(A) tail) and the negative-sense RNA template (antisense S-fragment) are very different in sequence. Thus, the nature of the recognition process by the RNA polymerase is clearly complex but is not yet defined. There are many different poly(A) containing mRNAs within the cell so presumably recognition of the positive strand FMDV RNA must include sequences away from the immediate 3′-terminus. The structure of the FMDV RNA polymerase has been determined by X-ray crystallography (Ferrer-Orta et al., 2004, 2006). Like other RNA polymerases, it folds into characteristic fingers, palm and thumb subdomains, with the presence of

an NH2-terminal segment that encircles the active site (see Chapter 6). The enzyme has to perform multiple functions; it not only has to polymerize nucleotides to make the RNA strands (both positive and negative) but it also has to perform the uridylylation of VPg. Thus, it has a diverse set of substrates to recognize. Interestingly the PV 3D molecule displays cooperativity in its polymerase activity in vitro (Pata et al., 1995) and may normally function as a multimer. The crystal structure of the PV 3D polymerase showed that there is considerable interaction between adjacent molecules in the crystal (Hobson et al., 2001). Whether these properties are relevant to the activity of the protein within an infected cell remains to be determined. The precursor 3CD does not have RNA polymerase activity but it does have RNA binding and protease activity. The PV 3CD product has been shown to bind to the cloverleaf structure at the 5′ terminus of the PV genome (Gamarnik and Andino, 1998) and it is also required in the cre-dependent VPg uridylylation assay (Paul et al., 2000; Rieder et al., 2000). Similarly, the FMDV 3CD is also required (in addition to 3D) for the uridylyation of

Figure 2.3  Distinct roles for positive-sense FMDV RNA. Following the entry of FMDV RNA into the cytoplasm of the cell, the RNA has to be translated to generate the viral proteins required for viral RNA replication, protein processing, capsid assembly and opposing host defence mechanisms. Newly synthesized positive sense (+) RNA genomes can be used in translation, RNA replication or packaged into new virions as illustrated. The negative sense (-) RNA is only used as a template for the synthesis of new positive strand RNAs.

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VPg (Nayak et al., 2005) but this requirement can be met, albeit less efficiently, by 3C alone (Nayak et al., 2006). PV 3CD is the protease required for the processing of the PV P1 capsid precursor whereas the PV 3C alone can process the P2 proteins (Ypma-Wong et al., 1988). In contrast, there seems to be no specific requirement for the FMDV 3CD protein in FMDV polyprotein processing. The FMDV 3C protease is sufficient to process the P1–2A capsid precursor (see, for example, Gullberg et al., 2013, 2014). Structure and function of the FMDV 3′UTR The 3′ untranslated region of FMDV RNA consists of two components, a region of about 100 nt of heterogeneous sequence and the poly(A) tail. The poly(A) tail of picornavirus RNA is included within the genome of the virus, this contrasts with cellular mRNAs in which this sequence is added as a post-transcriptional modification. There is rather little information about the role of the FMDV RNA 3′ UTR except that deletion of the unique (heterogeneous) sequence blocks infectivity (Saiz et al, 2001). It has been shown that the FMDV 3′ UTR sequence can stimulate the activity of the FMDV IRES (Lopez-de Quinto et al., 2002); furthermore, this effect is independent of the stimulation of IRES activity by poly(A) observed previously (Svitkin et al., 2001b). These results suggest further possible RNA–RNA interactions between the 5′ and 3′ UTRs or additional ‘bridging’ RNA–protein interactions. There is evidence for complex RNA–RNA interactions within the heterogeneous sequence of the PV and HRV 3′ UTRs (Mirmomeni et al., 1997; Melchers et al., 1997) and also for protein interactions with this element (Mellits et al., 1998). However, surprisingly, it has also been shown that the unique 3′ UTR sequence of PV RNA can be deleted without loss of virus viability (Todd et al., 1997) albeit that the virus replicated significantly less well than wt virus. In contrast, studies on the 3′ UTR of cardiovirus RNA, have shown evidence for three stem–loop structures, one of which is essential for virus viability (Duque and Palmenberg, 2001). It is possible that other sequences within the coding sequence (but towards the 3′ end of the

genome) are required to provide the specificity of PV RNA recognition by the replication machinery that would seem to be required, especially by the mutants lacking the usual 3′ UTR sequences. Studies have shown that the length of the poly(A) tail has an effect on the infectivity of PV RNA (Spector and Baltimore, 1974). This observation may reflect modification of the stability of the RNA or possibly a requirement for interactions between the 3′ and 5′ termini of the RNA potentially involving the poly(A) binding protein (PABP) that binds optimally to a sequence of at least 25 A residues. It should be noted that PABP interacts both with the translation initiation factor eIF4G (that binds directly to the FMDV IRES element, see above) and with the poly(rC) binding protein 2 that itself binds to the FMDV IRES (Stassinopoulos and Belsham, 2001) and presumably to the poly(C) tract. As indicated above, such interactions may serve to ‘circularize’ the RNA and it may be that such interactions affect the efficiency of RNA translation, at least in the early stages of infection prior to cleavage of eIF4G (see Svitkin et al., 2001b). Cleavage of eIF4G by the FMDV proteases can be expected to disrupt the circularization of the RNA but also removes competition from the cellular capped mRNAs for the translation machinery. It has been suggested that circularization of the PV RNA is required for RNA replication and is supposed to be achieved through the interaction of the poly(A) binding protein (bound to the 3′ poly(A) tail) with the poly(rC) binding protein that binds to the cloverleaf structure at the 5′ end of the genome (Herold and Andino, 2001). However, it is not entirely clear whether this would be achieved within the context of a cell containing many different polyadenylated mRNAs, each of which may have the poly(A) binding protein associated with it. As mentioned above, the cellular protein RHA interacts with the FMDV S-fragment (Lawrence and Rieder, 2009) and this protein co-immunoprecipitates with 2C and 3A which are components of the replication complex. Thus, there is a link between the S-fragment and the RNA replication mechanism. Furthermore, if the S-fragment does exist in a stable stem–loop structure, as predicted, then the 5′ end of the RNA would be close to the poly(C) tract which is likely to be bound to the poly(rC) binding protein, potentially achieving the same effect as proposed for the PV cloverleaf.

Translation and Replication of FMDV RNA |  33

Unsolved issues There are many questions about picornavirus biology that remain to be answered and some of these issues are outlined below. Interactions of viral proteins with cellular components The interactions of the IRES elements with the cellular protein synthesis machinery provides a good insight into the complexity of virus–host cell interactions but we are still a long way from understanding the mechanism of IRES action. It is apparent that cellular proteins that interact with virus components can determine virus tropism. There are obvious examples, such as receptor molecules on the cell surface, but clearly any cellular component that is required for the virus to replicate (either for RNA replication or protein production) may vary in its availability within different cell types. Hence, such factors may determine the competence of the cell to act as a host for the virus. These issues are important since they can determine the pathogenicity of the virus. The differential sensitivity of cattle and pigs to FMDVs with deletions within the 3A protein (O’Donnell et al., 2001) is a good example of an unexplained host dependent determinant of virus replication. Compartmentalization Molla et al. (1991) described a system for the in vitro synthesis of infectious PV using HeLa cell lysates. This system allowed the requirements for virus assembly and RNA replication to be analysed. No equivalent system has been reported for FMDV but Svitkin and Sonenberg (2003) have described an in vitro replication system for EMCV and this holds out the possibility of achieving this with FMDV RNA. The ability to produce infectious virus within a cell-free system does suggest that the requirement for compartmentalization of activities is limited. However, there are soluble and membrane-associated environments in this system and the membranes may create a protected environment. RNA packaging No specific packaging signals have been identified in any picornavirus RNA. It has been suggested that PV RNA synthesis and packaging into virions are closely linked (Nugent et al., 1999). However,

there are several aspects of this process that remain to be determined. To what extent do the capsid proteins assemble prior to virion assembly? It is well known that empty capsid formation can occur in the absence of virion RNA but this may, or may not, be a dead-end product. Are there discrete RNA replication complexes that are producing RNA genomes solely for packaging into virions? If so, how are these different from replication complexes that are producing RNA that will be translated and/ or replicated? A recent report, based on work with a plant RNA virus, has indicated the presence of multiple sequence elements within the viral genome that facilitate virus assembly (Patel et al., 2015). Concluding remarks Understanding of the structure and function of picornaviruses has grown considerably in recent years. However, the apparent simplicity of a picornavirus genome belies the great complexity of picornavirus biology. Moreover, different picornaviruses have their own individual properties that affect their interactions with the host cell. Therefore, a deep understanding of the consequences of FMDV genome organization in co-ordinating the synthesis of viral components that ultimately leads to productive infection and virus spread demands a collaborative effort involving molecular biology, cell biology and immunology. Acknowledgements Studies in the EMS laboratory were supported by grants BFU2011-25437 and BFU2014-54564 from MINECO, and by an Institutional grant from Fundación Ramón Areces. Studies in GJB’s laboratory were supported by the Danish Research Council (DFF). References

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Foot-and-mouth Disease Virus Proteinases and Polyprotein Processing Fiona Tulloch, Garry A. Luke and Martin D. Ryan

Abstract Foot-and-mouth disease virus encodes all of its proteins in a single, long, open reading frame which encodes a polyprotein. The full-length translation product (~ 2,330 amino acids) is not observed within infected cells, however, due to ‘processing’ of this polyprotein. The polyprotein undergoes extremely rapid co-translational, intramolecular, or ‘primary’, cleavages at three sites by the activities of the virus-encoded proteinases L and 3C, and a short oligopeptide sequence (2A) which mediates a ribosome ‘skipping’ activity – a translational ‘recoding’ event. The primary cleavage products then undergo ‘secondary’ proteolytic processing by a combination of inter- and intramolecular cleavages to produce the mature processing products. The L and 3C proteinases serve not only to cleave the virus polyprotein, but also to degrade specific host cell proteins thereby greatly enhancing virus replication and suppressing the innate immune response to infection. Introductory overview It was shown in the late 1960s that the multiplicity of poliovirus-specific proteins observed within infected cells – far exceeding the total coding capacity of the virus genome – was due to sequential proteolytic processing of a large precursor (Summers and Maizel, 1968) and, latterly, that this proteolytic processing could be inhibited by proteinase inhibitors (Summers et al., 1972). In the late 1970s the picornavirus 3C protein was identified as a virus-encoded proteinase (Gorbalenya et al., 1979; Palmenberg et al., 1979). Shortly thereafter a number of picornavirus complete genome

3

sequences were determined. These nucleotide sequencing data revealed a genome architecture common amongst entero-, rhino-, cardio- and aphthoviruses: all virus proteins were encoded in the form of a single, long, open reading frame (ORF) – a ‘polyprotein’. Bioinformatic analyses showed the close relationship between picornavirus 3C virus-encoded proteinases (3Cpro), but also between 3Cpro and cellular serine proteinases. Predictions based upon these bioinformatic analyses stimulated (and directed) much of the subsequent experimentation into catalytic mechanisms of picornavirus proteinases. The role of different residues in catalysis or substrate binding were confirmed by site-directed mutagenic analyses and the early bioinformatic predictions of secondary structural similarities with cellular proteinases were confirmed by the determination of the structures of picornavirus proteinases to atomic resolution by X-ray crystallography. Variations between the polyprotein structures of viruses within different genera were explained by the bioinformatic prediction and subsequent experimental confirmation of the presence of other picornavirus proteinases (entero- and rhinovirus 2Apro, aphthovirus Lpro; discussed below). In the case of FMDV it was shown that what had been assumed to be a specific proteolytic polyprotein cleavage (between the 2A and 2B proteins) was mediated by a novel translational ‘recoding’ event – mediated by the FMDV 2A oligopeptide. Subsequent research has revealed that these virus-encoded proteinases not only ‘process’ the virus polyprotein but, critically, have evolved to degrade a number of key cellular proteins thereby both enhancing virus replication and suppressing the innate immune response.

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FMDV polyprotein processing With regards to picornavirus polyprotein organization, the nature of the N-terminal and 2A regions represents the major differences between the various genera. The single, long ORF of FMDV encodes a polyprotein of some 2,330 aa, although the full-length translation product is observed neither within infected cells nor translation reactions in vitro – due to ‘primary’ proteolytic processing (Fig. 3.1A). In the case of the FMDV polyprotein, three primary polyprotein cleavages are observed. The first occurs between the FMDV L and 1A (capsid) proteins: FMDV L is a proteinase (Lpro), processing the polyprotein at this single site generating its own C-terminus. NOTE: In FMDV, the initiation of translation occurs at two sites, separated by 84nts, which gives rise to two forms of the L proteinase – Labpro and Lbpro (Clarke et al., 1985; Sangar et al.,

1987; Fig. 3.1C). The second ‘primary’ processing event occurs at the C-terminus of the 2A oligopeptide region. In FMDV 2A is very short – just 18aa and it has been proposed that this ‘cleavage’ is, in fact, the result of a translational ‘recoding’ event, rather than a proteolytic, mechanism (discussed below). In common with all other picornavirus polyproteins, the third ‘primary’ cleavage occurs between the 2C and 3A proteins, mediated by 3Cpro (Fig. 3.1A). The four products of FMDV primary polyprotein processing therefore comprise; Lpro, the capsid protein precursor [P1–2A] and the replicative protein precursors [2BC] and P3. Such primary processing reactions occur in an intramolecular manner (in cis). They are characteristically very rapid (co-translational) and, since the cleavage site and the proteinase are parts of the same molecule, the reaction is insensitive to dilution of

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Figure 3.1  Primary and secondary polyprotein cleavages. The polyprotein domain organization of enterovirus and FMDV are shown with capsid protein precursors shaded blue and replication proteins shaded green. The sites of the ‘primary’, co-translational, cleavages and the virus proteins responsible are indicated by the curved arrows. In the case of entero- and rhinoviruses, primary cleavages occur at the 1D/2A junction mediated by 2Apro, and at the 2C/3A junction mediated by 3Cpro. In contrast with enteroviruses, the FMDV polyprotein undergoes a primary ‘cleavage’ at the L/1A junction mediated by Lpro, the translational ‘recoding’ event at the 2A/2B site (vertical arrow). Like all other picornaviruses, proteolysis at the 2C/3A junction is mediated by 3Cpro (Panel A). Secondary processing of the primary cleavage products by 3Cpro gives rise to a series of alternative processing products (Panel B). The mature, individual, 3Cpro processing products are shown together with the two forms of Lpro, arising from alternative translation initiation sites, and the 1A/1B cleavage (vertical arrow) which occurs concomitantly with vRNA encapsidation by a very poorly understood mechanism (Panel C).

FMDV Proteinases and Polyprotein Processing |  45

translation reactions in vitro. Precursor forms spanning these primary cleavage sites are not observed. The [P1–2A], [2BC] and P3 precursor forms subsequently undergo ‘secondary’ processing mediated by 3Cpro (Fig. 3.1B). Secondary processing reactions occur in an intermolecular manner (in trans) and are characteristically slower. In contrast to primary cleavages, secondary polyprotein processing reactions are sensitive to dilution, are more sensitive to inhibitors and generally show a greater sensitivity to sequence variations flanking the scissile amino acid pair. Although sometimes referred-to as processing ‘intermediates’, this may be misleading since some of these proteins have specific activities in their ‘intermediate’ processing forms. The ‘mature’ processing products are shown in Fig. 3.1C. The cleavage of [1AB] to 1A + 1B occurs concomitantly with encapsidation of virus RNA (vRNA), by a poorly understood mechanism. In addition to cleaving the virus polyprotein, picornavirus proteinases have also been shown to degrade specific cellular protein targets (discussed below). The following sections are organized by the historical order in which their mechanisms of action became understood. The 3C proteinase A virus-specific proteolytic activity – in contrast with virus polyprotein processing by host cell proteinases – was first described for the cardiovirus encephalomyocarditis virus (EMCV; Lawrence and Thatch, 1975; Pelham, 1978) and subsequently

O1/K

1B ⇓ 1C ⇓ 1D ⇓ 2A DKKTE-/-FPSKE GIFPV-/-DARAE TTSAG-/-APVKQ TLNFDL Q Q M 2A q 2B ⇓ 2C ⇓ 3A ⇓ 3B1 ⇓ 3B2 -ESNPG PFFFS-/-RAEKQ LKARD-/-PIFKQ ISIPS-/-QPQAE GPYAG-/-LPQQE GPYAG-

O1/K

3Cpro ⇓ 3Dpol 3B2 ⇓ 3B3 ⇓ -LPQQE GPYAG-/-PVVKE GPYEG-/-EPHHE GLIVD-

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L s 1A -QRKLK GAGQS-/-GALLA R

mapped to protein 3C (Gorbalenya et al., 1979; Palmenberg et al., 1979). Picornavirus 3C proteinases mediate a primary cleavage between 2C and 3A, although one report indicated an alternative primary cleavage between proteins 2A and 2B of poliovirus (Lawson and Semler, 1992). In comparison with other picornavirus 3C proteinases, FMDV 3Cpro cleaves a wide range of amino acid pairs (E/G, Q/G, Q/T, Q/L, Q/I; Fig. 3.2). The primary [2BC]/P3 cleavage occurs at a conserved Q/I pair, whilst cleavages within P3 all occur at E – G pairs. Similarly, the cleavage site between 2B/2C is completely conserved, although in this case at a Q/L pair. Cleavages with the capsid proteins precursor [P1–2A] show a wider range of pairs; E/G, Q/G, E/T, Q/T, Q/L and Q/M. Not all such pairs present within the polyprotein are processed, however, – their position at the boundaries of polyprotein domains being the major determining factor. FMDV 3Cpro is 213aa long (predicted Mr = 23 kDa), whose N- and C-termini are defined by its own proteolytic activity. The 3Cpro-mediated secondary processing of the [P1–2A], [2BC] and P3 precursors is shown in Fig. 3.1B. Whilst the [P1–2A] precursor is processed to the end products [1AB], 1C and 1D (the [1AB] ‘maturation’ cleavage occurs concomitantly with vRNA encapsidation), a multiplicity of different products are generated during processing of P3, some of which are stable products. For example, protein 3CD and those comprising the 3BCD complex (3CD with 1, 2 or 3 3B proteins still attached) are all stable products

i

Figure 3.2  FMDV polyprotein cleavage sites. Cleavage at the L/1A site occurs at (K/R)/G pairs ( ). The capsid protein 1A/1B cleavage (↓) occurs concomitantly with vRNA encapsidation by an unknown mechanism. The primary 2A/2B translational recoding event (   ) occurs at a conserved G/P pair. 3Cpro-mediated processing (   ): the primary 2C/3A cleavage occurs at a conserved Q/I pair whilst secondary processing of P3 occurs only at E/G pairs and at the 2B/2C site at a conserved Q/L pair. A range of scissile pairs is cleaved during processing of the capsid proteins precursor [P1–2A] at the 1B/1C, 1C/1D and 1D/2A sites.

46  | Tulloch et al.

(Ryan et al., 1989). In the case of poliovirus, the processing of the P1 capsid protein precursor is mediated not by 3Cpro, but by 3CDpro ( Jore et al., 1988; Ypma-Wong et al., 1988). The 3Cpro from other genera are, however, able to process capsid protein precursors (Vakharia et al., 1987; Parks et al., 1989; Jia et al., 1991) and although the FMDV 3Cpro efficiently cleaves all 10 processing sites within the FMDV polyprotein (Bablanian and Grubman, 1993), processing of the capsid proteins precursor [P1–2A] was somewhat more efficient with 3CDpro (Ryan et al., 1989). Like FMDV, the EMCV 3C proteinase also retains proteolytic activity in 3Cpro-containing precursor forms ( Jackson, 1986; Parks et al., 1989). The catalytic mechanism of FMDV 3Cpro Early studies on 3Cpro using proteinase inhibitors showed rather confusing inhibitor profiles. Inhibition of proteolytic activity was observed with both serine and thiol proteinase inhibitors (Summers et al., 1972; Korant, 1972, 1973; Pelham, 1978; Gorbalenya and Svitkin, 1983; Korant et al., 1985; Baum et al., 1991). A breakthrough came when similarities between the large sub-class (trypsin-like) cellular serine proteinases and 3C proteinases were detected by sequence alignments (Gorbalenya et al., 1986, 1989; Bazan and Fletterick, 1988, 1990). These analyses predicted both a chymotrypsin-like (serine proteinase) fold and the identity of the residues which would form a catalytic triad analogous to that of serine proteinases (Fig. 3.3A). In the case of FMDV 3Cpro the putative catalytic triad would be composed of His46, Asp84, and, perhaps most interestingly, the active site nucleophile being Cys163 (rather than serine, as in chymotrypsin). Subsequent site-directed mutagenesis experiments confirmed the roles of these residues in rhinovirus (Cheah et al., 1990), polio (Hammerle et al., 1991; Kean et al., 1991; Lawson and Semler, 1991), hepatitis A ( Jia et al., 1991) and FMDV 3Cpro catalysis (Grubman et al., 1995). The structure of FMDV 3Cpro The resolution of the atomic structures of the 3C proteinases from human rhinovirus (HRV) 14 (Matthews et al., 1994), hepatitis A (Allaire et al., 1994; Bergmann et al., 1997), polio (Mosimann et al., 1997) and HRV 2 (Mathews et al., 1999)

confirmed the predicted chymotrypsin-like fold of the enzyme and catalytic mechanism. Detailed discussion of the structural and mechanistic relationships between these enzymes and cellular proteinases can be found in Dougherty and Semler (1993), Malcolm (1995), Babé and Craik (1997), Ryan and Flint (1997), Seipelt et al. (1999) and Skern et al. (2002). Resolution of the atomic structure of FMDV 3Cpro revealed a similar structural fold observed previously for other picornavirus 3C proteinases (Birtley et al., 2005 Sweeney et al., 2007; Fig. 3.3A). The characteristic catalytic triad reported in other picornavirus 3C proteinase structures was also observed in FMDV 3Cpro: His46, Asp84 and Cys163, although the latter residue was mutated to alanine to produce a proteolytically inactive form to facilitate expression, purification and crystallization – a strategy commonly adopted previously for other picornavirus proteinases. In the initial report, the absence of a β-ribbon structure overlaying the substrate-binding cleft, as observed in other picornavirus 3C proteinase structures, was proposed to contribute to the lower substrate binding specificity of FMDV 3Cpro, in that whilst other picornavirus 3C proteinases show a strong preference for glutamine at the P1 position (-Q/X- scissile pairs), the FMDV 3Cpro accommodates both glutamine and glutamate at this P1 position (-E/X- and -Q/X- scissile pairings). In FMDV, however, this β-ribbon structure appeared to be a disordered surface loop (Birtley et al., 2005). In the subsequent paper, however, a crystal form was reported in which residues comprising this loop now adopted a β-ribbon structure, similar to other picornavirus 3C proteinases (Sweeney et al., 2007). A recent publication showing the structure of FMDV 3Cpro complexed with a synthetic peptide (APAKQ/LLNFD – corresponds to the 1D/2A polyprotein cleavage site; Zunszain et al., 2010) showed that substrate binding produced an enzyme structural re-arrangement which formed an extended interaction with the substrate P4 to P2′ positions of the peptide APAKQ/LLNFD (underlined). Interestingly, the S1′ specificity pocket which accommodates the P1′ leucine (APAKQ/ LLNFD; underlined) formed only once the peptide substrate had bound (Zunszain et al., 2010). As observed previously for other picornavirus 3C proteinases, the peptide substrate bound to the

FMDV Proteinases and Polyprotein Processing |  47

A

BB

N-terminal helix

Cys163 catalytic triad

His46

Asp84

C-terminal helix

Figure 3.3 Atomic structure of FMDV 3C and L proteinases. Like other picornavirus 3C proteinases, FMDV 3Cpro (PDB Acc. No. 2J92) folds into a structure closely resembling that of the large class of serine (chymotrypsin-like) proteinases. C Although the FMDV 3Cpro active site nucleophile is cysteine (rather than serine), a close structural correspondence is observed between the catalytic triad of FMDV 3Cpro (Cys163-His46-Asp84) and the Ser-His-Asp triad of serine proteases (A). Again, similar to other picornavirus 3C proteinases, an extended exterior loop connects sheets βD2 and βE2 and is located between the two (anti-parallel) helices of the N- and C-termini. In the case of entero- and rhinovirus 3C proteinases, mutations affecting RNA binding clustered around a conserved –KFRDIR- motif within this domain linker, comprising the RNA-binding site. In the case of FMDV 3Cpro, a similar motif (–RVRDIT-; side-chains shown as sticks, together with the electrostatic surface) is present within the equivalent extended domain linker – which could comprise an FMDV 3Cpro RNA-binding site (B). The structure of FMDV Lpro (PDB Acc. No. 1QOL) is related to thiol (papain-like) proteinases, with the side-chains of the catalytic residues Cys51 and His148 shown as sticks. The cysteine residue was mutated to alanine for purposes of structural determination. The protruding C-terminal extension is seen on the opposite face (C). All structures were rendered using PyMol (DeLano Scientific).

FMDV enzyme in a linear, extended, conformation which oriented the P1 residue side-chain into the S1 binding pocket proximal to the catalytic triad. Whilst all the side-chains of the substrate P4-P4′ residues form contacts with FMDV 3Cpro to one extent or another, activity assays using a range of synthetic substrates, together with structural data of enzyme–peptide complexes, revealed a marked specificity for the P4, P2 and P1 positions – but a lower specificity for the P3 and P1′ positions (Zunszain et al., 2010). A case has been made for the development of FMDV antiviral drugs (3Cpro naturally being a target candidate) since one of the disease control

measures – emergency ring vaccination – involves a considerable lag-time in the sero-conversion of vaccinated animals and, therefore, the formation of a barrier to further disease transmission. However, research aimed at the development of such small molecule anti-proteinase drugs for entero- and rhinoviruses, for example, has not yet translated into the market place. FMDV 3Cpro: an RNA binding protein? An additional and quite unexpected property of this proteinase was discovered when mutations suppressing the effect of a four base insertion within the 5′ non-coding region of the RNA genome were

48  | Tulloch et al.

mapped within 3Cpro (Andino et al., 1990a). Subsequently it was demonstrated these mutations were affecting the binding of 3CDpro, rather than 3Cpro, to positive- strand RNA (Andino et al., 1990b, 1993). This vRNA-binding property of 3CDpro was demonstrated for rhinoviruses and hepatitis A. Although mapping to multiple sites, when the data were combined the mutations clustered to define an RNA binding site (Andino et al., 1990a,b, 1993; Leong et al., 1993; Walker et al., 1995; Blair et al., 1996, 1998; Kusov and Gauss-Muller, 1997). Mutations shown to affect RNA binding were located within the atomic structure on the opposite side to the catalytic site. The majority of these mutations were observed in a domain interconnecting the two lobes of the proteinase – located between the two (anti-parallel) helices of the N- and C-termini – and also clustered on the (exterior) loop connecting βD2 and βE2 and centred about the conserved entero-, rhinovirus –KFRDIR- motif of the domain linker. Interestingly, this interconnecting region lies in close proximity to the N- and C-terminal α-helices of the proteinase. In the case of FMDV 3Cpro, both the structure (exterior loop) and a similar sequence motif (–RVRDIT-, conserved amongst FMDVs) is similar to the RNA-binding site of entero- and rhinovirus 3C proteinases (Fig. 3.3B). Similarly, in FMDV 3Cpro this interconnecting region lies in close proximity to the N- and C-terminal α-helices. An RNA-binding activity has not been examined for FMDV 3CDpro, but the secondary structure of the FMDV 5′ terminal region of the non-coding region is quite different from enteroviruses, in that FMDV has a much longer stem–loop. The significance of this RNA-binding activity of picornavirus 3C/3CD proteinases has not been fully determined, but the conservation of the structure and the electrostatic nature of this region amongst 3C proteinases of viruses within different genera of picornaviruses support the notion that this is an important aspect of virus replication worthy of further investigation. The L proteinase The second viral proteinase of FMDV to be discovered was the Leader proteinase (Lpro), which was shown to cleave co-translationally at its own C-terminus (Burroughs et al., 1984; Strebel and Beck, 1986). The presence of a proteolytically active

leader protein at the N-terminus of the polyprotein is a feature possessed by the aptho- and erbovirus genera alone. Lpro contains two in-frame translation initiation codons (84 nt apart) which results in two forms being produced; Labpro and Lbpro (Clarke et al., 1985) The latter form undergoes self-cleavage, trimming the C-terminus by six to seven amino acids, via a carboxypeptidase B-like activity producing a product Lb’pro (Sangar et al., 1988). This C-terminal ‘trimming’ of Lbpro was reproduced by the truncation of sequences encoding Lbpro to remove the 6 C-terminal amino acids – termed sLbpro (Guarné et al., 1998, 2000) Note that this sLbpro form was used for structural studies (see below) and for comparative proteolysis analyses. Additional substrate specificity or functionality for sLbpro remains to be discovered, however, reports suggest Lbpro and sLbpro differ in cleavage efficiencies in trans (Cencic et al., 2007; Steinberger et al., 2014). The differential roles of these form of leader proteins – if any – produced during infection is unknown. Critically, both AUGs are conserved in all natural FMDV isolates (Sangar et al., 1987; Cao et al., 1995; Carrillo et al., 2005) suggesting their presence serves an as yet unknown function. Lpro generates its own C-terminus (Strebel and Beck, 1986), the cis cleavage occurring between Lys/Arg201 and Gly202. Both the Labpro and Lbpro forms have been shown to cleave at the L/P1 junction either in cis or in trans (Medina et al., 1993; Cao et al., 1995). Besides cleaving itself from the nascent viral polyprotein, Lpro cleaves the host cell translation factor eIF4G (Devaney et al., 1988; Medina et al., 1993). Proteolytic cleavage of eIF4G occurs between residues Gly479 and Arg480 – only seven amino acids upstream from the enterovirus 2A proteinase site at Arg486 and Gly487 (Kirchweger et al., 1994). The closely separated Lpro and 2Apro cleavage sites are both thought to lie in a hinge region between the two major domains of eIF4G. Cleavage activity within this region results in the separation of these two domains: the N-terminal portion required for eIF4E (binds the 5′-terminal 7meG host cell mRNA cap structure) and poly(A) binding protein (PABP), and the C-terminal domain which binds eIF3 and eIF4A (involved in unwinding secondary RNA structure in the mRNA and recruitment of the 43S pre–initiation complex; reviewed by Jackson and Wickens, 1997). This allows cap-independent translation of the viral

FMDV Proteinases and Polyprotein Processing |  49

genome to proceed via binding to the viral IRES, whilst progressively ‘shutting-off ’ cap-dependent translation of host cell mRNAs (Lamphear et al., 1993). Atomic structure and catalytic mechanism Amino acid sequence analysis of Lpro suggested a similarity to papain-like thiol-proteinases which comprise active site cysteine and histidine residues (Gorbalenya et al., 1991). This was later confirmed via inhibitor and site-directed mutagenesis studies, with Cys51 and His148 identified as active site residues (Kleina and Grubman 1992; Piccone et al., 1995a; Roberts and Belsham, 1995). Resolution of the atomic structure of Lbpro was solved via molecular replacement using the shorter sLbpro as a model. The crystal structure of Lbpro showed a compact globular form with a flexible C-terminal extension (CTE) comprised of 18 amino acid residues from D184 to K201 (Guarné et al., 1998, 2000). The active site is located between an α-helical domain and a β-sheet domain. The orientation of the histidine component of the active site is optimized by its interaction with Asp163, rather than an asparagine residue which performs this role in thiol proteinases such as papain. Lpro cleaves at its own C-terminus: the substrate binding site must, at some stage, therefore, accommodate its own C-terminus (the ‘P’ side residues of the scissile bond). This is consistent with the observed flexibility of the CTE in that it is disordered in both the crystal structure and in solution (NMR analyses; Cencic et al., 2007). Interestingly, the NMR data confirmed the formation of Lbpro homodimers in solution (in agreement with the crystal studies), which did not dissociate by increasing the ionic strength, dilution, or through binding to a peptide substrate corresponding to the eIF4GI cleavage site. Although complete dissociation of dimers (into monomers) was not observed following binding to the peptide substrate, shifts in NMR spectra suggested movement of the monomers which corresponded to the dimer interface, proposing Lbpro might function as a dimer under physiological conditions; however, a later study found Lbpro functioning as a monomer until enzyme concentrations reached > 2 µM (Santos et al., 2009). The authors of the previous study (Cencic et al., 2007) proposed dimerization may regulate the activity of

Lbpro by reducing the number of substrates cleaved via product inhibition. In the crystal structure of Lbpro, the C-terminus of one molecule is bound into the substrate binding site of the adjacent molecule. This gave an additional insight into the function of the enzyme since half of the substrate was bound into this site. The branched hydrophobic P2 residue (L/V) forms an important interaction with a deep hydrophobic cavity, although (unlike papain) Lbpro has appreciable binding pockets for the P1 and P1’ residues, which make a substantial contribution to its substrate binding specificity. Subsequent work has highlighted the substrate specificity of Lpro in comparison to other papain-like enzymes: Lbpro exhibits an extended substrate binding site (up to P7), with substitution of peptides at each position within the substrate affecting enzyme activity (Santos et al., 2009). High prime-side specificity has also been documented (Nogueira Santos et al., 2012). Replication characteristics of L-deleted viruses Despite the Lbpro form being the predominant form produced (Sangar et al., 1987; Cao et al., 1995), deletion of this region is not important for virus viability. Piccone and colleagues found that viruses lacking the portion of L directly after the Lbpro initiation codon replicated less efficiently in BHK-21 cells (Piccone et al., 1995b). This replication was only slightly slower than the wild-type (WT) virus, characterized by smaller plaque sizes, lower titres, slower host cell shut-off and slight attenuation in mice; however, when tested in cattle the ‘partially leaderless’ virus had greatly reduced pathogenicity and was less widely disseminated in the lung at 24 hours post infection (p.i.), with no lesions or virus detectable in secondary sites at 72 hours p.i. (Brown et al., 1996). In a later study (Mason et al., 1997) two of three animals inoculated with the leaderless virus did not develop lesions when challenged with WT virus, but showed mild signs of infection. The third inoculated animal developed some lesions, but these were less severe than in the un-inoculated control animal, which showed classical FMD. When this virus was later tested in swine, similar results to those observed earlier in cattle were found (Chinsangaram et al., 1998). The differences in replication of the leaderless virus within BHK cells and reduced virulence in animals reported in the above

50  | Tulloch et al.

studies appears to lie in the type I interferon (IFN) response. The inability of FMDV-infected cells to translate type I IFN mRNA, due to Lpro-mediated shut-off of host cell translation, makes a substantial contribution to the virulence of the virus. The BHK cells commonly used to propagate FMDV are, however, defective in their response to IFN. The 2A oligopeptide The second primary cleavage of the FMDV polyprotein occurs between the 2A and 2B proteins – markedly different from the 2A proteinase mediated polyprotein processing observed in the entero- and rhinoviruses NOTE: rhinoviruses are now included within the genus enterovirus (Fig. 3.1). Precursor forms spanning the FMDV 2A/2B cleavage site are not detected during native polyprotein processing. The 2A region of the FMDV polyprotein is very short (18aa), with a C-terminal sequence motif (-DxExNPG-) conserved amongst all FMDVs: the N-terminal proline residue of FMDV protein 2B is completely conserved. Indeed, this motif (together with the N-terminal proline of protein 2B) is now known to be conserved amongst 2A proteins of many picornavirus genera (e.g. Aphtho-, Aquama-, Avihepato-, Avisi-, Cardio-, Cosa-, Erbo-, Hunni-, Kunsagi-, Mischi-, Mosa-, Pasi-, Seneca-, Sicini- and Teschoviruses, plus the proposed new genera Limnipvirus and Potamipivirus) – indeed, some viruses have multiple iterations of the -NPGP- motif within what must be multifunctional 2A proteins. Cleavage at the 2A/2B site of FMDV or cardiovirus polyproteins was shown to require neither the L nor 3C proteinases (Clarke and Sangar, 1988; Roos et al., 1989; Ryan et al., 1989). Early studies showed the FMDV 2A region was not simply a substrate for a virus proteinase (Lpro, 3Cpro), and the very rapid, complete, ‘cleavage’ suggested it did not function as a substrate for a host cell proteinase. A series of in-frame deletions of the FMDV polyprotein were made such that the upstream or downstream context of 2A was altered (Ryan et al., 1991). These analyses suggested that the 2A/2B ‘cleavage’ was, indeed, associated with this 2A oligopeptide alone (plus the N-terminal proline of protein 2B). To test this hypothesis, the 2A sequence was used in the creation of an ‘artificial’ reporter gene polyprotein in which sequences encoding

chloramphenicol acetyl-transferase (CAT) were linked via FMDV 2A (plus the N-terminal proline of 2B) to β-glucuronidase (GUS) in a single ORF ([CAT-2A-GUS]). Analysis of this polyprotein system using in vitro translation systems showed, indeed, that the 20 amino acid FMDV sequence was able to mediate a co-translational cleavage producing the cleavage products [CAT-2A] and GUS (Ryan and Drew, 1994). In this heterologous context, therefore, the FMDV 2A sequence mediated a highly efficient cleavage at its own C-terminus – just as in FMDV polyprotein processing (-DxExNPG⇓P-). Consistent with these observations, other studies showed that deletion of the N-terminal 66% of the cardiovirus 2A did not abrogate cleavage at the 2A/2B site, and that mutations within the conserved – NPGP – sequence at the extreme C-terminus of EMCV 2A abolished cleavage activity (Palmenberg et al., 1992). Cleavage at the 2A/2B site of Theilers murine encephalitis virus (TMEV) was highly efficient when only 2A and 2B sequences were present (Batson and Rundell, 1991). Later it was shown that the C-terminal 19aa together with the N-terminal proline of 2B from either FMDV, EMCV or TMEV, when inserted into an artificial polyprotein, are able to mediate a co-translational ‘cleavage’ with high efficiency (~95%; Donnelly et al., 1997). Based upon the observed kinetics of the cleavage reaction, inability to inhibit the reaction with proteinase inhibitors plus data from site-directed mutagenesis of the 2A sequence, we have proposed that the 2A/2B cleavage is not a proteolytic cleavage (by either a virus or host cell proteinase), but the result of a translational ‘recoding’ activity – latterly termed as ‘ribosome skipping’ (Ryan et al., 1999; Donnelly et al., 2001a). Briefly, we have proposed that the nascent 2A sequence interacts with the exit pore of the ribosome to bring about a reorientation of the tRNA-peptidyl ester linkage precluding it from nucleophilic attack by the prolyl-tRNA. Hydrolysis of this bond would lead to release of the peptide such that elongation may continue from the prolyl-tRNA, producing two, discrete, ‘cleavage’ products (Fig. 3.4). The N-terminal proline of 2B is, therefore, a crucial part of the proposed mechanism. Site directed mutagenesis of this residue abrogates cleavage: it is completely conserved in all picornavirus 2B proteins which use this ‘cleavage’

FMDV Proteinases and Polyprotein Processing |  51 egress of prolyl-tRNA

Nascent pep1de

P

(2)

(1) exit tunnel

(3)

2A

OH OH G

E

P

A

E

G

P

OH

G

P

A

E

P

A eRF1/3

ingress of eRF1/3

eRF1/3

(6)

(5)

(4) OH G

E

P

A

OH

G OH

E

P

A

OH

OH

E

P

A eEF2

eEF2

(8)

(7) OH OH P

OH

E

E

P

A

P

P

(9) OH

A

E

P

P

A

Figure 3.4  2A-mediated translational ‘recoding’: Current Model. When the nascent FMDV capsid proteins [P1] upstream of 2A emerge from the ribosome, 2A is positioned in the ribosome exit tunnel (step 1). The nascent [P1–2A]-tRNAGly is translocated from the A- to P-site and prolyl-tRNA enters the A-site (step 2). The nascent 2A peptide interacts with the exit tunnel of the ribosome such that the C-terminal portion of 2A (-DVESNPG-) is sterically constrained within the peptidyl-transferase centre (PTC) of the ribosome. Nucleophilic attack of the ester linkage between [P1–2A] and tRNAgly by prolyl-tRNA in the A-site is inhibited – effectively stalling, or pausing, translation. The failure to form a new peptide bond leads to dissociation of prolyl-tRNA from the A-site of the ribosome (step 3), required for entry of release factors into the A-site (step 4). We have shown that this block is relieved by the action of translation release factors eRF1 and eRF3, hydrolyzing the ester linkage (step 5) and releasing the nascent protein (step 6). eRF1 leaves the complex, eRF3 being involved in this process (step 6). Two, mutually exclusive, outcomes may then arise: (i) translation terminates at the C-terminus of 2A, or (ii) prolyl-tRNA (re)enters the A-site (step 7), is translocated by eukaryotic elongation factor 2 (eEF2) from the A- to the P-site (step 8), allowing the next amino-acyl tRNA to enter the A-site to permit the synthesis of the sequences downstream of 2A (step 9).

mechanism and in ‘2A-like’ sequences from other viruses (see below). Preliminary studies showed the FMDV 2A sequence-mediated translational recoding in yeast (we now know this system is active in all eukaryotic cells tested to date). An interesting test of this hypothesis – using yeast expression systems – came from the analysis of an artificial polyprotein system in which the first protein bore a leader sequence. A single open reading frame was constructed

encoding three proteins; (i) DNαF – yeast proalpha factor with, instead of its native signal sequence, the signal recognition particle (SRP)-dependent signal sequence of dipeptidyl amino peptidase B (Dap2p) (ii) FMDV 2A and (iii) green fluorescent protein (GFP). Here, SRP binds to the Dap2p leader sequence, arrests translation, and leads to the nascent protein being translated in a ribosome: protein conducting channel complex (formed in the process of translocating proteins across the membrane into

52  | Tulloch et al.

the lumen of the endoplasmic reticulum – ER). In this situation, therefore, the nascent protein would not be accessible to cytosolic protein(ases) and the folding of the nascent protein would not occur until passing out of the protein conducting channel into the lumen of the ER. Analysis of the processing properties of this artificial polyprotein and the sub-cellular localization of the ‘cleavage’ products showed that (i) 2A-mediated ‘cleavage’ was unaffected; (ii) the first protein, DNαF, was translocated into the lumen of the ER; and (iii) GFP was located in the cytoplasm (de Felipe et al., 2003). That the FMDV 2A sequence was active in yeast enabled the power of yeast genetics, notably the extensive library of yeast mutants, to screen for host cell proteins that could be involved in the 2A translational recoding mechanism. These studies identified eukaryotic release (termination) factors eRF1 and eRF3 affecting this mechanism (Doronina et al., 2008a,b; Sharma et al., 2012). The translational model of 2A recoding activity was refined to propose these factors brought about the hydrolysis of the nascent peptide-tRNA ester linkage in a manner directly analogous to the ‘normal’ termination of translation, but that (i) these factors bound in a stop codon independent manner; and (ii) that once these factors exited the ribosome termination could either terminate at the C-terminus of 2A, or prolyl-tRNA could re-enter and re-initiate the synthesis of the downstream (replication) proteins (Fig. 3.4). In contrast with this model, a recent study using a translation system reconstituted with human translation factors showed, however, that translational recoding at the EMCV 2A/2B site did not require supplementation of the reaction with eRFs (Machida et al., 2014). Obviously, further work on the detailed mechanism of this translational recoding event is required! In summary, the huge expansion of the picornavirus sequence database shows that this type of translational recoding event at the 2A/2B protein junction is quite common amongst the different genera of the Picornaviridae, and that encoding a proteinase-type of 2A is (currently) found only in a few genera: certainly enteroviruses and quite possibly sapelo- and saliviruses. The number of genera – but perhaps more importantly the wider range of hosts (mammals, birds, fish, insects: also

see ‘2A-like’ sequences, below) of viruses encoding an ‘NPGP’ type of 2A –suggests that this form of 2A evolutionarily predates the appearance of a proteinase-type of 2A. The ‘translational’ model of 2A-mediated cleavage: implications for the biology of FMDV Our work on the cleavage of a [GFP-2A-GUS] polyprotein system using in vitro translation systems showed that a molar excess of the [GFP-2A] ‘cleavage’ product accumulated above the GUS product (Donnelly et al., 2001a). If the gene order was reversed ([GUS-2A-GFP]), more [GUS-2A] accumulated than GFP. We showed this was not due to different rates of protein degradation or non-specific dissociation of either the T7 RNA polymerase (during transcription of the template), or ribosomes (during translation). The molar excess was due to different levels of synthesis: the two different parts of the single ORF were being translated at different levels. This implies that in the FMDV polyprotein, more capsid proteins could be synthesized than replicative proteins. In a FMDV-infected cell at the latter stages of replication one might expect the depletion of cellular factors or, more probably, a reduction in activity due to phosphorylation/dephosphorylation, to become rate-limiting for virus replication. Indeed, a decrease in the rate of ribosome elongation is typical during the course of picornavirus infections (Summers et al., 1967; Hackett et al., 1978; Ramabhadran and Thatch, 1981). It could be that under these conditions, FMDV has a evolved a mechanism for ‘switching’ those remaining cellular resources present at the latter stages of replication into the synthesis of capsid proteins – and not replication proteins. If this were true, both particle yield and the kinetics of vRNA encapsidation could be increased – which might, in part, account for the rapidity of the growth and highly contagious nature of this amazing virus. ‘2A-like’ sequences At the outset of our work on FMDV polyprotein processing, this type of 2A sequence was known only for FMDV and cardioviruses. In addition to viruses within the range of picornavirus genera listed above, database probing with a [D-(V/I)-EX-N-P-G-P; ‘X’ = any amino acid] motif revealed

FMDV Proteinases and Polyprotein Processing |  53

the presence of translational recoding active ‘2A-like’ sequences in many other types of virus genome, both positive- and double-stranded RNA viruses – interestingly not within any DNA virus or plant virus genome of any type. Genomes encoding active 2A-like sequences include positive-stranded RNA viruses such as insect Ifla- and Tetra- and Dicistroviruses, plus double-stranded RNA viruses such as the mammalian type C rotaviruses, insect Cypoviruses and crustacean Totiviruses (Luke et al., 2008; reviewed in Luke and Ryan, 2013; Luke et al., 2014). Furthermore, we have identified active 2A-like sequences within the genomes of a range of organisms. Initially 2A-like sequences were detected within the non-LTR retrotransposon L1Tc present within the genomes of a number of trypanosome species (Heras et al., 2006). The expansion of genome sequence databases revealed that, indeed, such 2A-like sequences were present within a number of different evolutionary clades of nonLTR retrotransposons, but also non-LTRs present within the genomes of a range of different, notably marine, organisms (Odon et al., 2013). 2A-like sequences were also identified at the N-terminus of the innate immunity NOD-like receptor proteins (NLRs) of the purple sea urchin (Strongylocentrotus purpuratus), and we have shown that these active 2A-like sequences can also function as N-terminal signal sequences. If the 2A-like sequence mediates translational recoding (is ‘cleaved’ away from the downstream NLR protein) the protein localizes to the cytoplasm, but if the 2A-like sequence does not mediate translational recoding, it remains as an N-terminal feature, functions as an N-terminal signal sequence, and targets the protein to exocytic pathway (Roulston et al., in press). 2A and 2A-like sequences are, therefore, widely distributed amongst both virus and cellular genomes. It is fruitless to speculate as to the evolutionary origins or relationships between cellular (non-LTR retrotransposons/signal sequences) and virus 2A/2A-like sequences: given the shortness of these 2A/2A-like sequences, a polyphyletic origin is entirely feasible (even probable), but it is noteworthy that with a relatively modest number of point mutations a ‘classical’ signal sequence could acquire an additional translational recoding capacity – or vice versa!

The cleavage of host cell proteins Whilst picornavirus proteinases play a central role in the biogenesis of virus proteins from precursor forms, importantly they also cleave host cell proteins. Proteins involved in a range of cellular processes are degraded by these virus proteinases (Table 3.1). In many cases it is difficult to assess the contribution to replication efficiency or virulence of some these different cleavage events. This is of great interest, however, in understanding how persistent or chronic picornavirus infections are established (see Chapter 7 and Colbère-Garapin et al., 2010). The picture that has emerged is one of co-evolution – genetic changes occurring both in the host cell and in the virus (de la Torre et al., 1985, 1988, 1989). Furthermore, cell-lines may be generated expressing low levels of the cytotoxic 3Cpro (Lawson et al., 1989; Martinez-Salas and Domingo, 1995). Whilst defective-interfering (DI) genomes can be established quite readily for enteroviruses by passage in tissue-culture, this is not the case with FMDV. Defective non-interfering genomes are generated during the establishment of a persistent FMDV infection – genomes with deletions located within Lpro (Charpentier et al., 1996). Table 3.1 shows the host cell proteins degraded by FMDV L and 3C proteinases. In the case of FMDV Lpro, a crucial role is the ‘shut-off ’ host cell cap-dependent mRNA translation by the degradation of eIF4G (Kirchweger et al., 1994; Belsham et al., 2000). An early report described the Lpro degradation of cyclins A and B2 (Ziegler et al., 1995), although how this affects virus replication is not clear. Biochemical data have been reported, however, for other Lpro host factor targets. Gemin5 (first identified as a peripheral component of the survival of motor neurons – SMN – complex) was identified as an IRES-binding factor (Pacheco et al., 2009): increasing amounts of Gemin5 was shown to inhibit IRES-dependent expression in a bicistronic mRNA system. Subsequently it was shown that Gemin5 was degraded by Lpro (Piñeiro et al., 2012, 2013, 2015). There is an increasing body of evidence that Lpro is involved in the regulation of genes associated with the innate immune system, not only at the translational level, but also at the transcriptional level. Studies have shown a block in type I IFN

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Table 3.1 Cleavage of host-cell proteins by FMDV proteinases Host cell protein

Function

Proteinase

Reference

Cyclin B2 (Xenopus)

Control of cell cycle

L

Ziegler et al. (1995)

Cyclin A (human)

Control of cell cycle

L

Ziegler et al. (1995)

Gemin5

IRES-binding factor

L

Piñeiro et al. (2012)

p65/RelA

Subunit of NF-κB

L

de los Santos et al. (2007), de los Santos et al. (2009)

Ubiquitinated proteins

Modulate cellular processes (including immune responses)

L

Wang et al. (2011a)

RANTES expression

Inflammation and immune response

L

Wang et al. (2011b)

eIF4G

translation initiation factor

L 3C

Kirchweger et al. (1994), Belsham et al. (2000)

H3

Histone

(FMDV Grigera and Tisminetzky (1984), Falk et infection), 3C, al. (1990), Tesar and Marquardt (1990), 3C, 3ABC Capozzo et al. (2002)

eIF4A1

Translation initiation factor

3C

Belsham et al. (2000), Li et al. (2001)

NEMO

Essential for NF-κB activation

3C

Wang et al. (2012)

Sam68

Alternative splicing

3C

Lawrence et al. (2012)

expression – due to the inability of FMDV-infected cells to translate type I IFN mRNA (Chinsangaram et al., 1999) – as well as significant induction of interferon IFN-β and IFN stimulated gene (ISG) mRNA in cells transfected with ‘leaderless’ FMDV (de los Santos et al., 2006). Additionally, Lpro has been implicated in degradation of the p65/RelA subunit of nuclear factor kappaB (NF-κB) (de los Santos et al., 2007, 2009) and as a virally encoded deubiquitinase thought to be involved in degradation of host cell innate-immune proteins (Wang et al., 2011a,b). Interestingly, in addition to Lpro, FMDV 3Cpro has also been shown to degrade host cell translation factors. 3Cpro degrades eIF4G producing cleavage products different from those generated by Lpro (Belsham et al., 2000). 3Cpro also degrades the initiation factor eIF4A1, a DEAD-box helicase which undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation (Belsham et al., 2000; Li et al., 2001). Early studies showed histone H3, the most extensively modified of the five histone proteins, was degraded in FMDV-infected cells and subsequently it was demonstrated that this was mediated

by 3Cpro (Grigera and Tisminetzky 1984; Falk et al., 1990; Tesar and Marquardt, 1990; Capozzo et al., 2002). Again, the significance of these observations with regards to FMDV replication is not clear. FMDV 3Cpro also induces proteolysis of nuclear RNA-binding protein Sam68 (the Src-Associated substrate in Mitosis of 68 kDa, also called KHDRBS1 – KH domain containing, RNA binding, signal transduction associated 1). Sam68 localizes predominantly to the nucleus and its major function in the nucleus is to regulate alternative splicing by recognizing RNA sequences flanking the included/excluded exons. FMDV 3Cpro cleaves away the C-terminus containing the nuclear localization sequence (NLS) – causing the truncated form to redistribute to the cytoplasm. Knock-down of Sam68 in LFBK cells was shown to greatly reduce titres of FMDV and decrease IRES-driven in vitro translation. In the same study, binding of full-length Sam68 to the IRES of FMDV was observed, suggesting a role for this protein in enhancing translation of the vRNA genome (Lawrence et al., 2012). 3Cpro has been documented to antagonize the IFN-α/β response through proteolysis of nuclear transcription factor kappaB (NF-κB)

FMDV Proteinases and Polyprotein Processing |  55

essential modulator (NEMO). The cleavage site within NEMO was mapped to Gln383, resulting in removal of the C-terminal zinc-finger domain – a region essential for full activation of NF-κB, and subsequently interferon-stimulated gene (ISG) expression (Wang et al., 2012). Reporter assays showed that fragments of NEMO produced by 3Cpro proteolysis were inefficient at activating the IFN-β promoter, completely abolishing, or impairing expression. FMDV proteinases not only play a central role in the biogenesis of virus proteins, but are of great interest in disease control. Firstly, they may well form drug targets – it could be argued that in certain situations (such as contingency ring vaccination) drug administration could protect susceptible animals until a protective immune response is established. Secondly, it is apparent that establishment of persistent infections involves changes in the activity of the virus proteinases. Thirdly, over the past decade it has become clear that these virus-encoded proteinases have evolved to degrade not only key host cell proteins (e.g. eIF4G) involved in cellular processes to promote virus replication, but also to suppress the host cell innate immune system. References

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The Foot-and-mouth Disease Virion: Structure and Function Mauricio G. Mateu

Abstract X-ray crystallography, cryoelectron microscopy, nuclear magnetic resonance spectroscopy or a combination of methods have been used to determine the structure of foot-and-mouth disease virus (FMDV) virions, capsids and capsid components, and their complexes with receptors or antibodies. Interpretation and comparison of the structures solved, together with the results of many structure-based biophysical, biochemical and biological studies on the properties and functions of FMDV virions and their components, have greatly increased our understanding of FMDV biology in atomic detail. This knowledge is also facilitating the development of better anti-FMD vaccines, and may help the design of anti-FMDV drugs. The present chapter reviews the structure of the FMDV virion and many structural aspects related with different steps of the infectious cycle in which the virion participates: morphogenesis, maintenance of physical integrity, interaction with antibodies and escape from antibody recognition, recognition of cellular receptors, and viral genome uncoating. The production of FMDV empty capsids and the structure-based engineering of FMDV virions and empty capsids for the development of improved or novel anti-FMD vaccines are also reviewed. Introduction Excellent condensed overviews on the structure of foot-and-mouth disease virus (FMDV) have been published in the last years (Fry et al., 2005a; Fry and Stuart, 2010; Han et al., 2015). This chapter contains a considerably expanded and updated

4

review on the structural biology of the FMDV virion. The next section describes the atomic structure of FMDV. Later sections consider our current structure-based understanding of stages of the FMDV infectious cycle in which the virion participates. These later sections have been written as self-contained mini-reviews (based on the structural information provided next), so the reader may skip any of these sections without compromising readability of the others. They successively contemplate morphogenesis, the stage in the cycle where a virion begins its existence in a host cell; virus stability, interaction with antibodies and recognition by cell receptors, three critical aspects in the path of the virion towards its multiplication in another cell; and genome uncoating, the stage in the cycle where a virion ceases to exist. Applied research for the development of improved anti-FMD vaccines (production of empty capsids) or for increasing their stability (structure-based engineering of virions and capsids with increased thermostability) is summarized as a part of related sections reviewing FMDV morphogenesis or structural determinants of (in)stability. Excessive overlaps with Chapters 5 (receptors), 10 (immunology) and 13–14 (novel vaccines) have been avoided by focusing here only on those aspects more directly related to virus structure. Some subjects that are closer to my specific areas of expertise (e.g. antibody recognition, virion assembly and stability) are described in more detail. I have not been able to review here every facet and study related with this chapter’s subject. I apologize to the researchers whose structure-related work on FMDV have not been explicitly mentioned or directly referenced.

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Structure of the FMDV virion In the last ~35 years many high-resolution structures of spherical viruses, including different picornaviruses, have been obtained by X-ray crystallography or, in a few cases, by cryoelectron microscopy (cryo-EM). These studies have provided atomic-detail views of virus particles that reveal structural resemblances as well as many differences underlying their diverse properties and biological functions. The reader interested in placing work with FMDV in a more general structural context may want to consult recent structural virology books by Agbandje-McKenna and McKenna (2011), Rossmann and Rao (2012), or Mateu (2013a), or the overviews by Johnson and Speir (2008), Prasad and Schmid (2012), Harrison (2013), Castón (2013) or san Martín (2013). General overviews on picornavirus structure include those by Rossmann (2002) or Fry and Stuart (2010). Books and reviews on picornaviruses that contemplate structure–function relationships include those by Semmler and Wimmer (2002), Ehrenfeld et al. (2010), Tuthill et al. (2010) or Racaniello (2013). The first crystal structures of picornaviruses, those of the enteroviruses human rhinovirus (HRV) and poliovirus (PV), were respectively solved by Michael Rossmann’s and Jim Hogle’s groups as early as 1985 (Rossmann et al., 1985; Hogle et al., 1985). The structure of Mengovirus followed in 1987 (Luo et al., 1987) and, shortly thereafter, David Stuart and collaborators determined the first crystal structure of a FMDV (Acharya et al., 1989). The crystal structures of other FMDV virions and capsids followed, all of them solved by D. Stuart and coworkers as a part of collaborative studies. This extensive crystallographic work allowed atomic-detail comparisons of (i) virions of different serotypes, O (Acharya et al., 1989), C (Lea et al., 1994), A (Curry et al., 1996) and SAT1 (unpublished; Protein Data Bank ID 2wzr); (ii) virions of a same serotype (Lea et al., 1995; Fry et al., 2005b); (iii) neutralizing antibody-resistant virions versus their parent virions (Parry et al., 1990; Lea et al., 1995); (iv) reduced versus non-reduced type O virion (Logan et al., 1993); (v) FMDV variants adapted to different growth conditions (Curry et al., 1996); (vi) empty (RNA-free) capsid versus full virion (Curry et al., 1997); (vii) a virion complexed

with a cell receptor fragment versus the uncomplexed virion (Fry et al., 1999, 2005b); and (viii) engineered empty capsids of increased thermostability versus the parent capsids (Porta et al., 2013b; Kotecha et al., 2015). The crystal structures of complexes between FMDV-neutralizing antigen-binding antibody fragments (Fab) and an FMDV capsid peptide (the VP1 GH loop) that contains a major antigenic site and the natural cellular receptor binding site were determined by Ignacio Fita, Nuria Verdaguer and collaborators (Verdaguer et al., 1995, 1996, 1998; Ochoa et al., 2000). Structures of complexes between the same Fabs and the FMDV virion were obtained by Elizabeth Hewat and collaborators using cryo-EM (Hewat et al., 1997, Verdaguer et al., 1999). Studies on the solution structures of the VP1 GH loop have been carried out by a number of groups, and the interaction between the VP1 GH loop and a natural integrin receptor of FMDV using nuclear magnetic resonance (NMR) spectroscopy was studied by DiCara et al. (2007). General structure of the FMDV virion Comparison of the crystal structure of FMDV (Acharya et al., 1989; Lea et al., 1994; Curry et al., 1996) with those of other picornaviruses confirmed the expected broad structural similarities between animal viruses of a same family (summarized in this subsection). However, it revealed also unexpected structural differences (as outlined in the next two subsections) whose biological relevance has, in several cases, been investigated (reviewed in later sections). The small (30  nm in diameter), roughly spherical FMD virion (Fig. 4.1a) is formed by a nonenveloped, hollow protein capsid of icosahedral symmetry (Fig. 4.1b) that contains the genomic RNA molecule. As in all picornaviruses, the mature FMDV capsid is made of 60 copies of each of three major structural proteins termed VP1 (1D), VP2 (1B), VP3 (1C) (Fig. 4.1b), and a smaller polypeptide termed VP4 (1A). As in PV, HRV and most picornaviruses, VP4 is myristoylated at its N-terminus (Chow et al., 1987), and can be regarded as a long N-terminal extension of VP2 that is released by proteolytic cleavage during virion maturation (see ‘Morphogenesis of FMDV’, below). VP1, VP2 and VP3, each about 210–220 residues in length, share a

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Figure 4.1  (a) Structure of the FMDV virion. VP1, VP2 and VP3 are respectively coloured blue, green and red. (b) Icosahedral organization of the VP proteins in the FMDV capsid. VP1, VP2 and VP3 belonging to one biological protomer are coloured as in (a); cyan and violet thick lines outline two neighbouring pentamers. (Reproduced from Rincón et al., 2014.)

common fold consisting of a trapezoidal, eightstranded β-barrel (Acharya et al., 1989; Fry et al., 2005a) (Fig. 4.2a). The eight β-strands (labelled alphabetically as they appear in the polypeptide chain from the N-terminus to the C-terminus) form two fourstranded β-sheets (respectively including strands C, H, E, F, and B, I, D, G) that together adopt a wedgelike shape. These two β-sheets together make up a large part of two, relatively flat sides of the trapezoidal VP and of the capsid inner surface, where the VPs N-termini are also located (Fig. 4.2a). The β-strands are connected by loops of variable length (each labelled according to the two strands they connect, Fig. 4.2a). The relatively short BC, HI, DE and FG loops are located at the narrow end of the wedge, closest to a capsid symmetry axis (five-fold for VP1 and three-fold for VP2 and VP3), with the BC loop closest to the capsid outer surface, and the FG loop closest to the inner surface. The

Figure 4.2  (a) Schematic fold of VP1, VP2 and VP3 of FMDV. Single letters and letter pairs respectively identify β-strands and loops. Capsid surface is up (arrow). (b) Approximate positions of most surface-exposed loops and C-termini of VP1, VP2 and VP3 in one biological protomer in the FMDV capsid. The black circle indicates the approximate size of an antibody footprint drawn to the same scale as the viral capsid. (Reproduced from Sobrino, F. and Domingo, E. (eds). 2004. Foot-and-mouth Disease: Current Perspectives (Wymondham, UK: Horizon Bioscience); originally adapted from Harrison, S.C. 1989. Nature 338: 205–206, with permission).

longer GH, EF and CD loops, together with the protein C-terminus, make up a large part of the two other, more irregular sides of the trapezoidal VP structure, and of the capsid outer surface, with the GH and EF loops being the most exposed (Fig. 4.2a and b). The similar size and shape of the structurally homologous VP1, VP2 and VP3 allows these three proteins to fit quite closely as trapezoidal ‘bricks’ to build a capsid with pseudoT = 3 (P = 3) icosahedral symmetry (Figs. 4.1b and 4.2b). Five VP1 subunits are arranged around each capsid five-fold axis, and three copies of VP2 and three copies of VP3 alternate around each capsid three-fold axis. The VP4 polypeptide adopts an extended, partly disordered conformation, with the N-terminus close to a

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capsid five-fold axis and the C-terminus close to a capsid three-fold axis. The capsids of FMDV or any other picornavirus can be subdivided into 60 equivalent, roughly trapezoidal substructures, each containing one copy of each VP (Figs. 4.1b and 4.2b). The VPs are associated through extensive noncovalent interactions, with the largely extended VP4 polypeptides running along the inner surface. This substructure, termed the biological protomer, constitutes the building block from which the capsid is assembled (see ‘Morphogenesis of FMDV’, below). Each triangular facet of the icosahedral capsid (in which VP1, VP2 and VP3 are related by a pseudothree-fold axis) was defined as a crystallographic protomer. It is important to remember that it is the (trapezoidal) biological protomer, with its particular quaternary organization, and not the (triangular) crystallographic protomer, the one that has a free existence as a capsid building block. In FMDV and picornaviruses in general, five biological protomers associate around each capsid five-fold axis to form a higher-order, pentagonal capsid substructure termed the pentamer (Fig. 4.1b). The protomers in each pentamer are held together mainly by multiple non-covalent interactions that are, however, less extensive than those involved in intraprotomer interactions (Arnold and Rossmann, 1990; VIPER database, Carrillo-Tripp et al., 2009). The association of the N-termini of VP3 and VP4 around each five-fold axis contribute to connect the protomers in the pentamer. The picornaviral pentamer has a free existence as a capsid assembly intermediate (see ‘Morphogenesis of FMDV’, below). In the picornaviral capsid, including that of FMDV, the pentamers are noncovalently associated with neighbouring pentamers mainly through rather flat protein–protein interfaces, energetically weaker than the interprotomer interfaces within each pentamer (Arnold and Rossmann, 1990; VIPER database, Carrillo-Tripp et al., 2009). Formation of β-annuli made of VP2 N-termini, and of extended β-strands crossing the interpentamer interfaces contribute to connect the pentamers in the capsid. Also in common with other picornavirus structures, no ordered RNA could be observed in the crystal structure of any FMDV virion. In contrast to what was found for some other icosahedral plant

or animal viruses (e.g. the T = 1 parvoviruses), in most picornavirus structures no segments of the viral RNA molecule are seen to adopt similar, icosahedrally ordered folds that bind equivalent sites at the capsid inner wall. In some crystal structures of PV (Filman et al., 1989) or HRV (Arnold and Rossmann, 1990), a few nucleotides were tentatively identified in a similar location, and in a PV structure, nucleotide bases stacked with aromatic residues of VP4 were detected (Lentz et al., 1997). Other, disordered portions of VP4 might be interacting with segments of the RNA molecule. Icosahedrally ordered density corresponding to parts of the viral RNA and some capsid-RNA contacts have been identified in HRV virions and uncoating intermediates and in enterovirus 71 (Verdaguer et al., 2000; Wang et al., 2012; Pickl-Herk et al., 2013). Thus, the picornaviral RNA appears to be loosely tethered to the capsid, at least in enteroviruses. However, no ordered nucleotides could be observed in any FMDV structure so far. Positively charged polyamines that may help to neutralize the negative charge of the viral RNA have been detected in HRV and PV virions (Fout et al., 1984), and they may be expected to be present also in FMDV virions. Specific structural features of the FMDV virion The features of the FMDV virion described above are shared with PV, HRV and other picornaviruses. However, several structural features distinguish FMDV from other picornaviruses; some of them are unique. [Note: in this chapter, residues in FMDV capsids are identified by using four-digit numbers, in which the first digit identifies the capsid protein (VP1, VP2, VP3 or VP4) and the three other digits correspond to the residue number in the polypeptide chain (Lea et al., 1994)]. The smooth outer surface of the capsid The FMDV capsid is thinner than the capsids of other picornaviruses because the surface loops are generally shorter. The average thickness of the FMDV capsid is about 3.3 nm (75% of the HRV capsid thickness) if VP4 is excluded. The capsid surface is quite smooth (Fig. 4.1a), with no conspicuous topographic features such as the large depressions or protrusions found in other

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picornaviruses (the long and mobile VP1 GH loop being an outstanding exception). The biologically relevant, deep canyons or pits found in the capsids of enteroviruses or Mengovirus are filled in FMDV by the C-terminal segment of each VP1 subunit as it runs along the capsid surface. Absence of capsid pockets The 60 equivalent, large hydrophobic pockets behind the canyon floor in enterovirus capsids that can accommodate the so-called pocket factors, and which have critical roles for HRV and PV infections, do not exist in FMDV. The capsid five-fold axes Compared to other picornaviruses, the VP1 β-barrels in the FMDV capsid are more canted up towards the five-fold axes, which leads to a higher exposure of the short BC, HI, DE and FG loops of VP1 on the capsid surface, and a quite different local topography. As in other picornaviruses, the N-termini of five VP3 subunits form an internal β-annulus (cylinder) at each of the twelve five-fold axes in the FMDV capsid. In FMDV, this annulus delineates a largely hydrophobic channel (pore) with an average inner diameter of 10 Å that communicates the virus interior with the environment. VP3 residues including Phe 3003, Val3005 and Cys3007 form a constriction, with the side chains (sc) of Cys3007 acting as a diaphragm with an aperture about 6 Å in diameter. In PV, an inner layer formed by the VP4-linked myristyl groups and N-terminal amino acid residues of five VP4 subunits is observed below each five-fold axis, but these elements are not visible in any FMDV crystal structure; no ordered residues obstruct the five-fold axis pore (Acharya et al., 1989), although the myristyl groups could still form a cluster at the base of the five-fold axis which may interact with the VP3 β-annulus. Under non-reducing conditions, some of the five cysteines around each five-fold axis are linked by disulfide bridges, which could limit the dynamics of the pore region. The existence of these pores in the FMDV capsid and their basal free diameter is consistent with the penetration of caesium ions and small organic compounds into the virion. Interestingly, the RNA-alkylating agent N-dansylaziridine

inactivated FMDV at 37°C but not at 25°C, which suggests that the pores in the FMDV capsid may be conformationally dynamic and may open enough to allow penetration of this relatively large compound (Broo et al., 2001). The capsid three-fold axes In the FMDV virion the N-termini of three VP2 subunits form a short internal β-annulus at each of the 20 capsid three-fold axes, that fills a depression on the capsid inner wall. This β-annulus contributes to the association of three different pentamers together around each three-fold axis. The presence of a calcium ion bound to Glu2006 residues within the VP2 β-annulus was suggested (Acharya et al., 1989). Interpentamer β-sheets In the FMDV capsid extended, six-stranded β– sheets cross the interfaces between neighbouring pentamers. These sheets are formed by the C, H, E, F strands from VP3 of one pentamer, and the two strands that form a β-hairpin in the N-terminal segment of VP2 of the adjacent pentamer (Fry et al., 2005a). In HRV and PV, the equivalent extended β-sheets include a seventh strand contributed by the VP1 N-terminal segment. Serotype-specific disulfide bonds In serotype O FMDVs, but not in other serotypes, a disulfide bond is formed under non-reducing (extracellular) conditions between Cys1134 (located at the beginning of the VP1 GH loop) and Cys2130 in VP2, covalently linking VP1 and VP2 in each biological protomer. Reducing conditions reversibly break this bond, leading to conformational changes that involve the VP1 GH loop (see below), VP2 EF loop and VP3 GH loop. The highly mobile GH loop of VP1 In FMDV, the GH loop of VP1 (also called the ‘FMDV loop’) contains both a major antigenic and immunogenic region (see ‘Structural insights into the recognition and neutralization of FMDV by antibodies’, below, and Chapter 10) and the integrin receptor binding site (see ‘Structural insights into the recognition of cell receptors by FMDV’, below, and Chapter 5). This long loop encompasses approximately residues 1130–1160 (type O

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numbering). It came as an initial disappointment that, in every FMDV crystal structure solved (except that of reduced type O virions), most of the VP1 GH loop (residues 1137–1156 in type O viruses) is invisible due to structural disorder. Given the major functional relevance of, and extensive studies on this capsid element, its structure is reviewed in detail next. Important functional implications of the VP1 GH loop structure for virus–antibody and virus–receptor recognition are, respectively, contemplated in the corresponding sections.

Structure of the VP1 GH loop Structure of the unliganded VP1 GH loop as a part of the virion A structure for the unliganded form of the VP1 GH loop in the FMDV virion could be determined only for serotype O isolate (strain O1BFS) in which the serotype-specific VP1-VP2 disulfide bond had been broken by treatment with a reducing agent (Logan et al., 1993; Fig. 4.3a and c). The reduced virion remained infectious (Logan et al., 1993), although

Figure 4.3  Structure and location of the VP1 GH loop. (a) in a reduced type O virion (loop ordered in the ‘down’ orientation; Logan et al., 2003). (b) in a complex between a type C virion and the Fab fragment of neutralizing antibody SD6 (Hewat et al., 1997). In (a) and (b), left and right images respectively show ribbon diagrams of a pentamer (front view) and a protomer (side view) in the virion. The capsid VP subunits are coloured as in Fig. 4.1. (c) close-up view of the VP1 GH loop structure (ribbon diagram) in reduced type O virion. (Reproduced from Sobrino, F. and Domingo, E. (eds.). 2004. Foot-and-mouth Disease: Current Perspectives (Wymondham, UK: Horizon Bioscience)).

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its infectivity was markedly inferior to that of the non-reduced virion (Fry et al., 2005a). In the VP1 GH loop of reduced type O virion (Fig. 4.3c), a stretch of residues (1144–1146) in the N-terminal part of the VP1 GH loop adopts a β-strand conformation adjacent to β-strand C of VP2, extending the CHEF β-sheet of this protein. The central Arg-Gly-Asp (RGD) motif (residues 1145–1147), critical for integrin binding, adopts an open turn conformation very similar to those observed for the same motif in γ-crystallin, other integrin-binding proteins, and a complex between a RGD peptide and integrin αvβ3 (Xiong et al., 2002), which is also a receptor for FMDV. The segment that follows the RGD turn (residues 1148–1155) in the loop folds as a short 310-helix (Fig. 4.3c). The same overall conformation of the VP1 GH loop is adopted in type O viruses differing in up to four amino acid residues within the central 15-residue stretch in this loop (Lea et al., 1995). The immobilized VP1 GH loop fits a shallow depression on the surface of VP2 of the same biological protomer. Structure of the VP1 GH loop as an unliganded, functional synthetic peptide Studies by different groups showed that some synthetic peptides representing the sequence of the VP1 GH loop faithfully mimicked the antigenicity and immunogenicity of this loop in the virion (see ‘Structural insights into the recognition and neutralization of FMDV by antibodies’, below) and could be used as experimental FMD vaccines (Chapter 13). Those peptides could also efficiently inhibit binding of the virion to host cells and viral infection. Moreover, the effects of amino acid substitutions on recognition of the VP1 GH loop in the virion by antibodies could be also faithfully mimicked using synthetic peptides (see ‘Structural insights into the recognition and neutralization of FMDV by antibodies’). These results showed that the functionality of the VP1 GH loop is largely selfcontained, and have justified the extensive use of synthetic peptides to investigate in detail its structure and function. Studies of either linear or cyclized (conformationally restricted) synthetic peptides representing (partial) sequences of the VP1 GH loop of different FMDV variants have been carried out in solution, using circular dichroism, NMR spectroscopy and/ or molecular modelling (Siligardi et al., 1991;

Camarero et al., 1993; France et al., 1994; Pegna et al., 1996a,b; Haack et al., 1997; de Prat Gay, 1997; Petit et al., 1999; Furrer et al., 2001; DiCara et al., 2007; Wagstaff et al. 2012). In general, these studies revealed that the loop-mimicking peptides are essentially disordered in aqueous solution, but in structure-inducing solvents they have strong conformational propensities. In some studies, the conformational preferences found include a β-turn conformation in the RGD region, followed by a helical segment (France et al., 1994; Haack et al., 1997; DiCara et al., 2007; Wagstaff et al., 2012), similar to what was observed in the crystal structures of reduced type O virus and of VP1 GH loop–antibody complexes (next). Structure of the VP1 GH loop as a functional synthetic peptide bound to virus-elicited neutralizing antibodies A 15-mer synthetic peptide (A15) that represents the central sequence of the VP1 GH loop (residues 1136–1150) of a serotype C virus (strain C-S8) binds different virus-neutralizing monoclonal antibodies (MAbs) elicited against the whole virion. Comparative quantitative immunochemical analysis indicated that the conformation of the A15 peptide in a complex with a virus-induced neutralizing antibody should accurately mimic the conformation of the VP1 GH loop in a complex between the complete virion and the same antibody. This observation prompted the determination of the crystal structures of complexes between peptide A15 variants representing the sequences of two type C virus isolates, and the Fab fragments of neutralizing MAbs (SD6 and 4C4) that recognize overlapping continuous epitopes in the VP1 GH loop (Verdaguer et al., 1995, 1998; Ochoa et al., 2000; Fig. 4.4a). In every complex, the VP1 GH loop-mimicking peptide adopted a conformation that resembles closely that of the unliganded VP1 GH loop in the reduced type O virion (compare Figs. 4.3c and 4.4b). The peptide conformation is quasi-circular, being stabilized by several intrapeptide hydrogen bonds, van der Waals interactions and some buried hydrophobic area. The stretch immediately before the RGD motif (shorter by four residues in FMDV type C than in type O) is in an extended conformation. The RGD motif (residues 1141–1143 in type C) adopts an open

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Figure 4.4  (a) Structure (ribbon diagram) of the complex between the Fab fragment of FMDV-neutralizing MAb SD6 and the VP1 GH loop-mimicking peptide A15 (top; coloured yellow). The side chains of the RGD motif are depicted (Verdaguer et al., 1995). (b) Close-up ball-and-stick model of the VP1 GH loop peptide in the Fab–A15 complex. The RGD adopts an open turn conformation very similar to that in the unliganded VP1 GH loop in a reduced type O virus (compare Fig. 4.3c); Leucines at RGD+1 and RGD+4 are on the same face of a short helix, as in the NMR structural models of the VP1 GH loop of different serotypes under structure-inducing conditions (compare Fig. 4.9). (Reproduced from Sobrino, F. and Domingo, E. (eds). 2004. Foot-and-mouth disease: current perspectives (Wymondham, UK: Horizon Bioscience)).

turn conformation, almost identical to those in the unliganded VP1 GH loop of the reduced type O virion or in other integrin-binding proteins. A short helical stretch follows the open turn, in a direction that is essentially coincident with that of the longer helix in the VP1 GH loop of the reduced type O virion. Different conformations and orientations of the VP1 GH loop on the virion surface In the crystal structure of unperturbed (nonreduced) type O virion, bifurcated electron density for the disulfide bond linking Cys1134 with Cys2130 suggested the presence of multiple conformations for the disordered VP1 GH loop (Acharya et al., 1989). Comparison of the crystal structures of an antibody-resistant type O mutant virion with its parent virion (Parry et al., 1990) indicated that the VP1 GH loop could adopt two extreme orientations on the capsid surface, termed ‘up’ (closer to a capsid five-fold axis) and ‘down’ (closer to a interpentamer interface; Fig. 4.3a). Mutations in the VP1 BC loop (Parry et al., 1990) or disruption of the VP1-VP2 disulfide bonds (Logan et al., 1993) destabilized the ‘up’

conformation of the VP1 GH loop, leading to a change in the predominant orientation of the loop towards the ‘down’ conformation. Differences in the local conformations and orientations of the visible stretches at the beginning and end of the disordered VP1 GH loop between types O (Acharya et al., 1989; Parry et al., 1990), C (Lea et al., 1994) and A (Curry et al., 1996) virions indicated that this highly mobile, antigenic loop adopts different sets of conformations and orientations in different serotypes (Curry et al., 1996). The structures of complexes between type C FMDV and the Fab fragments of neutralizing MAbs SD6 and 4C4 were later solved by cryoEM (Hewat et al., 1997; Verdaguer et al., 1999). Fitting into the low-resolution electron density maps obtained by cryo-EM of the high-resolution crystal structures of unbound virion and A15-SD6 or A15–4C4 complexes provided quasi-atomic models of FMDV-antibody Fab complexes, and revealed the approximate positions the VP1 GH loop adopts in those complexes. In the type C virion–SD6 complex (Hewat et al., 1997; Fig. 4.3b), the VP1 GH loop was bent towards the ‘up’ position proposed for the non-reduced type O virion. In the type C–virion–4C4 complex the

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loop was oriented radially, fully protruding from, and very loosely connected with the rest of the capsid, with the RGD motif at its apex, farthest from the capsid surface. The fluctuations in position and conformation of the unliganded VP1 GH loop in serotype O FMDV have been simulated by all-atom molecular dynamics (MD) (Azuma and Yoneda, 2009). The coordinates of a crystallographic protomer were used, the simulation was started with the reduced state, the disulfide bond was formed in silico, and the atomic trajectories were followed on a nanosecond timescale. The results were compatible with the experimental observations using type O FMDV or type C FMDV–antibody complexes. In the simulations, three states of the VP1 GH loop were identified: (i) the initial folded state in the ‘down’ position; (ii) an intermediate state in which the unliganded loop is conformationally flexible and moves not as a hinged rigid body, but like a ‘tentacle’, protruding from the capsid surface as in the virion–antibody complexes; (iii) a final, equilibrium state close to the ‘up’ position, in which the loop still fluctuates somewhat between different conformations and orientations. A model for the structure and dynamics of the VP1 GH loop To recapitulate, the available evidence supports a model in which the long, dynamic, mobile VP1 GH loop has a strong intrinsic propensity to transiently adopt some conformational variations of a consensus strand-turn-helix motif. Several observations argue against a permanently, fully folded and stable intrinsic conformation of the unliganded VP1 GH loop on the unperturbed surface of virions of different serotypes (C, A and non-reduced type O): (i) the absence of a defined conformation of the VP1 GH loop as a free, either linear or conformationally restricted peptide in aqueous solution (consistent with the lack of a fully folded conformation for nearly any other protein-derived free peptide of similar length); (ii) the very loose connection of the VP1 GH loop in the virion with the rest of the capsid, especially in some functional (antibody- or receptor-binding) orientations; (iii) the results of all-atom MD simulations; (iv) a comparable MAb-binding affinity of free, disordered VP1 GH loop-mimicking linear peptides, and the VP1 GH loop as a part of the virion.

This latter observation suggests that the entropic penalty that must be paid for binding the free, conformationally disordered peptide to some antiFMDV antibodies must also be paid, with only a small ‘rebate’, when binding the VP1 GH loop as a part of the virion to those same antibodies. The somewhat lower entropic penalty in the latter case would be achieved through limited conformational restrictions (covalent linkage with the rest of the capsid). As with some intrinsically (partially) disordered proteins, binding the ligand (antibody or cellular receptor) or other capsid residues (in the reduced type O FMDV) would fully stabilize the transiently structured VP1 GH loop into one defined conformation. In addition to any internal conformational plasticity, it is clear that the VP1 GH loop undergoes extensive, global movements, and can populate very different orientations on the capsid surface depending on the specific virus variant and the conditions. Some functional consequences of the peculiar structure and dynamics of the VP1 GH loop are considered in ‘Structural insights into the recognition and neutralization of FMDV by antibodies’ and ‘Structural insights into the recognition of cell receptors by FMDV’ (below). Morphogenesis of FMDV As the pseudoT = 3 icosahedral capsid of FMDV (like those of other picornaviruses) is actually assembled from 60 identical protomers, it can also be regarded as a very simple T = 1 capsid made of 60 heterotetramers (each containing one copy of each capsid protein). There is no quasiequivalence, and no conformational switches are required during its assembly. However, in vivo morphogenesis of even the structurally simplest viruses is a complex process that involve a number of viral and cellular proteins, nucleic acid and other biomolecules, and that requires tight spatial and temporal coordination of multiple recognition events at the molecular and cellular levels (Mateu, 2013b; Almendral, 2013). Morphogenesis is still a poorly understood stage in the infectious cycle of FMDV, and of most icosahedral viruses. Steps in FMDV morphogenesis Most studies on picornavirus morphogenesis have been carried out with enteroviruses, and with

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PV in particular. In an excellent recent review by Jiang et al. (2014), Eckard Wimmer and associates recapitulated the experimental evidence obtained during several decades of studies on picornavirus morphogenesis, and proposed an updated, integrated model of enterovirus assembly (Fig. 4.5). The following paragraphs summarize this model and, if available, include evidence that indicates whether each step of the assembly process may be either similar or different for FMDV. Structurebased insights are emphasized. Step 1: Processing of the P1 (capsid) polyprotein In enteroviruses, the capsid precursor polyprotein P1 is released by site-specific cleavage mediated by protease 2Apro and myristoylated at the N-terminus. Release of P1 occurs in FMDV by a different process. FMDV protease 3Cpro releases polyprotein P1–2A, which includes a short peptide (2A) at the C-terminus that will be removed later. As with enteroviruses, the capsid polyprotein is myristoylated at the N-terminus. Descriptions of the post-translational processing of the FMDV P1–2A polyprotein and the proteinases involved are provided in Chapters 2 and 3.

Step 2: Folding and maturation of the protomeric capsid building block Chaperone Hsp90 may help the unprocessed enterovirus capsid protomer (sedimentation coefficient 5S), the elementary capsid building block, to fold into a conformation competent for site–specific cleavage by the viral protein 3CD. The 3CD protease activity breaks the peptide bonds between VP0, VP3 and VP1 to yield the assembly competent mature protomer. After the polyprotein is processed, Hsp90 dissociates. In FMDV, the P1–2A polyprotein is processed into VP0, VP3 and VP1 by 3Cpro to yield the mature protomer. A recent report (Newman et al., 2014) suggests that Hsp90 has a role also in FMDV morphogenesis, but related instead to promoting assembly of pentameric intermediates from mature protomers. The structures of both the unprocessed and processed forms of the capsid protomer have not been determined for any picornavirus. However, structural and other evidence led Hogle and Rossmann and their coworkers to suggest that the unprocessed picornaviral protomer is completely folded as a three-domain protein, with VP0, VP3 and VP1 already adopting their final β-barrel folds, as well as conformations, relative orientations, and intraprotomer interactions that closely resemble

Figure 4.5  A scheme of the model for enterovirus morphogenesis described by Jiang et al. (2014). See text for a detailed explanation. Steps where glutathione (GSH) is required are inhibited by L-buthionine sulfoximine (BSO). Pentamers are recruited by viral protein 2C to the replication complex made of several different viral proteins and bind the VPg-linked viral RNA (red line).

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those in the assembled virus particle (Hogle et al., 1985; Rossmann et al., 1987). In HRV, PV and other picornaviruses including FMDV, extensive intraprotomer interactions and intertwining of N- and C-termini of the VP subunits clearly indicate that the quaternary organization of the capsid building block corresponds closely to that of the trapezoidal biological protomer in the virion (Fig. 4.1b). For FMDV, further evidence was provided by specific MAbs that efficiently bind both the assembled FMDV virion and the unprocessed capsid protomer (Saiz et al., 1994; Goodwin et al., 2009). These antibodies recognize discontinuous epitopes in antigenic sites that can be formed in the biological protomer (Lea et al., 1994), but not in the crystallographic protomer. In addition, both the virion and the unprocessed protomer are similarly recognized by integrin αvβ6, a natural receptor of FMDV (Goodwin et al., 2009). Thus, different regions of the free unprocessed protomer and the mature protomer in the virion must display conformations similar enough to be recognized by proteins whose binding activity is very sensitive to minor structural changes in the ligand. As in HRV, PV and other picornaviruses, in the FMDV virion the VP termini that are joined in the unprocessed protomer are not contiguous in the virion structure. The C-termini of VP2 and VP3 are on the capsid outer surface, while the N-termini of VP3 and VP1 are in the capsid interior. In the folded unprocessed picornaviral protomer, the two covalent linkages between VP0 and VP3, and VP3 and VP1 remain on the surface, fully accessible to proteases. In FMDV at least, the cleavages may be dependent on the protomer local or global structure, as a mutation on the protomer surface, but not in the cleavage site, affected the processing of the VP3-VP1 linkage (Escarmís et al., 2009). After cleavage, some rearrangements of the N-terminal segments of VP3 and VP1 and the C-terminal segments of VP0 and VP3, and perhaps other minor local rearrangements, would suffice to achieve the final structure of the mature, assembly competent 5S protomer. Step 3: Assembly of the pentameric intermediate During picornavirus morphogenesis, mature 5S protomers form relatively stable 14S intermediates,

each made of five protomers (Putnak and Phillips 1981b) (Fig. 4.5). These pentamers can be found in cells infected with any picornavirus. For FMDV there is clear evidence for the progression from stable 5S protomers, to highly stable 12S pentamers, to complete viral particles (e.g. Grubman, 1984; Grubman et al., 1985). A high-resolution structure of the free pentameric assembly intermediate is not available for any picornavirus. However, a low-resolution (30 Å) structural model has been generated for the free pentamer of bovine enterovirus, based on EM imaging and three-dimensional reconstruction of negatively stained pentamers (Li et al., 2012). The model suggests that very large structural rearrangements may not occur during assembly of pentamers into enteroviral particles. In the crystal structures of all picornaviruses, highly complementary interprotomer interfaces are formed within each pentamer. In addition, the β-annulus formed by the N-termini of five VP3 subunits around each five-fold axis knits together the five protomers in the pentamer, and VP4-linked myristates and the N-termini of VP4 (as a part of VP0) enhance the assembly of PV pentamers (Ansardi et al., 1992). Likewise, in FMDV both VP4 and the myristyl group were shown to be important for the correct in vitro self-assembly of pentamers. Deletion of VP4 prevented the association of protomers; absence of the myristate led to association of protomers into capsid assemblyincompetent 17S particles, different from the assembly-competent 12S pentamers that were formed when VP4-containing, myristoylated protomers were used (Goodwin et al., 2009). Step 4: Assembly of immature viral particles In PV and other enteroviruses (but not every picornavirus), 75S empty capsids are formed during the infection process (Hummeler et al., 1962), and can also self-assemble in vitro (Putnak and Phillips, 1981a,b). Inhibition of RNA synthesis in infected cells by low guanidinium chloride concentrations led to the accumulation of procapsids that were converted into virus particles when RNA synthesis was restored (Fernandez-Tomas and Baltimore, 1973). These and other observations are consistent with the view that, in infected cells, the pentamers

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may self-assemble into RNA-free capsids, and the viral RNA would be later packaged in the preformed empty capsid. Other evidence, however, supports an alternative model of enterovirus assembly that is currently favoured. 14S pentamers (but not empty capsids) are able to bind RNA in vitro, which leads to their structural rearrangement (Nugent and Kirkegaard, 1996; Verlinden et al., 2000). After a block causing accumulation of pentamers in infected cells was removed, the pentamers could be chased into mature virions (Rombaut et al., 1990). The viral 2CATPase, a component of the membrane-associated viral RNA replication complex, was found to bind VP1 and/or VP3 and recruit the preassembled pentamers to the site of virus particle assembly (Liu et al., 2010). Recent studies revealed that the presence of an inhibitor of PV morphogenesis causing reduced glutathione depletion did not inhibit the formation of empty capsids, but these were unable to form virions (Ma et al., 2014). These and other observations support for PV the hypothesis that the capsid and viral RNA are simultaneously coassembled. In this model of enteroviral assembly, specific recognition between pentamers and the viral RNA molecule, and condensation of twelve pentamers around the RNA, perhaps with the help of 2CATPase, directly yields an immature, noninfectious 150S provirion ( Jiang et al., 2014) (Fig. 4.5). It has been suggested that the empty capsids formed in infected cells may constitute an offpathway assembly product that does not act as a direct precursor of the assembled virion, but that could act as a physiological reservoir of capsid proteins. Nonetheless, the possibility that an on-pathway empty capsid is formed first and the enteroviral RNA is later packaged into the preassembled capsid has not been entirely ruled out. Many other important aspects of this step in enterovirus morphogenesis remain unclear. For example, picornaviral RNA packaging signals have been searched with no success to date, except for the Aichi virus of the genus Kobuvirus (Sasaki and Taniguchi, 2003); it is unclear whether the RNAbound VPg could interact with capsid proteins during morphogenesis; etc. ( Jiang et al., 2014). As for other picornaviruses, FMDV empty capsids are naturally formed in infected cells (Rowlands et al., 1975), and can also be assembled inside cells (reviewed in Dong et al., 2014) and

cell-free translation systems in vitro (Grubman 1984; Grubman et al., 1985). Early studies on FMDV morphogenesis by Eduardo Palma and colleagues using pulse-chase experiments revealed a precursor–product relationship between capsid precursors, procapsids (empty capsids) and virions, with an observed rate of virion formation from procapsids that was identical to the observed rate of empty capsid assembly from capsid precursors (Gomez-Yafal and Palma, 1979). These results led the authors to propose a model in which the FMDV empty capsid is self-assembled first, and the viral RNA is packaged later in the preformed capsid, using some unknown mechanism. The authors also considered the alternative possibility that empty capsids could serve as a reservoir of capsid proteins and could dissociate into pentamers which, in turn, would coassemble with the RNA to form the provirion. However, they suggested that for FMDV this latter possibility is unlikely, based on the observed quantitative transformation of empty capsids into virions, and on stability considerations. Further studies are required to decide whether FMDV empty capsids are (on-pathway) productive intermediates of virion assembly, or (off-pathway) dead-end products or capsid protein reservoirs. Step 5: Maturation of the provirion The noninfectious, relatively stable enterovirus provirion is converted into a mature, infectious 150S virion by a RNA-dependent, probably autocatalytic cleavage of VP0 into VP4 and VP2 (Fig. 4.5). This maturation process entails a substantial conformational rearrangement of the PV particle, including repositioning and organization of the VP4 and VP2 termini (Hogle et al., 1985), which provide further interactions between pentamers and likely contributes to stabilize the mature virion. His2195, conserved in all picornaviruses, was identified as being essential in PV for efficient cleavage of the VP4–VP2 linkage; mutations of this residue led to assembly of VP0-containing, RNA-containing, immature 150S particles that were highly unstable (Basavappa et al., 1994; Hindiyeh et al., 1999). Morphogenesis of FMDV also includes as a final step the cleavage of VP0 into VP4 and VP2 to yield an infectious mature virion. As in other picornaviruses, this process entails a considerable conformational reorganization of the virion, including the repositioning and reorganization of

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VP2 and VP4 termini and the formation of secondary structure elements (β-annuli at the three-fold axes and extended β-sheets) that reinforce the connection between pentamers (Acharya et al., 1989; Lea et al., 1994). No stable, non-infectious FMDV provirions have been detected so far, but a combination of mutations around the VP4–VP2 cleavage site led to production of VP0-containing, non-infectious provirions (Knipe et al., 1997). Empty capsids that are naturally produced during infection by PV or other picornaviruses lack viral RNA, and usually contain uncleaved VP0. In contrast, in a serotype A FMDV empty capsid used for structural studies, most VP0 subunits had been cleaved into VP4 and VP2 (Curry et al., 1995). However, this cleavage likely occurred downstream of the cleavage site observed in mature virions. Comparison of the crystal structure of this FMDV empty capsid (Curry et al., 1997) with that of PV suggested that the RNA-dependent, autocatalytic, His2195-mediated mechanism proposed for VP0 cleavage in PV operates also in FMDV. A structural comparison of the empty capsid and the corresponding virion (serotype A) (Curry et al., 1997) allowed an appraisal of the structural effects the RNA exerts on the mature capsid structure (after VP0 processing). The differences observed are confined to the interior of the capsid. The N-terminus of VP1 and C-terminus of VP4 are less well ordered in the empty capsid. This higher disorder suggested a more loose association of these segments that help connect the capsid subunits, protomers, and pentamers, and a role of the viral RNA in the stabilization of some intersubunit interfaces, including those between pentamers. An (incomplete) model for FMDV morphogenesis To recapitulate, the limited evidence available tends to support a model for FMDV morphogenesis that resembles the model proposed for enterovirus assembly by Jiang et al. (2014) and schematized in Fig. 4.5, with some important differences and uncertainties: 1

2

The unprocessed P1–2A polypeptide folds as a three-domain (VP0, VP3, VP1) protein, the immature biological protomer, which constitutes the elementary capsid building block. The immature protomer is made competent

3

4

5

6

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for assembly upon its proteolytic processing by 3Cpro that converts the three domains into individual proteins VP0, VP3 and VP1. The three proteins remain non-covalently associated in the mature protomer, whose conformation closely resembles that of the immature protomer, except for the repositioning of the released, solvent-exposed termini of VP0, VP3 and VP1. Five mature protomers associate to form a highly stable pentameric intermediate. The formation of pentamers that are competent for self-association into complete particles involves the establishment of many protomer– protomer interactions, including association of the VP3 N-termini in a β-cylinder around the central five-fold axis, and requires the participation of the VP4 N-termini and myristate groups. This morphogenetic step may be helped by chaperone Hsp90. Twelve assembly-competent pentamers associate to form complete empty capsids. These viral RNA-free capsids present an outer surface that is quite similar to that of the virion, but differ from the latter in some structural features of its inner surface. RNA-containing, non-infectious virus particles are formed. FMDV empty capsids may constitute on-pathway assembly intermediates into which the viral RNA is encapsidated by an unknown mechanism; however, evidence obtained with enteroviruses favours the possibility that picornavirus empty capsids are dead-end products or reservoirs of capsid proteins. If this pathway occurs, assembled empty capsids should eventually dissociate again into pentamers, and free pentamers (or partially disassembled empty capsids) would associate with the newly synthesized viral RNA to form non-infectious provirions in a single coassembly plus encapsidation step. The non-infectious, unstable provirion immediately matures into a relatively stable, infectious virion; this process involves the RNA-dependent, probably autocatalytic cleavage of VP0 into VP4 and VP2, and a substantial conformational reorganization of parts of the inner capsid surface in the viral particle, that may contribute to stabilize the mature virion.

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Structural and functional dissection of intersubunit interfaces involved in FMDV assembly A molecular dissection of the two different intersubunit interfaces consecutively formed during FMDV assembly, based on the crystal structure of a model FMDV virion (type C isolate C-S8) (Lea et al., 1994), revealed a fundamentally different structural organization and functional response to mutation (Mateo et al., 2003; Rincón et al., 2015). Interprotomer interfaces in each pentamer Contact analysis of the interface between two neighbouring protomers within a pentamer (Rincón et al., 2015) revealed that about 90 residues belonging to either protomer participate in the association. Interprotomer main chain-main chain (mc-mc) interactions included those involved in the formation of the five-strand β-cylinder at the five-fold axis. Formation of secondary structure elements does not automatically involve in itself a substantial stabilization of the folded state of a protein, or the associated state of a protein complex, as the connecting mc-mc hydrogen bonds only replace those formed with water in the unfolded or dissociated state. Thus, the actual direct stabilizing effect of the β-cylinder may largely depend on the strength of interactions and buried hydrophobic area brought about by the sc of the residues in the cylinder. Mutation Cys3007Val in the cylinder, that eliminates any disulfide bonds at the capsid pores, had no detrimental effect on virion assembly, infectivity, or stability against thermal inactivation; in contrast, mutation Asp3009Ala nearby was lethal, unless compensated by fixation of another mutation nearby (Mateo et al., 2007). Excluding the β-cylinder, each protomer– protomer interface within a pentamer, although narrow and very elongated, is essentially made up of a central, buried hydrophobic region flanked by solvent-exposed polar residues, as in most protein–protein interfaces studied. Dissection by alanine scanning of the individual role in virus infectivity of many of the sc (beyond Cβ) at these interprotomer interfaces showed that most of them, even those involved in presumably strong interactions, are not required for virus infectivity (Rincón et al., 2015).

Interpentamer interfaces in the virion Contact analysis of the interfaces between pentamers (Mateo et al., 2003) revealed that about 60 residues participate in the association of each biological protomer in a pentamer with other protomers belonging to two neighbouring pentamers around a three-fold axis (Fig. 4.2b). Interpentamer mc–mc interactions include those involved in connecting β-strands in neighbouring pentamers, or in the β-annulus at each three-fold axis that knits together three pentamers. Again, the actual stabilization achieved by these elements may depend on the strength of the interactions established by the sc in these elements. These interactions remain undefined around the three-fold axes, as for the three-fold annulus only the backbone could be traced in the crystal structure of any FMDV. The interpentamer interfaces, quite unlike the interprotomer interfaces within the pentamer, are essentially made up of polar residues, with no substantial hydrophobic central region. Dissection by alanine scanning of the individual role in virus infectivity of nearly every sc (beyond Cβ) that participates in these interpentamer interfaces showed that the vast majority of them are critically required for virus infectivity, probably because they individually exert important roles in capsid assembly and/or stability (Mateo et al., 2003). It has been tentatively suggested that the different structural organization of these interfaces, and their different functional sensitivity to mutation, could be the result of different selective pressures: during the viral cycle pentamers need not be disassembled into protomers (Rincón et al., 2015). Thus, selection may have favoured energetically strong intrapentamer interfaces that are quite insensitive to comparatively small energetic effects caused by individual mutations during virus variation in the field. During virus morphogenesis in the host cell, this high-energy association would ensure the maximum availability of stable pentamers to reach the critical concentration required for efficient assembly of many viral particles. In contrast to individual pentamers, FMDV virions must be disassembled for genome release. Thus, the virus could have evolved polar, energetically weak interpentamer interfaces for facilitating the acidinduced endosomal uncoating of the genome (see ‘Structural insights into uncoating of the FMDV

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genome’, below). The trade-off of such energetically weak interfaces would be an extreme sensitivity to disruption by even single mutations that remove a few interpentamer interactions. This sensitivity, however, may be not detrimental to rapid FMDV evolution: the virus manages to accept the introduction of many individual lethal mutations at the interpentamer interfaces through fixation of compensatory mutations that frequently occur at a few ‘compensation hotspots’ in the viral capsid (Luna et al., 2009). Production of FMDV empty capsids for the development of novel anti-FMD vaccines The early discovery that empty capsids are formed during FMDV morphogenesis, and that they share the same antigenic and immunogenic characteristics as the virion (Rowlands et al., 1975) led the development of different approaches to produce FMDV empty capsids, either for fundamental studies or as a basis for novel vaccines. The design of alternative anti-FMD vaccines is a quite intense area of applied research (Rodriguez and Grubman, 2009; Chapters 13 and 14). Empty capsid-based vaccines would avoid any risk of virus escape or deficient inactivation during vaccine production, while preserving the full immunogenicity and antigenic spectrum of current virion-based vaccines; capsid-based vaccines would also allow differentiation between vaccinated and infected animals (DIVA assays). The development of procedures to obtain substantial amounts of recombinant FMDV empty capsids in different cell types (mammalian, insect or bacterial) or transgenic plants for vaccination purposes started about 25 years ago, and is being actively pursued today (Roosien et al., 1990; Lewis et al., 1991, Abrams et al., 1995; Li et al., 2008, 2011; Pan et al., 2008; Cao et al., 2009, 2010; Lee et al, 2009; Polacek et al., 2013; Gullberg et al., 2013; Guo et al., 2013; Porta et al., 2013a,b; reviewed by Dong et al., 2014). Such strategies are recapitulated here. DNA vaccines based in the use of recombinant adenovirus carrying the FMDV capsid polyprotein P1–2A and 3C genes for expression of immunogenic empty particles directly in the vaccinated animals (reviewed by Grubman et al., 2010; Fowler and Barnett, 2012) are described in Chapter 14.

Production of FMDV empty capsids using baculovirus-based expression systems In 1990, Just Vlak and collaborators described a procedure based on the introduction of the P1–2A and 3C protease coding regions of FMDV in a baculovirus expression vector to produce in insect cells small amounts of 70S particles that resembled FMDV empty capsids (Roosien et al., 1990). In recent years, a number of articles have described other baculovirus-based approaches to produce FMDV empty capsids. Li et al. (2008, 2011) used recombinant baculovirus containing the FMDV P1–2A and 3C coding regions to obtain empty capsids in silk worms. The capsid preparations obtained protected bovines against viral challenge. Cao et al. (2009) constructed recombinant baculovirus to simultaneously express P1–2A and 3C of FMDV in insect cells. The 70S empty capsids obtained were characterized in immunoassays and imaged by EM, and were shown to induce anti-FMDV neutralizing antibodies in guinea pigs. Cao et al. (2010) avoided the need to use the 3C protease by expressing separate VP0 and VP1–2A-VP3 that were processed and assembled into capsid-like particles in insect cells. Porta et al. (2013a, 2013b) described the expression of FMDV empty capsids in insect cells using recombinant baculoviruses expressing P1–2A–3C with a number of modifications to reduce 3C activity, thus decreasing its toxic effects and increasing empty capsid production. Empty capsids were obtained in yields high enough to be purified and crystallized. They were characterized immunochemically and by EM and genetically modified to increase their stability (see next section), and their structure was solved by X-ray crystallography (Porta et al., 2013b; Kotecha et al., 2015). The purified empty capsids were shown to induce high levels of neutralizing antibodies in guinea pigs and bovine, and to protect cattle against challenge with virus (Porta et al., 2013b). Ruiz et al. (2014) described a modified baculovirus-based expression system that has also been used to produce FMDV empty capsids in insect larvae, leading to high yields (about 30 micrograms/g of insect biomass) that compared to those obtained by Li et al. (2008, 2011) and Porta et al. (2013a,b).

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Production of FMDV empty capsids using vaccinia virus-based expression systems In 1995, Graham Belsham and associates described the use of a recombinant vaccinia virus containing the P1–2A and 3C coding regions for production in mammalian cells of FMDV 70S empty capsids that were characterized by EM and immunoassays with anti-FMDV MAbs directed against discontinuous epitopes (Abrams et al., 1995). Very recently, different modifications of vaccinia virus-based expression systems have been described to substantially increase empty capsid yields. Polacek et al. (2013) showed that cotransfection using a plasmid encoding P1–2A and lower amounts of a plasmid encoding 3C was sufficient to obtain efficiently processed capsid proteins. Gullberg et al. (2013) described two alternatives to reduce 3C activity in the transfected cells: the use of a mutated 3C (Cys142Ser) with reduced specific activity; or the use of internal ribosome entry site (IRES) elements with reduced ability to direct internal initiation of translation to produce lower levels of 3C. They showed that P1–2A was efficiently expressed and processed, and that 70S empty capsids were produced. EM imaging was used to obtain a three-dimensional reconstruction of the assembled empty capsids at low (36 Å) resolution, which showed the expected size, icosahedral symmetry and general morphology. The capsids bound the receptor integrin with the same activity as the intact virion, and were recognized by anti-FMDV antibodies. Porta et al. (2013a,b) also produced FMDV empty capsids in mammalian cells using a vaccinia virus-based expression system in which the 3C activity was modulated. Production of FMDV empty capsids in Escherichia coli In 1991, Marvin Grubman and associates obtained similar FMDV 70S particles by expressing the P1–2A polyprotein and 3C protease in E. coli, and showed that these particles were recognized by anti-FMDV MAbs against discontinuous epitopes, were immunogenic in guinea pigs and swine, and protected the later against viral challenge (Lewis et al., 1991; Grubman et al., 1993). Lee et al. (2009) simultaneously expressed in E. coli SUMO protein fused to VP0, VP1 and VP3 of FMDV. The three

fusion proteins formed a complex, and after specific proteolytic cleavage, capsid-like particles were observed by EM. Production of FMDV empty capsids in transgenic plants Pan et al. (2008) introduced the P1–2A and 3C coding regions of FMDV into a plant binary vector and transformed tomato plants using Agrobacterium tumefaciens. Guinea pigs vaccinated with transgenic plant extracts were protected against a challenge infection. To recapitulate, critical advances have been made on the production of FMDV empty capsids using different expression systems. Production in insects or cultured insect cells, or in cultured mammalian cells, respectively, using recombinant baculovirus or vaccinia virus-based systems in which 3C activity has been adequately modulated has been thoroughly investigated and shows great promise. However, there are still several important issues that must be solved to allow the use of recombinant empty capsids as a basis for antiFMD vaccines. They include the need to establish a routine, large scale production and purification of FMDV empty capsids, and the fact that empty capsids are even more thermosensitive than virions (Doel and Baccarini, 1981). To address this latter issue, that also concerns current virion-based vaccines, both FMDV virions and empty capsids have been engineered to increase their stability against heat-induced dissociation (see next section). Structural determinants of FMDV (in)stability Compared to other picornaviruses, FMDV virions and empty capsids are remarkably labile. FMDV virions rapidly lose infectivity when subjected to mild acidification, moderate heating, subzero temperatures or very high pressures or, at a slower rate, even when stored at 4°C. The weak acid stability of the FMDV virion plays a critical role in infection; its weak thermal stability may also be a selective trait; its heat-induced dissociation into pentamers constitutes a serious problem in FMD control by vaccination. Identifying the structural determinants of the very low physical stability of FMDV is critically important not only for a better understanding

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of morphogenesis and genome uncoating, but also for the development of improved vaccines and antiviral approaches. It is important to stress that acid stability and thermal stability of FMDV particles are not necessarily linked: (i) empty capsids can be more acid-resistant than full virions, but they are even less thermostable (Doel and Baccarini, 1981; Curry et al, 1995); (ii) FMDV virions from different strains and different sensitivity to acid-induced inactivation showed similar sensitivity to thermal inactivation (Maree et al., 2013); (iii) an engineered FMD virion with a highly increased resistance against thermal dissociation into pentamers was, however, as sensitive as the parent virion against acid-induced dissociation (Rincón et al., 2014). Likewise, experimental evidence shows that for the FMDV virion, stability against thermal inactivation of infectivity, and stability against thermal dissociation into subunits, do not necessarily occur at the same time and may be uncoupled: (i) both at 4°C and at 42°C, purified FMDV virions are inactivated much faster than they are dissociated into pentamers (Mateo et al., 2008); (ii) some engineered virions with a much increased resistance against dissociation into pentamers are, at most, only slightly more stable than the parent virion against thermal inactivation at 42°C (Mateo et al., 2008). FMDV sensitivity to acidification Investigation of the structural basis of the acid lability of FMDV particles is especially important for a better understanding of the genome uncoating step during the infectious cycle (Chapter 5). Histidine residues close to interpentamer interfaces as determinants of acid lability Of all picornaviruses, the FMDV virion is the most sensitive to dissociation into pentamers under acidic conditions. The acid sensitivity varies somewhat between different virions, but in nearly all reported cases the pH at which half of the viral particles lose integrity (pH50) in vitro is close to neutrality (around pH 6–7 for natural virions, depending on viral isolate and experimental conditions). This acid sensitivity must be highly disadvantageous for the virion in the environment. However, as the number of virions shed by an infected animal can be quite large, and very few virions are enough to

productively infect another animal, the relatively low survival rate in even mildly acidic environments may not impose an insurmountable negative selection pressure. In contrast, such extreme acid sensitivity appears to have a critical adaptive value by facilitating uncoating of the viral genome in the mildly acidic environment of endosomes. Inspection of the crystal structure of FMDV revealed a relatively high density of histidine residues lining the pentamer interfaces. The pKa value of the sc of a free histidine is close to the range of pH at which dissociation of the FMDV virion into pentamers occurs. Thus, it was suggested that interpentamer electrostatic repulsions involving protonated histidines could underlie the acid sensitivity of this virion (Acharya et al., 1989). Subsequent structural and biochemical analysis of virions and capsids of several serotype A isolates led to the suggestion that electrostatic repulsions close to the capsid two-fold axes between protonated His3142 in each pentamer and the intrinsic dipole of an α-helix (residues 2189–2198) in the neighbouring pentamer could influence the acid lability of the FMDV virion (Curry et al., 1995, 1997). Twomey et al. (1995) analysed the positions of charged residues around histidines at the interpentamer interfaces and suggested that His3142 and His3145 could be major determinants of acid lability. Simplified titration calculations, using a model composed of two crystallographic protomers related by a two-fold symmetry axis, reproduced the decrease in stability of the FMDV capsid at acidic pH, but not the difference in sensitivity between two type A viruses (van Vlijmen et al., 1998; Schaefer et al., 1998). According to these calculations, only residues within 15 Å of the interface could influence acid lability, with His3142 and His3145 in serotype A (corresponding to histidines 3141 and 3144 in serotype A and 3140 and 3143 in serotype C) being major contributors, as previously suggested. The calculations predicted that the specific interaction between His3142 and the helix dipole in the neighbouring pentamer contributes about 20% of the total destabilization energy. Mutational analysis was used to ascertain the effects of replacements of His3142 in the assembly and stability of FMDV recombinant empty capsids produced in mammalian cells. A His to Arg mutation prevented capsid assembly, while a His to

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Asp (charge reversal) mutation allowed assembly of empty capsids which showed a significantly increased stability against acid-induced dissociation into pentamers (Ellard et al., 1999). This result provided experimental evidence for a electrostatic destabilization of pentamer–pentamer interactions at acidic pH determined by His3142 in the FMDV capsid. Structural determinants of decreased or increased acid resistance of selected FMDV mutants Infectious FMDV mutants with either decreased or increased resistance against acid-induced inactivation of infectivity have been readily isolated from viral populations of different serotypes subjected to the appropriate selection pressure (Twomey et al., 1995; Martín-Acebes et al., 2010, 2011; VázquezCalvo et al., 2014; Caridi et al., 2015). A series of studies by Francisco Sobrino’s group have identified a substantial number of capsid residues that modulate acid stability of FMDV, and provided new tools to study FMDV uncoating (Vázquez-Calvo et al., 2012a,b; see ‘Structural insights into uncoating of the FMDV genome’, below, and Chapter 5). FMDV mutants with increased sensitivity to acid inactivation were readily selected by impairing acidification in the endosomes of host cells treated with NH4Cl (Martín-Acebes et al., 2010; Caridi et al., 2015). Individual mutations responsible of increased acid lability were mapped to VP2 or VP3 residues located very close to the interpentamer interfaces, or within a stretch of residues (1011– 1022) in the VP1 N-terminal segment. Of the five mutations close to the interfaces, all preserved the electric charge, but most led to the introduction of a bulkier sc (e.g. Ala3118Val or Ala3116Val). The 6 mutations at the VP1 N-terminus were chemically and sterically varied, but all of them affected residues clustered at some distance from the interpentameric interfaces. FMDV mutants with increased resistance against acid inactivation were selected by incubation of viral populations under mildly acidic conditions (Martín-Acebes et al., 2011; Vázquez-Calvo et al., 2014). The two mutations individually responsible of the acid-resistance phenotype were Thr1017Asp at the VP1 N-terminus (very close to mutations responsible for the opposite, acid-labile phenotype) and His2145Tyr. In both cases, the stabilizing effect

could be traced to an increased resistance against acid-induced dissociation of the virions into pentamers. Experiments carried out with the mutant virion Asn1017Asp and the control virion showed that increased resistance against acidification may be achieved without a significant reduction in infectivity and biological fitness at physiological pH, at least in cultured cells (Martín-Acebes et al., 2011). Also, neither mutation altered the antigenic specificity of the virion. Both mutations involve an increase in negative charge and in bulkiness of the sc, and are located at a considerable distance from the interpentameric interfaces and from each other. Residues mutated in an acid-resistant variant of a different serotype selected in an earlier study are also located far from the interpentameric interfaces (Twomey et al., 1995). Inspection of the FMDV virion structure suggested some tentative explanations for the virion-destabilizing or stabilizing effects at acidic pH of the selected mutations. For example, the destabilizing mutations located close to the interfaces could exert their effect by locally distorting the interfaces and altering the charge distribution around the pH-sensing histidines, leading to increased electrostatic repulsion between pentamers at acidic pH. The destabilizing or stabilizing mutations located at the VP1 N-termini could provoke some local rearrangement of these segments, which could propagate to the interfaces, leading to either increased or decreased repulsion between pentamers at acidic pH. These and other tentative structural explanations proposed remain to be tested. A role of the viral RNA in determining acid lability of the FMDV virion FMDV empty capsids are between 0.3 and 0.7 pH units more resistant to acid-induced dissociation than the corresponding RNA-filled virions (Curry et al., 1995). In contrast, they appear to be less resistant to dissociation than the virions at nonacidic pH and moderate temperatures (Doel and Baccarini, 1981). Thus, the RNA itself must contribute, either directly or indirectly, to the increased acid lability of the virion relative to the empty capsid. The interpentamer interfaces in the virion and empty capsid are nearly identical, but they differ in the organization or degree of disorder of internal features that involve VP terminal segments (Curry et al., 1997).

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A tentative explanation is that this reorganization could modify the electrostatic potential close to the interpentamer interfaces specifically at acidic pH, thus increasing the electrostatic repulsion between pentamers in mildly acidic conditions; a possible change in the dielectric constant has also been invoked (Curry et al., 1995). FMDV sensitivity to thermal inactivation of infectivity The mechanism by which FMDV subjected to moderate heating irreversibly loses infectivity without particle dissociation is still unclear. Early observations suggested that mild heating, even at physiological temperature (37°C), facilitates degradation of the RNA genome inside the virion by an endogenous viral nuclease (Brown and Wild, 1966; Newman and Brown, 1997). In addition, more recent studies showed that many mutations that involve substitution of different residues in the FMDV capsid either decrease or increase the resistance of the virion to thermal inactivation of infectivity (e.g. Mateo et al., 2003, 2008; MartínAcebes et al., 2011). Likewise, shortening the viral RNA led to increased resistance against thermal inactivation of infectivity (Ojosnegros et al., 2011). High pressure and subzero temperatures also inactivate the FMDV virion without disrupting the viral particle (Ishimaru et al., 2004). As one possible unifying explanation, it could be tentatively suggested that any of these factors may influence the energy barrier of a same unidentified, inactivating conformational rearrangement of the virion without capsid disruption. There is of course the possibility that virion inactivation may occur by several different mechanisms triggered by different agents. FMDV sensitivity to thermal dissociation into pentamers, and engineering of FMDV virions and capsids with increased thermostability for the development of improved vaccines Sensitivity of FMDV particles to heat-induced dissociation is a problem for FMD control by vaccination Current commercial anti-FMD vaccines based on chemically inactivated virions (Chapter 12) are economically produced using time-tested methods,

and usually confer adequate protection of animals. Unfortunately, a number of problems severely limit its safety and efficacy. One problem arises from the high sensitivity of the FMDV virion to heat-induced dissociation into poorly immunogenic pentamers (Doel and Baccarini, 1991; Doel and Chong, 1982), leading to unacceptable reductions in vaccine potency during storage and transportation. An expensive cold chain is routinely implemented, but this chain is frequently disrupted, eventually leading to vaccine failure. Different methods of chemical thermostabilization of FMD vaccines have been tested along the years, but so far they appear to be inadequate, insufficient and/or inconsistent enough to tolerate a significant relaxation of the cold chain. Empty capsid-based vaccines are being seriously considered and investigated as potential novel vaccines against FMD (see above). Unfortunately, there are still several important issues with this approach, and an important one is the fact that the FMDV empty capsid is even more sensitive than the virion to heat-induced dissociation (Doel and Baccarini, 1981). Engineering FMDV virions with increased thermostability for improving current anti-FMD vaccines The demand to improve the thermostability of current anti-FMD vaccines and, eventually, of potential empty capsid-based vaccines led our group to attempt the rational engineering of FMDV virions with increased stability against thermal dissociation into pentamers, in order to develop genetically stabilized anti-FMD vaccines that could be prepared using current production procedures, but that are less dependent on a fail-safe cold chain. The thermostabilizing mutations found could also be introduced in recombinant empty capsids in future developments of capsid-based vaccines of adequate thermostability. As a result, Mateo et al. (2008) first provided proof of concept that fully infectious FMDV virions with highly increased stability against thermal dissociation into pentamers can be rationally engineered, providing a solid starting point for the thermostabilization by genetic methods of current anti-FMD vaccines (see comments by Hedge et al., 2009). In a successful approach, different amino acid substitutions to increase the net electrostatic

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attraction between pentamers were introduced at or close to the interpentamer interfaces in a serotype C FMDV model virion (Fig. 4.6a). Residues chosen for replacement included those adequately positioned and whose sc established limited interpentamer interactions that could be removed (by mutation to Ala) without causing substantial reductions in viral infectivity (Mateo et al., 2003). Mutations Ala2065His and Asp3069Glu/ Thr2188Ala separately led to dramatic increases in virion stability against heat-induced dissociation into pentamers (Mateo et al., 2008). The half-life of the two purified mutants was much (severalfold) higher than that of the parent virion, both under storage at 4°C (see graph in Fig. 4.6a) or at high ambient temperatures, e.g. 42°C. Both mutants preserved the full infectivity and antigenic specificity of the parental virion, and were genetically fairly stable during propagation in cell culture (enough for large-scale production during vaccine preparation) (Mateo et al., 2008). One of the thermostable virions (Ala2065His) was tested for acid-induced dissociation and found to be as sensitive as the parent virion, consistent with its normal infectivity (as required for vaccine production). Moreover, these mutants could be normally inactivated by binary ethyleneimine without losing their higher structural thermostability (Rincón et al., 2014). Identification of structural determinants of the sensitivity of FMDV to thermal dissociation The engineered thermostable virions described above were used to investigate the thus far unknown structural determinants of the sensitivity of FMDV virions and capsids to heat-induced dissociation into pentamers. Rincón et al. (2014) found that charge screening at very high ionic strength had little effect on the stability against thermal dissociation of the purified engineered virions, but actually increased that of the parent virion. This observation suggested that the mutations introduced in the thermostable mutants were increasing the net electrostatic attraction between pentamers by reducing electrostatic repulsions. Carboxylate groups close to the Ala2065His stabilizing mutation and to the interpentamer interface were identified in the parent FMDV structure (Lea et al., 1994), and individually removed in the parent, thermolabile virion by isosteric charge-to-neutral

site-directed mutations. Single removal of most of the six targeted solvent-exposed carboxylates (Fig. 4.6a, top left image) preserved viral infectivity and increased as predicted the stability of the virion against thermal dissociation into pentamers (Rincón et al., 2014). Introduction of the Ala2065His mutation into one of these thermostable variants (Asp3195Asn) led to no further increase in stability, consistent with the hypothesis that His2065 (with a raised pKa due to its interactions) acts by neutralizing repulsions between Asp3195 and other acidic residues nearby. Simplified electrostatic potential calculations were qualitatively consistent with the experimental results and the repulsive effect of the identified carboxylates (Fig. 4.6a, bottom left image), supporting the use of such calculations to guide the electrostatic stabilization of protein complexes (Rincón et al, 2014; see comments by Sivertsson and Itzhaki, 2014). Engineering empty capsids with increased stability for developing safer anti-FMD vaccines Some of the virion-thermostabilizing mutations identified using the electrostatics-based strategy reviewed above (Mateo et al., 2008; Rincón et al., 2014) were introduced in the FMDV empty capsid. As expected, they increased also the resistance of the empty capsid against thermal dissociation into pentamers (Rincón and Mateu, unpublished observations). Very recently, a consortium of groups has described two additional strategies aimed at the engineering of recombinant empty capsids with increased thermostability (Porta et al., 2013b; Kotecha et al., 2015). As only empty capsid-based vaccines were (in principle) contemplated in their designs, the choice of adequate mutations was based only on their possible effects on capsid assembly and stability; there was no need to consider possible unwanted effects on virus infectivity or genetic stability, unlike the original approach followed by Mateo et al. (2008). The strategy followed by Porta et al. (2013b) was based in the introduction of disulfide bonds between pentamers, an approach that had been successfully used before for thermostabilization of phage MS2 under nonreducing conditions (Ashcroft et al., 2005). The FMDV structure was manually inspected to identify geometrically

(A)

(B)

Figure 4.6  Rational strategies for thermostabilizing FMDV virions and/or empty capsids. (A) An electrostatics-based approach for stabilizing FMDV particles without impairing infectivity and genetic stability of the virion (Mateo et al., 2008; Rincón et al., 2014). Top left image: two neighbouring pentamers are coloured cyan or violet. Inbuilt electrostatic repulsions between the carboxylates of some acidic residues (coloured yellow) close to the interpentamer interfaces contribute to the low resistance of FMDV particles against thermally induced dissociation into pentamers (right image). Individual removal of the negative charges of any of these carboxylates through isosteric mutations (Asp to Asn or Glu to Gln), or partial neutralization by introducing a positive charge through mutation of Ala2065 (coloured green) to His, led to large increases in resistance against thermal dissociation. The plot at right shows the kinetics of dissociation into pentamers of parent (circles) and engineered Ala2065His mutant (triangles) virions during storage at 4°C in phosphate-buffered saline. After 40 days, about 90% of the engineered mutant virions were still intact, while only 10% of the wt virions remained undissociated (Mateo et al., 2008; see text). Bottom left image: the destabilizing effect of the above carboxylates was predicted by simple electrostatic potential calculations using an ensemble of all VP proteins that surround the interface between two pentamers (6 VP2 (green), 6 VP3 (red) and 2 VP4 (yellow) subunits. (B) Molecular dynamics (MD)-guided approach for stabilizing FMDV capsids based on mutations close to the two-fold axis predicted to increase affinity between pentamers (Kotecha et al., 2015). Top left, the approximate area of two neighbouring pentamers used for simplified MD calculations is delimited by a dotted line. Residue at position 93 very close to the two-fold axis of VP2 in three different serotypes (O, SAT2, A) (labelled) was the main target. Introduction of stacking interpentamer interactions by appropriate replacements of this residue led to large increases in thermostability of viral particles against dissociation into pentamers (Kotecha et al., 2015). Establishment of interpentamer disulfide bonds by introducing cysteine at this same position led also to stabilization of type A empty capsids (Porta et al., 2013). Bottom: 1.5 ns MD trajectory. Left image shows large conformational deviations from the starting model in an unrestrained MD simulation; right image depicts initial (red), middle (white) and final (blue) conformations along a trajectory in a restrained MD simulation. (Images in panel (A) reproduced from Rincón et al. 2014 and Mateo et al, 2008; panel (B) reproduced from Kotecha et al., 2015; with permission).

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adequate positions. Substitution His2093Cys (see Fig. 4.6b) was chosen and introduced in a recombinant empty capsid (serotype A) to form a disulfide bond between the Cys2093 residues of neighbouring pentamers across each of the capsid two-fold axis. Parent and mutant empty capsids were produced both in mammalian cells (using a vaccinia virus-based expression system), and to higher yields in insect cells (using a baculovirusbased expression system). The crystal structures of both recombinant empty capsids were determined. The geometry of the disulfide bond was found to be correct. No other differences were observed, except that VP4 was fully disordered in the mutant. The capsids produced in mammalian cells were analysed in qualitative stability assays. Heating at 56°C for 2 hours disrupted the purified parent capsids, while most mutant capsids were not dissociated. Likewise, acidification to pH 5.2 for 15 minutes dissociated the parent capsids, while the mutant capsids remained intact. Thus, introduction of the disulfide bonds led to capsids with increased stability against dissociation by both heat and acid. Both capsids induced comparable antibody titres in cattle, and protected two (parent capsid) or three (mutant capsid) out of four animals against infection with the homologous virion. The strategy followed by Kotecha et al. (2015) used simplified all-atom MD simulations to predict changes in binding energy across the interpentamer interface caused by pre-selected mutations. A panel of potentially stabilizing amino acid substitutions within VP2, close to the capsid two-fold axis, were first manually chosen based on inspection of FMDV crystal structures. The somewhat different structural contexts and residues in the capsids of different serotypes (O, SAT2, A) were considered. Final MD simulations were performed using simplified models which included only those atoms from two crystallographic protomers related by a two-fold axis, and located within 13 Å of the interpentamer interface (Fig. 4.6b). Positional restraints were increased with the distance of the atoms to the interface. Based on the stabilizing effects predicted by the simulations, some chosen mutations were actually introduced in recombinant empty capsids produced in mammalian or insect cells. Using infectious clones, the mutations were also introduced in virions, which presented small-plaque phenotypes (suggestive of clearly reduced biological fitness in

cell culture), but yielded titres comparable to those of the parent viruses. The relative stability of the mutant virions was determined in temperature gradients, both at neutral and slightly acidic pH. Four out of five mutations tested in type O and two out of three mutations tested in type SAT2 thermostabilized the virions to different extents. The stability of one of the most stable type O mutants (Ser2093Tyr) was additionally determined by incubation at fixed times and temperatures. Albeit a controlled comparison is not possible, similar analyses of particle integrity on prolonged storage at 4°C suggests that the thermostabilizaton achieved by Ser2093Tyr in a type O virion in this study compares to that found in the previous study by Mateo et al. (2008) by Ala2065His or Asp3069Glu/Thr2188Ala in a type C virion using a different approach. The stability of three type O and one type A mutant empty capsids were also determined, again by incubation at fixed times and temperatures. All were more thermostable than the parent virus and also showed increased acid resistance. The structures of the thermostable mutants type O Ser2093Tyr, type A His2093Phe and type SAT2 Ser2093Tyr were determined by X-ray crystallography or cryo-EM. Overall, the structures revealed the expected stacking interactions between the substituted aromatic residues, consistent with the predicted role of these interactions in stabilizing the viral particles. Immunization of calves with freshly prepared vaccines based on type O or type SAT2 inactivated virions (parent and Ser2093Tyr mutant) yielded similar neutralizing antibody titres. Immunization of guinea pigs with vaccines based on SAT2 inactivated virions (parent and Ser2093Tyr mutant) and stored for 1 or 6 months at 4°C resulted in higher neutralizing antibody titres for the thermostabilized mutant. To recapitulate, different structure–based rational approaches based on removal of electrostatic repulsions between pentamers, introduction of disulfide bonds, or introduction of additional stacking interactions at the interpentamer interfaces led to substantial stabilization of a considerable number of FMDV virions and empty capsids of different serotypes (C, A, O and SAT2) against heat-induced dissociation into pentamers (Mateo et al., 2008; Porta et al., 2013b; Rincón et al., 2014;

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Kotecha et al., 2015). Other desirable features were tested for some mutants only. When tested: (i) the thermostabilizing mutations introduced in virions had no or moderate (small plaque) detrimental effects on infectivity; (ii) the virions were genetically fairly stable (allowing their largescale production); (iii) the virions conserved their increased thermostability after chemical inactivation; (iv) the viral particles were found to conserve the antigenic specificity and/or were as immunogenic as the parent; (v) they conferred protection against infection; and (vi) they conserved their immunogenic potency as vaccines for a longer time than their parents. Structural insights into the recognition and neutralization of FMDV by antibodies Antibodies are major effectors in the protection of host animals against FMD (Chapter 10). Thus, actions for better control of this disease, including immunological surveillance, updating of current vaccines and development of new vaccines, may greatly benefit from a deep understanding of the mechanisms of virus recognition by neutralizing antibodies and virus escape, in the context of the high genetic variability and quasispecies structure of FMDV populations (Chapter 7). As a consequence, in the last four decades these aspects have been intensively studied, mainly using polyclonal antibodies and MAbs, virus variants of different serotypes, antibody-escape virus mutants and synthetic peptides representing viral sequences. Structural aspects of the recognition of FMDV by antibodies and the mechanisms of virus escape have been thoroughly reviewed by Mateu (1995) and Mateu and Verdaguer (2004). These reviews have not been fundamentally outdated by later studies. Here a more condensed, updated overview on this subject is provided. Structural basis of the interaction of virus-neutralizing antibodies with a major antigenic site in the VP1 GH loop A large number of functional and structural studies by different groups have focused in the VP1 GH loop as a major, independent antigenic element. These studies provided a uniquely detailed view of

the interaction between virus-neutralizing antibodies and a particular, important antigenic region in a virion, and revealed some remarkable mechanisms of virus escape without compromising viral function. The VP1 GH loop is a major antigenic region of FMDV As a part of the pioneering work on FMDV by Fred Brown and collaborators, it was found that trypsin treatment of virions (serotypes O or A) greatly diminished their infectivity, attachment to host cells, antigenicity and immunogenicity (Wild and Brown, 1967; Wild et al., 1969). Trypsin cleavage led to excision of a capsid peptide fragment from type O virus particles (Strohmaier et al., 1982), and it was concluded that this peptide contains both the cell receptor binding site and a major antigenic region in the FMDV virion, a conclusion fully confirmed later. Determination of the FMDV structure (Acharya et al., 1989) showed that this peptide corresponds to the VP1 GH loop in the virion (see ‘Structure of the FMDV virion’, above). Chemically synthesized peptides that represent (a part of) the sequences of the VP1 GH loop of different FMDV serotypes were efficiently recognized by serum antibodies elicited against the virus, and could themselves elicit a potent antiviral humoral response (Kaaden et al., 1977; Bachrach et al., 1979; Strohmaier et al., 1982; Pfaff et al., 1982; Bittle et al., 1982). Amino acid substitutions in the VP1 GH loop of viruses of different serotypes, subtypes or variants, when introduced in those synthetic peptides, mimicked the specificity of the immune response elicited by the complete virion in animal models (Bittle et al., 1982; Clarke et al., 1983; Rowlands et al., 1983). Geysen et al. (1984, 1985) used variant synthetic hexapeptides with all possible single replacements to explore in detail the antigenicity of this region of the FMDV capsid using polyclonal antibodies. An interesting observation was that conserved residues in the RGD(L) sequence, later identified as the cell attachment site, were antigenically important in the three serotypes tested. Unfortunately, these peptides proved to be too short to reproduce complete epitopes, and thus provided an incomplete, biased portrait of consensus residues involved in polyclonal antibody binding.

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Discontinuous and continuous epitopes that involve the VP1 GH loop Later antigenic analyses of the VP1 GH loop generally used neutralizing MAbs elicited against virus particles to characterize individual epitopes. Antigens tested included virions and/or VP1 of field isolates of known sequence, and VP1 GH loop-mimicking peptides of nested or overlapping sequences, eventually containing single or multiple amino acid substitutions. In addition, many mutant viruses that escaped recognition by neutralizing MAbs were selected under antibody pressure, and the escape mutations were identified by sequencing. Comparisons of the crystal structures of different FMDVs provided detailed structural insights into the epitopes identified. The epitopes thus detected in viruses of different serotypes (O, A, C) differ in fundamental ways. In serotype O FMDV (strain O1BFS), the VP1 GH loop is involved in discontinuous epitopes (i.e. epitopes formed by residues that are located far apart in the primary structure, but that come spatially close in the folded protein). These epitopes include residues from both the VP1 GH loop and the VP1 C-terminal segment (Parry et al., 1985, 1989). Remarkably, some escape mutations selected by those MAbs mapped in the VP1 BC loop. The same mutations inhibited the interaction between the virion and serum antibodies elicited against a VP1 GH loop-mimicking peptide, whose epitopes could not include residues outside that loop. Disruption of VP1 GH loop epitopes by residues located elsewhere was noted also in another study with a type O virus (Krebs et al., 1993). Structural studies provided an explanation for those puzzling results. In the crystal structure of the parent O1BFS virion, the disordered, mobile VP1 GH loop in the ‘up’ position is close to the VP1 C-terminus and BC loop. In contrast, in the structure of an escape virion with a mutated VP1 BC loop, the VP1 GH loop appeared to be mainly towards the ‘down’ position (Fig. 4.3a), far away from the VP1 C-terminus and BC loop (Parry et al., 1990). The authors proposed that mutations in the VP1 BC loop stabilize the ‘down’ conformation of the VP1 GH loop away from its original position close to the VP1 C-terminus, thus disrupting the discontinuous epitopes that involve these

two VP1 stretches (Parry et al., 1990). Similar conformation-dependent mechanisms of escape from neutralization was proposed for two different epitopes in serotype A viruses (Thomas et al., 1988; Bolwell et al., 1989a,b; Curry et al., 1996). Unlike serotype O virus, multiple continuous epitopes (i.e. formed by residues within one short peptide segment) within the VP1 GH loop of serotypes C or A viruses defined an independent antigenic site (termed site A for type C viruses) (Mateu et al., 1987, 1988, 1989, 1990; Bolwell et al., 1989a). These epitopes are true native epitopes, as they are defined by neutralizing MAbs elicited against the virion. In type C viruses the VP1 C-terminus also contain continuous epitopes that define another antigenic site (site C). Site C and site A are topologically independent, as binding to the virion of a type A MAb did not sterically inhibit binding of a type C MAb, and vice versa (Lea et al., 1994). Functional and structural dissection of continuous epitopes that define a major antigenic site in the VP1 GH loop Because of the remarkable structural compactness of folded proteins, most B-cell epitopes on their surfaces are discontinuous. Some authors even suggested that true continuous B-cell epitopes may not exist in folded proteins. Thus, a full characterization of the seemingly continuous epitopes defined by neutralizing antibodies elicited against the FMDV virion was conceptually important. Definitive proof of the existence of continuous B-cell epitopes in FMDV (and other pathogens) has also a clear biotechnological relevance when evaluating the potentiality of peptide-based vaccines against FMD or other diseases. A uniquely extensive and detailed series of structure–function studies on epitopes within the VP1 GH loop was started by Esteban Domingo’s group using serotype C FMDV, and soon became a multidisciplinar collaboration that involved a considerable number of additional groups. It was found that 15-mer to 21-mer linear peptides encompassing a core sequence of the type C VP1 GH loop approached, on a molar basis, the reactivity of the loop in the virion when bound to the same antibodies. The same single or multiple replacements in the peptide and the virion caused similar variations

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in the binding of several virus-neutralizing MAbs (Mateu et al., 1989, 1992). Thus, free linear peptides proved to be excellent quantitative mimics of the antigenic activity and specificity of the VP1 GH loop for binding multiple antibodies. Individual replacements at any of nine positions within a 10-residue stretch affected the recognition by each MAb tested (Verdaguer et al., 1998). These and other results allowed a precise functional definition of multiple continuous epitopes within a 15-residue segment (1136–1150) of the VP1 GH loop of serotype C FMDV (Mateu et al., 1989, 1990; Verdaguer et al., 1995, 1998; Mateu, 1995). The RGD motif and residue 1146 at position +3 downstream of the RGD (RGD+3), within the contiguous short helical stretch were critical for binding all MAbs tested, while some replacements of some other residues severely affected binding of only a few MAbs (Mateu et al., 1990; Novella et al., 1993; Verdaguer et al., 1995, 1998). Thus, despite a substantial overlap of the epitopes, and the critical roles of some residues within site A in binding most antibodies, the individual epitopes in the VP1 GH loop defined by different MAbs are not functionally identical. Comparisons on the recognition of variant VP1 GH loop peptides by polyclonal sera obtained from convalescent or vaccinated swine indicated that the antibodies elicited against the VP1 GH loop in a natural host recognize epitopes that are similar to those recognized by neutralizing MAbs, supporting the biological relevance in the field of continuous epitopes quite similar to those structurally and functionally characterized in detail using neutralizing MAbs (Mateu et al., 1995b). The crystal structures of complexes between the Fab fragments of MAbs SD6 or 4C4 and the A15 peptide (Verdaguer et al., 1995, 1998; Ochoa et al., 2000; see also the section on the structure of the VP1 GH loop, above) (Fig. 4.4) showed that SD6 contacts 10 out of the 15 residues in peptide A15, and 4C4 contacts 8 out of the 11 A15 residues ordered in the complex. Thus, in both cases the structural epitope consisted of an almost continuous stretch of amino acid residues. The conformations of the VP1 GH loop in the SD6-A15 or 4C4–A15 complexes are very similar to each other. The conformation of a variant A15 peptide that represents the sequence of a different

field isolate of serotype C was also very similar to the conformation of the reference (C-S8) A15 peptide. Comparison of the unliganded and A15-bound structures of Fabs SD6 and 4C4 suggested that peptide recognition involves substantial conformational rearrangements in the antibody paratope for a better accommodation of the peptide (Verdaguer et al., 1996, 1998). Some substantial local differences in the mc torsion angles were, however, observed between the Fabbound peptides, particularly around the RGD motif. Changes in the torsion angles in Gly1142 (in the RGD triplet) compensated the differences in other torsion angles, explaining the overall structural similarity despite the local differences (Verdaguer et al. 1995, 1998; Ochoa et al., 2000). Consistent with the structural similarities between SD6- and 4C4-bound peptides, many specific peptide–antibody interactions, including critical contacts with the RGD motif, were shared by the three complexes. However, in agreement with the local structural differences observed between the bound peptides, and between the antibody paratopes, some interactions did not occur in every complex. The crystallographically defined contact epitope (i.e. the residues involved in interactions with the antibody paratope) and the immunochemically defined functional epitope (i.e. the residues that affect the MAb-binding affinity) accurately matched each other, even in details at the residue level (Verdaguer et al., 1995, 1998; Ochoa et al., 2000). The pseudoatomic models of the SD6- or 4C4– virion complexes provided a direct view on how these antibodies interact with the VP1 GH loop in the context of the complete viral particle (Hewat et al., 1997; Verdaguer et al., 1999; Fig. 4.3b). The orientation of the loop is very different in both complexes (see section on the structure of the VP1 GH loop, above), providing further evidence of the extreme mobility of this functionally critical loop in FMDV. In both complexes, the antibodies appear to interact almost exclusively with the VP1 GH loop, irrespective of the different orientations of the latter, and make no other contacts with the capsid. This observation provided additional structural evidence for the true continuous nature of the epitopes defined in the FMDV virion by these neutralizing antibodies.

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A model of the interaction of the VP1 GH loop with virus-neutralizing antibodies To recapitulate, the evidence supports the following model for the interaction of the VP1 GH loop in the native virion with virus-induced neutralizing antibodies. As already discussed, the unliganded loop is very loosely connected to the rest of the capsid, and can sample a wide range of similar conformations and widely different orientations, behaving much like a free linear peptide whose ends had been immobilized and conformationally restricted. In viruses of some serotypes (type C and, to a limited extent, type A) the different predominant orientations tend to exclude any interaction of the VP1 GH loop residues with other capsid residues. As a consequence, host antibodies can recognize this loop as an independent antigenic region. Because of the limited surface area of the VP1 GH loop and its propensity to adopt a protruding, convex fold on the virion surface, binding of antibodies with concave paratopes (as observed for MAbs SD6 and 4C4) is favoured because it maximizes contact area, providing higher binding affinity. This situation strongly resembles the recognition of free peptides by anti-peptide antibodies that also have concave paratopes, instead of the flatter paratopes typical of antibodies that recognize the abundant, relatively flat discontinuous epitopes in proteins. As also discussed above, the unliganded VP1 GH loop in the native virion may not be fully folded in a stable conformation. The limited intraloop enthalpic interactions and internal hydrophobic effect may barely compensate the reduction in conformational entropy on folding of this ‘independent’ loop. This would lead to a very small free energy difference between its folded and unfolded states that could be easily overcome, even by thermal fluctuations. Recognition of the VP1 GH loop by an antibody (or by an integrin receptor; see section ‘Structural insights into the recognition of cell receptors by FMDV’, below), would provide the additional enthalpic interactions and increased hydrophobic effect required to finally overcome the entropic cost of fully stabilizing the unliganded loop into a relatively rigid strand–turn–helix conformation, for which this loop has a strong intrinsic conformational propensity. This scenario also explains why free, linear peptides (Mateu et al., 1989; Verdaguer et al., 1995)

or (even better) cyclized peptides (Valero et al., 2000) representing the VP1 GH loop sequence are excellent qualitative and quantitative antigenic and immunogenic mimics of this antigenic site (and also potent inhibitors of virion-cell and virion-integrin recognition). Both as a part of the virion or as a free peptide, binding of the VP1 GH loop to antibodies or integrins may be achieved at the cost of comparable reductions in the conformational entropy of the unliganded antigen. If the unliganded VP1 GH loop on native virions were already fully and stably folded (instead of only transiently folded), the binding affinity of anti-VP1 GH loop antibodies to free peptide mimics should be much lower (because of the much higher entropic penalty), which was not observed. The model for antibody recognition of the VP1 GH loop of serotype O virions differs in some points from the model just described for serotype C (and possibly A) viruses. In native type O virions, the preferred ‘up’ orientations of the VP1 GH loop on the capsid surface may place this loop particularly close to, and may be transiently interacting with, the VP1 C-terminus. Thus, antibodies that recognize both VP1 segments as one discontinuous epitope with a larger surface area are favoured in type O viruses over antibodies that would bind continuous epitopes within either the VP1 GH loop or the VP1 C-terminus. This situation provides type O viruses with the selective advantage of escaping neutralization through an additional mechanism: mutations outside the epitopes that favour a different orientation of the VP1 GH loop separate it from the VP1 C-terminus, leading to the physical disruption of the epitopes (Parry et al., 1990; Logan et al., 1993). Immunodominance of the VP1 GH loop in FMDV Multiple observations have revealed that the VP1 GH loop constitutes one, but not the only major immunogenic region in FMDV (summarized and extensively referenced in Mateu and Verdaguer, 2004; see next subsection). Moreover, the degree of immunodominance of the VP1 GH loop is dramatically variable. For example, immunoaffinity fractionation of anti-type C FMDV sera from convalescent or vaccinated pigs revealed that the percentage of the virus-neutralizing activity corresponding to antibodies that bind antigenic site A in

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the VP1 GH loop was generally high (respectively, 60% or 30% on average), but ranged from about 90% to 2%, depending on the animal and other variables (Mateu et al., 1995a). Other antigenic sites in the FMDV virion The early available evidence led some authors to consider the VP1 GH loop as the only major immunogenic region in FMDV. However, later results showed that, as in other picornaviruses, several antigenic regions exist in the FMDV virion, and are indeed major contributors to its antigenicity and immunogenicity (reviewed in Minor 1990; Mateu 1995; Usherwood and Nash, 1995; Mateu and Verdaguer, 2004). Multiple functional antigenic sites in FMDV Lack of cross-neutralization activity between antibody escape mutants and MAbs used to select them has been used to define several functionally independent antigenic sites in serotype O (McCullough et al., 1987a; Xie et al., 1987; Stave et al., 1988; Pfaff et al., 1988; McCahon et al., 1989; Kitson et al., 1990; Crowther et al., 1993; Barnett et al., 1998; Aktas and Samuel, 2000; Asfor et al., 2014); serotype A (Thomas et al., 1988; Baxt et al., 1989; Saiz et al., 1991; Mahapatra et al., 2011); serotype C (Lea et al., 1994); and serotype Asia 1 (Sanyal et al., 1997; Butchaiah and Morgan, 1997; Marquardt et al., 2000; Grazioli et al., 2013). As expected partly from the limited numbers of MAbs used and the sequence and structural differences between FMDV virions, the functional sites identified with some serotypes or isolates were not observed with others, or are not fully equivalent. However, considering the ensemble of results it is apparent that the antibody escape mutations used to define functionally independent antigenic sites tend to cluster in several separate regions of the capsid surface (Mateu, 1995; Mateu and Verdaguer, 2004) (Fig. 4.7). It is generally considered that serotype O virions contain five functionally independent sites (sites 1–5) identified in cross-neutralization assays. Site 1 is defined by mutations in the VP1 GH loop (site 1a) and the VP1 C-terminus (site 1b). Sites 2, 3 and 4 are respectively defined by mutations in the VP2 BC or EF loops, the VP1 BC loop, or the VP3 BB

(A)

(B)

Figure 4.7 Location on the FMDV virion (A) and biological protomer (B) of most residues where antibody-escape mutations have been identified in viruses of serotypes O, A or C. Green-coloured residues correspond to the mobile VP1 GH loop, which is shown in the position adopted in the type C virion-Fab SD6 complex (Hewat et al., 1997). Blue-coloured residues identify residues corresponding to antigenic sites different from the VP1 GH loop. Clustering of escape mutations at the VP1 GH loop, close to the five-fold axis, or close to the three-fold axis is apparent, and serves to define a consensus between serotypes of three major antigenic areas (topological antigenic sites) in FMDV. However, scattering of escape mutations over a major part of the smooth capsid surface is also apparent. (Reproduced from Sobrino, F. and Domingo, E. (eds). 2004. Foot-and-mouth disease: current perspectives (Wymondham, UK: Horizon Bioscience)).

‘knob’. Site 5 is defined by a specific mutation in the VP1 GH loop. In serotype C virions, functionally independent sites involve the VP1 GH loop (site A), the VP1 C-terminus (site C), a turn at the beginning of the VP1 C-terminal segment (site D1), the VP2 BC loop (site D2), and the VP3 BB ‘knob’ (site D3). In a serotype A virus (Thomas et al., 1988), functionally independent sites were defined by the VP1

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GH loop (Group 1), the VP1 C-terminus (Group 2), and the VP2 BC, VP3 BC, HI, EF loops and BB ‘knob’ (Group 3); similar (but not identical) groupings could be made for the sites detected in other type A isolates. In a serotype Asia1 virus (Grazioli et al., 2013), four functionally independent sites respectively involved the VP1 GH loop, VP2 BC loop, VP3 BB ‘knob’ and VP3 C-terminus. Topologically independent antigenic sites in FMDV The definition of functionally independent antigenic sites based on cross-neutralization assays is widely used, convenient and useful, but has some limitations. These limitations arise especially when one wishes to understand FMDV-antibody recognition in structural terms, or for some applications (e.g. the design of novel anti-FMD vaccines). Evidence obtained with FMDV and other viruses (Mateu, 1995; Mateu and Verdaguer, 2004) has shown that: 1

2

As more MAbs and escape mutants were included in the assays, antigenic sites originally defined as functionally independent based on lack of cross-neutralization had to be redefined as a single functional site. This was the case, for example, with sites 2 and 4 in FMDV type O that were later functionally connected (Barnett et al., 1998). Mutual steric inhibition between pairs of Fabs in competition experiments for binding the virion showed that antibodies that recognize functionally independent sites actually bind structurally overlapping epitopes in the viral particle, and thus define only one topological antigenic region. For example, functionally independent antigenic sites D1, D2 and D3 in FMDV type C are contained a single topological site, site D, at the junction between VP1, VP2 and VP3 on the surface of each biological protomer (Lea et al., 1994). Functionally independent sites 1 and 5 in serotype O FMDVs are defined by escape mutations located very close within the VP1 GH loop; it is likely that they could be functionally connected if more escape mutants and MAbs were used to test cross-neutralization, and that they constitute a single topological antigenic site.

Fab competition experiments to define topologically independent sites in FMDV have been carried out with FMDVs of serotype A (A10Holland; Thomas et al., 1988) and serotype C (C-S8; Lea et al., 1994). The results of these experiments, the locations of all of the escape mutations found for FMDV of any serotype, and the relatively large size of an antibody footprint (Fig. 4.2b), support the existence of at most three physically separate antigenic regions on each protomer in the generic FMDV virion (compare Figs. 4.2b and 4.7). These consensus sites were termed structural antigenic sites I, II and III in Mateu and Verdaguer (2004). Site I involves the mobile VP1 GH loop and the VP1 C-terminus; depending on virus strain or serotype, and the relative locations of these two elements, they behave as structurally independent sites containing continuous epitopes, or as a single site made of discontinuous epitopes. Site II contains discontinuous epitopes that are between the three-fold axis and the junction between VP1, VP2 and VP3 on the protomer surface. Antigenic structural elements may predominantly include the VP2 BC loop and/or the VP3 BB ‘knob’, and/or also the VP2 HI and EF loops, VP3 BC and GH loops, and a turn at the beginning of the VP1 C-terminal segment. Not all of these elements are involved in all site II contact epitopes, which probably overlap only to a limited extent. Site III contains discontinuous epitopes located close to the five-fold axis that collectively involve at least the VP1 BC and HI loops. Functional or topological antigenic sites are both operational definitions, because they depend on the use of limited panels of escape mutants and/ or MAbs as probes. A quasi-continuum of epitopes may exist on the smooth FMDV capsid surface (as observed with some thoroughly analysed protein antigens). Indeed, many short peptides representing the sequence of many segments of VP1, VP2 or VP3 bound anti-FMDV antibodies, which suggested that their epitopes are scattered all over the virion surface (Meloen et al., 1986). The three different topological (structural) antigenic sites I, II, III proposed could be viewed as wide areas of each protomer that constitute three centres of higher epitope density. However, no clear borders would exist between them (Fig. 4.7), unlike antigenic areas in enteroviruses that are more clearly separated by high protrusions and deep canyons (Minor, 1990;

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Mateu, 1995). Thus, thermostabilized FMDV capsids may prove to be a best option in case one wishes to develop non-infectious anti-FMD vaccines able to reproduce the full immunogenicity of the FMDV virion. Biological functions of the FMDV virion severely constrain viral strategies for escaping antibody recognition The extensive studies on the recognition of the VP1 GH loop of serotype C FMDV by virus-neutralizing antibodies (see above) provided a paradigm for the presence and consequences of functional restrictions to mutation in a viral antigenic site (reviewed in Mateu, 1995; Domingo et al., 2003; Mateu and Verdaguer, 2004). Several of the 10 contiguous residues in the SD6 contact epitope and overlapping epitopes in antigenic site A within this loop were never found mutated in a very large collection of SD6-escape mutants (Mateu et al., 1989; Martínez et al., 1997), and were highly conserved in many type C field variants (Martínez et al., 1991). It was found that most of these residues are required for integrin-mediated attachment of the virus to cells and viral infectivity (Mateu et al., 1996; see also next section). Thus, residues critically involved in antibody recognition are kept invariant in the face of antibody pressure through the action of negative selection (Verdaguer et al., 1995; Mateu, 1995, Mateu and Verdaguer, 2004). Accordingly, when the negative selection pressure on these residues was removed by creating a binding site for a different receptor elsewhere in the capsid after virus propagation in cell culture, a completely different spectrum of SD6-escape mutants was obtained (Martínez et al., 1997; Ruiz-Jarabo et al., 1999; Domingo et al. 2003). Many of these variants contained mutations in residues critically involved in binding to integrins (no longer used as receptors for these variants), that had not been found in SD6-escape mutants derived from the parental virus or in type C field variants. Restrictions to variation within neutralization epitopes were also found for residues not critically involved in receptor recognition. Single substitutions of highly conserved residues 1145 (RGD + 2) and, especially, 1146 (RGD + 3) in antigenic site A within the VP1 GH loop of serotype C FMDV

had limited or no effect on attachment to cells and infectivity (Mateu et al., 1996). Moreover, they abolished recognition by nearly all tested site A MAbs and anti-site A antibodies present in sera from host animals. Remarkably, analyses of many type C field viruses isolated in two continents over six decades revealed that only very rarely the viruses had ‘chosen’ to evade the antibody response through a single mutation at those two ‘ideal’ residues. The general route apparently followed to escape neutralization in the field involved accumulation of substitutions at other site A positions (Martínez et al., 1991) despite the fact that, individually, replacements at these latter positions had a limited potential to evade a polyclonal response against this antigenic site (Mateu et al., 1990, 1995b). Viral competition experiments revealed that antibody escape mutations in the VP1 GH loop of FMDV, or in other antigenic sites, are frequently acquired at the expense of reduced biological fitness (Domingo et al., 2003). The rare occurrence of antigenically drastic mutations in the VP1 GH loop during circulation of type C FMDV in the field may be due to considerably reduced biological fitness, perhaps because of a somewhat reduced affinity for the integrin receptor (Mateu et al., 1996; see also next section). Restrictions to sequence variation at the FMDV capsid surface are not limited to the VP1 GH loop at all. In fact, indirect evidence suggested restrictions to variation also in site D, the other major antigenic site identified in type C (Lea et al., 1994; Mateu et al., 1994; Mateu 1995; Mateu and Verdaguer, 2004) and in other antigenic sites of FMDV (Mateu, 1995). Further genetic and antigenic analysis suggested that antigenic variation of FMDV type C in the field in two continents over six decades has occurred mainly through accumulation of mutations confined to very few, highly exposed positions on the capsid surface. These positions are remarkably coincident with those mutated in MAbescape variants that define different functional antigenic sites (Martínez et al., 1992; Mateu et al., 1994; Feigelstock et al., 1996; Mateu and Verdaguer, 2004). As the effects of more mutations introduced in the FMDV capsid by site-directed mutagenesis of infectious clones are tested, it is becoming more clear that the vast majority of non-natural capsid mutations are detrimental for viral infectivity and/

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or biological fitness; the same is being found for other animal viruses. The capsid of FMDV and of small, nonenveloped animal viruses in general may have been streamlined through evolution to occupy quite narrow peaks of higher fitness in the sequence space. Only a few, relatively small ‘hotspots’, even on the viral capsid surface, may tolerate a relatively large spectrum of amino acid substitutions without causing unacceptable reductions in biological fitness. In this scenario, the structurally confined, functional antigenic sites defined by escape mutations correspond to small capsid surface ‘hotspots’ where amino acid substitutions are better tolerated by the virus. For the same reason, these same ‘hotspots’ probably constitute the places most frequently ‘used’ by FMDV (as well as other picornaviruses and nonenveloped viruses) to escape the antibody response during circulation in the field. It has long been noted that these ‘hypervariable’ regions generally correspond to quite exposed tips of long loops that abound in the nonenveloped capsids of many animal viruses. It is tempting to speculate that exposed loops on those capsids may have been enlarged by positive selection partly to act as ‘decoys’ to confront the immune system. These relatively long, mutation-tolerant loops promote antibody binding, but provide at the same time an easy escape route, because they can accept mutations that impair antibody recognition without severely compromising virus viability and fitness. Neutralization of FMDV infectivity by antibodies Despite the wealth of structural and functional information on the recognition of FMDV by different antibodies, not much is known on the mechanisms of antibody-mediated FMDV neutralization. Early work by Baxt et al. (1984) revealed that some anti-FMDV MAbs neutralize infectivity mainly through aggregation of viral particles, and others act by blocking virus attachment to cells; one neutralizing MAb caused, however, little aggregation and had no effect on attachment. A different MAb could neutralize FMDV by triggering some conformational change in the virion that facilitated RNA release (McCullough et al., 1987b). The observed binding of Fabs SD6 and 4C4 to the RGD and other studies on FMDV attachment to cells and neutralization by these Fabs (Verdaguer

et al., 1997, 1999) show that antibodies that bind the VP1 GH loop do neutralize FMDV infectivity by binding some of the same residues that bind the integrin receptor (including the RGD), and sterically inhibiting attachment to cells. Structural insights into the recognition of cell receptors by FMDV FMDVs isolated in the field, and most strains adapted to growth in tissue cultures, infect cells by binding any of several integrins of the αv subgroup (αvβ1, αvβ3, αvβ6, αvβ8), with αvβ6 probably being the primary cognate receptor in host animals. In addition, some FMDVs propagated in cultured cells have acquired the capacity to bind HS proteoglycans as alternative receptors. Some evidence indicates that other receptors may also mediate FMDV infection of cells, but they have not been identified yet. Molecular aspects of the recognition of cell receptors by FMDV and virus entry into cells are extensively covered in Chapter 5. For other recent reviews on structural and/or functional aspects of FMDV receptors see Jackson et al., 2003; Fry et al., 2005a; Ruiz-Sáenz et al., 2009; Fry and Stuart, 2010; Han et al., 2015. Here only a few aspects regarding the important, but still rather limited structural knowledge gained so far on FMDVreceptor recognition are summarized. FMDV-integrin receptor recognition After the VP1 GH loop was identified as a receptor binding site (Wild and Brown, 1967; Wild et al., 1969; Strohmaier et al., 1982; Acharya et al., 1989; previous section), RGD-containing peptides (Surovoi et al., 1988; Fox et al., 1989; Baxt and Becker 1990) and FMDV mutants (Mason et al., 1994; McKenna et al., 1995; Rieder et al., 1996) were used to show that the conserved RGD motif within that loop is involved in receptor recognition. Subsequently, Barry Baxt, Peter Mason and collaborators identified the RGD-binding integrin αvβ3 as a receptor for FMDV (Berinstein et al., 1995; Neff et al., 1998). Later, a series of studies by Terry Jackson, Andrew King and collaborators identified other RGD-binding integrins as additional receptors of FMDV: αvβ6 ( Jackson et al., 2000b), αvβ1 ( Jackson et al., 2002) and αvβ8 ( Jackson et al., 2004) (Chapter 5).

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Unfortunately, no structure of any complex of FMDV virion or VP1 GH loop-mimicking peptide with any integrin has been determined to date. Current structural knowledge on FMDV-integrin recognition derives mainly from: (i) The crystal structure of the ectodomain of integrin αvβ3 bound to a RGD peptide (Xiong et al., 2002); (ii) studies on the interaction of VP1 GH loop-derived peptides or FMDV mutants with host cells expressing different integrins, and/or with isolated integrins; (iii) NMR studies on the conformation and interactions of VP1 GH loop-mimicking peptides either

free or bound to integrin αvβ3 (DiCara et al., 2007; Wagstaff et al., 2012). The results of these studies are summarized next. The crystal structure of the ectodomain of integrin αvβ3 bound to a RGD peptide provided the first view on an integrin-RGD ligand (Xiong et al., 2002; compare Fig. 4.8b). The bound RGD adopts an open turn conformation. The peptide is bound at the ectodomain apex, inserted in a crevice between two domains, one from the α subunit and the other from the β subunit. The Arg sc is involved in salt bridges, and the Asp sc is at the centre of a network

(A)

(B) Figure 4.8  Structural aspects of the recognition between the FMDV virion and cellular receptors. (A) Structure of a complex between a type O virion and its heparan sulfate receptor (Fry et al., 1999). The left image shows a blue-colour spacefilling model of one pentamer in the virion bound to HS; the right image shows a blue-colour ribbon diagram of one protomer in the virion bound to HS. The HS receptor is represented as a ball-and-stick model and coloured green. (B) A docking model of a complex between a type C virion and integrin αvβ3 (Verdaguer et al., 2004). The model was obtained by docking the integrin structure bound to the RGD tripeptide (Xiong et al., 2002) onto the structure of the virion with the VP1 GH loop in the position observed in a virion-neutralizing Fab complex (Hewat et al., 1997). The left image shows a front view of one pentamer (blue-colour spacefilling model) docked to five integrin molecules (green-colour ribbon models); the right image shows a side view of one protomer (blue) docked to an integrin molecule (green). The RGD peptide is shown as a ball-and-stick model. (Reproduced from Sobrino, F. and Domingo, E. (eds). 2004. Foot-and-mouth disease: current perspectives (Wymondham, UK: Horizon Bioscience)).

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of polar interactions, including coordination with a Mn2+ ion. The extended conformation of the central Gly forms a bridge at the interface between the two integrin subunits. The RGD conformation in the peptide–integrin complex is very similar to that observed in the unliganded VP1 GH loop in reduced type O FMDV (Logan et al., 1993), and in the A15 peptide–antibody complexes (Verdaguer et al., 1995, 1998; Ochoa et al., 2000). Thus, it can be presumed that, in a complex between the FMDV virion and the αvβ3 receptor, the RGD in the VP1 GH loop would establish interactions with this integrin similar to those observed in the RGD peptide–αvβ3 complex. A complex between the VP1 GH loop of FMDV presented on the hepatitis B core surface and integrin was visualized by EM at low resolution (Sharma et al., 1997) but yielded no structural details on the loop–integrin interaction. Docking models of a complex between FMDV and αvβ3 were later obtained by combining the structural models of the FMDV-VP1 GH loop peptide–Fab complex and of the integrin–RGD complex, relying on the very similar RGD conformations common to those models for a best superposition (Verdaguer et al., 2004). The final models (Fig. 4.8b) suggest that integrin αvβ3 can only bind the virion through the VP1 GH loop when the latter is in a highly exposed orientation, similar to the ones that allow binding to antibodies. This docking model is not precise enough to predict which other capsid residues (apart from the RGD), either in the VP1 G-H loop or elsewhere, could establish interactions with the integrin. Functional studies on the interaction between variant peptides or virus mutants with cells and isolated integrins have revealed specific determinants of the interaction. The three residues in the RGD triplet are strictly required, with very few exceptions (Chapter 5). Studies on virus or peptide binding to purified integrins confirmed the critical role of the RGD motif for recognition by individual integrins ( Jackson et al., 1997; Sharma et al., 1997). In quite exceptional occasions, FMDV propagation in cell culture led to variants that could still use integrins but had modified RGD motifs, such as RSGD (Rieder et al., 2005) or KGE (Berryman et al., 2013). In addition to the RGD triplet, other amino acids at the helix located immediately to the C-terminus

of the RGD have been identified as being important for recognition between FMDV of different serotypes and integrin receptors. Serotype A mutant FMDVs with enhanced ability to bind BHK cells showed substitutions at residues RGD+1 and +7 within the helical stretch (Rieder et al., 1994). In a systematic analysis to dissect the individual role of VP1 GH loop residues in infection of cultured BHK cells by a type C FMDV, each residue in the VP1 GH loop-mimicking A15 peptide (see above) was individually replaced by any of 16 other amino acids. The inhibitory activity of each of the variant peptides on virus infection was tested. The inhibitory action of A15 on FMDV infectivity had been previously shown to be due to the efficient and specific inhibition of virus attachment to cells (Hernández et al., 1996), which validated this approach to study the determinants of the interaction between the virion and the receptor on the cell membrane. The results revealed a nearly absolute requirement for R, G and D of the RGD motif, and of the highly conserved Leu residues located at positions RGD + 1 and RGD + 4 in the helical stretch contiguous to the RGD turn (and to a minor extent, the less conserved residue at RGD + 2) (Mateu et al., 1996). More recently, inhibition by peptides of FMDV binding to cells expressing different integrins and infection revealed that Leu residues at RGD + 1 and RGD + 4 are specifically relevant for recognition of type O FMDV by integrins αvβ6 and αvβ8 (Burman et al., 2006). Analysis of the recognition of variant peptides by integrin αvβ6 identified the same RGD+1 and RGD+4 residues as important determinants for a stable serotype O peptide–integrin interaction (DiCara et al., 2007, 2008). Differences in the sequence at the RGD + 1 and RGD + 4, and other positions in the segments flanking the RGD motif mediate the specificity of different FMDV isolates/serotypes for different integrin receptors ( Jackson et al., 1997, 2000a, 2002; Burman et al., 2006; DiCara et al., 2007). Apart from the RGD motif itself, the conformation adopted by the VP1 GH loop when bound to an integrin receptor is not known. However, the strong propensity of this loop to adopt a strandturn-helix conformation, either in unliganded form or bound to antibody (see above; Figs. 4.3a and b and 4.4) suggests that it may adopt a similar conformation when bound to an integrin. This

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global fold is also preserved in different FMDV serotypes and isolates despite large variations in loop sequence (including insertions/deletions), and some local differences in conformation that are adequately compensated through different intraloop interactions. Consistent with this view, introduction of different conformational restrictions on VP1 GH loop-mimicking peptides by different cyclizations led to qualitatively similar effects on antibody and cell recognition (Mateu et al., 1996). It seems reasonable to hypothesize that the strong propensity of this loop to adopt a conserved strand–turn–helix conformation may have been biologically selected precisely to allow productive binding of FMDV, irrespective of serotype, to integrin receptors. It had been noted that in the VP1 GH loop-derived peptides complexed to antivirus antibodies, the Leu residues at RGD + 1 and RGD + 4 are located on the same face of the helical stretch adjacent to the RGD turn (Mateu et al., 1996; Fig. 4.4b) and make hydrophobic contacts with the antibody paratope (Verdaguer et al., 1995, 1998). In a remarkable structure–function study by DiCara et al. (2007), NMR spectroscopy was used to probe the conformation and interactions of VP1 GH loop-mimicking peptides with integrin αvβ6. The results revealed that (i) in the presence of structureinducing solvents, the VP1 GH loop peptides do adopt turn-helix conformations similar to those determined for the unliganded loop in reduced type O virus (Fig. 4.3c) and in loop peptide-antibody complexes (Fig. 4.4b). (ii) the RGD forms the turn at the tip of the loop; downstream residues form a helical stretch with residues RGD+1 and RGD+4 are presented as adjacent residues on the exterior face of the loop; (iii) In the peptide-αvβ6 integrin complex, these two residues bind very closely to the integrin surface, possibly through a hydrophobic interaction with the integrin; (iv) Other residues in the loop may influence the propensity to adopt a helical conformation, and thus indirectly contribute to the strength of the interaction between the VP1 GH loop and the integrin. In a further study, NMR structures and dynamics of a VP1 GH loop peptide (both in linear and cyclic forms) and other RGD-containing, αvβ6-binding peptide in the presence of trifluoroethanol were analysed (Wagstaff et al., 2012) (Fig. 4.9). The results suggested that αvβ6 specificity requires the formation of a structurally

rigid helix preceded by a RGD motif exhibiting slow internal motion. To recapitulate, the strong intrinsic propensity of the VP1 GH loop of FMDV to adopt a strandturn-helix conformation appears to be the result of a selective pressure to preserve binding to αvβ6 and other receptor integrins on the surface of host cells. The conformation of this loop is, then, similar when bound as an ‘independent’ capsid element to integrins or antivirus antibodies, or when it is immobilized in reduced type O FMDVs. Both the RGD motif and residues at positions RGD+1 and RGD+4 in the exterior face of the helical element adjacent to the RGD turn in the folded VP1 GH loop may directly interact with αv integrins including αvβ6, the principal receptor of FMDV in host animals (Monaghan et al., 2005; Burman et al., 2006; Chapter 5), and critically determine affinity and specificity by influencing structure and conformational dynamics. Other residues in the VP1 GH loop, especially within the helical segment, appear to have some role in binding integrins or modulating conformational propensities of the loop. FMDV-HS receptor recognition Jackson et al. (1996) found that a serotype O FMDV strain that had been originally adapted to grow in tissue culture could use HS as a receptor to efficiently infect cultured cells. HS is a negatively charged, glycosaminoglycan component of proteoglycans found on the membranes of many cell types. It was subsequently found that propagation of FMDV strains of different serotypes in cultured cells sometimes lead to the selection of variants with a high affinity for HS that show increased virulence and expanded tropism in cell culture, but that are attenuated in cattle (Sá-Carvalho et al., 1997; Baranowski et al., 1998, 2000; Escarmís et al., 1998). The ability of the adapted viruses to bind HS was acquired through only one or two mutations on the capsid surface that generally involved an increase in positive charge (e.g. His3056Arg in type O strains). The crystal structures of HS bound to two FMDV strains (of serotypes O and A) propagated in cultured cells (Fry et al., 1999, 2005b) provided direct information on the interaction between FMDV and this alternative receptor (Fig. 4.8a). In both cases, the HS binding site is located in a concavity close to the junction between VP1, VP2 and VP3 at the centre of the biological protomer. The

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(A)

(B)

(C)

(D)

Figure 4.9  NMR structures (closest to the mean) for peptides mimicking the VP1 GH loop of FMDV O1 BFS and other RGD-containing, integrin αvβ6-binding peptides. (A, B) two cyclized forms of the VP1 GH loop peptide. (C) linear VP1 GH loop peptide. (D) linear LAP2 peptide (derived from the latency-associated peptide from transforming growth factor β1). For each peptide the assigned NOE contacts, hydrogen-bond restraints are indicated at right (Reproduced from Wagstaff et al., 2012, with permission).

HS site overlaps with relatively variable spots on the capsid surface that form a part of the topologically mapped antigenic site D in type C viruses and in which escape mutations that define antigenic sites D3 in type C and 4 in type O viruses were identified (see previous section). In the complex, a carbohydrate motif made of up to five sulfated residues is bound to the capsid,

making contacts with some nine amino acid residues belonging to any of the three capsid proteins. In both complexes, Arg3056 (a residue responsible for adaptation of type O viruses to bind HS) is involved in ionic interactions with sulfate groups, while most contact residues are not conserved in the two HS-bound virus isolates analysed. Residues at the beginning of the VP1 C-terminal

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segment also contact HS, and it has been suggested that interactions of other residues within this exposed segment and other spatially close capsid residues may indirectly contribute to shape the HS binding site. Binding of HS occurs with very little local or global structural reorganization of the virus capsid. No evidence for a physical or spatial connection between the integrin binding site (the mobile VP1 GH loop) and the HS binding site was apparent, thus providing structural support for the observed independent function of the two sites in binding the respective receptors on host cells. Structural insights into uncoating of the FMDV genome There is abundant evidence that, after FMDV binds and integrin receptor in a susceptible cell, the virion is internalized via clathrin-mediated endocytosis and delivered to endosomes, where the acidic pH mediates uncoating of the viral genome (reviewed by Vázquez-Calvo et al., 2012; Chapter 5). Unfortunately, the mechanism of RNA uncoating is one of the less understood steps of the FMDV infectious cycle. Binding the receptor does not compromise the integrity of the virions (Baxt and Bachrach, 1980). Natural integrin receptors could be artificially bypassed by redesign of virus-cell recognition in the laboratory, providing alternatives for targeting the endosomes (Mason et al., 1993, 1994; Rieder et al., 1996). These and other observations indicate that natural FMDV receptors allow specific virus-cell recognition, and may also play a role in internalizing the viral particle and facilitating entry into the endosome, but they are not involved in genome uncoating. Shortly after entry into cells, the 140S virion dissociates into 12S pentameric subunits and releases the viral RNA (Cavanagh et al., 1978; Baxt and Bachrach 1980, 1982; Baxt, 1987). Weak bases and ionophores that block acidification in endosomes inhibit infection by FMDV (Carrillo et al., 1984, 1985; Baxt, 1987). Internalized FMDV particles colocalize with markers of endosomes (Berryman et al., 2005; O’Donnell et al., 2005), and expression of a dominant negative mutant of the Rab5 quinase impairs FMDV infection ( Johns et al., 2009). All these observations indicate that viral RNA

uncoating occurs in endosomes (Vázquez-Calvo et al., 2012). The biochemical and structural studies on the acid lability of the FMDV virion and capsid in vitro (see above), and the limited evidence obtained so far on FMDV uncoating in the cell (Vázquez-Calvo et al., 2012), support a simple model in which the moderately acidic environment in endosomes directly leads to the complete dissociation of the viral capsid into pentameric subunits, and both VP4 and the viral RNA molecule are just released in the endosomal cavity. However, this proposed mechanism leaves as an open question how the released RNA is protected from acid degradation in the endosome, and how it is transferred to the cytosol. Studies on acid-dependent viral entry have revealed that acid-mediated uncoating of some viral genomes in endosomes may lead to disruption of the latter, allowing the direct release of the viral nucleic acid (and the endosomal content) in the cytosol. Alternatively, some viral RNAs may be translocated into the cytosol through the formation of pores in endosomal membranes. Both mechanisms have been documented for HRV (Vázquez-Calvo et al., 2012). For enterovirus like HRV and PV, it is hypothesized that pores in the endosomal membrane are formed after an acidinduced conformational change of the virion in the endosome, which leads to the externalization of five myristoylated, hydrophobic VP4s and the N-terminal segment of one VP1 molecule through a capsid opening (a pore or fracture). The externalized hydrophobic segments would be inserted in the endosomal membrane, forming a channel through which the viral RNA could be directly translocated from the viral capsid cavity into the cytosol (Vázquez-Calvo et al., 2012, and references therein). The FMDV uncoating model based on the pHinduced dissociation of the capsid and the direct release of the viral RNA into the endosomal lumen excludes the channel-based translocation mechanism proposed for enteroviruses: this mechanism requires preservation of the integrity of the viral particle until the RNA has been transferred to the cytosol through the pore generated in the endosomal membrane. However, a few observations suggest that uncoating of the aphthovirus genome may be more complex. FMDV provirions obtained

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by mutation, in which the VP0 to VP4+VP2 cleavage did not occur, could still adsorb to cultured cells and were as acid-sensitive as the non-mutated infectious mature virions, but they were non-infectious (Knipe et al., 1997). Thus, acidification may not be enough for allowing a productive infection. Recent studies by David Rowlands and collaborators on equine rhinitis A virus (ERAV), an aphthovirus closely related to FMDV (Tuthill et al., 2009; Groppelli et al., 2010) are opening a new perspective on aphthovirus uncoating. As for FMDV, productive infection by ERAV is dependent on clathrin-mediated endocytosis and endosome acidification (Groppelli et al., 2010). Remarkably, when the ERAV virion was subjected in vitro to a weakly acidic treatment under conditions that stabilize uncoating intermediates in PV, it first released the viral RNA, becoming a relatively unstable 80S empty particle before being disassembled into free pentamers. The crystal structure of the ERAV empty particle is very similar to that of the virion, but significant rearrangements and a higher disorder were observed in several internal loops and VP termini, that were proposed to destabilize the empty particle relative to the virion (Tuthill et al., 2009). The authors tentatively suggested that acidification of ERAV in the endosomes, and perhaps of FMDV, could lead to the transient formation of an unstable viral particle. Such particle could act as an uncoating intermediate to release the viral RNA through a channel-mediated mechanism similar to that proposed for enteroviruses, before dissociating into pentamers (Tuthill et al., 2009). Further experiments are needed to test this enticing possibility.

suggestions the viral structures have induced await verification by performing appropriate biophysical, biochemical or biological experiments. Conversely, structural interpretations of the results of some biochemical or biological analyses have not yet been verified by solving the appropriate viral structures. The organization of the viral RNA inside the capsid, the conformational dynamics of FMDV particles, the mechanisms of FMDV assembly, integrin recognition, and viral genome uncoating are still little understood. Our limited current knowledge on the structural biology of FMDV compromises the rational design of improved vaccines and the development of antiviral approaches. Continued structure–function work may be instrumental to effectively control and even eradicate FMD, an old but re-emerging disease (Chapter 18) that still inflicts severe economic losses to many countries, and poses a permanent threat to the world trade economy.

Concluding remarks Structural and structure-based studies have unveiled in atomic detail the molecular basis of many properties and biological functions of FMDV particles. As a result, our knowledge of FMDV morphogenesis, particle stability, interaction with antibodies, recognition of cellular receptors and entry into cells, variation and evolution, etc. has been greatly increased. Structure–function studies are also facilitating the development of improved or new anti-FMD vaccines, and may help the design of future anti-FMDV drugs. However, it must be recognized that much remains to be done. Some of the hypothesis or

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Acknowledgements The author acknowledges M.A. Fuertes for help with figures. I wish to express my gratitude also to former and present members of my group, and to our many collaborators in the study of relationships between structure, properties and function of viruses. Present work in the author′s laboratory is funded by grants from MINECO/FEDER EU (BIO2012-37649 and BIO2015-69928-R), and by an institutional grant from Fundación Ramón Areces. M.G.M. is an associate member of the Institute for Biocomputation and Physics of Complex Systems, Zaragoza, Spain. References

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disease virus capsids and their stabilities as a function of pH. J. Mol. Biol. 275, 295–308. Vázquez-Calvo, A., Saiz, J.C., McCullough, K.C., Sobrino, F., and Martín-Acebes, M.A. (2012a). Acid-dependent viral entry. Virus Res. 167, 125–137. Vázquez-Calvo, A., Caridi, F., Rodríguez-Pulido, M., Borrego, B., Saiz, M., Sobrino, F., and Martín-Acebes, M.A. (2012b). Modulation of foot-and-mouth disease virus pH threshold for uncoating correlates with differential sensitivity to inhibition of cellular Rab GTPases and decreases infectivity in vivo. J. Gen. Virol. 93, 2382–2386. Vázquez-Calvo, A., Caridi, F., Sobrino, F., and Martín-Acebes, M.A. (2014). An increase in acid resistance of foot-and-mouth disease virus capsid is mediated by a tyrosine replacement of the VP2 histidine previously associated with VP0 cleavage. J. Virol. 88, 3039–3042. Verdaguer, N., Mateu, M.G., Andreu, D., Giralt, E., Domingo, E., and Fita, I. (1995). Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction. EMBO. J. 14, 1690–1696. Verdaguer, N., Mateu, M.G., Bravo, J., Domingo, E., and Fita, I. (1996). Induced pocket to accommodate the cell attachment Arg-Gly-Asp motif in a neutralizing antibody against foot-and-mouth-disease virus. J. Mol. Biol. 256, 364–376. Verdaguer, N., Fita, I., Domingo, E., and Mateu, M.G. (1997). Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding. J. Virol. 71, 9813–9816. Verdaguer, N., Sevilla, N., Valero, M.L., Stuart, D., Brocchi, E., Andreu, D., Giralt, E., Domingo, E., Mateu, M.G., and Fita, I. (1998). A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation. J. Virol. 72, 739–748. Verdaguer, N., Schoehn, G., Ochoa, W.F., Fita, I., Brookes, S., King, A., Domingo, E., Mateu, M.G., Stuart, D., and Hewat, E.A. (1999). Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation. Virology 255, 260–268. Verdaguer, N., Blaas, D., and Fita, I. (2000). Structure of human rhinovirus serotype 2 (HRV2). J. Mol. Biol. 300, 1179–1194. Verdaguer, N., Fita, I., Reithmayer, M., Moser, R., and Blaas, D. (2004). X-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein. Nat. Struct. Mol. Biol. 11, 429–434. Verlinden, Y., Cuconati, A., Wimmer, E., and Rombaut, B. (2000). Cell-free synthesis of poliovirus: 14S subunits are the key intermediates in the encapsidation of poliovirus RNA. J. Gen. Virol. 81, 2751–2754. Wagstaff, J.L., Rowe, M.L., Hsieh, S.J., DiCara, D., Marshall, J.F., Williamson, R.A., and Howard, M.J. (2012). NMR relaxation and structural elucidation of peptides in the presence and absence of trifluoroethanol illuminates the critical molecular nature of integrin αvβ6 ligand specificity. RSC Adv. 2, 11019–11028.

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Wang, X., Peng, W., Ren, J., Hu, Z., Xu, J., Lou, Z., Li, X., Yin, W., Shen, X., Porta, C., et al. (2012). A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat. Struct. Mol. Biol. 19, 424–429. Wild, T.F., and Brown, F. (1967). Nature of the inactivating action of trypsin on foot-and-mouth disease virus. J. Gen. Virol. 1, 247–250. Wild, T.F., Burroughs, J.N., and Brown, F. (1969). Surface structure of foot-and-mouth disease virus. J. Gen. Virol. 4, 313–320.

Xie, Q.C., McCahon, D., Crowther, J.R., Belsham, G.J., and McCullough, K.C. (1987). Neutralization of foot-and-mouth disease virus can be mediated through any of at least three separate antigenic sites. J. Gen. Virol. 68, 1637–1647. Xiong, J.P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S.L., and Arnaout, M.A. (2002). Crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155.

Foot-and-mouth Disease Virus Receptors: Multiple Gateways to Initiate Infection

5

Paul Lawrence and Elizabeth Rieder

Abstract Since its discovery over 100 years ago as the causative agent of foot-and-mouth disease (FMD), research has been directed at understanding the biology of the foot-and-mouth disease virus (FMDV) so as to be able to control this devastating and highly contagious disease of cloven-hoofed livestock. Given its persistence and high rate of transmission, FMDV threatens worldwide livestock and related industries and has the potential for significant negative impacts on broader economies. A considerable amount of knowledge has been amassed in the last several decades on FMDV replication, structural biology, and the functionality of its RNA genome and encoded proteins. As a result, new technologies have now afforded the means to control this disease both with new generation vaccines and antiviral therapies. Despite these advances, many of the molecular features of the FMDV genome that determine virulence remain unclear. Developing detailed molecular knowledge of virus–host interactions and identifying mechanisms that might influence pathogenesis and host range will be essential to more effectively countermeasure FMD in the future. This chapter focuses on the cellular receptor molecules that have been identified for FMDV that affect organ and host tropism, as well as the non-receptor proteins and viral factors known to influence either host range or virulence of the virus. Introduction Recent outbreaks of foot-and-mouth disease (FMD) in both developed and developing countries have re-emphasized the need for new and innovative approaches for worldwide control of this

animal pathogen. Given its transmissibility, broad host range, serological diversity, and that infected animals can also become persistent carriers, FMD represents one of the most feared diseases of susceptible livestock including cattle, pigs, sheep, and goats (Bachrach, 1968; Sutmoller et al., 2003). The identification of factors that both promote genetic diversity and restrict the occurrence of FMD in certain animals is crucial to understanding the pathogenesis of the disease. The fact that the aetiologic agent, foot-and-mouth disease virus (FMDV), is able to infect numerous cloven-hoofed livestock with differences in severity of symptoms, has opened new avenues of research into host and viral factors, which control both the virulence of individual isolates and their host range. In theory, any viral structural and non-structural (NS) protein, elements within the genome, and host proteins and membranes that participate in viral replication can be considered to be virulence and/or host range factors (Mason et al., 2003). Such elements may also be implicated in the high variability and rapid adaptability of the viral quasispecies (Domingo et al., 2003; Haydon et al., 2001). The past several decades of FMDV research have seen considerable progress in elucidating the molecular biology of its replication, cell recognition, virus structure–function relationships, and immunological aspects of the host response to infection or vaccination. Progress has also been made in understanding the pathogenesis of the disease, though many aspects of the FMDV life cycle remain elusive. FMDV is the prototypic member of the Aphthovirus genus of the Picornaviridae family of viruses, and is represented by seven different serotypes (A, O, C, Asia1, SAT1, SAT2, and SAT3) with

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varying global distributions. The virus possesses a single-stranded positive-sense RNA genome of approximately 8.5  kilobases (kb) in length. The coding sequence spans approximately 7000 nucleotides comprising a single open reading frame (ORF), from which a polyprotein precursor is translated and subsequently cleaved into a series of intermediate and mature viral proteins. Translation is initiated from one of two potential AUG start codons upstream of the leader proteinase (Lpro), thus producing one of two possible isoforms: Lab or Lb. Classically, the coding sequence downstream of Lpro has been sub-divided into three regions P1, P2, and P3. P1 encodes the four structural proteins: 1A (VP4), 1B (VP2), 1C (VP3), and 1D (VP1). The virus NS proteins are encoded by P2 (2Apro, 2B, and 2C) and P3 (3A, 3B1, 3B2, 3B3, 3C protease or 3Cpro, and the 3D RNA-dependent RNA polymerase or 3Dpol). The single ORF is flanked on both sides by highly structured 5′ and 3′ untranslated regions (UTRs). The representative stable secondary structures found in the UTRs have been implicated in viral protein synthesis and RNA replication, though the functions of some structures remain unknown. The 3′ UTR possesses a poly-adenylated (poly-A) tail, encoded within the genome. One of three copies of a small peptide (VPg or 3B), of viral origin, is covalently attached to the 5′-terminal nucleotide. Additionally, two closely adjacent stem loop structures have also been predicted in the 3′ UTR in between the termination of the P3 coding region and the poly-A tail. The 5′ UTR is approximately 1200 nucleotides in length and contains a crucial cis-acting replication element (cre) required for genome replication, and a region to direct internal ribosomal binding (internal ribosomal entry site or IRES). Additional RNA elements of unknown function in the 5′ UTR include an approximately 360 nucleotide segment that folds into a long stem–loop (called the S-fragment), a segment of greater than 100 nucleotides containing mostly cytosine residues (poly-C tract), and three or four RNA pseudoknots (see Mason et al., 2003, and references therein). As stated above, the mono-cistronic FMDV genome encodes a single polyprotein, which undergoes a series of proteolytic cleavages yielding a total of 14 structural and NS proteins (Grubman et al., 1984; Robertson et al., 1985). Cleavage occurs as a result of the activity of

two auto-proteinases (Lpro, 2Apro), and a third proteinase, 3Cpro, which performs the majority of the polyprotein cleavages (Kleina and Grubman, 1992; Klump et al., 1984; Strebel and Beck, 1986; Vakharia et al., 1987). The VP0 precursor molecules are cleaved by an unknown mechanism to produce VP2 and VP4, transforming the particles into mature (infectious) virions. In cells, the presence of viral receptors is the first level of susceptibility to productive infection, while NS viral proteins create conditions within the cell, which favour replication of the viral RNA and impose a second level of susceptibility. FMD had been effectively controlled in developed countries for many years through the implementation of rigorous import controls and vaccination. However, within the past several years, outbreaks of FMD have occurred in countries such the United Kingdom, Taiwan, Japan, and South Korea, with unusual host restrictions. These events have emphasized both the need for control of the disease and the inadequacies of the current vaccine (see Grubman and Baxt, 2003, and references therein). Traditional inactivated whole virus vaccines generally induce protection by seven days post-inoculation (Doel et al., 1994; Graves et al., 1968; Sellers and Herniman, 1974), which is adequate for countries in which the disease is endemic. However, in emergency outbreak situations, it is important to either block or reduce virus shedding as rapidly as possible to contain the outbreak. Future containment strategies will need to employ antiviral therapy in conjunction with vaccination for effective control of an outbreak. In order to develop effective antiviral agents, it is imperative to identify specific viral or host targets and to understand how they may be exploited. This article addresses only those elements where available data indicates their involvement in either virulence or host range within susceptible animals. Readers will find clear descriptions of recent advances made in the understanding of FMDV– host interactions, focusing on the cognate receptor molecules for FMDV and other non-receptor molecules implicated in the attachment, uptake, and propagation of the virus. Other sections in this book examine elements involved in viral replication and the pathogenesis of the disease, so we will discuss these only as they pertain to the issues we are considering.

FMDV Receptor and Non-receptor Interactions |  109

Viral receptors The fundamental first step in the life cycle of any virus is represented by the initial attachment of the virus particle to the host cell surface. For nonenveloped viruses, this typically proceeds via a high affinity interaction between a virus capsid protein and a host cell receptor molecule present on the extracellular surface of the plasma membrane. Once the virion is bound to the cell surface, a series of events cascade, whereby the virus particle is internalized and begins virus translation and nucleic acid replication inside the host cell. Many viruses are not limited to one receptor molecule, which has clear evolutionary advantages. The capacity to engage multiple points of attachment enhances the potential for adhering to a host cell and potentially expands the tropism of the virus beyond a limited range of cell and tissue types. In the case of FMDV, the virus initiates infection via attachment to one of several candidate receptor molecules. Depending upon which gateway FMDV selects to infect a host cell, the replication cycle will proceed via a distinct set of steps. It has been generally accepted for many years that viral receptors play a role in both tissue and organ tropism, which affects the pathogenesis of disease (Crowell, 1981; Evans and Almond, 1998; Schneider-Schaulies, 2000; Wimmer, 1994). The wide variety of FMDV serotypes and subtypes are reflective of changes in amino acid residues scattered across the surface of the virion including residues surrounding the cell receptor recognition site (Baxt et al., 1989; Bittle et al., 1982; Kitson et al., 1990; Mateu, 1995; Mateu et al., 1994; McCullough et al., 1987; Pfaff et al., 1982; Rieder et al., 1994b; Rowlands and Brown, 2002; Strohmaier et al., 1982; Xie et al., 1987). Despite this molecular diversity, all of the known FMDV serotypes induce similar clinical manifestations upon infection of susceptible species, namely fever and vesicular lesions of the epithelium of the mouth, tongue, nose, muzzle, feet, and teats (Alexandersen et al., 2003). Therefore, all of the virus serotypes and variants have developed a common mechanism allowing for host cell attachment and entry via recognition of receptors on the cell surface, which might be similar in different species. In cell culture, FMDV binds rapidly to a limited number of receptor sites, and cross-competition studies revealed that all of the serotypes appear to

utilize a common primary receptor in vitro (Jackson et al., 1997, 2000b, 2002, 2004; Neff et al., 1998), while some bind to an alternative second receptor, present in much higher abundance on the cell surface (Baranowski et al., 2000; Baxt and Bachrach, 1980; Baxt et al., 2002; Fry et al., 1999; Jackson et al., 1996; Sa-Carvalho et al., 1997; Sekiguchi et al., 1982). Moreover, evidence has been amassed suggesting that a small subset of FMDV variants can utilize a third alternative receptor (Baranowski et al., 2000; Berryman et al., 2013; Chamberlain et al., 2015; Lawrence et al., 2013; Zhao et al., 2003). Since different cell lines can express a unique repertoire of cellular receptor molecules, cell culture systems can be employed to define the range of receptor affinities available to a particular virus variant. As will be further expanded upon below, the receptor range for an FMDV isolate can be initially defined by its capacity to replicate within cell lines. This cell culture based method for screening FMDV receptor affinity is a key tool described in many of the studies reviewed below. A list of different cell lines frequently used to propagate multiple FMDV isolates and variants along with receptor expression profiles has been compiled in Table 5.1. Primary FMDV receptor: integrins In early studies on virion binding to cultured cells, it was shown that limited trypsin digestion of virions resulted in the generation of non-infectious virions, which were unable to bind to cells in culture (Barteling et al., 1979; Baxt and Bachrach, 1982; Cavanagh et al., 1977; Moore and Cowan, 1978). Additional studies with trypsin-treated virus revealed a single cleavage site at Arg144 of VP1 (Robertson et al., 1983), which was subsequently shown to be located within the βG-βH loop of VP1 (G-H loop) (Acharya et al., 1989, 1990; Jackson et al., 2003; Logan et al., 1993). These results strongly suggested that this region of the virion interacted with the cellular receptor. This residue in VP1 is part of an Arg-Gly-Asp (RGD) tripeptide sequence, which was subsequently shown to be a recognition sequence for a family of cell surface receptors called integrins (Pierschbacher et al., 1985; Pierschbacher and Ruoslahti, 1984a,b; Tamkun et al., 1986). Integrins are type I membrane proteins containing

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Table 5.1 Cell lines used in the propagation of FMDV Cell line

Receptors expressed

LFBK

αVβ1, αVβ3, αVβ6, αVβ8b, HS, JMJD6

LFBK-αvβ6 αVβ1, αVβ3, αVβ6c, αVβ8b, HS, JMJD6

Animal source

Type of FMDV Propagated

Reference

Swine

All known serotypes and variants Swaney et al. (1988), Lawrence et al. (2013)

Swine

All known serotypes and variants Larocco et al. (2013, 2014)

BHK-21

αVβ1, αVβ3, αVβ6, αVβ8, Hamster HS, JMJD6

All known serotypes and variants

IBRS2

αVβ8, HS, JMJD6

Swine

All known serotypes and variants Burman et al. (2006), Johns et *reduced affinity for αVβ8 if al. (2009) RGDR in VP1 G-H loop

CHO K1

HS, JMJD6

Hamster

VCRM4, SIR mutants

Neff et al. (1998), Lawrence et al. (2013)

CHO 677

JMJD6

Hamster

C-S8c1p100, Chinese type O1 variant, A-SIR #42, JMJD6-FMDV

Lidholt et al. (1992), Baranowski et al. (2000), Zhao et al. (2003), Lawrence et al. (2013, 2016a,b)

Confirmed expression. Inferred expression (Kraft et al., 1999). c Molecule heavily expressed. a

b

two subunits (α and β) non-covalently bound at the cell surface, and are involved in cell adhesion, cell migration, thrombosis, lymphocyte trafficking, and can act as cell signalling molecules (Hynes, 1987, 1992, 1999, 2002). Currently, 18 α-subunits and 8 β-subunits have been identified, which combine in various combinations to generate 24 integrins. However, only 8 of these 24 integrins use the RGD tripeptide as a recognition sequence (Table 5.2) (Hynes, 1999; Ruoslahti, 1996). This sequence is highly conserved within all FMDV serotypes and subtypes despite being located within the highly variable G-H loop sequences (Knowles and Samuel, 2003). Biochemical evidence that the RGD sequence interacted with the viral receptor came from studies showing that small peptides containing this sequence could interfere with attachment of virus to cultured cells (Baxt and Becker, 1990; Fox et al., 1989). Definitive evidence of the involvement of the G-H loop in binding was obtained by reverse genetic methods, which showed that by either mutating or deleting the RGD sequence in infectious cDNA clones, virions were produced that were non-infectious, unable to bind to cultured cells, and could not cause disease in susceptible animals (Leippert et al., 1997; Mason et al., 1994a; McKenna et al., 1995; Rieder et al., 1996).

αvβ3 The first identification of an integrin receptor for FMDV was made through a comparison with a human picornavirus, coxsackievirus A9 (CAV9). This virus utilizes the integrin αvβ3 as a receptor, via an RGD sequence in a C-terminal extension of VP1 (Chang et al., 1989, 1992; Roivainen et al., 1991, 1994), but has also been shown to utilize a non-integrin co-receptor (Hughes et al., 1995; Roivainen et al., 1994, 1996), the 70 kDa MHC class I associated heat-shock protein, GRP78 (Triantafilou et al., 2000b 2002). Other human picornaviruses, which utilize αvβ3 as a cellular receptor, are the parechoviruses and echovirus 9 ( Joki-Korpela et al., 2001; Nelsen-Salz et al., 1999; Pulli et al., 1997; Triantafilou et al., 2000a). Competition binding studies between FMDV and CAV9 revealed that CAV9 was able to block the binding of FMDV to monkey kidney cells, and in addition, antibodies to the αvβ3 integrin were also able to inhibit virus adsorption (Berinstein et al., 1995). These results were confirmed genetically whereby cells which did not express this integrin, and were not susceptible to FMDV infection, became permissive for viral infection upon transfecting cDNAs encoding either human (Neff et al., 1998) or bovine (Duque and Baxt, 2003; Neff and Baxt, 2001; Neff et al., 2000) αvβ3 integrin. These studies also suggested that α5β1 integrin, which is naturally expressed in

FMDV Receptor and Non-receptor Interactions |  111

Table 5.2 Integrins recognizing the RGD sequence Recognition Integrin motifa

Principal ligands

Tissue and cell distribution

αVβ1

RGD

Fibronectin

Malignant cells, smooth muscle, CNS

αVβ3

RGD, RLD/ KRLDGS

αVβ5

FMDV receptor in vitro

Reference

Yes

Duque and Baxt (2003), Jackson et al. (2002)

Vitronectin, Vascular endothelium, fibronectin, fibrinogen, smooth muscle, vWFc osteoclasts, epithelial cells

Yes

Berinstein et al. (1995), Duque and Baxt (2003), Neff et al. (1998, 2000)

RGD

Vitronectin

Airway epithelium, CNS, keratinocytes

No

Duque and Baxt (2003)

αVβ6

RGD, DLXXLb

Fibronectin, tenascin

Epithelial cells

Yes

Duque and Baxt (2003), Jackson et al. (2000b)

αVβ8

RGD

Vitronectin

Epithelial cells, human airway, CNS

Yes

Jackson et al. (2004), Burman et al. (2006), Johns et al. (2009)

αIIbβ3

RGD

Fibrinogen, vitronectin, Platelets fibronectin, vWF

α5β1

RGD

Fibronectin

Many cell types and organs NOC

RGD

Fibronectin, vitronectin, tenascin

Lung parenchyma, smooth muscle

α8β1

? Neff et al. (1998)

?

Ruoslahti (1996). Kraft et al. (1999). c Isolated immobilized α5β1 can bind virus (Jackson et al., 2000a), but does not mediate infection when expressed on cells in tissue culture (Jackson et al., 2000b; Neff et al., 1998). a

b

the cell lines used for these studies, was unable to function as an FMDV receptor in cell culture even though the virus was shown to bind to the isolated immobilized integrin ( Jackson et al., 2000a). When a similar experiment was performed with type O1BFS, however, virus was able to replicate in cells which were not transfected with the αvβ3 integrin, and neither antibodies to α5β1 nor RGD peptides were able to inhibit viral replication (Neff et al., 1998, 2000), suggesting that this virus was not utilizing an integrin receptor. Interestingly, when plasmids encoding either human or bovine homologues of αvβ3 were transfected into αvβ3-negative cells, the bovine homologue was more efficient in mediating infection than its human counterpart. In addition, this phenomenon appeared to be a function of the bovine β3 subunit (Neff et al., 2000). To gain insight into increased activity of this subunit, chimeric bovine/human β3 subunits were generated and analysed for receptor activity. Surprisingly, the increased efficiency of the bovine β3 subunit mapped, not to the subunit ligand-binding domain (LBD), but to the C-terminal region of the ectodomain (Neff et al., 2000). This region has a high number of cysteine residues, which is common to

all integrin β subunits (Calvete et al., 1991; Green et al., 1998; Horton, 1997; Hynes, 1992). The cysteine-rich region has been implicated in integrin activation (Faull et al., 1996; Kashiwagi et al., 1999; Yan et al., 2000; Yan and Smith, 2000, 2001), and sequence differences between the human and bovine β3 subunit in this region, which include one less cysteine in the bovine subunit (Neff et al., 2000), may explain the increased activity of the bovine receptor. While these studies identified a receptor for the virus, they left many unanswered questions. In susceptible species, FMDV infects primarily epithelial cells. Initial sites of viral replication are the lung and pharyngeal areas followed by rapid dissemination to oral and pedal epithelial sites (Alexandersen et al., 2001, 2003; Brown et al., 1992; Brown et al., 1995, 1996; Burrows et al., 1981; Sutmoller and McVicar, 1976; Zhang and Kitching, 2001). In humans, αvβ3 is generally expressed on vascular endothelium and smooth muscle cells (Brooks et al., 1994; FeldingHabermann, 1993; Liaw et al., 1995). Studies of αvβ3 expression in susceptible animals (cattle and pigs) revealed it is expressed in epithelium of a number of organs, mainly of the small intestine,

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kidney, bile duct (Merono et al., 2002; Singh et al., 2001), and also in endometrium (Kimmins and MacLaren, 1999). Thus, it was presumed that other integrin receptors might be involved in disease pathogenesis (Neff et al., 1998). Of all of the other RGD-dependent integrins, αvβ5 has a tissue distribution in humans, which is the most consistent with tissues affected by FMDV in susceptible species (Goldman and Wilson, 1995; Kim et al., 1994). Despite this, studies utilizing antibody inhibition or transient expression of recombinant αvβ5 have demonstrated that it is not a functional receptor for FMDV (Berinstein et al., 1995; Duque and Baxt, 2003; Jackson et al., 2000b). αvβ6 The hypothesis, that there must be more than one integrin receptor for FMDV, was proven correct when it was shown by Jackson and co-workers that the epithelial integrin αvβ6 was utilized as an FMDV receptor ( Jackson et al., 2000b), followed in rapid succession by studies from the same laboratory implicating both αvβ1 ( Jackson et al., 2002) and αvβ8 ( Jackson et al., 2004) as receptors for the virus (Table 5.2). Interestingly, a study using cell lines transiently expressing different integrin heterodimers indicated that different FMDV serotypes may exhibit preferential binding to different integrin receptors (Duque and Baxt, 2003). In that study, serotype A FMDVs showed efficient binding to αvβ3 and αvβ6, but only moderately so with αvβ1. In contrast, serotype O FMDVs exhibited greater efficiency binding αvβ1 and αvβ6 than αvβ3. Subsequent studies examining the role of αvβ6 in the pathogenesis of FMDV have suggested that αvβ6 may be the more relevant integrin heterodimer for the propagation of virus in the animal host. Investigations of the level of integrin expression in tissue at the sites of FMDV replication in both pigs and cattle have repeatedly shown higher levels of αvβ6 relative to αvβ3 (Monaghan et al., 2005). Indeed, the production and examination of soluble secreted bovine αvβ3 and αvβ6 revealed that both serotype A and O FMDVs bind to αvβ6 with greater affinity than αvβ3 (Duque et al., 2004). Moreover, pre-incubation of virus with soluble αvβ6, but not αvβ3, significantly impeded the progression of infection by blocking virus adsorption in cell culture. In addition, serial passaging of FMDV in cell culture in the continuous presence of soluble αvβ6

acted as a selective pressure leading to the emergence of soluble integrin resistant (SIR) FMDV mutants that subverted the anti-adsorptive effects of the soluble αvβ6 treatment (Lawrence et al., 2013). Cumulatively, these findings strongly imply that, as a receptor molecule, αvβ6 plays a greater role in FMDV pathogenesis than αvβ3. αvβ1 Shortly after the identification of αvβ6 as another integrin heterodimer that would support FMDV attachment to host cells, αvβ1 was also reported to function as an FMDV receptor molecule ( Jackson et al., 2002). Subsequent infectivity assays utilizing transient expression of the cloned bovine homologues of αvβ6 and αvβ1 confirmed these results (Duque and Baxt, 2003), but did not examine the efficiency of these receptors compared with the human homologues. As described above, different efficiencies of receptor usage between serotypes A and O FMDV was observed. With respect to αvβ1, it appears that serotype O viruses utilize αvβ1 with relatively higher efficiency than serotype A viruses (Duque and Baxt, 2003). Exchanges of LBDs between the different β subunits suggested that the subunit LBD appeared to play a role in the efficiency of receptor usage, but exchanging the G-H loop of the type A virus with one from type O (Rieder et al., 1994a) did not change the receptor utilization of the type A virus, suggesting that sequences outside of the loop were responsible for these results (Duque and Baxt, 2003). αvβ8 Later, Jackson and co-workers reported that integrin heterodimer αvβ8 was a fourth receptor molecule for FMDV ( Jackson et al., 2004). Interestingly, the swine based IBRS2 cell line is enriched in αvβ8 and not in the other three FMDV integrin receptors (Burman et al., 2006; Johns et al., 2009). Thus, this adds another cell culture system with which to characterize FMDV variants that have greater preference for this integrin heterodimer over others (Table 5.1). The RGD+1 site has been described as an important determinant of FMDV receptor affinity, and is frequently occupied by a leucine (L) residue, though occasionally arginine (R) and methionine (M) have been found to occupy this site in isolated viruses. Viruses displaying an RGDR sequence in the VP1 G-H loop were observed to have reduced

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affinity for αvβ8 as a receptor molecule in a study employing IBRS2 cells for virus characterization (Burman et al., 2006). As was observed between FMDV serotypes A and O with αvβ1, αvβ3, and αvβ6, a serotype-specific differential integrin receptor preference for αvβ6 was also detected in the SIR mutant investigation (Lawrence et al., 2013). It is not clear why FMDV should need four integrin receptors to cause disease, or if it utilizes all, or some, of the integrins in the susceptible hosts. Infection with different FMDV strains may cause different degrees of disease severity, but there are generally no differences in clinical symptoms within a species. There are, however, differences in the clinical course of disease among different susceptible species. Additionally, FMDV can utilize integrin receptors from humans, hamsters, and monkeys, species not known to be susceptible to the disease. While the virus can utilize the bovine αvβ3 integrin more efficiently than the human homologue (Neff et al., 2000), no comparisons of homologues of the other known integrins from different species have been conducted. Table 5.2 includes the known tissue distribution of the RGD-binding integrins in humans. Each of the integrins identified as receptors for FMDV are expressed in tissues that are not involved in disease pathogenesis. Also, as described above, different serotypes of FMDV exhibit altered efficiencies of integrin utilization, and all serotypes tested utilized the αvβ6 integrin with relative high efficiency (Duque and Baxt, 2003). The possibility exists that the virus uses different receptors during various stages of the disease. That is why definitive information on integrin expression in the susceptible species would be helpful in correlating integrin utilization in tissue culture with sites of viral replication in vivo. We were able to generate cDNAs encoding the bovine αv, β1, β3 and β5, subunits from primary bovine tongue keratinocytes (Duque and Baxt, 2003). In contrast, β6 sequences could not be amplified from a number of different FMDV target organs, but was amplified from bovine thyroid cells (Duque and Baxt, 2003), which are highly sensitive to FMDV infection (House and House, 1989; Snowdon, 1966). However, the bovine thyroid gland does not appear to be a target organ for infection as evidenced by the absence of FMDV antigen in thyroids from infected animals (D. Gregg and B. Baxt, unpublished). It is interesting to note that bovine mononuclear cells in the lung and

spleen express αvβ3 (Singh et al., 2001) leading to speculation that such cells might be a mechanism of disseminating virus from sites of initial replication to secondary replication sites through lymphoid organs, although it is doubtful that virus can productively replicate in lymphoid cells (Alexandersen et al., 2003; Baxt and Mason, 1995; Rigden et al., 2002). As with other viruses, FMDV is subject to a variety of environmental stresses that function as selective pressures forcing the virus to adapt. The most perilous adaptation for a virus is a shift in receptor tropism. Any mutation rendering the virus incapable of binding to its traditional receptor without a corresponding mutation allowing for attachment to another site on the host cell surface will condemn the virus to a non-infectious state left to drift into non-existence in the extracellular milieu. Many strategies have been developed in the laboratory setting to drive virus-receptor plasticity, and viruses have evolved natural back up strategies when the primary cell receptor is limiting or unavailable. In the next section, artificial and natural FMDV alternative receptors will be described with particular focus on the FMDV secondary receptor and the elusive third FMDV receptor. Artificial FMDV receptors Using modern cloning technologies and online repositories of genomic sequence information, affinities for specific receptor molecules have been rationally designed and engineered into laboratory viral constructs. For example, an alternative FMDV receptor has been engineered by joining the antigen-binding domain of an FMDV-specific antibody to a cell-surface receptor specific for another virus. This was accomplished by cloning the antigen-binding portion of an anti-FMDV type A12 monoclonal antibody (mAb) in the form of a single chain antibody (scAb) (Mason et al., 1996), and fusing it to ICAM-1, the receptor for the major group of human rhinoviruses (Rieder et al., 1996). When Chinese hamster ovary (CHO) cells were transfected with this engineered receptor molecule, they supported productive infection of type A12 virus, while non-transfected CHO cells were resistant to infection. This receptor was only functional for viruses carrying the epitope recognized by the scAb, and the RGD sequence was unnecessary for infection with these viruses

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(Rieder et al., 1996). Using cells expressing this receptor, large amounts of type A12 virus, lacking the RGD sequence, were produced, which were shown to be non-infectious in cells normally susceptible to infection with RGD-containing viruses (Rieder et al., 1996), and unable to infect cattle (McKenna et al., 1995). Alternative FMDV receptor: heparan sulfate In 1996, it was demonstrated that serotype O1 FMDV could utilize heparan sulfate (HS), a ubiquitous cell-surface glycosaminoglycan (GAG) consisting of a sulfated polysaccharide covalently linked to a protein core, as an alternative receptor molecule ( Jackson et al., 1996). While the involvement of co-receptors in FMDV infection had not been ruled out, the data indicating that the integrin receptor or alternative receptors like HS were sufficient for binding the virion to cells, argued against the existence of co-receptors in cultured cells. The results of Jackson et al. (1996) correlated well with earlier studies suggesting that some FMDVs utilized a second receptor, present in high copy number on cultured cells (Baxt and Bachrach, 1980; Sekiguchi et al., 1982), a property descriptive of an HS receptor. In addition, these results helped to explain earlier findings that type O1BFS replicated in cells which did not express the αvβ3 integrin (Neff et al., 1998). Critical amino acid substitutions associated with HS receptor usage To further examine the role of HS in viral infection, genetically defined derivatives of a serotype O1 Campos (Sa-Carvalho et al., 1997) which are similar to the viruses (O1Kaufbueren and O1BFS) utilized by Jackson et al. (1996) were used. This study demonstrated that passage of FMDV in cell culture resulted in selection of viruses capable of growing in CHO cells. This adaptation appeared to be mediated by selection of an arginine (R) residue at position 56 of VP3 (here referred to as 3056R) (Sa-Carvalho et al., 1997). The added positive charge on the virion surface would mediate electrostatic binding to negatively charged HS. Additionally, heparin inhibited plaque formation by this virus (3056R), but had no effect on plaque formation by a second O1Campos variant with a histidine (H) at this position (3056H) and more

resembled viruses isolated from infected animals in the field (Sa-Carvalho et al., 1997). The cDNAs encoding the capsids of these two variants were inserted into a type A12 infectious cDNA clone and used to produce viruses exhibiting the phenotypes of the original variants (Sa-Carvalho et al., 1997). Receptor utilization of these viruses, and type O1BFS, was examined in wild-type (K1) and GAG-deficient (677) CHO cells, and was compared with type A12. Cells were transfected with cDNAs encoding human αvβ3 integrin subunits, resulting in cell lines expressing specific integrin and GAG receptors (Neff et al., 1998). Viral replication in this panel of CHO cells showed that tissue culture adapted type O1BFS and the 3056R viruses only replicated in cells expressing HS, independent of αvβ3 expression. A 3056R virus with an RGD → KGE mutation also only replicated in the HS-expressing cells, indicating that this virus was not utilizing another RGD-dependent integrin. In contrast, the A12 and O1 3056H viruses only replicated in cells expressing the transfected integrin cDNAs, and did not need HS for replication. Furthermore, a type O1 3056H virus containing an RGD → KGE mutation was unable to replicate in cells expressing either HS or the integrin, identical to results obtained with RGD-mutated type A12 (Mason et al., 1994a). Therefore, while the ‘field-type’ virus utilizes an integrin as a receptor, tissue culture adaptation of type O1 virus results in the utilization of HS as an alternative secondary receptor. The tissue culture adapted phenotype results from genetic changes at amino acid residue 56 in VP3, distant from the VP1 RGD motif, and may also be dependent on residue 134 in VP2 (Sa-Carvalho et al., 1997). The importance of these residues in binding HS was directly supported by X-ray crystallographic structural data of types O1BFS and A10 virus crystals infused with HS (Fry et al., 1999; Jackson et al., 2003). This showed amino acid residue 3056R of both viruses made contact with two sulfate groups within HS. In addition, a conserved R residue at position 135 of VP2 (2135R) also made contact with one of the HS disaccharides. This latter residue is next to K134 in VP2, which we showed was necessary for the tissue culture adapted phenotype (Sa-Carvalho et al., 1997). Interestingly, two residues at the C-terminus of VP1 (residues 1193K and 1195H) of both type O1BFS and A10 also make contact with HS (Fry et

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al., 1999; Jackson et al., 2003). It had been previously reported that removal of C-terminal residues 201–211 of VP1 of type O1, or antibodies directed against this region, inhibited virus attachment to cells in culture (Fox et al., 1989). It is still unclear if this region mediates binding to the integrin or HS receptors, although the virus–HS complex showed no interactions between HS and this region of VP1 (Fry et al., 1999). Studies described above with type O1Campos variants showed the importance of the virus–integrin interaction in vivo. Inoculation of cattle with either the HS-binding 3056R variant or the integrin-binding 3056H variant revealed that the 3056H virus was highly virulent in cattle, while the 3056R virus was at least 100,000-fold less virulent in cattle (Sa-Carvalho et al., 1997). Moreover, two cattle inoculated with large amounts of HS-binding virus eventually showed signs of FMD. Virus isolated from these animals could no longer replicate in CHO cells, interact with HS in vitro, and had amino acid substitutions at either residue 56 of VP3 (R → C), or at residue 134 of VP2 (K → E), which removed positive charges from the virion surface (Sa-Carvalho et al., 1997). Finally, in the context of the full-length infectious clone of O1 Campos (Bra/58 strain), the substitution of H56R in VP3 was reinforced as the critical amino acid leading to cell culture adaptation and virus attenuation, in the absence of the K134E VP2 substitution described above (Borca et al., 2012). In this study, viruses identical genetically except for H or R at position 56 in VP3 grew similarly in cell culture, but showed significantly different degrees of pathogenicity in cattle and pigs. The VP3-H56 virus exhibited the characteristic clinical course of FMD in both host species, while the VP3-R56 counterpart only showed disease symptoms if the R reverted to an H or C. Additionally, in a recently described soluble integrin study, the H56R mutation in VP3 was consistently acquired in every serotype O SIR mutant that adopted HS binding in lieu of integrin receptors in the continued presence of soluble bovine αvβ6 (Lawrence et al., 2013). Interestingly, variants of serotype A SIR mutants that developed an affinity for HS binding exhibited amino acid substitutions exclusively in VP1, with a single L150R in the RGD + 4 position for A-SIR #45 (Lawrence et al., 2013). In another instance, the cell culture adaptation of PanAsia-1 field

strains of FMDV serotype O viruses (O/Fujian/ CHA/9/99wt and O/Tibet/CHA/6/99wt) involved the acquisition of several amino acid substitutions in capsid proteins VP4, VP2, and VP1. However, the positively charged VP1 (E103K in O/ Fujian/CHA/9/99tc) and neutral VP2 (L2080N in O/Tibet/CHA/6/99tc) amino acid substitutions were determined to be critical for adopting HS as receptor (Bai et al., 2014). The acquisition of positively charged residues has been also found associated with cell culture passaged type C viruses (Baranowski et al., 2000, 2001a,b; Martinez et al., 1997). FMDV serotypes SAT1, SAT2, and SAT3 are notoriously difficult to adapt to cell culture (Preston et al., 1982). Studies with cell culture adapted SATs identified positively charged amino acid substitutions in the F-G (SAT1) and D-E (SAT2) loops of VP1, positions proximal to the five-fold axis of symmetry on the virus particle (Maree et al., 2011; Maree et al., 2010). A recent report describing the cell culture adaptation of a vaccine variant of FMDV serotype A (A/IRN/87) identified positively charged amino acid substitutions in a novel location in the VP2 that was different from the previously determined HS-binding sites in the depression between VP1, VP2, and VP3 as well as the five-fold symmetry axis (Chamberlain et al., 2015). Interestingly, two isolated variant viruses, termed A+ and A-, have been described where the latter virus possessed a large deletion within the G-H loop of VP1 that encompassed the highly conserved RGD motif for integrin binding and yet remained infectious in cell culture (Fowler et al., 2011) and in cattle (Fowler et al., 2014). Furthermore, this unique set of mutations in VP2 allowed for growth of the A- virus on both CHO K1 (HS-positive) and CHO 677 cells (HS-negative) indicating that this virus can utilize a pathway to infection that is both integrin and HS independent (Chamberlain et al., 2015). Moreover, type C1 variants have also been isolated which appear to bind to host cells and replicate in both an integrin and HS-independent manner, suggesting the possibility of a third natural receptor system for FMDV (Baranowski et al., 2000; Baranowski et al., 1998). Such non-integrin non-HS targeted type C viruses were obtained after continuous serial passaging in cell culture for 100 passages (FMDV C-S8c1p100) or 213 passages (FMDV C-S8c1p213).

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Alternative FMDV receptor: unidentified FMDV receptor(s) Studies with both type A and C FMDVs have inferred the existence of a third as yet uncharacterized FMDV receptor (Baranowski et al., 2000; Berryman et al., 2013; Chamberlain et al., 2015). In 2003, it was reported that a tissue culture adapted derivative of a type O1 virus, isolated in China, was able to replicate in tissue culture in both an integrin and HS-independent manner and cause mild disease in pigs (Zhao et al., 2003). This phenomenon appeared to be related to a group of amino acid residues surrounding the five-fold axis of the virion. Furthermore, genetically engineered non-RGD variants of field isolate Asia1/JS/CHA/05 were able to produce disease in inoculated animals similar to parental virus without compensatory mutations (Li et al., 2011a). Thus, the possibility was established that non-integrin receptors might also be involved in disease pathogenesis in animal host. As mentioned in the sections pertaining to the primary integrin receptor and the secondary HS receptor, among the soluble integrin resistant mutant viruses one type A derived mutant (A-SIR #42) exhibited the capacity to efficiently replicate on the CHO 677 cell line, thus indicating an integrin-independent and HS-independent pathway of attachment and uptake (Lawrence et al., 2013). Using soluble integrin as a selective pressure, this FMDV variant was selected after only three passages, which contrasts significantly with the 100–213 passages required to develop such type C variants. The A-SIR #42 mutant contained mutations E95K/S96L in the VP1 capsid protein upstream of the conserved RGD motif. An infectious clone was developed incorporating the E95K/ S96L mutations in VP1 with the RGD → KGE substitutions to ablate integrin interaction (Lawrence et al., 2016b) and (Mason et al., 1994b). The resulting virus was viable, and the mutations remained stable; therefore this construct was used to evaluate the significance of these mutations to the growth adaptation in CHO 677 cells. Recent investigations developed evidence suggesting that Jumonji C-domain-containing protein 6 ( JMJD6) might serve as an alternative FMDV receptor in CHO 677 cells (Lawrence et al., 2016a,b). Previously, JMJD6 has been described as a RNA-binding protein with attributed arginine demethylase activity (Chang et al., 2007).

Moreover, JMJD6 was observed by immunofluorescent microscopy to re-localize from a cell membrane/cytosolic subcellular distribution to a predominantly nuclear location following infection with FMDV in LFBK cells (Lawrence et al., 2014). The same redistribution pattern of JMJD6 was detected during infection with three non-HS and non-integrin FMDV variants in CHO 677 cells: (i) A-SIR #42, (ii) KGE-containing infectious clone of A-SIR #42, and (iii) Chinese type O1 cell cultureadapted mutant (Lawrence et al., 2016b; Zhao et al., 2003). The sub-cellular localization of JMDJ6 has remained a controversial topic. In particular, this protein was first described as a phosphatidylserine receptor (PSR) for phagocytic cells to recognize and engulf cells undergoing apoptosis (Fadok et al., 2000; Fadok et al., 2001). Later reports detected the protein predominantly in the nucleus (Cikala et al., 2004; Cui et al., 2004; Tibrewal et al., 2007). Subsequently, it was shown that surface bound JMJD6 could be stimulated to be internalized from the cell membrane and distributed to the cell nucleus (Zakharova et al., 2009). Given its trafficking pattern during the course of FMDV infection, JMJD6 was explored as an alternative receptor involved in the CHO 677 susceptibility to non-HS and nonintegrin mutant FMDVs. Using the KGE-containing infectious clone derived from A-SIR #42 (referred hereafter as JMJD6-FMDV) described above, JMJD6 redistribution from the cell surface/cytoplasm to the nucleus was detected using several different JMJD6-specific antibodies. Moreover, JMJD6-FMDV infection of CHO 677 cells could be abrogated by pre-treatment of the cell culture with JMJD6 antibodies or by pre-treatment of the JMJD6-FMDV with purified soluble JMJD6, but not by pre-treatment with heparin or soluble αvβ6. Co-immunoprecipitation experiments and in silico modelling suggested that the modified VP1 of JMJD6-FMDV bound to JMJD6. In light of these in vitro findings, animal studies were performed in cattle, which were inoculated with either JMJD6FMDV or the parental A24 Cruzeiro by either the intradermolingual (Hoffmann et al., 2001) or aerosol route (Lawrence et al., 2016a). The resulting disease symptomology from JMJD6-FMDV was indistinguishable from the parental virus if introduced to the animal via the IDL route, but FMD pathogenesis was not observed with either virus

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if they were introduced to the animal via aerosol. Importantly, tissue cross-sections were examined by immunofluorescent microscopy for co-localization of FMDV antigen and JMJD6. In tissue from animals infected with the wild-type virus, VP1 could occasionally be observed in JMJD6-positive cells. However, in strong contrast, in tissue from JMJD6-FMDV-infected animals, VP1 was detected exclusively in JMJD6-positive cells. Based on the cumulative findings, it was proposed that JMJD6 could function as an alternative FMDV receptor both in vitro and in vivo (Lawrence et al., 2016a,b). Attachment is only the first stage of the virus–host cell interaction. Without subsequent internalization, the FMDV particle would be left tethered to the cell surface in a precarious state, unable to commandeer the host cell translation machinery within the cytoplasm to begin the production of viral proteins necessary for assembly of more virus capsids and replication of the RNA genome. In the next section, we discuss what has been delineated regarding the receptor-mediated endocytosis of FMDV. Mechanisms of FMDV host cell entry Studies of the entry and uncoating of FMDV into infected cells have suggested that the processes have some similarities and differences with other Picornaviruses. In context of enteroviruses, the first step in the uncoating occurs prior to entry of the virion through the membrane. The interaction with the receptor causes a conformational rearrangement of the virion resulting in the release of VP4 and the externalization of the N-terminal extension of VP1 to form an altered (A) particle (Carson, 2014; Everaert et al., 1989; Fricks and Hogle, 1990; Lonberg-Holm and Whiteley, 1976; Organtini et al., 2014). A-particles express new antigenic determinants and have a lower sedimentation rate (135S) in sucrose gradients than the native virus (160S) (De Sena and Mandel, 1977). These particles are hydrophobic resulting from the presence of bound saturated fatty acid ‘pocket factors’ and have an affinity for liposomes (Fricks and Hogle, 1990), an interaction which results in the conversion of the A-particle to an 80S particle and the release of the RNA into the cell (Belnap et al., 2000a). While there is still controversy regarding

the exact role of the A-particle in enterovirus entry and uncoating (Dove and Racaniello, 1997), the initial conformational change which generates this particle is mediated by the interaction of the virion with the cellular receptor, which binds into a pit or canyon on the surface of the virion (Belnap et al., 2000b; He et al., 2000; Hogle et al., 1985; Kolatkar et al., 1999; Luo et al., 1987; Muckelbauer et al., 1995; Rossmann et al., 1985; Xiao et al., 2001), as determined from studies using soluble-secreted enterovirus receptors (Gomez Yafal et al., 1993; Kaplan et al., 1990; Tsang et al., 2001). In contrast to enteroviruses, A-particles have not been detected in cells following FMDV infection. After adsorption to the cell surface, the 140S virion breaks down into 12S pentameric subunits releasing the RNA (Baxt, 1987; Baxt and Bachrach, 1980, 1982; Cavanagh et al., 1978). This breakdown does not occur on the cell surface, since particles which are eluted from the cell after adsorption are fully infectious and still sediment at 140S (Baxt and Bachrach, 1980). The intracellular uncoating of FMDV occurs within an acidic endocytic vesicle (Baxt, 1987; Berryman et al., 2005; Carrillo et al., 1984, 1985; O’Donnell et al., 2005). FMDV provirions were generated by a site-directed mutation in VP0, resulting in an inability to perform the maturation cleavage of VP0 to VP2 and VP4. The provirions were analysed, and found to be non-infectious, could adsorb to cells in culture, and were acid-sensitive (Knipe et al., 1997). Thus, breakdown of 140S virus to pentameric subunits, by itself, does not lead to productive infection, suggesting there are additional steps subsequent to the virion breakdown. Internalization of vitronectin by αvβ3 has been reported to require the ligation of a second integrin, α5β1 via the β3 cytoplasmic domain (Blystone et al., 1995; Pijuan-Thompson and Gladson, 1997). However, deletions to either the αv or the β3 cytoplasmic domains were found to not affect the ability of αvβ3 to function as a viral receptor (Neff and Baxt, 2001). This contrasted with the results of Miller and colleagues (Miller et al., 2001), who showed that deletion of the β6 cytoplasmic domain abolished the ability of the αvβ6 integrin to mediate FMDV infection, while still retaining the ability to bind the virus. However, this study did not directly examine the internalization of the virus by these cytoplasmic

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domain-deleted receptors. It is possible that cytoplasmic domain-deleted αvβ3 internalizes FMDV by either interacting with another cell surface molecule, or by an integrin-independent mechanism, similar to that which has been described for the internalization of cyclic RGD peptides by this integrin (Castel et al., 2001). Internalization of parechoviruses or adenoviruses, mediated by αvβ3 or αvβ5, was shown to be clathrin-mediated and required dynamin ( JokiKorpela et al., 2001; Meier et al., 2002; Wang et al., 1998). Similarly, subsequent studies have demonstrated that integrin-mediated entry of FMDV occurs by the same mechanism. Immunofluorescent microscopy experiments elegantly demonstrated that FMDV particles bound to host cells via the integrin primary receptor underwent receptormediated endocytosis through clathrin-coated pits (CCPs) (Berryman et al., 2005; O’Donnell et al., 2005). Consistent with these observations, the internalization of FMDV bound integrin molecules was sensitive to the effects of chemical inhibitors of the CCP uptake pathway including chlorpromazine and hypertonic sucrose. In contrast, cell culture adapted FMDV particles (O1 Campos with a 3056R substitution), which utilize the HS secondary receptor, were visualized by immunofluorescent microscopy to enter host cells through the caveolae-mediated uptake pathway (O’Donnell et al., 2008). Cell culture adapted FMDVs were endocytosed via caveolae as evidenced from the co-localization of VP1 with the signature marker of caveolae: caveolin-1. The caveolae containing FMDV eventually merged with endosomes consistent with the observed co-localization with endosomal markers. Additionally, chemical uptake inhibitors of caveolae-dependent internalization (such as nystatin) effectively blocked the progression of FMDV infection through the HS receptor. Similarly, knockdown in caveolin-1 expression through RNA interference also impeded infection by cell culture adapted FMDVs. Finally, in the context of the JMJD6-involved attachment and entry pathway, the JMJD6-FMDV variant appeared to be internalized through the CCP-dependent pathway. Productive infection as measured by non-structural protein translation (3Dpol) was blocked when hypertonic sucrose or chlorpromazine were present, but not nystatin. Similarly, JMJD6-FMDV failed to affect

the redistribution of JMJD6 in the presence of hypertonic sucrose and chlorpromazine, but not nystatin as observed by immunofluorescent microscopy (Lawrence et al., 2016a). That both the integrin- and JMJD6-involved pathways were sensitive to CCP inhibitors is consistent with findings suggesting that αvβ3 (and potentially αvβ6) and JMJD6 occupy a similar membrane microdomain seen in studies with C. elegans implicating JMJD6 as a PSR involved in recognition of PS on the surface of apoptotic cells (Hisatomi et al., 2003; Hsu and Wu, 2010; Wang et al., 2003). Therefore, it can be inferred that the uptake pathway employed by FMDV is pre-determined by the receptor molecule utilized by the virus for the initial attachment step. Collectively, the information reviewed to this point supports a model where FMDV is internalized in complex with its cognate receptor, it is delivered to an acidic vesicular compartment, where capsid de-stabilization leads to the delivery of the FMDV RNA genome to the host cell cytoplasm where the bulk of its replication cycle transpires. These critical post-attachment and entry steps are driven by a series of non-receptor protein interactions involving FMDV proteins and RNA elements that are described further in the last section of this chapter. FMDV interactions with non-receptor host factors: roles in infection, host range, and virulence Beyond the initial engagement between the FMDV capsid with the host cell surface receptor molecule(s), a variety of non-receptor host factors play important roles in the progression of infection, host range, and virulence. The limited coding capacity of small RNA viruses necessitates that the virus acquire and re-purpose host factors in its sub-cellular replication environment to efficiently complete a replication cycle. Significant progress has been made in the understanding of the molecular and cellular biology of FMDV replication, where multiple host proteins have been identified as important contributors to the progression of FMDV infection (summarized in Table 5.3). The molecular communication between the virus components and the host cell machinery can be broadly divided into two categories: viral protein-host

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protein interactions and viral RNA-host protein interactions. Viral proteins Viral proteases: Lpro and 3Cpro Infection of cultured cells with FMDV induces a rapid shut-off of host cell macromolecular synthesis. Both the 3Cpro and Lpro proteins are involved in these events. Lpro is a papain-like protease (Kleina

and Grubman, 1992; Piccone et al., 1995a; Roberts and Belsham, 1995; Skern et al., 1998), and is auto-catalytically cleaved from the polyprotein at its C-terminus (Guarne et al., 1998; Strebel and Beck, 1986). It is the N-terminal component of the polyprotein, and the region of the genome encoding this protein contains two in-frame AUG initiation codons that result in the generation of two L proteins, Lab and Lb (Robertson et al., 1985; Sangar et al., 1987). Both forms of the protein are

Table 5.3 Non-receptor molecules involved in FMDV life cycle Point of interaction

Contribution to FMDV infection

Reference

RBP

S-fragment, 2C, 3A, PABP1

RNA replication

Lawrence et al. (2009)

Sam68

RBP

IRES, 3Cpro, 3Dpol

RNA replication

Lawrence et al. (2012), Rai et al. (2015)

JMJD6

RBP

VP1, RHA

Site of attachment, Lawrence et al. (2014, 2016a,b) RHA demethylation

Molecule

Type of molecule

RHA

PABP1

RBP

Poly-A tail

–

Rodriguez-Pulido et al. (2007)

PCBP2

RBP

IRES

Affects translation

Pacheco et al. (2008)

Gemin5

RBP

IRES

Inhibits translation

Fernandez-Chamorro et al. (2014)

PTB

RBP

IRES

Promotes translation

Niepmann et al. (1997)

Beclin1

Autophagy

2C

Promotes translation

Gladue et al. (2012)

Vimentin

Autophagy

2C

Membrane rearrangement

Gladue et al. (2013)

DCTN3

Dynactin component

3A

Intracellular movement

Gladue et al. (2014)

N-Myristoyl transferase

Lipid modifier

VP4

Capsid assembly

Belsham et al. (1998)

LYPLA/APT1

Lipid modifier

–

–

Guo et al. (2015)

eIF-4G

Translation factor

Lpro

Block cellular translation

Devaney et al., 1988 Kirchweger et al., 1994

NF-KB, IRF3, IRF7, RANTES

Transcription factors

Lpro

Modulation of innate immunity

De los Santos et al. (2007), Wang et al. (2011a,b)

PKR, OAS

IFN-induced genes

Lpro

Antiviral activity

Chinsangaram et al. (1999)

Histone H3

DNA packaging

3Cpro

Block overall cellular transcription

Grigera and Tisminetzky (1984), Tesar and Marquardt (1990), Falk et al. (1990)

eIF-4A and eIF-4G Translation factors

3Cpro

Block cellular translation

Belsham et al. (2000)

Ebp1

RBP

IRES

Transcription regulation

Monie et al. (2007)

NEMO

Transcription 3Cpro Complex Adaptor

Modulation of innate immunity

Wang et al. (2012)

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synthesized in infected cells (Clarke et al., 1985), though data strongly suggest that Lb may be the biologically relevant protein in vivo (Cao et al., 1995; Piccone et al., 1995a). This protease is responsible for inhibition of cellular protein synthesis in infected cells. Unlike most host mRNAs, actively translated viral mRNA does not contain a 7-methyl-G cap structure at its 5′ end (Grubman and Bachrach, 1979) and initiates protein synthesis internally, at the IRES, by a capindependent mechanism (Belsham and Brangwyn, 1990; Jang et al., 1988; Kuhn et al., 1990; Pelletier and Sonenberg, 1988). Lpro inhibits cap-dependent mRNA translation by cleaving the protein synthesis initiation factor, eIF4G (Devaney et al., 1988; Kirchweger et al., 1994), which bridges the mRNA cap to the 40S ribosomal subunit. In contrast, initiation of FMDV RNA translation only requires the Lpro generated C-terminal eIF4G cleavage product, which binds to the FMDV IRES and interacts with the 40S ribosomal subunit (Lopez de Quinto and Martinez-Salas, 2000; Saleh et al., 2001). The proteolytic activity of Lpro was inhibited in both a cell-free translation system and in cell culture (Kleina and Grubman, 1992) by the compound E-64 and its membrane-permeable analogue E-64d; both of which have been shown to specifically inhibit thiol proteases (Hanada et al., 1978a; Hanada et al., 1978b). Inhibition of Lpro effectively blocked virus capsid assembly, presumably because N-terminal myristoylation of VP4 and processing of P1–2A by 3Cpro were blocked. Animal experiments with genetically engineered Lpro-deleted (leaderless) type A12 virus showed Lpro to be a virulence determinant, which was thought to be due to the inability of the virus to shutoff cellular protein synthesis. While leaderless virus replicated at only a slightly lower rate than wildtype virus in BHK-21 cells (Piccone et al., 1995a), it was markedly avirulent when injected into cattle and pigs and was unable to spread to co-housed animals (Chinsangaram et al., 1998; Mason et al., 1997). Post-aerosol exposure, wild-type virus infected cattle had histologically altered respiratory bronchioles and virus-specific in situ hybridization (ISH) signals in bronchioles by 24 h. By 72 hours these animals developed clinical disease including fever and vesicles on the feet and positive ISH signals in epidermal sites corresponding to visible lesion development. In contrast, cattle infected

with leaderless virus showed no clinical disease at 72 hours and no pulmonary changes at either 24 or 72 h. These animals had only limited positive virus-specific ISH signals in respiratory bronchioles by 24 hours and no evidence of lesions or ISH signals in epithelial tissue by 72 hours (Brown et al., 1996). Thus, the leaderless virus did not appear to replicate well at the site of primary infection and was not able to spread to other sites within the host. It was subsequently shown that infection with wild-type or leaderless virus induced the synthesis of type I interferon (IFN) mRNA in both tissue culture (Chinsangaram, 2001; Chinsangaram et al., 1999), and in lung mononuclear cells from aerosol exposed cattle (Brown et al., 2000). In tissue culture, however, IFN-associated antiviral activity was only detected in leaderless virus infected cells (Chinsangaram, 2001; Chinsangaram et al., 1999). This latter observation can be attributed to the inability of the leaderless virus to inhibit host translation, including IFN synthesis, and the production of IFN within the infected animal probably inhibited initial amplification and spread of the virus. In contrast, wild-type virus infection blocks IFN mRNA translation allowing the virus to rapidly spread to neighbouring cells, and systemically, prior to the induction of the adaptive immune response. A similar leaderless virus containing the capsid-coding region from serotype O1Campos in the genetic background of type A12 was also avirulent in cattle, but caused a mild disease in swine and was transmitted to a naive animal (Almeida et al., 1998). The main function of viral 3Cpro is to perform most of the cleavages of the viral polyprotein (Clarke and Sangar, 1988; Vakharia et al., 1987). However, it has also been implicated in cleavage of some host cell components. 3Cpro has been shown to cleave another member of the cap-binding complex, eIF4A (Belsham et al., 2000), and thus may also be involved in the inhibition of cellular protein synthesis. It also cleaves eIF4G late in infection, although at sites different from those cleaved by the Lpro (Belsham et al., 2000). Histone H3 is cleaved in FMDV-infected cells (Grigera and Tisminetzky, 1984), and 3Cpro was later shown to be responsible for this cleavage, suggesting that it is also responsible for the inhibition of host cell transcription (Falk et al., 1990; Tesar and Marquardt, 1990). Additionally, 3Cpro was demonstrated to cleave histones as

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a complex with viral non-structural proteins 3A and 3B, and the 3ABC complex concentrates in the perinuclear area of BHK-21 cells suggesting that residues in the 3A protein target the complex to this site enabling the 3C cleavage of histones (Capozzo et al., 2002). It is interesting to note that poliovirus 3Cpro does not cleave histone H3 (Tesar and Marquardt, 1990), but has been implicated in the cleavage of RNA polymerase II transcription factors (Clark et al., 1991; Clark et al., 1993; Yalamanchili et al., 1997). Recently, the multi-functional nuclear RNAbinding protein Sam68 (68 kDa Src-associated protein in mitosis) was identified as a host factor involved in the FMDV life cycle (Lawrence et al., 2012). Sam68 was observed to be proteolyzed over the course of FMDV infection in cell culture. The in vitro co-incubation of Sam68 and 3Cpro also resulted in the proteolysis of Sam68. The functional significance of the Sam68 cleavage to the progression of FMDV infection remains unclear. Capsid protein VP4 As described in the introduction, the P1 region of the FMDV genome encodes four capsid proteins: VP4, VP2, VP3, and VP1. In the discussion of host receptor interactions, much has been discussed of the involvement of VP1, VP2, and VP3 in the engagement of cell surface molecules for attachment of virion. VP4 is unique among the capsid proteins in that it is buried inside the capsid after assembly, and it is the only viral protein identified to date that is post-translationally modified by a fatty acid modification (Belsham et al., 1991). The PV VP4 protein also possesses an established myristoylation motif, which targets the protein for the attachment of the 14-carbon myristate group on VP4 molecules (Krausslich et al., 1990; Marc et al., 1990, 1991; Moscufo et al., 1991). For PV, the myristoylation of VP4 has been found to be absolutely required for proper proteolytic processing of the virus capsid, proper capsid assembly, and viral infectivity (Krausslich et al., 1990; Marc et al., 1990, 1991; Moscufo et al., 1991). In contrast, it was reported that co-translational myristoylation of VP4 was dispensable for FMDV capsid assembly (Belsham et al., 1991). However, in recombinant expression systems, myristoylation of VP4 is required for capsid production (Abrams et al., 1995; Goodwin et al., 2009).

Two isoforms of N-myristoyltransferase (NMT) have been characterized as the cellular proteins responsible for protein myristoylation with NMT isoform A being the preferred enzyme for PV VP4 myristoylation (Boutin et al., 1993). However, the involvement of either NMT isoform in the modification of FMDV VP4 has yet to be directly demonstrated. Further study will be necessary to understand the role of NMT and the significance of VP4 myristoylation in FMDV infectivity and virulence. Non-structural protein 2B The coding region for the FMDV non-structural protein 2B represents one of the most conserved regions in the entire FMDV genome with 117 of 154 amino acid residues being invariant (Carrillo et al., 2005). In other picornaviruses, 2B has been described as a ‘viroporin’ protein that oligomerizes to form a transmembrane pore through cellular membranes (Agirre et al., 2008; Ao et al., 2014; Madan et al., 2007; Martinez-Gil et al., 2011; Sanchez-Martinez et al., 2012), though until recently evidence of this has remained elusive with respect to FMDV. Examination of the 2B amino acid sequence and molecular modelling experiments have suggested that the FMDV 2B protein possesses two transmembrane regions, and as such implies that 2B interacts with host cell membranes (Ao et al., 2015; Carrillo et al., 2005). In vitro experiments confirmed that, like other known viroporin proteins, the FMDV 2B protein could both oligomerize and disrupt the cellular Ca2+ concentration in cells transfected with FMDV 2B. Moreover, FMDV 2B has been found to co-localize with the endoplasmic reticulum, and could induce the cellular autophagy pathway (Ao et al., 2015). Although amantadine treatment, a known viroporin inhibitor, has been shown to decrease virus titres, the mechanism by which these 2B interactions benefit FMDV infection remains to be defined. Non-structural protein 2C Recently, it was determined that FMDV nonstructural protein 2C from serotypes O1 Campos and A24 Cruzeiro interacts with the host protein Beclin1, a key regulator protein in the host cell autophagy pathway (Gladue et al., 2012). This specific interaction was first detected by the yeast two-hybrid assay and corroborated by

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both co-immunoprecipitation and observed co-localization through immunofluorescent microscopy. This finding is consistent with a previous report that FMDV 2C co-localized with the LC3 autophagy marker along with non-structural proteins 2B and 3A. This same study showed that viral replication was impeded through inhibitors of the autophagy pathway while an autophagy inducer increased viral yields (O’Donnell et al., 2011). The authors suggest that this interaction promotes FMDV replication by subverting the fusion of lysosomes with autophagosomes. Moreover, FMDV was demonstrated to induce the formation of autophagosomes, which appeared to facilitate infection by the virus but not at the level of RNA replication (Berryman et al., 2012). This study showed a significant departure from what had been observed with other picornaviruses, which utilize autophagosomes as sites of RNA replication, and whose formation is concomitant with nonstructural protein production, particularly 3A and 3D polymerase. In contrast, neither FMDV 3A nor 3Dpol were detected in association with autophagosomes triggered by FMDV infection, but rather FMDV VP1 was found to co-localize with these vesicles. Additionally, formation of autophagosomes could be induced by UV-inactivated FMDV and empty FMDV capsids. This latter point suggested that the induction of autophagosomes by FMDV occurred during the process of cell entry, and happened irrespective of whether the virus particle bound the primary integrin receptor or the alternative HS receptor (Berryman et al., 2012). Additionally, the 2C protein was also identified as a binding partner for the cellular protein vimentin (Gladue et al., 2013). Although the significance of this interaction remains to be elucidated, vimentin appears to form a structure surrounding FMDV 2C that is eventually dissipated. While increased levels of vimentin did not affect virus yields, FMDV replication was impeded separately by treatment with a vimentin disrupting agent and the overexpression of a dominant-negative isoform of vimentin. These results suggest that the 2C–vimentin interaction may be essential for virus replication. Non-structural protein 3A Several lines of evidence have shown that nonstructural protein 3A is an important host-range determinant for FMDV in both tissue culture and

susceptible animals. This 153 amino acid protein is cleaved from the C-terminus of 2C and N-terminus of 3B by 3Cpro. It is considerably longer than 3A proteins found in enteroviruses, and consists of an N-terminal hydrophilic region followed by a hydrophobic domain of about 28 residues that allows for association with intracellular membranes where RNA replication occurs (O’Donnell et al., 2001). The precise role of the protein in FMDV replication is still being delineated. However, FMDV 3A forms a complex with the 3B protein (Falk et al., 1992; O’Donnell et al., 2001), and the enterovirus 3AB forms a complex with 3CDpro and binds to a cloverleaf structure at the 5′ end of the viral RNA (Xiang et al., 1995). The 3A protein was reduced in size by a 19- or 20-codon deletion in the C-terminal portion of the protein (Giraudo et al., 1990) when FMDV was serially passaged in embryonated eggs, during an attempt to produce an attenuated vaccine virus (Giraudo et al., 1987; Sagedahl et al., 1987). The role of 3A in both viral virulence and host range was demonstrated during studies of the FMDV responsible for an outbreak of FMD in Taiwan in 1997 (designated O/Taw/97). This outbreak was unusual in that only pigs, and not cattle, were affected, and the disease had an unusually high mortality rate (Dunn and Donaldson, 1997; Huang et al., 2000). Molecular characterization of the virus revealed that it contained a 10-codon deletion in the C-terminal half of the 3A protein (Beard and Mason, 2000), similar to the deletion found in FMDV passaged in chick embryos. The role of this deletion in the bovine-attenuated phenotype was confirmed using reverse genetic analysis (Beard and Mason, 2000; Pacheco et al., 2003a), and subsequent analysis of viruses circulating in the region suggested that in addition to the deletion, mutations in the 3A protein in the region surrounding the deletion, may also be responsible for the observed phenotype (Knowles et al., 2001). The molecular basis for the porcinophilic phenotype appears to be related to a reduction in viral RNA synthesis, which is manifested to a greater degree in bovine cells than in swine cells (O’Donnell et al., 2001). The 3A proteins from either the porcinophilic or a bovine-virulent isolate co-localized to RNA replication complexes in either bovine or porcine cells, and also caused a disruption of the Golgi apparatus (O’Donnell et al., 2001).

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However, as yet, there is no clear picture, as to why the 3A deletion should affect FMDV replication in bovine cells more than in swine cells. Additionally, a single amino acid change in 3A was responsible for adaptation of FMDV to guinea pigs, though the mutation was located in a different region than the deletion associated with the porcinophilic phenotype (Nunez et al., 2001). The 3A protein has also been associated with altered PV host range in vitro (Lama et al., 1998). Intriguingly, deletions ranging from 10–50 amino acids in length within 3A have been repeatedly shown to be well-tolerated by the virus in vitro and to a lesser extent in vivo (Biswal et al., 2015; Li et al., 2014, 2010, 2011b; Pacheco et al., 2003b). Indeed, the resilience of multiple serotypes of FMDV to mutations within both the 3A and 3B proteins has inspired some to pursue using such alterations in the coding sequence as a platform for future FMDV marker vaccines, where deletions and substitutions would provide the capability to differentiate between infected and vaccinated animals (DIVA) (Li et al., 2010, 2011b, 2014; Pacheco et al., 2010; Uddowla et al., 2012). Recent studies have demonstrated that the 3A protein also interacts with the host factor DCTN3 (Gladue et al., 2014). DCTN3, along with cellular dynein, are components of the dynactin complex, which participates in various intracellular movements including organelle transport. Interestingly, the dynactin complex has been shown to contribute to vimentin filament organization, and as stated above, FMDV 2C interacts with vimentin. This suggests that these protein–protein interactions function cumulatively to benefit FMDV replication. Importantly, mutations ablating the binding of 3A to DCTN3 were highly deleterious to the progression of FMDV infection in both cell culture and in cattle. This protein–protein interaction was shown to be an important virulence factor in that a mutant of O1 Campos containing mutations that interfered with the 3A–DCTN3 interaction exhibited difficulty replicating in primary bovine cell culture and delayed onset of disease in cattle, and virus collected from lesions frequently possessed amino acid substitutions in 3A that restored the DCTN3 interaction (Gladue et al., 2014). Cumulatively, it can be stated that the 3A protein is multifactorial, playing a critical role in both the virus host range and virulence.

Non-structural proteins 3B1, 2, 3 The FMDV 3B protein (also known as VPg) is a short protein covalently linked to the 5′ end of the RNA genome (Grubman, 1980; Sangar et al., 1977). FMDV is unique among the family Picornaviridae in that three non-identical copies of 3B (3B1, 3B2 , and 3B3) are encoded in tandem in the genome (Forss and Schaller, 1982). Viruses genetically engineered to lack either one 3B coding sequence, or two inactive 3B coding sequences were able to replicate, although the level of viral RNA synthesis was reduced (Falk et al., 1992). Genetically engineered FMDVs encoding only one 3B gene were infectious when transfected into BHK-21 cells, though these RNAs were impaired in the ability to replicate in porcine and bovine cells, and virus progeny produced only mild disease in pigs (Pacheco et al., 2003a). More recently, individual deletions and transposon-mediated insertions within the 3B proteins from an infectious clone of a field strain A 24 Cruzeiro strain (Rieder et al., 2005) resulted in mutant viruses that exhibited no impairment to growth in cell culture, RNA synthesis, protein maturation, and presented clinical disease consistent with wildtype virus (Pacheco et al., 2010). RNA-dependent RNA polymerase 3Dpol The FMDV RNA dependent RNA polymerase 3D has been referred to as the virus infection associated antigen (VIAA), as it is one of the most highly immunogenic and highly conserved non-structural proteins encoded by FMDV. Extensive research has demonstrated the criticality of this viral protein in the development of a robust immune response with regard to prospective FMDV vaccine platforms. Recently, the aforementioned host factor Sam68 was found to co-precipitate with 3Dpol in infected cells (Rai et al., 2015). Using cell-free extracts, the Sam68– 3Dpol interaction was demonstrated to contribute to the replication of the viral RNA genome. Importantly, when Sam68-targeted siRNA constructs were introduced into cell culture, subsequent FMDV infection was stymied significantly with a reduction in viral titres of approximately 3 logs (Lawrence et al., 2012), suggesting this host factor interaction is a critical component of FMDV infection.

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Viral genetic elements S-fragment Upstream of the poly-C tract at the extreme 5′ terminus of the FMDV genome exists a large stem loop structure that has been postulated to be critical for replication of the FMDV RNA genome. RNA pull-down experiments indicated a large ~ 120 kDa unidentified RNA-binding protein pulled down with the S-fragment portion of the 5′ UTR (Serrano et al., 2006). A similarly large RNA-binding protein, RNA Helicase A (RHA), was also described as a critical host factor involved in virus RNA replication of the positive-sense single-stranded RNA genome of hepatitis C virus (Isken et al., 2007). Through a combination of RNAi-induced knockdown of RHA and biochemical and microscopy experiments, RHA was confirmed to be a critical host factor in the replication cycle of FMDV, and the first known RNA-binding protein to interact with the S-fragment (Lawrence and Rieder, 2009). Intriguingly, FMDV infection triggered the subcellular rearrangement of RHA from the nucleus to the cytoplasm, and this redistribution was determined to be contingent upon the de-methylation of arginine repeats in the C-terminus of the protein. The de-methylation was later determined to be caused by the arginine de-methylase, JMJD6, which is also an RBP (Lawrence et al., 2014). During the course of FMDV infection, RHA co-precipitated with viral proteins 2C and 3A as well as cellular PABP, which were also observed to localize in close proximity to each other via immunofluorescent microscopy (Lawrence and Rieder, 2009). Although the functional significance of these interactions remains to be fully delineated, 2C, 3A and the S-fragment are all purportedly involved in RNA replication, and the helicase activity of RHA would be of benefit to melting the interaction between the positive and negative RNA strands during viral RNA replication. Poly-C tract The poly-C tract comprises over 90% C-residues with a small number of U and A residues. This segment is over 100 bases in length; however, the length of the poly-C tract can be extremely variable (Costa Giomi et al., 1984) and, in one case, a tissue culture adapted virus has been shown to have a poly-C length of over 400 bases (Escarmis et al., 1992). Although an early study suggested that the

length of the poly-C tract was associated with virulence (Harris and Brown, 1977), other studies have been unable to correlate poly-C length with this property of the virus (Costa Giomi et al., 1984). It has been shown that the poly-C length of natural viral isolates are elongated by repeated passage in cell culture (Escarmis et al., 1992), as is the length of this segment in genetically engineered viruses (Rieder et al., 1993). It is possible to replicate virus with essentially no poly-C, and while this virus is virulent in suckling mice, it has a much higher particle/PFU ratio than viruses containing longer poly-C tracts (Rieder et al., 1993). The virulence of this virus in susceptible animals has not been determined. cre Just upstream from the IRES there is a short hairpin loop structure termed the cre (Mason et al., 2002). The cre, which has been identified in the genomes of other picornaviruses, has a stem–loop with a conserved AAACA sequence in the loop region. In contrast to other picornaviruses, where the cre is located within different regions of the ORF, the cre of FMDV is located within the 5′ UTR. However, it has been shown to be positionally independent (Mason et al., 2002), and also has been demonstrated to be functional in trans (Tiley et al., 2003). The cre is essential for RNA genome replication (see Grubman and Baxt, 2003, and references therein). Although the cre has not been shown to be involved in virulence or host range, its role in replication makes it a good candidate for antiviral intervention. IRES The FMDV IRES contains a high degree of secondary and tertiary structure (Belsham and Brangwyn, 1990; Kuhn et al., 1990). The IRES element for Aphthoviruses, similar in structure to the cardiovirus IRES, is about 450 nucleotides in length and has been modelled into a five-domain structure (Pilipenko et al., 1989). IRES elements contain a pyrimidine-rich region at their 3′ ends immediately preceding the AUG translation initiation codon and FMDV contains pyrimidine-rich regions directly upstream of each of the alternative AUG initiation codons (Belsham and Brangwyn, 1990; Kuhn et al., 1990; Pilipenko et al., 1992). The FMDV IRES interacts with a number of cellular proteins, including initiation factors important for

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normal cellular mRNA translation. A host factor of 57 kDa, the polypyrimidine tract binding protein (PTBP), was shown to interact with at least two regions of the IRES (Luz and Beck, 1990; Pilipenko et al., 2000). Deletion of these two sites inhibited both the binding of the protein and in vitro translation (Luz and Beck, 1991). Another host factor has been identified which is required for FMDV IRES-driven translation, but not for translation of cardiovirus mRNA (Pilipenko et al., 2000). This 45 kDa protein, IRES-specific trans-acting factor (ITAF45), along with PTBP, is required for the formation of the 48S translation–initiation complex (Martinez-Salas et al., 2001; Pilipenko et al., 2000). More recently, Sam68, previously shown to interact with virus proteins during poliovirus infection (McBride 1996), was found to interact with the FMDV IRES (Lawrence et al., 2012), and showed a preference for a UAAA motif within domain 4 (Rai et al., 2015). Similar to RHA, Sam68 was observed to undergo a partial redistribution from the nucleus to the cytoplasm, and was found to interact with viral 3Cpro and 3Dpol. In a cell-free system, Sam68 was found to promote FMDV RNA replication (Rai et al., 2015), and this role is likely functionally important as RNAi-induced Sam68 knockdown significantly diminished virus titres (Lawrence et al., 2012). Additionally, Gem-associated protein 5 (Gemin5) was identified in large proteomic screens to interact with the IRES of FMDV, an association that was demonstrated to inhibit viral translation (Pineiro et al., 2013). Therefore, the functionality of this large RNA structure in the FMDV 5′ UTR can be significantly modulated through a variety of host protein interactions. It has been suggested that IRES elements, and possibly the host factors that bind to them, effect pathogenicity and virulence of other Picornaviruses (Evans et al., 1985; Kawamura et al., 1989; Malnou et al., 2002; Moss et al., 1989; Ochs et al., 2003; Skinner et al., 1989). In FMDV, a virus rescued from persistently infected BHK-21 cells had two mutations within the IRES, which the authors suggest might have resulted in increased virulence of the virus in tissue culture (Martinez-Salas et al., 1993). Novel binding proteins Recently, Guo et al. (2015) used a stable isotope labelling with amino acids in cell culture (SILAC)

based approach to identify an array of previously unreported host proteins as potential co-factors with FMDV upon infection of BHK-21 cell line. The SILAC assay revealed several host proteins that were up-regulated and several that were down-regulated upon infection of host cells with FMDV. These proteins are of diverse functions, and in many cases, their relative significance to the progression of FMDV infection has yet to be determined. A few of the reported proteins were further examined for potential roles in FMDV infection, virulence, and host range. One novel protein demonstrated to be up-regulated in response to FMDV infection by the SILAC method was: LYPLA/APT1, which has been characterized as a protein acyl thioesterase responsible for the removal of palmitoyl groups (Veit and Schmidt, 2001). Since reversible protein palmitoylation is frequently associated with membrane interactions, this is of interest to FMDV research as FMDV infection is known to promote the reorganization of membranes in the cytoplasm of infected cells with implications for viral RNA replication. The many proteins detected in this study provide attractive targets for future research into the complex network of FMDV non-receptor protein interactions. Final remarks While significant progress has been made in elucidating the molecular biology of FMDV, many unanswered questions remain regarding FMDV pathogenesis, virulence, and host range in susceptible animals. The delineation of the roles that multiple receptors play in disease as well as the identification of the as yet undescribed partner molecules involved in the processes of viral attachment and entry will continue to be of great interest. Moreover, the developing knowledge of the plasticity of these interactions and the shifting of downstream pathways will contribute to the rationale design of novel vaccine platforms and antiviral therapeutics. In addition, further analysis of the interactions between viral proteins, genome segments, and intracellular host components, in addition to their role in the virus life cycle, will increase both our understanding of the pathogenesis of FMD, and help to determine ways for rational control of this disease.

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Acknowledgements The authors wish to thank Dr Barry Baxt for generously donating his time for fruitful discussions and commentary during the preparation of this chapter. References

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The RNA-dependent RNA Polymerase 3D: Structure and Fidelity Cristina Ferrer-Orta and Nuria Verdaguer

Abstract RNA viruses typically encode their own RNAdependent RNA polymerase (RdRP) to ensure genome replication within the infected cell. RdRP function is critical not only for the virus life cycle but also for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses for a rapid adaptation to changing environments. In this chapter, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused in the FMDV polymerase 3D. The RdRP 3D also catalyses the covalent linkage of UMP to a tyrosine on the small protein VPg. Uridylylated VPg then serves as a protein primer for the initiation of RNA synthesis. Different crystal structures of FMDV 3D catalytic complexes enhanced our understanding of template and primer recognition, VPg uridylylation and rNTP binding and catalysis. Such structural information is providing new insights into the fidelity of RNA replication, and for the design of antiviral compounds. Introduction Foot-and-mouth disease virus (FMDV) as the rest of picornaviruses possesses a positive singlestranded RNA genome, with a small peptide (VPg or 3B) linked to its 5′-end. The FMDV genome is replicated via a negative-sense RNA intermediate by the viral RNA-dependent RNA polymerase (RdRP), termed 3D. This enzyme uses VPg as a primer to initiate the replication process. The structure and function of 3D has been studied extensively in the past decades in FMDV as well as in other

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picornaviruses. To date, the crystal structures of the 3D polymerase have been reported for six different members of the enterovirus genus [including poliovirus (PV), coxsackievirus B3 (CVB3), enterovirus 71 (EV71) and the human rhinoviruses HRV1B, HRV14, andHRV16], for FMDV and for the Cardiovirus EMCV, either isolated or bound to different substrates. These structures provided high resolution snapshots for a range of conformational states associated to both initiation of RNA synthesis and the replication elongation processes (see Ferrer-Orta et al., 2006a, 2009, 2015a, for a review). All these enzymes share closed ‘right hand’ architecture with the fingers, palm and thumb subdomains encircling seven sequence and structural motifs (designated A to G) that have been shown to play different roles (Fig. 6.1A and B). Four of these domains (A–D) are common to all hand-shaped polymerases (Poch et al., 1989) and are located in the palm domain, which consists in four antiparallel β-strands and two α-helices. The catalytic aspartic acid of motif A is located on one of these β-strands, while the GDD triplet of motif C is placed within the turn connecting two other β-strands (Fig. 6.1C). The strictly conserved aspartic acids of motifs A and C are essential for the coordination of the catalytic Mg2+ ions (Steitz, 1998) (Fig. 6.1C). Motif B forms a ‘loop-α-helix’ structure located at the base of the template channel, with the α-helix packed against the β-sheet core containing the catalytic residues. This extended α-helix possesses a conserved Asn residue that facilitates the correct positioning of a second aspartic acid residue of motif A directly involved in the recognition of the 2′OH of the incoming rNTP, and essential for the discrimination of ribonucleotides versus desoxyribonucleotides. In addition, the N-terminal loop of

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Figure 6.1  (A) Ribbon diagram of the structure of FMDV 3D polymerase in complex with a template-primer RNA (PDB: 1WNE). The 3D molecule (grey) is shown in the conventional orientation with the seven conserved structural motifs of palm and fingers domains depicted in different colours and explicitly labelled. The RNA bound to the central cavity is shown in orange. (B) Structure of the ternary complex FMDV 3D-RNA-RTP (PDB: 2E9R). Side view of the polymerase surface (grey) that has been cut to expose the three channels by which the different substrates and products of the polymerization reaction go in (template and rNTP channels) or leave the active site (central channel). The conserved structural motifs are coloured as A, the template and primer strands of the bound RNA molecule are shown in orange. (C) (Top panel) conformation and interactions in the 3D active site on RTP binding (PDB: 2E9R). The 3D active site is in open conformation with the incoming nucleoside analogue (stick representation in atom type colour with carbons in blue) stacked to the 3′ terminus of the primer (orange) and base paired to the template acceptor base. The position of the RTP base is further stabilized by interactions with residues of motifs A (red), and the motif B loop β9α11. The triphosphate moiety is hydrogen bonded to different residues of motifs A and F (blue) and interacts with one metal ion (light blue ball). The bottom panel show an example of a picornavirus 3D palm in closed conformation. This corresponds to the PV 3D-RNA-CTP ternary complex after nucleotide incorporation and pyrophosphate release (PDB 3OL7). The catalytic aspartic acids of motif A and C, as well as, the motif B residues interacting with the incoming nucleotide are shown as sticks. The two metal ions are shown as balls in light blue. (D) Structure and interactions in the template channel of FMDV 3D–RNA complexes, the molecular surface of the polymerase is shown in grey with the acidic residues of the active site in red and the RNA depicted as sticks in orange (wt 3D–RNA complex; PDB 1WNE). The RNA corresponding to the FMDV 3D N-terminal mutant K18E is superimposed in green (PDB 4WZM). The left and right insets show close-ups of the interactions involving the N-terminal residues (from M16 to K20) responsible of the largest conformational changes occurring at the template channel entrance.

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this motif (β9-α11 in FMDV) is directly participating in template binding and translocation of the nascent dsRNA (Ferrer-Orta et al., 2007; Gong and Peersen 2010; Garriga et al., 2013; Sholders and Peersen, 2014). Until recently, the only function associated with motif D was scaffolding for the palm domain. Recent NMR data provided evidences that motif D is highly dynamic and that this dynamics appears to be essential for the closure of the catalytic complex when the correct nucleotide is placed in the active site (Yang et al., 2012). Motifs E and F are located at the boundary of the palm and thumb domains (motif E) and in the fingers domain (motif F). These motifs participate in primer and rNTP binding, respectively. Finally motif G, is also located in the fingers and forms the entry of the template channel (Fig. 6.1A and B). Furthermore, mutational analyses together with the structural characterization of the mutant enzymes in FMDV and also in PV have demonstrated that some substitutions in residues located far from the active site have significant effects on catalysis and fidelity. All of these observations suggest that nucleotide binding and incorporation are modulated by a long-distance network of interactions (Pfeiffer and Kirkegaard 2003; Thompson and Peersen 2004; Arnold et al., 2005; Agudo et al., 2010; Ferrer-Orta et al., 2010; Moustafa et al., 2011; Ferrer-Orta et al., 2015b). Structural and functional studies of VPg binding and uridylylation Picornaviruses initiate RNA replication by the successive addition of two uridine-monophosphate molecules to the Tyr-3 residue of VPg. VPg uridylylation is catalysed by 3D using as template a cis-replicating element (cre) that is located at different positions of the RNA genome, in the different picornaviridae genera (e.g. the FMDV cre is located in the 5′-UTR of the genome). The picornaviral proteins: VPg, 3D and 3C, alone or in the 3CD precursor form, together with the viral RNA cre elements comprise the so-called ‘VPg uridylylation complex’ responsible of VPg uridylylation in vivo. The extensive structural and biochemical studies provided several different models for the interactions established between VPg and 3D or 3CD in the uridylylation complex, however, the precise mechanism of uridylylation in picornavirus remains

uncertain (see Paul and Wimmer 2015 for an extensive review). Biochemical and structural studies performed for different members of the family: PV (Lyle et al., 2002), HRV16 (Appleby et al., 2005), FMDV (Ferrer-Orta et al., 2006b), CVB3 (Gruez et al., 2008) and EV71 (Chen et al., 2013) revealed three distinct VPg binding sites on 3Dpol (Fig. 6.2A). Strikingly, whereas the most picornaviruses express only a single VPg protein, FMDV possesses three similar but not identical copies of VPg: VPg1, VPg2 and VPg3 (Forss et al., 1982), all of which are found linked to viral RNA (King et al., 1980). Although not all the copies are needed to maintain infectivity (Falk et al., 1992, Pacheco et al., 2003), there are no reports of naturally occurring FMDV strains with fewer than three copies of 3B, suggesting that there is a strong selective pressure towards maintaining this redundancy (Carrillo et al., 2007). The structures of two complexes between FMDV 3D and VPg1 were determined by X-ray crystallography. They depicted both uridylylated and non-uridylylated forms of VPg (Ferrer-Orta et al., 2006b). Both structures revealed that the VPg occupies the central cleft of the polymerase, with the hydroxyl group of Tyr-3 positioned so as to mimic the free 3′-hydroxyl group of a nucleic acid primer at the active site of 3D (Fig. 6.2B and C). Several amino acid contacts between 3D and VPg, predicted to be important for initiation of RNA synthesis, were confirmed by site-directed mutagenesis of 3D polymerase and by using chemically synthesized mutant versions of VPg. Twelve out of 16 3D residues that were identified as interacting with VPg, are strictly conserved among different picornaviral polymerases, suggesting that they may play an important role during the critical VPg-uridylylation step (Ferrer-Orta et al., 2006b). In the 3D–VPg-pU complex, the hydroxyl group of the Tyr-3 side chain was found covalently attached to the α-phosphate moiety of the uridinemonophosphate (UMP) molecule. Two divalent cations, together with the catalytic aspartic acid residues of motifs A and C, participate in the uridylylation reaction, following a similar mechanism to that described for the nucleotidyl transfer reaction in other polymerases (Steitz, 1998). A cluster of positively charged residues of motif F also participate in the uridylylation process, stabilizing Tyr3 and UMP in a proper conformation for the catalytic reaction (Ferrer-Orta et al., 2006b) (Fig. 6.2C).

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Figure 6.2  (A) Comparison of identified VPg binding sites in picornavirus 3Ds. As all reported structures of picornavirus 3Ds share high structural similarities, we used the structure of the FMDV 3D–VPg-UMP complex (PDB: 2F8E) as a representative model. The molecular surface of the FMDV 3D polymerase (grey) is shown in two different views: the conventional orientation, as if looking into a right hand (left) and a side view (right). The bound VPgs of FMDV (pale blue), CVB3 (yellow; PDB: 3CDW) and EV71 (red; PDB: 4IKA) are shown as sticks. The 3D residues essential for VPg binding in FMDV, PV and EV71 are coloured as blue, wheat and magenta, respectively, in the surface representation. (B) Structure and interactions of the FMDV 3D–VPg-UMP complex. Top-down view of the FMDV polymerase represented with its molecular surface (grey) that been cut to expose the central channel where the primer protein VPg (pale-blue) binds. The conserved structural motifs, lining the central cleft of the polymerase are shown as cartoons in different colours (Motif A red, B green, C yellow, E cyan, F blue and G magenta). The VPg residue Tyr3 is located at the active site, positioning its hydroxyl group as a molecular mimic of the free 3′ hydroxyl group of a nucleic acid primer for nucleotidylylation. In the structure, the hydroxyl group of Tyr3 side chain was found covalently attached to an UMP molecule (orange) by a phosphodiester linkage. Two metal ions (green spheres) participate in the uridylylation reaction. (C) Detail of the interactions in the 3D active site. Metal 1 bridges the catalytic aspartate of motif C and the O– of tyrosine side chain, now covalently bonded to the α-phosphate of UMP. Metal 2 coordinates the O1 oxygen of phosphate α and the hydroxyl group of the conserved serine of motif B. The positively charged residues of motif F also participate in the uridylylation process.

This ‘front-loading’ mode of VPg binding to the FMDV 3D polymerase is compatible with a cis mechanism of VPg uridylylation. The VPg priming

model described for FMDV was further supported by the crystal structures of HRV16 3Dpol (Appleby et al., 2005) and of the PV 3CD precursor (Marcotte

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et al., 2007). In the latter structure, the extensive crystal packing contacts found between symmetryrelated 3CD molecules and the proximity of the N-terminal domain of 3C to the VPg binding site, in the way that VPg was positioned in the FMDV 3D–VPg complex, suggests a possible role of the contacting interfaces in forming and regulating the VPg uridylylation complex (Marcotte et al., 2007). After VPg nucleotidylylation, a number structural rearrangements of the 3D polymerase occur, marking the transition from initiation to the elongation phase of RNA synthesis. There is experimental evidence supporting possible structural differences in 3D when involved in the priming reaction versus elongation of RNA. i.e. The nucleoside analogue 5-fluorouridine triphosphate (FUTP) is a potent inhibitor of VPg uridylylation but not of RNA elongation (Agudo et al., 2008). A second binding site for VPg was found in the structure of the CVB3 polymerase (Gruez et al., 2008). The VPg fragment solved, corresponding to the C-terminal half of the peptide, was bound at the base of the thumb sub-domain in an orientation that did not allow uridylylation by its own 3D carrier (Fig. 6.2A). This binding mode, partially agreed with previous data reported for PV, showing that a number of amino acids located in motif E of poliovirus (PV) were required for VPg or their precursor, 3AB, binding and affecting VPg uridylylation (Lyle et al., 2002). In light of these data, authors proposed that a VPg bound at this position might be either uridylylated by another 3D molecule or playing a structural stabilizing role within the uridylylation complex (Gruez et al., 2008). Finally, a third VPg binding site was discovered in the structure of EV71 3D–VPg complex. In this complex, VPg is anchored at the bottom of the palm domain of the polymerase, showing a V-shape conformation that crosses from the front side of the catalytic site to the back side of the enzyme (Fig. 6.2A). As in the previous viruses, the mutational analyses of the interacting residues evidenced a reduced binding of VPg to the EV71 3D, also affecting uridylylation (Chen et al., 2013; Sun et al., 2012). However, the structure of the EV71 3D–VPg complex showed that the VPg Tyr3 is buried at the base of the polymerase palm (Fig. 6.2A) indicating that a conformational change should occur to expose the side chain of Tyr3 for the uridylylation reaction.

The important sequence homology between the picornaviral VPg sequences (Ferrer-Orta et al., 2006b; Sun et al., 2014) and the high similarities existing in the 3D polymerase structures prompts to hypothesize that the three VPg binding sites observed in the different crystal structures would reflect distinct binding positions of VPg to both 3D or its precursor 3CD at different stages of the virus replication initiation process. As mentioned above, the FMDV genome codes for three VPg molecules, all of them are present in naturally occurring viruses. Structural elements regulating the replication elongation process The replication elongation process can be roughly divided in three steps, including nucleotide selection, phosphodiester bond formation and translocation to the next nucleotide for the subsequent round of nucleotide addition. A number of structures during RNA elongation in FMDV were obtained in the presence of natural nucleotides and the nucleotide analogues FUTP and RTP, using different RNAs as template-primer molecules (Ferrer-Orta et al., 2007). The structures revealed the critical polymerase residues involved in the correct positioning of the template and primer nucleotides and those responsible of the recognition and binding of the incoming nucleotide for catalysis (Fig. 6.1). In all complexes analysed, the single stranded 5′-end of the template extends across the face of the fingers domain towards the active site cleft, contacting with different residues in the polymerase N-terminus and motifs G and F, which drive the template chain towards the active site cavity. The acceptor base of the template (position t + 1) is located adjacent to the nucleotide binding pocket, sitting above the active site and stacked on the upstream duplex and ready for the binding of the incoming rNTP (Fig. 6.1D). Furthermore, the downstream t + 2 template nucleotide is also pointing towards the active site cavity, stacked with the t + 1 nucleotide. The interactions established between t + 2 and the 3D N-terminal residue R17 substantially contribute to maintain the observed template orientation (Fig. 6.1D). In the active site, the 3′-hydroxyl group of the primer strand interacts directly with the catalytic aspartic acid D338 of motif C (Ferrer-Orta et al., 2004; 2007).

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The structures of the ternary complexes 3D-RNA-rNTP, where the incoming nucleotides were trapped close or at the polymerase active site, showed the central role of amino acid D245 of motif A, N307 of the motif B helix, and S298, G299 and T303, of the motif B N-terminal loop (β9-α11), in nucleotide recognition and correct positioning of the sugar in the ribose-binding pocket (Fig. 6.1C). Structural comparisons of the different FMDV elongation complexes revealed that the loop β9-α11 is able to adopt different conformations in response to different template and incoming nucleotides, being the most flexible element of the active site of the FMDV polymerase (Ferrer-Orta et al., 2009). In all these complexes the polymerase active site was trapped in an open conformation, characterized by a partially formed three-stranded β-sheet of the palm domain motifs A and C and the catalytic residue of motif A, D240, not properly oriented for the catalytic reaction. In this open conformation, the incoming rNTP reaches the active site and establishes base-pairing interactions with the template base (t+1) but catalysis has not taken place. In addition, only one metal ion was found in contact with the triphosphate moiety of the incoming nucleotide (Fig. 6.1C) (Ferrer-Orta et al., 2007). The structural analyses of other replication– elongation complexes in the enteroviruses PV and CVB3 trapped the polymerase active site in a closed conformation. This conformation was achieved by the realignment of the palm β-strands that include motifs A and C after correct nucleotide binding, resulting in the repositioning of the motif A aspartate to allow interactions with the two metal ions required for catalysis (Fig. 6.1C) (Gong et al., 2013; Gong and Peersen 2010). Furthermore, growing amounts of biochemical and structural data in PV also indicated that additional conformational changes in motif D, including the repositioning of a strictly conserved lysine (K369 in FMDV; Fig. 6.1C) determined both efficiency and fidelity of nucleotide addition (Castro et al., 2007, 2009; Yang et al., 2012). This lysine is presumed to coordinate the export of the PPi group from the active site once catalysis has taken place, triggering the end of the reaction cycle and allowing enzyme translocation (Shen et al., 2012). A number of crystal structures have been solved in a post-translocation state (Gong et al., 2013; Gong and Peersen 2010), including those of the

FMDV 3D after the incorporation of standard nucleotides or the mutagenic nucleoside analogue FUTP (Ferrer-Orta et al., 2007). However, no structures of translocation intermediates are currently available for picornaviral polymerases and the precise mechanism is not yet known. Very recently, structural and functional data in PV indicate that steric clashes between the motif-B loop and the template RNA would also promote translocation (Sholders and Peersen 2014). Structural comparisons between different 3D–RNA–rNTP complexes indicate that some interactions are common to standard nucleotides and to nucleotide analogues, while others are specific for a particular analogue (Ferrer-Orta et al., 2007; Fig. 6.1C). This structural information is essential to understand the molecular basis of template-copying fidelity and may help in the design of new mutagenic nucleotides targeted specifically to viral RdRPs. Structural and functional studies with the 3D polymerase of ribavirin-resistant mutants Viral RdRPs are considered to be low fidelity enzymes, generating mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. One consequence of the low fidelity of RdRPs is the enhanced sensitivity of the viral population to accumulate additional mutations during replication. A new strategy against RNA viruses consists in using mutagenic nucleotides. The objective is to provoke an excessive number of mutations, to deteriorate the viral functions to the point that the virus cannot survive (reviewed in Domingo, 2005). One of the mutagens used in research on lethal mutagenesis is ribavirin (1-β-d -ribofuranosyl-1-H-1,2,3-triazole-3-carboxamide), extensively employed in clinical practice. Unfortunately, viral mutants that are resistant to ribavirin have been selected, thus facilitating escape from lethal mutagenesis. Ribavirin-resistant mutants have been isolated both in PV and FMDV and the resistance phenotype mapped in 3D (Pfeiffer and Kirkergaard, 2003; Sierra et al., 2007). Interestingly, the amino acid substitutions that confer ribavirin-resistance are different for the two viruses, whereas G64S is located within the fingers domain of PV 3D M296I

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resides in the motif B loop β9-α11 of FMDV. Biochemically, the two mutant enzymes showed significant differences, the purified mutant polymerase 3D(G64S) displayed an increase in copying fidelity relative to wild type enzyme, at the cost of producing mutant spectra of lower complexity than wt virus (Pfeiffer and Kirkergaard et al., 2005; Vignuzzi et al., 2006). In contrast, the 3D(M296I) mutant FMDV restricted the incorporation of ribavirin monophosphate (RMP) into the RNA through subtle rearrangements in the loop β9-α11, close to the polymerase active site that did not have a significant effect on the rate of misincorporation of the standard nucleotides (Sierra et al., 2007; Arias et al., 2008; Ferrer-Orta et al., 2010). A third mechanism of ribavirin-resistance in FMDV was elucidated by the functional and structural characterization of a mutant obtained the in the presence of increasing concentrations of the mutagen (from 800 μM to 5000 μM), resulting in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in 3D, 3D(SSI) (Agudo et al., 2010). The in vitro polymerization assays performed with the purified 3D(SSI) enzyme indicated that the main biological effect of these substitutions was to attenuate the consequences of the mutagenic activity of ribavirin through the alteration of the pairing behaviour of RTP (Agudo et al., 2010). Structural comparisons of the wild type and mutant polymerases suggested that the amino acid substitutions altered the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The observed structural changes affect mainly the 3D N-terminal residues M16-K20 which appear completely reorganized, resulting in the formation of a hydrophobic pocked where the t + 2 nucleotide binds (Fig. 6.1D) (Agudo et al., 2010). Surprisingly, similar changes in the template binding channel together with rearrangements in the β9-α11 loop were also observed in the characterization of other FMDV 3D mutants, harbouring substitutions at the 3D N-terminal residues K18 and K20 which belong to a nuclear localization signal of the enzyme. All these mutants exhibit low RNA binding activity, low processivity and alterations in nucleotide recognition, including increased incorporation of RMP compared with the wild-type enzyme (Ferrer-Orta et al., 2015b). Besides facilitating specific contacts with the RNA

template, the dynamic nature of residues lining the template channel should permit the access of the t + 1 nucleotide into the 3D catalytic site. The base of the template channel is built mainly by residues of the β9-α11 loop that are involved in interactions with the t + 1 nucleotide, as well as, with the incoming rNTP (Ferrer-Orta et al., 2007). Altogether these data prompts to hypothesize that the rearrangements in the template channel and the β9-α11 occur in a concerted manner and that these concerted changes would regulate both RNA replication processivity and fidelity. Conclusions The ever growing availability of structures of picornavirus 3D catalytic complexes provided an increasingly accurate picture of the functional steps and regulation events underlying viral RNA genome replication. Data currently available provides high-resolution pictures for a range of interactions associated to VPg binding and uridylylation and for the different conformational states associated to template and primer binding, rNTP recognition and binding, catalysis and chain translocation. Such structural information is providing new insights into the fidelity of RNA replication, and for the design of antiviral compounds. Protein primed mechanism of replication initiation mediated by VPg appears to be a process that involves more than one VPg binding site in 3Dpol possibly at different stages of the virus replication initiation process. Although the number of 3D-VPg structures available show individual snapshots of the process, to obtain a global picture of the assembly, regulation and dynamics of complete replication initiation complexes, requires further analyses of high order assemblies formed not only by the polymerase and VPg but also involving different viral and host proteins, protein precursors and RNA templates. Such structures should provide a more detailed view of the molecular events underlying the initiation of picornavirus genome replication. The structures of a large number of replication– elongation complexes of FMDV 3D as well as other picornaviruses captured subtle conformational changes associated with nucleotide selection, active site closure and chain translocation. Among these movements, the motif B-loop β9-α11appears to

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play a crucial role assisting in the positioning of the template and incoming nucleotides in the active site, in chain translocation and presumably in the discrimination between natural nucleotides and mutagenic analogues as ribavirin. The structural and functional studies with a number of FMDV 3D mutants resistant to ribavirin suggest that subtle rearrangement in the template channel, mainly affecting the polymerase N-terminus, may occur in concert with the conformation changes in the β9-α11 loop and that these concerted changes would regulate both RNA replication processivity and incorporation of correct versus incorrect nucleotides. Acknowledgements Núria Verdaguer acknowledges funding from the Spanish Ministry of Economy and Competitiveness (BIO2011–24333 and Maria de Maeztu action MDM-2014-0435). References

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by engineering RNA mediated crystal contacts. PLOS One. 8, e60272. Gong, P., and Peersen, O.B. (2010). Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 107, 22505–22510. Gruez, A., Selisko, B., Roberts, M., Bricogne, G., Bussetta, C., Jabafi, I., Coutard, B., De Palma, A.M., Neyts, J., and Canard, B. (2008). The crystal structure of coxsackievirus B3 RNA-dependent RNA polymerase in complex with its protein primer VPg confirms the existence of a second VPg binding site on Picornaviridae polymerases. J. Virol. 82, 9577–9590. King, A.M., Sangar, D.V., Harris, T.J., and Brown, F. (1980). Heterogeneity of the genome-linked protein of foot-and-mouth disease virus. J. Virol. 34, 627–634. Lyle, J.M., Clewell, A., Richmond, K., Richards, O.C., Hope, D.A., Schultz, S.C., and Kirkegaard, K. (2002). Similar structural basis for membrane localization and protein priming by an RNA-dependent RNA polymerase. J. Biol. Chem. 277, 16324–16331. Marcotte, L.L., Wass, A.B., Gohara, D.W., Pathak, H.B., Arnold, J.J., Filman, D.J., Cameron, C.E., and Hogle, J.M. (2007). Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase. J. Virol. 81, 3583–3596. Moustafa, I.M., Shen, H., Morton, B., Colina, C.M., and Cameron, C.E. (2011). Molecular dynamics simulations of viral RNA polymerases link conserved and correlated motions of functional elements to fidelity. J. Mol. Biol. 410 159-181. Pacheco, J.M., Henry, T.M., O’Donnell, V.K., Gregory, J.B., and Mason, P.W. (2003). Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-and-mouth disease virus. J. Virol. 77, 13017–13027. Paul, A.V., and Wimmer, E. (2015). Initiation of protein-primed picornavirus RNA synthesis. Virus Res. 206, 12–26. Pfeiffer, J.K., and Kirkegaard, K. (2005). Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLOS Pathog. 1, e11.

Pfeiffer, J.K., and Kirkegaard, K. (2003). A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. Proc. Natl. Acad. Sci. U.S.A. 100, 7289–7294. Poch, O., Sauvaget, I., Delarue, M., and Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO. J. 8, 3867–3874. Shen, H., Sun, H., and Li, G. (2012). What is the role of motif D in the nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus? PLoS Comput. Biol. 8, e1002851. Sholders, A.J., and Peersen, O.B. (2014). Distinct conformations of a putative translocation element in poliovirus polymerase. J. Mol. Biol. 426, 1407–1419. Sierra, M., Airaksinen, A., González-López, C., Agudo, R., Arias, A., and Domingo, E. (2007). Foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe. J. Virol. 81, 2012–2024. Steitz, T.A. (1998). A mechanism for all polymerases. Nature 391, 231–232. Sun, Y., Guo, Y., and Lou, Z. (2014). Formation and working mechanism of the picornavirus VPg uridylylation complex. Curr. Opin. Virol. 9, 24–30. Sun, Y., Wang, Y., Shan, C., Chen, C., Xu, P., Song, M., Zhou, H., Yang, C., Xu, W., Shi, P.Y., et al. (2012). Enterovirus 71 VPg uridylation uses a two-molecular mechanism of 3D polymerase. J. Virol. 86, 13662–13671. Thompson, A.A., and Peersen, O.B. (2004). Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase. EMBO. J. 23, 3462–3471. Vignuzzi, M., Stone, J.K., Arnold, J.J., Cameron, C.E., and Andino, R. (2006). Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439, 344–348. Yang, X., Smidansky, E.D., Maksimchuk, K.R., Lum, D., Welch, J.L., Arnold, J.J., Cameron, C.E., and Boehr, D.D. (2012). Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition. Structure 20, 1519–1527.

Quasispecies Dynamics Taught by Natural and Experimental Evolution of Foot-and-mouth Disease Virus

7

Esteban Domingo, Ignacio de la Higuera, Elena Moreno, Ana I. de Ávila, Rubén Agudo, Armando Arias and Celia Perales

Abstract Foot-and-mouth disease virus (FMDV) exhibits high mutation rates and replicates as complex and dynamic mixtures of related mutants termed viral quasispecies. Here we review the basics of quasispecies theory, how FMDV helped to establish a link between theory and experimental observations, and some biological implications of quasispecies for the biology of FMDV. Topics covered include the relationship between genetic and antigenic heterogeneity, the escape strategy for long-term survival, memory in viral quasispecies, consequences of bottleneck events, evolution of host cell recognition, epidemiological fitness, evidence against the molecular clock hypothesis in virus evolution, internal interactions within mutant spectra, new antiviral strategies based on the error threshold concept, and a salient evolutionary transition towards genome segmentation when FMDV was subjected to many serial passages at high multiplicity of infection. The chapter illustrates that FMDV has been an excellent model system to unveil the potential of genetically variable viruses to find molecular pathways for survival under adverse environmental conditions. Introduction: genetic variation of RNA viruses Viruses that have RNA as genetic material (or use RNA as a replicative intermediate) and some DNA viruses of limited genetic complexity share a salient feature that conditions their biology: error-prone replication. Due to absence or low efficiency of proofreading and post-replicative repair functions acting on replicating RNA, many viruses display extensive intra-host genetic heterogeneity, and the

genetic composition of the viral populations varies as the infection progresses. The terms mutant spectrum, distribution, swarm, or cloud have been used to refer to such a population structure. As shown in the schematic representation of Fig. 7.1, a mutant spectrum can modify its genomic composition without modification of the consensus (or average) sequence of the genome population. Such mutant distributions are termed viral quasispecies, defined as collections of related but non-identical genomes subjected to a continuous process of genetic variation, competition and selection and that often act as a unit of selection (what is selected is not necessarily an individual genome type but a set of related genomes). Quasispecies theory was formulated by M. Eigen, P. Schuster and their colleagues as a model for error-prone replication and self-organization of simple replicons that might have been primitive life forms (Domingo and Schuster, 2016; Eigen, 1971, 1993, 1996; Eigen and Biebricher, 1988; Eigen and Schuster, 1979; Nowak, 1992). As other theoretical descriptions of evolutionary dynamics, quasispecies stands on Darwinian principles (Page and Nowak, 2002). The theory emphasizes mutation as an essential component of the replication dynamics, not as an occasional event during replication. The initial theory was formulated considering steady-state equilibrium conditions achieved with replicons of infinite population size. Recent theoretical formulations have extended quasispecies to finite populations replicating in variable fitness landscapes, thus bridging the gap between the initial quasispecies theory and experimental systems (see the different chapters in Domingo and Schuster, 2016). Intra-host virus variation is the first step of virus diversification in nature, and a relevant ingredient

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Figure 7.1  A simplified representation of an evolving viral quasispecies that maintains an invariant consensus sequence. Genomes are depicted as horizontal lines and mutations as symbols on the lines. Genomes represented by discontinuous lines are not perpetuated due to an excess of mutations; the invariant consensus sequence is represented by a horizontal line at the bottom of each distribution. In reality, viral populations consist of hundreds or thousands of genomes in each replicative unit, as evidenced experimentally with many viruses. Figure reproduced from Domingo et al. (2012), with permission from the American Society for Microbiology, Washington DC, USA.

of viral pathogenesis. As a classic example, simian immunodeficiency virus (SIV) evolved in monkeys from an initial phase without disease symptoms into a phase with AIDS-like pathology. Naive monkeys developed disease when they were inoculated with virus expressed from clones retrieved from the disease phase but not from the asymptomatic phase of the infection (Kimata et al., 1999). The result means that evolution of SIV played a direct role in disease development. Recent observations on the molecular epidemiology of pathogenic viruses have documented genetic changes that have modified their virulence profile. Genetic heterogeneity and diversification are also major challenges for disease prevention by vaccination and antiviral treatment because of the selection of mutants that are not significantly affected by the vaccine-induced immune response or by antiviral agents (reviewed in Domingo, 2016; Domingo and Schuster, 2016). FMDV, the most important viral pathogen in veterinary medicine, fully participates of quasispecies dynamics and its biological consequences, affecting viral pathogenesis, virus persistence and difficulties for disease prevention and control. Phenotypic plasticity of FMDV was suggested by early studies on variations of acid and thermal

lability of virus particles, differences in plaque morphology, and atypical pathogenic manifestations of viral isolates. Indeed some FMDV variants, often depending upon their passage history in cell culture, could be associated with fulminant or degenerative forms of myocarditis, neuropathology or diabetes; atypical symptoms of viral infections have been described for many viral pathogens (Bachrach, 1968; Domingo et al., 1990; Holland et al., 1992, and references therein). Therefore, contrary to an extended belief that goes back to early times of influenza virus research, viral diseases are not necessarily ‘invariable’ manifestations of ‘variable’ viruses. This point was addressed by J. Holland and colleagues who wrote the following statement: ‘Therefore, the acute effects and subtle chronic effects of infections will differ not only because we all vary genetically, physiologically, and immunologically, but also because we all experience a different array of quasispecies challenges. These facts are easily overlooked by clinicians and scientists because disease syndromes are often grossly similar for each type of virus, and because it would appear to make no difference in a practical sense. However, for the person who develops Guillain– Barré syndrome following a common cold, or for the individual who remains healthy despite many

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years of HIV-1 infection, for example, it may make all difference in the world’ (Holland et al., 1992). New findings in the last decade have unveiled a degree of population complexity of FMDV which is even greater than suspected a few years ago. In this chapter we first review briefly the molecular basis of genetic diversity of viruses, its consequences for virus population dynamics, and, finally, observations with FMDV on intra-population interactions and new antiviral strategies.

number of substitutions per nucleotide and year (s/nt/y) that are incorporated in the consensus sequence of an evolving viral population. If a horizontal time axis were added to the scheme of Fig. 7.1, a zero evolutionary rate (invariant consensus sequence) would be calculated despite a dynamic mutant spectrum. Rates of evolution can be calculated within a single host or among multiple hosts (during virus spread in an outbreak, epidemic or pandemic).

Molecular basis of genetic variation of RNA viruses Viruses exploit the same mechanisms of variation that operate in all life forms on earth, namely mutation, recombination and genome segment (chromosome) reassortment (Fig. 7.2). The main difference between complex organisms and molecular parasites such as viruses is the extent to which such molecular mechanisms alter their genetic material while maintaining biological competence, and the time frame in which the alterations and their consequences can be observed. These differences are due to interconnected parameters which include: mutation rate, genome complexity and population size, number of replication cycles per unit time and opportunity of dual (or multiple) infections of the same cell for recombination and reassortment (Domingo, 2016). Next we examine some of these parameters.

Recombination and reassortment Viruses may also vary genetically by recombination and segment reassortment in the case of viruses with segmented or multipartite genomes (Fig. 7.2). Genome segment reassortment consists in the formation of new constellations of viral genomic segments from two (or several) parental viruses that coinfect the same cell. The typical example is reassortment in influenza virus type A, associated with antigenic shift and occasionally with new influenza pandemics (Fig. 7.2C). Following the early genetic evidence of recombination in poliovirus by P.D. Cooper and colleagues, the studies by A.M. King et al. provided the first molecular evidence of RNA recombination using FMDV as model system (King et al., 1982) (Fig. 7.2D). Recombination rates are quite variable among RNA viruses, and one of the determining factors is polymerase processivity (capacity to maintain the copying of the same template molecule), that may be linked to fidelity of nucleotide incorporation. During picornavirus infections, recombination frequencies between closely related genomes have been estimated in 10–20% of the progeny (King, 1988; Lai, 1992). Mutation, reassortment and recombination can contribute individually or conjointly to adaptive genome variation in viruses (reviewed in Domingo, 2016).

Mutation and antigenic variation Mutation rates calculated for several RNA and DNA viruses using genetic and biochemical methods are in the range of 10–3 to 10–5 substitutions per nucleotide copied (s/nt) (Batschelet et al., 1976; Drake and Holland, 1999; Sanjuan et al., 2010). These rates are 105- to 106-fold higher than those estimated for replicating cellular DNA under normal conditions. Mutation frequencies (the proportion of mutated residues present in a population of genomes) are only indirectly related to mutation rates. The latter are determined by biochemical events dictated essentially by the replication machinery while the mutation frequency is a population parameter dependent on relative replication capacity of the different mutants that coexist at any given time. There is no direct correlation between average mutation rates and frequencies and the evolutionary rate generally measured as the

Some consequences of quasispecies dynamics The main implications of quasispecies in virology can be summarized as follows: (i) it provides a definition of wild type not as a fixed, reference genome but as a collection of related genomes that may change in response to environmental alterations; (ii) it provides a reservoir of phenotypic variants for adaptation; (iii) it explains positive interactions

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Figure 7.2  Scheme of main types of genetic variation in viral genomes. (A) Mutation due to miscopying of template residues by viral replication complexes. (B) Hypermutation (high frequency of biased mutation types) often due to cellular editing activities. (C) Genome segment reassortment. (D) Homologous, replicative (left), and non-replicative (right) recombination. (E) Genome segmentation as documented with FMDV (described in ‘Fitness gain and limits to fitness gain. A transition towards genome segmentation’). Figure reproduced from Domingo et al. (2012), with permission from the American Society for Microbiology, Washington DC, USA.

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of complementation or negative interactions of interference that are established within mutant spectra; (iv) it suggests new antiviral strategies, including lethal mutagenesis. Generalizing a statement by J. Gomez and colleagues on hepatitis C virus: ‘An RNA virus is constantly redefining itself both genetically and phenotypically’ (Gomez et al., 1999). FMDV has been an excellent model system to investigate quasispecies mechanisms and lethal mutagenesis as an antiviral strategy. Quasispecies dynamics as studied with FMDV Early work on FMDV variation FMDV genome diversity was evidenced in the early molecular analyses of the viral RNA. Nucleic acid competition-hybridization using genomic FMDV RNA, and T1 oligonucleotide fingerprinting yielded nucleotide sequence identities of 40–70% between RNAs from FMDVs of serotypes A, O and C, and greater than 70% identity between RNAs of FMDVs of different subtypes within serotypes A and O (Dietzschold et al., 1971; Robson et al., 1977). Nucleotide sequence differences among FMDV RNAs of the same serotype were also detected (Frisby et al., 1976; Robson et al., 1979). In the beginning of nucleotide sequencing (deduced from analysis of partial and complete ribonuclease digests of 32P-labelled RNA) only limited sequence identity was observed in the 40 nucleotides adjacent to the 3′-terminal polyadenylate (poly A) of FMDVs A-61, O-VI and C-997 (Porter et al., 1978). Evidence was obtained of FMDV genetic heterogeneity within infected animals that was not compatible with multiple infections of an animal, thus providing the first evidence of viral quasispecies for an animal virus in vivo (Domingo et al., 1980). Nucleotide sequence heterogeneity of FMDV RNA in vivo has been documented by molecular cloning and Sanger sequencing as well as by new generation deep sequencing (Logan et al., 2014; Sarangi et al., 2015; Van Borm et al., 2015; Wright et al., 2011) (see Chapter 11). The rapid generation of mutants from a single genome was demonstrated by passage of biological clones (derived from a single genome) of natural FMDV isolates in cell culture (Sobrino et al., 1983). The results showed (i) the generation

of a spectrum of related mutants upon passage of a biological clone in two cell lines; (ii) a dynamics of competition among newly generated mutants, and dominance of different genomic sequences in parallel lineages; and (iii) adaptation to the cell culture environment, evidenced by an increase in the capacity to produce progeny in cell culture. These observations extended previous results obtained with bacteriophage Qβ, and reinforced a link between virus evolution and quasispecies theory (Domingo et al., 1978, 1985; Holland et al., 1982). What traditionally was termed ‘an FMDV isolate’ is in reality ‘a pool of variants’. Variants are rarely neutral since fitness (replicative capacity) variations can be quantified among components of mutant spectra and between ensembles of mutants, both in vivo and in cell culture. Genetic and antigenic flexibility of FMDV Genetic heterogeneity among circulating FMDVs has been extensively documented for any of the serotypes. Antigenic heterogeneity is a consequence of genetic changes when they affect amino acids located at antigenic sites of the virus or that can affect the antigenic sites (Mateu, 1995; Mateu et al., 1987, 1988) (see Chapter 4). FMDV subtyping was discontinued because with monoclonal antibodies (MAbs) as diagnostic tools it was observed that many new isolates could constitute new subtypes. An epitope involved in neutralization of FMDV subtype C3 was generated by a single amino acid replacement at the corresponding site of FMDV subtype C1 (Hernández et al., 1992), thus illustrating the short evolutionary distances among some subtypes. Amino acid replacements at a major antigenic site, found as minority components in the mutant spectra of natural isolates, altered substantially the interaction with antibodies (Mateu et al., 1989), providing evidence of quasispecies as phenotypic reservoirs of viruses (Schuster, 2010). Field observations were supported by experimental evolution designs. Antigenic variants were isolated following experimental persistent infections of cattle (Gebauer et al., 1988) or acute infections of swine (Carrillo et al., 1990), initiated with biological clones of FMDV. The determination of the three-dimensional structure of FMDV of different serotypes by D.I. Stuart and colleagues provided new insights into the

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mechanisms of antigenic variation of FMDV (Acharya et al., 1989; Curry et al., 1996; Lea et al., 1994, 1995). Comparison of amino acid sequences of a major antigenic site located within the G-H loop of capsid protein VP1 [a protruding and mobile loop that includes also an integrin-recognition domain (Acharya et al., 1989)] of serotype C viruses, together with virus reactivity with MAbs, revealed two mechanisms of antigenic diversification of FMDV in nature: (i) a gradual increase in antigenic distance brought about by amino acid replacements at two hypervariable regions within the antigenic site, and (ii) an abrupt antigenic change, associated with a replacement at some critical positions within the same antigenic site that resulted in loss of several epitopes (Martínez et al., 1991b). These two mechanisms are similar to antigenic drift and shift, extensively characterized for human influenza viruses, but without participation of genome segment reassortment. FMDV has at least four different antigenic sites some of which may interact with each other, depending on the viral serotype, and by mechanisms which are not well understood (Baranowski et al., 2001b; Feigelstock et al., 1992; Mateu, 1995; Mateu et al., 1994; Parry et al., 1990). Furthermore, there are non-additive effects of amino acid substitution in antigen–antibody recognition (Mateu et al., 1992), thus contributing to complex antigenic profiles of FMDV isolates. A general point is in place here. High mutation rates do not entail a correspondingly high antigenic diversity. Within the Picornaviridae family the number of serotypes varies remarkably: there is a single serotype of Mengo virus or of hepatitis A virus (HAV), three of poliovirus, seven of FMDV and around one hundred of human rhinoviruses. Yet in all these viruses the frequency of monoclonal antibody (MAb)-escape mutants is similar and in the range of 10–3 to 10–5, and the specific frequency values are dependent on the nature of each individual epitope rather than on the degree of antigenic diversity attained by the virus in nature. Several mechanisms have been proposed to account for differences in the number of viral serotypes among viruses that share similarly high mutation rates. They include the presence in some viruses of dominant and invariant antigenic sites that obscure the contribution of other variable antigenic sites, differences in the procedures used

for serotype classifications, history of long-term virus circulation in nature, or constraints to variation of some antigenic sites [reviewed in (Domingo, 2016)]. The number of serotypes may reflect the constraints that operate in viruses to tolerate amino acid replacements that affect their antigenic profile. Antigenic variation in the absence of immune selection: coevolution of antigenicity and cell tropism While selection by antibodies and cytotoxic T-cells probably plays a major role in antigenic variation of viruses, evidence with several viruses, including FMDV, suggests that antigenic variation is not necessarily the result of immune selection (Bolwell et al., 1989; Curry et al., 1996; Díez et al., 1990; Domingo, 2016; Domingo et al., 1993, 2001; Haydon and Woolhouse, 1998; Holguín et al., 1997; Martínez et al., 1991a; Sevilla and Domingo, 1996). The underlying mechanism is the acceptance of amino acid replacements generated at random at the antigenic sites because surface residues of viral capsids and envelopes are less subjected to structural constraints than internal residues in the same proteins. Following its generation by mutation, an antigenic variant may rise to dominance by the following mechanisms: 1

2

The relevant antigenic site may perform functions additional to the interaction with components of the immune response. In particular amino acid residues of antigenic sites may also be involved in recognition of cellular receptors (Baranowski et al., 2001a, 2003; Verdaguer et al., 1995, 1998; reviewed in Domingo, 2016). The use of an entirely new receptor by a virus may render the obsolete receptor-binding site highly tolerant to variation with obvious consequences when the old receptor recognition residues overlap with an antigenic site. These mechanisms underlie a possible coevolution of antigenicity and host cell tropism, with consequences for the adaptive potential of the virus (Baranowski et al., 2003; Domingo, 2016). An additional mechanism for antigenic variation in the absence of immune selection – which does not necessitate that residues of an antigenic site are involved in other functions – relates to the classical concept of ‘hitch-hiking’

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of mutations. It proposes that an antigenic variant may rise to dominance due to fluctuations in mutant distributions associated with random drift or positive selection targeted at sites other than the antigenic sites (Domingo et al., 1993; Haydon and Woolhouse, 1998). Again, the tolerance of antigenic sites to amino acid replacements facilitates random changes at those sites that are then hitch-hiked by any event that drives replication of a genome carrying the change. Therefore, antigenic variation of a virus is not necessarily the result of positive selection by components of the immune response. Flexibility of receptor usage The repertoire of amino acid substitutions that became dominant at the major antigenic site A of FMDV C-S8c1 was different when the virus was passaged in BHK-21 cells in absence or presence of antibodies directed to that site (Borrego et al., 1993). Passage in absence of antibodies resulted in an expansion of host cell tropism that made dispensable the RGD triplet involved in recognition of integrin receptors (Baranowski et al., 1998, 2000). This permitted isolation of FMDV mutants with RED, RGG and even GGG instead of RGD at the G-H loop of capsid protein VP1 (Martínez et al., 1997; Ruíz-Jarabo et al., 1999) (Fig. 7.3). Since the RGD plays also a critical role in virus binding to neutralizing antibodies (Ochoa et al., 2000; Verdaguer et al., 1995, 1999), the mutants lacking RGD were antigenically altered (Ruíz-Jarabo et al., 1999). FMDV C-S8c1 passaged in BHK-21 cells could use at least three cell entry pathways involving either integrins, heparan sulfate or a third, unidentified component (Baranowski et al., 2000, 2001a; Chamberlain et al., 2015). Dispensability of the RGD is not restricted to FMDV in cell culture. Replication of FMDV C3 Arg-85 in partially immune cattle resulted in selection of mutants with amino acid substitutions within the RGD or at neighbouring positions, suggesting a coevolution of antigenicity and host cell tropism in vivo (Taboga et al., 1997; Tami et al., 2003). Dispensability of the RGD was also shown with FMDV O/CHN/90, a type O FMDV used for vaccine production in China (Zhao et al., 2003). It is now widely accepted that (i) in many viral systems there is an overlap between antigenic sites

Figure 7.3  Expansion of host cell tropism of FMDV. Biological clone FMDV C-S8c1 infected BHK-21 cells but not several other cell lines. Upon subjecting the virus to one hundred serial passages in BHK-21 cells at a MOI of 1 to 5 PFU/cell, the viral population expanded its host cell tropism and infected several cell lines indicated at the tip of the thick arrows. Figure based on the results reported by Ruiz-Jarabo et al. (2004) and reproduced from Domingo (2016), with permission from Academic Press, Elsevier, Amsterdam.

and receptor recognition sites; (ii) a virus can use different receptors and coreceptors; (iii) a receptor can be shared by different microbial pathogens; (iv) a phylogenetic position of a virus or a set of disease manifestations do not predict the use of a receptor class; (v) one or a few amino acid substitutions at surface residues of viral particles may modify receptor recognition; and (vi) different compartments (tissues or organs) within an organism do not necessarily express the same set of surface molecules that can act as viral receptors; concerted expression of sets of receptors and co-receptors is one of the determinants of viral pathogenesis [for justification of these statements and literature references, see (Baranowski et al. (2001a, 2003) and Domingo (2016)]. Variations in cell tropism and host-range of FMDV can also be mediated by alterations of non-structural proteins. Deletions or point mutations in 3A have been associated with FMDV attenuation or adaptation to alternative host species (Beard and Mason, 2000; Giraudo et al., 1990; Núñez et al., 2001). The current view is that the interplay between structural and non-structural proteins and their variant forms can influence

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virus–host interactions, rendering an understanding of viral pathogenesis a challenging endeavour. Diversification in the field: power and limitations of phylogeny; epidemiological fitness Phylogeny connects the evolutionary history of related virus isolates, and it is a basic technique for molecular epidemiology and virus classification. Its main weakness is that a phylogenetic position is a poor predictor of important viral features such as pathogenic potential. In other words, modifications of virulence may occur without alteration of the position of a virus in a phylogenetic tree. Despite being based on specific nucleotide sequences, trees for viruses should be regarded as consisting of a cloud of points at the tip of the branches, to recognize the simplification implied by the connection one virus–one sequence. Many different genomic sequences from the same cloud display different phenotypic traits. Phylogenies express the dominance of viral lineages in the field but not its causes. The term epidemiological fitness was coined to describe an ensemble of properties (often difficult to specify) that determine the prevalence of some variants at the epidemiological level. The standard fitness concept, that is, the relative replication (multiplication) capacity of virus variants in a given environment is just one of several parameters that contribute to dominance in the field. Epidemiological fitness encompasses several features in addition to intracellular replication capacity: stability of viral particles in infected animals and in the environment, transmissibility, host range, symptomatology – including capacity of produce subclinical infections that delay virus detection – and others that apply to all pathogenic viruses (Domingo, 2016). In Chapter 18, B.W.J. Mahy and G.J. Belsham describe the spectacular expansion of FMDV O PanAsia since its first isolation in India in 1990. This virus replaced other circulating strains in many countries during the 1990s, and produced in the year 2000 an outbreak in Japan, a country that had been free of the disease since 1908! FMDV O Pan Asia is an example of high epidemiological fitness due to mechanisms that are not understood. In support of the continuous capacity of FMDV to diversify while maintaining its dominance, a virus termed

PanAsia-II and some of its sub-lineages have been associated with recent outbreaks in the Middle East and West Eurasia (see Mahy and Belsham, this book). It is interesting that some lineages and sublineages are endowed with the capacity of extensive dominance, while others such as FMDV serotype C are nearly extinct. At least for the time being. The molecular clock controversy: intra-host versus inter-host evolutionary rate Early work on the rate of FMDV evolution during a disease outbreak in a limited geographical area provided evidence that the rate of evolution (defined as the accumulation of mutations in the consensus sequence of sequential isolates as a function of time; see ‘Mutation and antigenic variation’) is not constant. The rate varied not only among viral genomic regions of the same isolates but also depending on the time interval between the viral isolations on which calculations were based (Sobrino et al., 1986). Values ranged from 104 TCID50 have been reported (Anderson et al., 1979; Dawe et al., 1994b). Individual animals may remain persistently infected for at least 5 years (Condy et al., 1985) but it is probable that a significant number of animals fail to maintain infection for a prolonged period of time because the proportion of persistently infected animals falls after reaching a peak in the 1–3 year age group (Hedger 1976; Anderson, E.C. and Knowles, N.J., personal communication, 1994). The frequency and titre of the virus recovered decrease over time (Vosloo et al., 2007) and it has also been shown that persistently infected buffalo can clear infection under experimental conditions over a 15-month period ( Juleff et al., 2012a). A further site of viral persistence in buffalo has been shown to be germinal centres of lymphoid tissues ( Juleff et al., 2012a,b), similar to cattle where it was shown that virus locates to and is maintained in a non-replicating state in the light zone of germinal centres within the dorsal soft palate, pharyngeal tonsil, palatine tonsil, lateral retropharyngeal lymph

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Cows in breeding herds produce calves from about 4-5 years of age. At birth calves are free of FMD infection

Most calves are born in mid-summer (but a few throughout the year). Soon after birth they acquire passive immunity via colostrum; passive immunity probably lasts for 2-6 months

Buffalo calves lose passive immunity more-or-less synchronously, i.e. a large cohort of susceptible calves is recruited into herds in winter/spring

TRANSMISSION OF SAT VIRUSES TO OTHER SPECIES EFFICIENT TRANSMISSION On first infection young buffalo excrete large quantities of SAT viruses for 2-3 weeks although clinical signs are mild or absent

Environmental contamination with FMD viruses

Breeding herds transmit infection to other cloven-hoofed species in the close vicinity (e.g. cattle & antelope)

INEFFICIENT TRANSMISSION Following recovery from acute infection >50% of infected calves become persistently infected (carriers) for months to years, i.e. low levels of virus present in the oro-pharynx.

Carriers only transmit during close contact over extended periods

Probably ensures viral survival in buffalo herds in inter-epidemic periods.

BREEDING HERDS PERIODICALLY PROVIDE A

CARRIERS RARELY TRANSMIT TO SPECIES

POTENT SOURCE OF INFECTIVITY, INCLUDING OF THE

OTHER THAN BUFFALO

IMMEDIATE ENVIRONMENT.

Figure 9.2  Diagrammatic representation of the theory for transmission of SAT serotype FMD viruses within breeding herds of African buffalo in southern Africa (where buffalo breed seasonally) and transmission of these viruses from acutely infected- (efficient pathway) and carrier buffalo (inefficient pathway) to other cloven-hoofed species. Source: Foot-and-mouth disease: Southern Africa, Bulletin #4, March 2014 (http://www.afrivip.org/ node/3857/zip_download).

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node and mandibular lymph node following primary infection ( Juleff et al., 2008). The authors postulated that it is possible that maintenance of non-replicating FMD virus in these sites represents a source of persisting antigens and contributes to the generation of longer-lasting antibody responses against neutralizing epitopes of the virus than can be generated by inactivated vaccines. More than one type of SAT virus may be maintained by individual buffalo (Hedger, 1972; Anderson et al., 1979). This is despite the fact that high levels of antibody to the viruses concerned occur in pharyngeal secretions (Hedger, 1976; Francis and Black, 1983). It was demonstrated that persistently infected buffalo are refractory to re-infection with the same strain of virus (Hedger et al., 1972). Genetic diversity of FMD viruses derived from buffalo in sub-Saharan Africa Genomic analysis of viruses isolated from domestic and wildlife species for the three SAT serotypes divides each serotype into a number of topotypes, i.e. genotypes that occur within a specific geographic region (Bastos et al., 2001; Samuel and Knowles, 2001). To date, nine topotypes have been assigned for SAT-1 (Sangula et al., 2010; Sallu et al., 2014), 14 for SAT-2 (Ayelet et al., 2009; Habiela et al., 2010) and six for SAT-3 (Bastos et al, 2003) (Table 9.1). Since a number of these studies included historical viruses, some topotypes may be extinct as no recent, related isolates have been found. The number of viruses recovered from wildlife in most regions of sub-Saharan Africa is limited, the exception being southern Africa, making accurate inferences on genetic relationships difficult. Outside southern Africa, viruses from wildlife generally fall within topotypes previously described for domestic animals, but there is evidence, for SAT-1 at least, that unassigned topotypes are present (Ayebazibwe et al., 2010b; Kasanga et al., 2014; Wekesa et al., 2015). In southern Africa, where most detailed genetic analyses have been performed, SAT-type viruses have been shown to be constantly evolving in buffalo populations (Vosloo et al., 1996; Bastos et al., 2001, 2003), as would be expected from what is known of the quasispecies dynamics of FMD viruses (Domingo et al., 2003). In

addition, because buffalo populations have been fragmented for many years now, different buffalo populations can be differentiated on the basis of SAT-type viruses recovered from persistently infected animals representative of those populations (Vosloo et al., 2001). Even within the buffalo population of the KNP (approximately 30,000 individuals), clear intratypic differences in the genomes of SAT-1, -2 and -3 viruses obtained from different regions of the KNP have been shown (Vosloo et al., 1995, 2001; Bastos et al., 2000, 2001, 2003). The fragmentation of southern Africa’s buffalo population occurred as a result of the drastic reduction (approximately 10,000-fold) in buffalo numbers following the Great Rinderpest Pandemic of 1887–1904 (Rossiter, 1994) as well as human expansion into formerly deep rural areas. That is likely the reason why some small populations within the southern-most buffalo distributional range are free of FMD (Esterhuysen et al., 1985). The present geographic isolation of discrete buffalo populations in ‘conservation islands’ probably explains the locality-specific distribution of viral topotypes apparent today. High mutation rates (Vosloo et al., 1996, 2007) and continuous, independent virus cycling within discrete buffalo populations (Condy et al., 1985) probably account for the extensive intratypic variation evident today. For all three SAT serotypes in southern Africa it has been demonstrated that the genetic differences between viruses from different topotypes is such that outbreaks should be traceable to specific countries, game parks and even to specific regions within game parks (Bastos, 2001; Bastos et al., 2001, 2003; Vosloo et al., 2001, 2002a,b). However, some countries such as Botswana and Zimbabwe have more than one topotype within their borders and if uncontrolled movement of buffalo occurs, distribution of topotypes will become commingled (FMD Bulletin, 12 December 2011). The high levels of genetic diversity will most likely be reflected in antigenic differences and it has been shown that for vaccination to be effective, the viruses incorporated into vaccines need to be antigenically related to viruses circulating in the field (Hunter et al., 1996; Hunter, 1998). Therefore, the uncontrolled movement of buffalo within the southern African region could have severe implication for the control of FMD (Maree et al., 2014).

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Table 9.1 Summary of the historical topotype distribution across countries for SAT-1, SAT-2 and SAT-3. The current topotype distribution is not certain due to lack of sample submission from both livestock and wildlife for further characterization (Information kindly provided by NJ Knowles from records at the World Reference Laboratory, The Pirbright Institute) Serotype

Topotype

Countries

SAT-1

NWZ/I

Kenya, Malawi, Mozambique, Tanzania, Zambia, Zimbabwe (north-west)

SEZ/II

South Africa, Swaziland, Zimbabwe (south-east)

WZ/III

Botswana, Namibia, South Africa, Zambia, Zimbabwe (west)

IV

Uganda

V

Niger, Nigeria

VI

Israel, Nigeria, Sudan, Yemen

EA-2/VII

Uganda

EA-3/VIII

Uganda

SAT-2

IX

Ethiopia

I

Botswana, Malawi, Mozambique, South Africa, Zimbabwe (south-east)

II

Botswana, Namibia, Zimbabwe, Zambia

III

Botswana, Namibia, Zambia, Zimbabwe

IV

Bahrain, Burundi, Ethiopia, Kenya, Malawi, Tanzania, Uganda, Yemen, Zambia

V

Ghana, Nigeria, Senegal

VI

Gambia, Senegal

VII

Cameroon, Egypt, Eritrea, Ethiopia, Libya, Mali, Mauritania, Niger, Nigeria, Oman, Palestinian Autonomous Territories, Saudi Arabia, Sudan, Uganda

VIII

Burundi, Democratic Republic of the Congo, Rwanda

IX

Kenya

X

Democratic Republic of the Congo, Uganda

XI

Angola

XII

Uganda

XIII

Ethiopia, Sudan Sudan

SAT-3

XIV

Ethiopia

SEZ/I

South Africa, Zimbabwe (south-east)

WZ/II

Namibia, Botswana, Zimbabwe

NWZ/III

Malawi, Zimbabwe (north-west)

ZAM/IV

Zambia

EA/V

Uganda

VI

Uganda

Knowledge of the occurrence and distribution of SAT virus lineages and topotypes in buffalo populations has enabled the transmission of SAT viruses from buffalo to other species to be unequivocally proven (Bastos et al., 2000; Brückner et al., 2002; Vosloo et al., 2002b; Thomson et al., 2003a). Further details are provided below. This confirms the early observations made by J.B. Condy and R.S. Hedger into the association between the occurrence of FMD in cattle and the distribution and

behaviour of buffalo harbouring SAT-type viruses (Condy et al., 1969; Hedger et al., 1969, 1972; Condy, 1971, 1979; Hedger, 1972, 1976; Condy and Hedger, 1974; Hedger and Condy, 1985). Ability of acutely and persistently infected buffalo to transmit SAT viruses to cohorts Transmission of SAT-type viruses between individual buffaloes appears to occur in two ways: (1)

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contact transmission between acutely infected and susceptible individuals that is likely to account for the majority of infections and (2) occasional transmission between persistently infected buffalo and susceptible individuals (Fig. 9.2). Although precisely how persistently infected buffalo transmit the infection to susceptible cohorts is unknown (Thomson, 1996), a possibility for which the evidence remains tenuous is sexual transmission (Bastos et al., 1999; Thomson et al., 2003a). In a separate study in buffalo 3–5 years of age, virus was isolated from only one of 108 sheath washes and one of 23 testes samples. No virus was isolated from the reproductive tracts of 25 adult female buffalo (W. Vosloo, B. Botha and C.I. Boshoff, unpublished results). It is also not known whether these animals were actively or persistently infected. However, these results indicate that the presence of virus in the reproductive tract of buffalo is a rare occurrence. Therefore, unless further and more convincing evidence for sexual transmission can be produced, it can only be considered an interesting theory. The close contact that occurs between buffalo in breeding herds, especially among juveniles, is likely to ensure efficient transmission of SAT-type viruses, bearing in mind the levels of excretion that have been demonstrated in naive animals infected for the first time (Gainaru et al., 1986). Transmission between persistently infected buffalo and their cohorts has been shown (Condy and Hedger, 1974) but is less efficient than that involving acutely infected animals that excrete large quantities of virus (Thomson et al., 1985). Generally, these infections are ‘silent’ because buffalo generally suffer few ill effects from infection (see below). Therefore, infection can only be inferred from serology or other laboratory-based investigations. To better understand the complex interactions between breeding herds of buffalo and SAT viruses, the above factors were incorporated into a stochastic model for the behaviour of a single SAT virus in a breeding herd of buffalo numbering up to 500 individuals. Included were variable transmission coefficients for spread of the infection within subgroups of the herd (such as family sub-groups) as well as within the herd at large (Thomson et al., 1992). Using values for the transmission coefficients that are plausible based on current understanding, the model predicted that each year new infections in the herd would rise to a peak

following loss of maternally derived immunity in the calves and then fall as the number of susceptible individuals decreased. Furthermore, periods of time where no new infections occurred within the herd were predicted resulting in the infection ‘dying out’. The only way in which viral circulation could be re-established was through the introduction of new acutely infected individuals at a time when a new cohort of susceptible young animals had been created by the next batch of calves losing their maternal immunity or by re-activation of the virus by transmission of the virus from a ‘carrier’ to the new group of susceptible calves (Thomson et al., 1992). It should be remembered, however, that, in reality, transmission of SAT viruses within buffalo herds is more complex than this because such herds usually maintain all three SAT serotypes simultaneously. It is likely therefore that there is a period of time each year, probably covering 2–3 months, when significant numbers of young buffalo in breeding herds will be excreting SAT viruses that pose a threat of infection for other species that may come in close contact with buffalo breeding herds. Transmission of SAT viruses from buffalo to other species In the KNP outbreaks of FMD among impala Aepyceros melampus within the Park are a regular occurrence, although strangely, other species are rarely affected. This could be due to the low minimum infectious dose (approximately 1 TCID50 was shown to be capable of infecting adult impala by the respiratory route – R.G. Bengis and G.R. Thomson, unpublished data) required to infect impala, in contrast to cattle and sheep that require 10–25 (Gibson and Donaldson, 1986; Donaldson et al, 1987). Since 1938 there have been ~ 60 recorded outbreaks of FMD in impala the KNP and of the 25 outbreaks recorded between 1976 and 2008, 62% were caused by SAT-2, followed by SAT-1 (28%) and a single outbreak due to SAT-3. These outbreaks lasted 4.4 months on average (range 1–13 months) (Vosloo et al., 2009). Unlike the situation in buffalo, these outbreaks have been identified by the fact that, usually, at least a proportion of impala developed clear, although transient, clinical signs of FMD. Sequence analysis of the SAT-2 viruses involved has shown that these outbreaks were causally distinct (Vosloo et al., 1992; Bastos et al., 2000). Infection and attack

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rates have varied in outbreaks of FMD in impala that have been studied, with the latter sometimes much lower than the former, indicating that subclinical infection is common (Keet et al., 1996), as has been seen in experimentally infected impala (R.G. Bengis and G.R. Thomson, unpublished data). Serological evidence has indicated that impala in other regions of sub-Saharan Africa, where impala are abundant, have also been infected but clinical disease has not been reported (Anderson et al., 1993) except for anecdotal, unconfirmed reports (S. Cleaveland, personal communication, 2015). That outbreaks of FMD in wildlife have only been recorded in the KNP could be due to the fact that surveillance in this Park is more thorough than elsewhere. Between 1997 and 2007 a serological survey was performed on impala in the KNP in three defined locations located in the north, centre and south of the Park. Animals were sampled on a three-monthly basis in each of the locations, involving collection of blood for serum preparation as well as examination for clinical disease. About 40 randomly selected animals were darted with a tranquillizer at each sampling. In the most northern sampling site, no serological or clinical evidence of infection was found, while in the central site, serological data indicated that both SAT-1 and SAT-2 infections had occurred on at least three occasions, although no virus was recovered because lesions were not detected. However, mixed infection in impala herds has been confirmed by virus isolation previously (Vosloo et al., 2009). The most southern sampling site had serological evidence of at least six SAT-2 outbreaks. A regression model indicated that summer and autumn were the highest risk periods associated with sero-prevalence, which was in contrast with clinical data that indicated most outbreaks occur at the end of the dry season (Bengis et al., 1994; Bastos et al., 2000; Vosloo et al., 2006). Vosloo et al. (2009) postulated that this discrepancy could be explained by a slow progression of infection from the dry season into summer, i.e. the wet season. This slow progression of infection in impala herds had previously been shown by Keet et al. (1996). Females and older animals also had a higher risk of sero-positivity that could be explained by impala herd structure and behaviour (Vosloo et al., 2009). It has been observed that contact between buffalo and impala constitutes  iC3b), promoting phagocytosis. The iC3b fragment can also interact

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with mononuclear phagocytes, neutrophils and NK cells through CR3 – the β2 integrin CD11b/CD18, which also binds to ICAM-1 – and CR4 – the β2 integrin CD11c/CD18. B-lymphocyte activity can also be enhanced by activated products from the complement cascade. Together with iC3b, the fragments C3d and C3dg generated from C3 can bind to CR2 (CD21) as co-receptors for activating B-lymphocytes (C3d, C3dg > iC3b). Activated complement components also enhance leucocyte chemotaxis and inflammatory responses – further promoting enhanced pathogen destruction; the best-described examples are the anaphylatoxins C3a, C4a and C5a. The biological impact on the law of mass action Interaction of antibody with the virus must be of minimum avidity for immune protection to ensue, dependent on the specificity of the antibodies and maturation of the adaptive immune system responding to virus infection or vaccination. The avidity of that reaction ensures a relative degree of stability, within the law of mass action, inducing the necessary conformational alterations in the antibody Fc portions, to facilitate interaction with phagocyte FcRs and complement activation. The complexes influence cytokine induction – particularly IFN-α – promoting immune cell functions including DC maturation. Yet, elevated levels can be more regulatory, including inducing apoptosis in cells. Overall, the outcome of antibody interaction with FMDV is determined by how MΦ and DCs interact with immune complexes in vivo, and what levels of complexes likely interact with a particular cell at any particular moment in time. The outcome of MΦ and DC interaction with FMDV immune complexes MΦ and DCs can destroy FMDV in immune complexes (Baxt and Mason, 1995; Guzylack-Piriou et al., 2006a; Harwood et al., 2008; Lannes et al., 2012; McCullough et al., 1986, 1988, 1992b; Ostrowski et al., 2005; Rigden et al., 2002; Summerfield et al., 2009). Such observations relate to the central importance of antibody in immunological protection against FMDV, reported by several groups (Black et al., 1984; Lavoria et al., 2012; McCullough et al., 1986, 1992a; Oliveira et al., 2005; Ostrowski et al., 2007; Pay and Hingley, 1987; Rieder et al.,

1994; Scicluna et al., 2001; Steward et al., 1991; Van Maanen and Terpstra, 1989). The protective immune response involves uptake and ultimate destruction of the virus by MΦ and DCs, likely elaborated by induction of IFN-α production by pDCs (Baxt and Mason, 1995; Guzylack-Piriou et al., 2006a; Harwood et al., 2008; Lannes et al., 2012; McCullough et al., 1986, 1988, 1992b; Ostrowski et al., 2005; Rigden et al., 2002). With live virus in the immune complexes, an abortive replication in the DCs provides RNA for important PAMP signalling to activate the innate defence mechanisms of DCs, both pDCs (Guzylack-Piriou et al., 2006a; Lannes et al., 2012; Reid et al., 2011; Summerfield et al., 2009), and skin DCs (Bautista et al., 2005). Importantly, porcine DCs remain immunocompetent and functionally active following uptake of immune complexes carrying live FMDV (Bautista et al., 2005), and stimulate FMDV-specific Thlymphocytes (Harwood et al., 2008). Cytotoxic effector immune responses Related to the role of phagocytes in destroying virus infectivity within immune complexes, the phagocyte FcR can also engage antibody bound to the surface of infected cells. This requires that the virus infection express viral proteins on the cell surface, which is not as obvious with a non-enveloped virus as with an enveloped virus. Nonetheless, immune defence against a non-enveloped virus such as FMDV does not preclude the presence of virus infection-derived proteins at the infected cell surface. If these induce humoral immunity, phagocyte-dependent attack of virus-infected cells through antibody-dependent cellular cytotoxicity (ADCC) becomes a reality (Biburger et al., 2014). However, it is unlikely that inactivated or peptide/ protein-based vaccines that do not replicate would induce such antibody specificities. Even vector vaccines are unlikely to induce such specificities, which is more reliant on an intact virus genome. Phagocytes are not alone in their capacity to destroy infected cells, although the other cytotoxic immune processes are not reliant on antibody reaction with infected cells. Natural killer (NK) cells target viral antigens on infected cell surfaces, but do not require involvement of MHC molecules (in contrast with antigen-specific Tc-lymphocytes – see below) (Lugli et al., 2014; Waggoner et al., 2015). NK cell activity does relate to that of

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Th- and Tc-lymphocytes in their innate-adaptive communications through production of IFNγ. NK cell activity has been identified in infected cattle (Patch et al., 2014), but could not be demonstrated following vaccination – in line with NK cells attacking antigens expressed on virus-infected cells. Yet, a non-MHC restricted FMDV-specific cytolytic activity has been identified in NK-lineage cells isolated from vaccinated cattle (Amadori et al., 1992). NK cell responsiveness to the presence of FMDV infection of pigs was also reported (Toka et al., 2009a), and activated NK cells will efficiently lyse FMDV-infected targets (Toka et al., 2009b) (see ‘Ranking innate immune defences’). Detection of NK cell-like activity following vaccination suggests a virus-derived protein as the NK cell target, rather than an infected cell-derived entity. Nonetheless, the contribution of such NK activity to protection or viral clearance remains unclear. The other main component of antigen-specific cytotoxic immune defence is associated with the Tc-lymphocytes, generated by MHC Class I presentation of antigen (Fig. 10.10). Vaccination could generate such cells, through cross-presentation (see ‘Considerations for MHC Class II processing’), and certainly infection should induce such immunity. However, cellular cytotoxic responses (NK or Tc-lymphocyte) would have to target infected cells early after infection by FMDV, prior to virus killing of the infected targets. Induction of FMDV-specific effector Tc-lymphocytes has been difficult to evaluate, possibly due to the short life-span of infected cells to provide appropriate targets. Virus infection was also reported to induce a rapid reduction of MHC class I expression on susceptible cells (SanzParra et al., 1998), which would impair effector Tc-lymphocyte responsiveness. On the contrary, down-regulation of MHC Class I molecules on infected cells would facilitate detection of viral antigens on the cell surface by NK cells. Despite these scenarios, anti-FMDV Tclymphocyte activity has been identified. Peptides containing T-cell epitopes have been described in terms of porcine MHC Class I-dependent (SLA-I) presentation (as well as MHC Class II dependency) (Fig. 10.20), as well as their ability to activate different T-lymphocyte subsets, including CD4–CD8+ porcine Tc-lymphocytes (Blanco et al., 2000). Vaccination of pigs and cattle with recombinant adenovirus vaccines expressing the

P1 precursor polypeptide of FMDV induced cellular rather than humoral immunity (Sanz-Parra et al., 1999a,b). Additional studies on adenovirusvectored FMDV vaccination (Patch et al., 2011), as well as fowlpox virus-based and inactivated FMDV vaccines (Guzman et al., 2008), showed induction of Tc-lymphocyte activity. A partial protection in the absence of detectable humoral immunity was also related to Tc-lymphocyte activity (Patch et al., 2013). Although only partial protection was noted, this would fit to the expected role of effector cellular immunity, particularly if that were due to Tc-lymphocytes. While antibody would be most effective at preventing virus spread and therefore the acute phases of infection, cytotoxic immunity would be more efficient at removing foci of infected cells and finally clearing the infection. In the absence of antibody, an initial infection by the virus would be possible, but the infected cells may ultimately succumb to the cytotoxic defence, although the acute phase of infection would still be observable. Overall, current evidence suggests that cytotoxic immune defences are important components of effector immunity, even when humoral immunity is the dominant entity in the protective immune response, as with FMDV. A continued study of the relative role played by cell-mediated immunity against FMDV, and the means of induction through vaccination should provide the necessary information for designing new vaccines generating a more compete immune defence. Immunological memory induction: in balance with effector immunity When effective immune defence has surmounted the antigenic and infectious threat from FMDV, the immune system enters the phase of regulation to dampen down activities within the innate and adaptive compartments. Concomitantly, the adaptive immune system develops a memory immune defence. This allows for a certain retention of the expanded antigen-specific lymphocyte clones, together with their increased reactivity derived from maturation – ‘fine tuning’ for recognition of the epitopes – in response to several rounds of antigen stimulation. By such means, there are more lymphocytes available for responding to a subsequent encounter with the virus, and their responsiveness is more specific and of higher affinity for the virus epitopes. This should be the main

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Figure 10.20 (a) SLA-dependency of FMDV peptide-induced specific lymphoproliferation. Anti-SLA class I (white bars) and -SLA class II (grey bars) MAb blocking of the lymphoproliferation obtained with peptide VP4–0 [20–34], compared with peptide in the absence of the MAb (black bars). The percentage of inhibition obtained with each MAb is shown. (b–e) Relative responses of cytotoxic CD4–CD8+ Tc cells (b), memory/activated helper CD4+CD8+ Th cells (c), non-Th/Tc CD4–CD8– cells (d), and naive CD4+CD8– Th cells (e), by measurement of IL-2 receptor (CD25) expression (dark grey histograms) in cultures stimulated with a BT tandem peptide. Light grey histograms show the CD25 expression in control unstimulated cultures. Adapted from Blanco et al. (2000).

aim of vaccination (Sallusto et al., 2010). There is both central memory – associated with lymph nodes, spleen and bone marrow – and peripheral memory – in dermal and mucosal tissues – enhancing the capacity of adaptive immune defences to respond in a rapid and specific manner for eliminating any future threat, before infection can create problems for the host. The influence of FcR signalling on effector versus memory immunity One important signal for the shift from active generation of effector cells to immunological memory

development is the replacement of antigen by antigen/antibody complexes (Kurosaki et al., 2010; McHeyzer-Williams et al., 2012). This is due to the progression of the effector immune defences into production of the antibody now reacting with the antigen. As mentioned above in ‘Effector humoral immune defence’, when low affinity FcγRII and FcγRIII on phagocytes bind antibody-complexed antigen, the cytoplasmic portion of the FcRs is modified, resulting in enhanced phagocytosis. An additional consequence of antibody/antigen complex formation is the interaction with the FcRs on B-lymphocytes (Kurosaki et al., 2010). In

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addition to FcγRII and FcγRIII involved in positive signalling of the cell, a second isoform of the FcγRII termed FcγRIIB is involved in a suppressive response (Nimmerjahn and Ravetch, 2007; Ravetch, 1997). These FcRs are also present on DCs, whereby their relative roles in induction and regulation of immune responses are found (Nimmerjahn and Ravetch, 2007). B-lymphocytes also carry FcαR for binding immune complexes of antigen with IgA. Similar to FcγRIIB, cross-linking of FcαR by immune complexes provides a suppressive signal (Nimmerjahn and Ravetch, 2007; Ravetch, 1997; Tsujimura et al., 1990). With the activating FcRs – such as FcγRIIA and FcγRIII – the outcome is ITAM activation in the cytoplasmic tail of the FcR, leading to a Syk kinase- and PI3 kinase-dependent activation pathways (Nimmerjahn and Ravetch, 2007). In contrast, ligation of the inhibitory FcγRIIB induces ITIM activation in the cytoplasmic tail of this FcR, leading to an SH2-domain containing inositol 5′ phosphate (SHIP)-dependent inhibitory signalling pathway (Nimmerjahn and Ravetch, 2007). FcγRIIB is involved in deletion and/or inactivation of self-reactive B-lymphocytes, in both the bone marrow and the periphery. With plasma cells, the FcγRIIB triggering induces apoptosis, thus controlling plasma cell homeostasis. An important structure on the antibody molecule influencing the selection of the FcR isoform for binding is the sugar moiety. For example, fucose in the antibody sugar moiety at asparagine residue 297 influences binding to human FcγRIII but not FcγRII (Nimmerjahn and Ravetch, 2007). In contrast, terminal sugar residues of sialic acid rather than galactose are important for regulating antibody activity in vivo. When these sialic acids are at high levels, impaired binding to FcRs is noted, but terminal sialic acids are still important as noted by their reduced levels in autoimmune diseases (Nimmerjahn and Ravetch, 2007). This is a combination of reduced binding of the sialic acid-rich antibodies to FcRs together with actively promoting an antiinflammatory response. It should be noted that induction of memory B-lymphocytes is not separable from activation of the B-lymphocytes into antibody-producing plasma cells. Memory development will occur during the active phase of antibody induction (Kurosaki et al., 2015; McHeyzer-Williams et al., 2012; Shlomchik

and Weisel, 2012; Takemori et al., 2014; Yoshida et al., 2010). Switching B-lymphocyte differentiation from antibody-producing, short-lived plasma cells to memory B-lymphocyte development (including long-lived plasma cells) involves modifications of the surface interactions between B- and Th-lymphocytes, and the cytokines involved. Activation via FcγRIIA or FcγRIII involves IFN-γ, TNFα and the complement product C5a as important cofactors; with FcγRIIB ligation, the cytokines TGF-β and IL-4 become more prominent cofactors (Nimmerjahn and Ravetch, 2007). In addition, immune complexes are also reported to influence memory cell development. When captured on the follicular dendritic cells (FDC), germinal centre B-lymphocytes are observed to scan these FDC when ‘laden’ with immune complexes (McHeyzerWilliams et al., 2012). Yet, this process appears to be more relevant to selection of high-affinity BCR-bearing B-lymphocytes, and thus refining the humoral immune defence. Indeed, memory B-lymphocyte generation can occur in the absence of antigen-containing immune complexes held by the FDC (Anderson et al., 2006). Memory B-lymphocytes Memory B-lymphocyte development can be driven by activated Th-lymphocytes (Kurosaki et al., 2015; McHeyzer-Williams et al., 2012; Shlomchik and Weisel, 2012; Takemori et al., 2014; Yoshida et al., 2010). This can lead to immunological memory development, either dependent or independent of the germinal centres; the independent processes can actually occur prior to germinal centre formation (Takemori et al., 2014). Memory B-lymphocytes can also be found outside the lymphoid organs, for example in the bone marrow wherein long-lived ‘memory’ plasma cells have been described (Manz et al., 1998; Yoshida et al., 2010). These cells are particularly important to effector immunity, with their long duration of antibody production, which may remain over a lifetime (Yoshida et al., 2010). They are distinct from the short-lived plasma cells in extrafollicular foci of antibody production (Kurosaki et al., 2015), seen by their elevated expression of anti-apoptotic Bcl-2 (Yoshida et al., 2010). An active effector process does not need to be particularly advanced before memory development is initiated. It has been suggested that within 3 days of immunization/vaccination, Th-lymphocytes

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interacting with processed antigen presented on DCs will interact with antigen-activated B- lymphocytes via CD40–CD40L interaction, determining the differentiation of the B-lymphocytes into memory or germinal centre cells for antibody production (Takemori et al., 2014). Memory B-lymphocytes can be found of IgM+, IgG+, IgA+ and IgE+ phenotype (Takemori et al., 2014). All show the characteristics of being long-lived, an essential quality for ensuring durable immunity, but it appears that IgM+ memory cells are more long-lived than the IgG+ cells (Kurosaki et al., 2015). It has been suggested that this reflects difference in their self-renewal activities. The IgG+ cells may require more regular BCR-mediated signalling, for example by FDCs acting as a depot for antibody–antigen complexes. There is also the reported influence of DC-derived BAFF and APRIL, which do not affect survival of the memory IgG+ cells, but the memory IgM+ cells may require these cytokines similarly to naive B-lymphocytes (Kurosaki et al., 2015). A major involvement in memory cell development is this CD40-CD40L cognate interaction and its durability. While durable T–B conjugates facilitate B lymphocyte differentiation into germinal centre B-cells, shorter conjugate formation is more likely to drive the B-lymphocytes into the germinal centre-independent memory cell pool; as for the germinal centre-dependent memory cells, these can derive from stochastic differentiation of germinal centre B-lymphocytes (Kurosaki et al., 2015). Therein, metabolic programming and autophagy appear to be major factors for the generation and maintenance of memory B-lymphocytes; expression of genes regulating autophagy initiation and autophagosome maturation are detected in these memory cells, leading to elevated levels of basal autophagy (Chen et al., 2014). Germinal centre-dependent memory B-cell development requires immunological help from the follicular Th-lymphocytes (Tfh-lymphocytes), as does germinal centre B-lymphocyte differentiation. While the germinal centre-independent memory B-cell development also requires immunological help, this is from Th-lymphocytes that are not Tfh-lymphocytes (Kurosaki et al., 2015; Takemori et al., 2014). An additional signal involved in the germinal centre-dependency of memory B-cell development is IL-21, signalling from which has

a major impact on germinal centre B-lymphocyte proliferation and differentiation into memory cells. An important distinction between the germinal centre-dependent and -independent memory B-lymphocytes is their affinity for interacting with antigen, and therefore responsiveness upon antigen re-encounter. The germinal centre-independent cells have lower affinity than the germinal centredependent cells (Takemori et al., 2014). Overall, the generation of different forms of memory B-lymphocytes affords the immune defences with a variety of responses upon antigen re-encounter. Regardless of the differences in their affinity of reaction, their relative persistence provides a durable response upon re-encounter with antigen or pathogen. The differences in affinity can facilitate broadening the responsiveness upon challenge with a modified pathogen, while the higher affinity IgG+ memory cells would require more self renewal through re-encounter. Both these characteristic are important in the context of vaccination, and should be analysed in terms of both the vaccine and adjuvant employed in any formulation. Memory T-lymphocytes As with the antigen-specific B-lymphocytes, clones of specific T-lymphocytes can be expanded by antigen presentation of DCs into both effector cells and memory cells. The Tfh-lymphocytes mentioned above as important for germinal centre-dependent B-lymphocyte responses, including memory, also differentiate into memory cells (Hale et al., 2015). The memory Tfh-lymphocytes can develop from the Th1, Th2 and Th17 lineages (Fig. 10.2). The cytokine profiles involved in the T-lymphocyte expansion from naive cells will define the memory subset to develop. IL-12 and Type I IFN are important for Th1 development, IL-4 for Th2 and IL-6 plus IL-1β and TGFβ for Th17. The presence of IL-1, IL-21 and ICOS (inducible co-stimulator) will influence this differentiation towards the memory context (Hale et al., 2015). Related to the extrafollicular B-lymphocytes, such as the long-lived memory plasma cells, there are also memory T-lymphocytes found in the tissues, inducing both Th- and Tc-lymphocyte subsets (Schenkel and Masopust, 2014). These tissue resident memory cells can be related to the central memory (Tcm) and effector memory (Tem) cells (Sallusto et al., 2010; Sallusto et al., 1999).

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The differential expression of chemokine receptors by these cells determines their localization in the body, and therefore their role in expanding immune responsiveness. The Tcm-lymphocytes are associated with secondary lymphoid organs, wherein they respond rapidly to antigen presented on DCs, differentiating into effector cells, which migrate to other sites of the body, including the tissues. Temlymphocytes behave more like the induced effector cells, responding to antigen by rapidly executing their effector functions. Akin to the activated Tcmlymphocytes, they may recirculate between blood and non-lymphoid tissues, but unlike the resting Tcm-lymphocytes they do not home to secondary lymphoid organs. Certainly, upon recall response to antigen the activated Tcm-lymphocytes can migrate from the lymphoid organs into the tissues, wherein they may well generate Tem-lymphocytes as well as the effector cells. Development of the memory T-lymphocytes is associated with antigen clearance, which is understandable for regulating an active response and providing the clonal expansion and affinity maturation to enable a more rapid recall response upon re-encounter with the antigen. The expression of PD-1 and ICOS receptors, together with up-regulation of FR4 found upon development of effector Tfh-lymphocytes from naive T-lymphocytes, is reversed as the cells develop into memory Tfh-lymphocytes(Hale et al., 2015). While these memory cells retain the CXCR5 expression of effector cells, the loss of CCR7 expression from the effector cells returns the memory phenotype. It has been concluded that understanding the derivation and preservation of memory Tfh-lymphocytes, and how they recall their gene expression programmes upon re-encounter with antigen is critical for formulating informative vaccination strategies (Hale et al., 2015). These authors proposed that analyses should determine how memory Tfh-lymphocytes could be modulated by vaccine immunisations, with the aim of enhancing the durability and robustness of immunological memory. Similar proposals have been made concerning better understanding of how vaccination influences the Tcm-lymphocytes and Tem-lymphocytes (Sallusto et al., 2010; Schenkel and Masopust, 2014). When provided for the Tfh-lymphocytes, there will be an extension to enhancing long-lived antibody responses, while combination of increased

knowledge on Tcm-lymphocytes and Tem-lymphocytes will expand vaccine efficacy into the realm of the effector T-lymphocyte immunity (Sallusto et al., 2010). Immune defence against FMDV: conclusions The induction of long lasting and rapid protective immunity is the primary aim for successful vaccination against infectious diseases. With respect to FMDV, the major arm of the immune defences involved in protection is that based on specific antibody together with the increased efficiency of macrophage phagocytosis and destruction of virus/antibody complexes. For the induction of effective immune defences against FMDV, the critical player therein is the dendritic cell (DC). DCs are the key controllers required for antigen presentation to T-lymphocytes and antigen delivery to B-lymphocytes, resulting in stimulation of the antigen-specific immune responses. In the context of vaccination, adjuvants play an important role in this process, particularly through induction of potent local cytokines and chemokines, which regulate the trafficking and function of DCs, MΦ and lymphocytes. Current vaccines are certainly efficient at interacting with DCs and initiating effective immune responses and defences against FMDV. Considerable detail is available on the actual epitopes on the virus involved in the antigen processing events for T-lymphocytes, and the antigen stimulation events for promoting the B-lymphocytes and antibody-based immunity. FMDV also impacts of innate cell activity through its interactions with these cells, particularly the MΦ and DCs. This latter area is critical for furthering our understanding of how vaccines can be employed to modulate DCs, promoting the most efficient immune defence possible. In this context, current vaccine formulations and vaccination protocols promote systemic immunity, but with a lack of mucosal immunity. This runs the risk of permitting airborne virus to establish a local infection in the dorsal soft palate, even with vaccinates. Generating mucosal immunity could circumvent such a threat. Although oronasal immunization is the classical method for such induction, it is not always a practical solution with livestock. Evidence has demonstrated how to

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generate mucosal immunity following parenteral immunization, and is a major area of current research in vaccinology. Further studies on DCs with respect to their targeting through vaccination and homing to mucosal surfaces, are major themes currently under scrutiny in the furtherance of studies on the immunology of efficacious vaccination. Consequently, successful vaccination leading to induction of efficacious immune defence provides for removal of the FMDV infectious threat. This requires initial interaction with specific antibody to form immune complexes, completed through phagocyte involvement, as well as DCs – particularly pDCs. Activating both humoral (B-lymphocyte) and T-lymphocyte immunity promotes effector immune defences, together with expansion of antigen-specific lymphocyte clones into memory cells. Not only do memory cells reside in the lymphoid organs wherein naive cells first responded to antigen, they also migrate into other sites of the body including the tissues. Thereby, immunological memory provides both expanded clones of antigen-specific cells and expanded immune defence network, with increased affinity of reaction with the antigen to promote rapid and durable immune protection. Accordingly, effective immunological protection requires a sequence of immune reactions: (i) Uptake of antigen (virus or vaccine) by DCs to (ii) process into antigenic peptides for association with MHC Class II molecules for (iii) activating antigen-specific Th-lymphocytes, which provide immunological help for (iv) antigen-specific Tc-lymphocytes activated by DCs presenting antigenic peptides in association with MHC Class I molecules, and (v) antigen-specific B-lymphocytes receiving antigen delivered by DCs, (vi) these activated B-lymphocytes differentiating into plasma cells producing antibody, which (vii) complexes with the virus (opsonization) – with or without complement involvement – providing (viii) modified antibody Fc and activated complement fragments, which in turn (ix) enhance phagocytosis of the virus by MΦ and

neutrophils via interaction of the complexes with cell surface FcRs or CRs, leading to (x) consequential signalling of the phagocytes to internalize the complexes, (xi) promoting endosomal association of the complexes together with maturation of the endosomal system to (xii) degrade the virus leading to destruction of its infectivity and therefore (xiii) immune protection, which is subsequently enhanced by (xiv) expansion of the antigen-reactive lymphocyte clones to provide (xv) more rapidly responding memory of both B- and T-lymphocytes, ensuring (xvi) maintenance of a rapid and long-lasting immune protection. Overall, these are the components that need to be considered in the context of vaccination and therefore development of efficacious vaccines and formulations with defined immunomodulatory adjuvants. References

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Laboratory Diagnostic Methods to Support the Surveillance and Control of Foot-and-mouth Disease

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Anna Ludi, Valerie Mioulet, Nick J. Knowles and Donald P. King

Abstract Foot-and-mouth disease (FMD) infects clovenhoofed livestock and is globally one of the most widespread epizootic animal diseases. FMD is difficult and expensive to control since the causative virus, FMD virus (FMDV), is highly contagious and spreads rapidly through susceptible animals. FMD remains endemic and impacts upon the rural livelihoods in many countries in Africa and Asia, from where it can spread to cause sporadic outbreaks in countries that are normally free from disease, such as the recent episodes that have occurred in East Asia (South Korea and Japan) and Europe (Bulgaria). These outbreaks reinforce the concerns about how readily the disease can pass across international borders, and stimulate the development and improvement of new assays for the detection and characterization of FMDV. Focusing on the recent revolution in molecular and sequencing technologies, this chapter reviews the range of approaches that are increasingly being employed by FMD Reference Laboratories to support national programmes and regional roadmaps for the identification, surveillance, control and eradication of FMD. Introduction Foot-and-mouth disease (FMD) is one of the most widespread epizootic transboundary livestock diseases, and is generally considered to be one of the most infectious animal diseases. FMD affects domestic cloven-hooved livestock (cattle, pigs, sheep, goats) and a number of wildlife species in the order Artiodactyla, including African and Asian buffalo, wild boar, deer, camelids and antelopes. The highly contagious nature of FMDV means

that the virus spreads rapidly through susceptible animals as was graphically illustrated during the epidemic that occurred in the United Kingdom in 2001. During an 8-month period, the virus spread to more than 2000 farms. The subsequent control and eradication of disease is estimated to have cost the national economy £8 billion, and resulted in the slaughter of more than 6 million animals (Scudamore and Harris, 2002). FMD outbreaks continue to occur in FMD-free countries such as in Miyazaki Prefecture, Japan, the Burgas Region of Bulgaria during 2010–11, and in South Korea during 2014– 16, where sporadic cases are still being reported. These outbreaks provide a constant reminder of the ease by which the disease can be spread and cross international borders, and highlight the continued risk of FMD introduction into FMD free countries. Accurate and timely diagnostic tests provide essential support to programmes that detect, monitor and eradicate the disease from an affected region or country. Indeed, data from the 2001 epidemic in the UK indicates that early recognition of FMD on farms is crucial, and is the single most important factor that can limit the size of subsequent ‘downstream’ outbreaks (McLaws and Ribble, 2007). The diagnostic process usually begins with field investigation carried out by farmers or veterinarians who are trained to recognize the typical clinical signs of FMD, and is followed by confirmation of the presence of infectious virus or FMDV-specific antibodies in samples using laboratory methods. Laboratory diagnostic confirmation is particularly important for FMD in small ruminants since clinical signs are often unapparent and diagnosis based solely on clinical observation can be difficult (Watson, 2004; Teifke et al., 2012). The routine laboratory-based assays used for FMD diagnosis

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include virological methods that detect virus, viral antigen or genome, and are supplemented by nucleotide sequencing methods used for strain characterization purposes. Serological approaches detect FMDV-specific antibodies; these methods inform on prior infection and vaccination status and are widely used for diagnostic and surveillance purposes. In addition to tests that are used in centralized laboratories, there is increasing emphasis on developing diagnostic technologies that can be deployed close to the animals with suspected clinical signs. These assays are variously referred to as ‘point-ofcare tests’ (POCT), ‘pen-side’, ‘portable’, ‘on-site’ or ‘field’ tests, though perhaps a more accurate generic term would be ‘point of decision’ tests. Recent developments in this area include simple-to-use lateral-flow devices (LFD) for the detection of FMDV, as well as new hardware PCR platforms and disposable isothermal assays for deployment into the field for use by non-specialists. Virological tests Virus-detection assays aim to detect FMDV in clinical or preclinical specimens collected from animals, and include (1) virus isolation in cell culture systems to propagate ‘live’ FMDV and other viruses (such as swine vesicular disease virus) causing signs of vesicular disease in susceptible species, (2) immunoassays such as enzyme-linked immunosorbent assays (ELISAs) to detect and characterize FMD viral antigen in clinical specimens and material from cell culture tests, and (3) molecular tests such as real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect viral genome (RNA). Biopsy samples (and fluid) collected from vesicular lesions represent the ideal material for virus detection assays and sequencing protocols, while the utility of tests for other types of clinical specimens such as blood/sera, milk, swabs and oesophageal– pharyngeal fluid ‘probang’ samples is governed by ‘diagnostic windows’ in the respective hosts (Alexandersen et al., 2003). Highly sensitive in-vitro cell cultures are used to isolate and propagate FMDV, and are widely considered to be the ‘gold-standard’ diagnostic test for FMD diagnosis; these include primary bovine thyroid cells (BTy; Snowdon, 1966) and

a permanent pig kidney cell line (IB-RS-2; De Castro, 1964). A goat tongue cell line (ZZ-R 127; Brehm et al. 2009) has also been shown to be highly sensitive to infection with wild-type and celladapted strains of FMDV, and has proved useful for isolation from a variety of clinical samples from animals experimentally inoculated with FMDV (Fukai et al., 2013). Molecular approaches are now being explored in attempts to develop improved cell lines that are suitable for FMD diagnosis. Work in this area has recently yielded a continuous bovine kidney cell line which stably expresses both subunits of αVβ6 integrin, the principal cell receptor for FMDV (LFBK- αVβ6: LaRocco et al. 2013). These cells were found to be more sensitive to infection with FMDV material of animal origin (vesicular fluid, tissue homogenates) than BHK, IB-RS-2 and MVPK cells. Although cell cultures systems can be highly sensitive, testing of samples is relatively slow (taking between 1–4 days), results can be influenced by the host-specificity of certain FMDV strains (depending upon the particular cell culture systems used), and are critically impacted by the quality of the individual specimens submitted to laboratories. Furthermore, the investment in sterile facilities and technical expertise required for the maintenance of cells (and regular sourcing of animals certified to be FMD-free in the case of BTy cells) makes virus isolation still inaccessible to the vast majority of local and smaller laboratories. Cytopathic effect in susceptible cells is not sufficient alone to provide a conclusive diagnosis of FMD. An antigen detection ELISA is typically used to assign the serotype and verify FMDV presence. These assays have lower analytical sensitivity compared to virus isolation when used to detect FMDV in clinical epithelial samples, and are not suitable for blood, milk and swab samples. Typically these assays utilize characterized polyclonal antisera in an antigen-capture (sandwich) format (Ferris et al. 1988); although review and production of new antibody reagents is required to detect new FMDV strains as they continuously emerge in endemic settings. Recent advances with the antigen-ELISA test have involved the use of a purified recombinant αVβ6 integrin as a trapper coupled with type-specific monoclonal antibodies (Ferris et al., 2005, 2011). Amplification of specific nucleic acid sequences using reverse-transcription polymerase chain reaction (RT-PCR) is now widely used for the laboratory

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detection of FMDV. Molecular assays are suitable for the diverse range of different sample types that might be submitted for laboratory investigation (including tissues, blood, swabs, oesophageal/pharyngeal (OP) scrapings, faecal samples and milk). Over the past 15 years, improvements have been made to RT-PCR protocols used for the detection of FMDV. Initially, assays that targeted conserved regions of the genome [3D: Meyer et al. 1991; Rodrıguez et al., 1994; and 5′ untranslated region (5′ UTR): Reid et al. 2000] utilized agarose gel electrophoresis for the detection of amplified products. However, these labour-intensive procedures carry a high risk of generating false positives due to carry-over of PCR amplicons and are therefore not generally considered ideal for routine testing of large numbers of samples. Real-time RT-PCR (rRT-PCR) assays have now largely replaced agarose gel based assay formats. These more rapid fluorescence-based approaches are highly sensitive enabling simultaneous amplification and quantification of FMDV specific nucleic acid sequences. In addition to enhanced sensitivity, the benefits of these closed-tube rRT-PCR assays over conventional endpoint detection methods include a reduced risk of cross-contamination and an ability to be scaled up for high-throughput applications. In common with the approaches used in other human and animal disease diagnostic laboratories, assays that exploit the 5′-nuclease (TaqMan®) system for routine diagnosis are the most widely used for FMD (Reid et al. 2002; Callahan et al. 2002). In order to increase assay throughput and minimize error associated with human operator, these assays can be automated using robots for nucleic acid extraction (Reid et al. 2003). Together with the implementation of quality assurance systems (such as ISO/IEC 17025), these improvements have increased the acceptance of the rRT-PCR assays as the front-line test for routine diagnostic purposes (Reid et al., 2009). Other real-time detection chemistries have also been explored including the use of modified MGB probes (Moniwa et al. 2007; McKillen et al. 2011), hybridization probes (Moonen et al. 2003), Primer-probe energy transfer (PriProET; Rasmussen et al. 2003) and RT-linear-after-the-exponential PCR (LATE PCR; Reid et al. 2010); however, these formats are not widely employed since they do not typically provide any tangible advantages over the well-established TaqMan® system.

As more viral genome data become available, there is now focus on the development of serotype (or lineage)-specific rRT-PCRs to supplement the pan-serotypic molecular tests used for FMD diagnosis. These tools can be used in FMDendemic countries for the identification of the serotypes of the causative virus strains, providing important information for vaccine selection, and for tracing the source of outbreaks. Conventional RT-PCR procedures using primers corresponding to the VP1 (1D) coding region and to the 2A/2B coding regions for serotyping of FMDV have been reported (Vangrysperre and De Clercq, 1996; Callens and De Clercq, 1997). However, subsequent studies have shown that these agarose gel-based RT-PCR assays have relatively poor sensitivity and specificity due to the genetic diversity within FMDV serotypes. While these procedures can be used in conjunction with Ag-ELISA and VI to provide additional information, they are insufficiently sensitive to replace them for primary diagnosis of FMD (Reid et al., 1999). A conventional RT-PCR procedure for differentiation of FMDV serotypes native to India using multiple primers based mostly on nucleotide sequences of viruses circulating in that geographical area was described by Giridharan et al. (2005). These studies demonstrated the potential for using tailored molecular tools to identify specific serotypes as an alternative or addition to pan-serotypic assays for detection of FMDV. More recently, lineage specific RT-PCRs and rRTPCR have been reported for South East Asia (Le et al., 2012), West EurAsia (Reid et al., 2014; Jamal and Belsham, 2015), and exotic viral incursions in North Africa (Ahmed et al., 2012; Knowles et al., 2014). Going forward into the future, it is possible to imagine that a ‘diagnostic tool-box’ of RT-PCR specific assays will be available for researchers and diagnosticians in the different global FMD endemic pools. Serological approaches Serological tests are widely used to monitor the immune status of animals exposed to FMDV or FMDV vaccines. Approaches used include enzyme linked immunosorbent assays (ELISAs) and virus neutralization tests (VNTs), although complement fixation tests (CFT) are still used in a limited number of laboratories. One particular application

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of these serological assays is to identify animals in a vaccinated herd that have been infected with FMDV. This so called DIVA (differentiating infected from vaccinated animals) principle exploits differences in the antibody (humoral) responses generated in vaccinated animals compared to those animals naturally infected with FMDV (whether or not they have been vaccinated). High-quality FMDV vaccines are purified to contain structural protein (SP) viral capsid components from which most of the viral non-structural proteins (NSP) have been removed. In contrast, during natural infection with FMDV, viral NSP are expressed that elicit a corresponding immune response that can be detected using diagnostic approaches (Fig. 11.1). There are a number of commercially available tests, and in-house assays that detect NSP-specific antibody responses including 3ABC, 2B, 2C, 3B, 3B2, 3D. The strength of the NSP antibody responses in individual vaccinated animals can vary according to the extent of virus replication. Therefore, when the comparative performance of five 3ABC assays and one 3B tests were evaluated (Brocchi et al., 2006), the ability of these tests to detect vaccinated animals that have been subsequently exposed to FMDV varied considerably (from 38% to 74%), although these sensitivity values were higher when only carrier animals were

Vaccinated

Vaccine: purified to remove NSPs

Vaccinated and infected

NSPs SPs Virus replication

Abs against Structural Proteins

Abs against Structural and NS Proteins

Figure 11.1 The principle of using non-structural proteins (NSPs) tests to differentiate between vaccinated and infected animals. Both structural (SP) and NSP antigens induce the production of antibodies in infected animals. In contrast, vaccinated animals that have not been exposed to replicating virus will only develop antibodies to the viral capsid (SP) antigens.

included in the analysis (48–89%). The specificity of all these assays in vaccinated cattle exceeded 96% (Brocchi et al., 2006). Study designs usually focus on younger animals ( 6 PD50) during emergencies, particularly in naive populations, while standard vaccines (up to 3 PD50) would mainly be recommended for use during regular vaccination programs. However, this distinction could be doubtful considering the low precision of the PD50 method (Goris et al., 2007). Since, according to dose–response studies, vaccines formulated with a higher antigen concentration result in higher potency (EMEA, 2004), this division may be more precise by determining the antigen mass. Studies in pigs demonstrated that after a single vaccination with oil based vaccine, neutralizing antibodies were first detected between 4 and 7 days after vaccination (Eble et al., 2004; Salt et al., 1998; J. Filippi, unpublished). Peak antibody levels were reported between 21 and 60 days post vaccination, which persisted for at least five to six months (Liao et al., 2003; Selman et al., 2006; J. Filippi, unpublished). Protection from FMD virus challenge was observed for as long as seven months post vaccination (Cox et al., 2003). In sheep and goats, high titres of neutralizing antibodies were detected after a one-dose vaccination with single and double oil emulsion vaccines at 15–60 days which persisted for more than 5 months (Cox et al., 2003; Hunter, 1996; Patil et al., 2002b; Selman et al., 2006; Späth et al., 2008). Regarding early protection and effect on virus transmission, animals vaccinated with emergency vaccines and exposed to FMD virus between 2 and 7 days after vaccination were protected from challenge, preventing virus transmission in ruminants and pigs. These responses have been extensively reviewed (Cox and Barnett, 2009). In addition, challenge studies in cattle performed at seven days post vaccination with standard vaccines demonstrated satisfactory protection. Moreover, no transmission was recorded either during the convalescent period or during the persistent state (Duffy et al., 2012; Golde et al., 2005; Quattrocchi et al., 2014). Similar experiments in calves demonstrated that vaccination seven days before FMD virus inoculation induced protection, blocked virus shedding and avoided environmental contamination by FMD virus secretions and excretions, thus preventing new infections (Bravo de Rueda et al., 2015). It should be noted that protection outcome may be influenced by the route of inoculation, virus dose,

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time of exposure (in case of contact challenge) and match of challenge (Cox and Barnett, 2009). Influence of maternally derived antibodies in the immune response Maternally derived antibodies (MDA) play a key role in the protective mechanism against infectious diseases during the early life of animals; however, they may partially suppress the response to early active immunity induced by vaccines. The quantity of MDA transferred to individuals varies greatly and depends on many factors, such as the degree of immunity of the mother and the quantity and time of uptake by the neonate after birth (EMEA, 2007). Early studies with aqueous FMD vaccines documented failure to induce antibody responses in calves with MDA (Graves, 1963; Nicholls et al., 1984). Conversely, later reports described the benefits of high quality oil vaccines for immunization of calves born to regularly vaccinated cows in the presence of high levels of MDA. In spite of certain suppression of the post vaccinal response due to MDA, vaccination of calves at early age prevented the decline in antibody levels. In fact, vaccination of 20- to 40-day-old calves induced antibody responses, resulting in the detection 3 months later of significantly higher levels than those in nonvaccinated calves (Spath et al., 1995). Other studies indicated that vaccinating 30- to 90-day-old calves born to systematically vaccinated cows resulted in high antibody levels for two months after first vaccination (Aznar et al., 2011). Secondary responses have been shown in boosted calves which received their first dose in the presence of high levels of MDA (Aznar et al., 2011; Smitsaart et al., 1996). Considering these results and from a practical point of view, the recommendation would be to vaccinate all the calves irrespective of age along with the whole herd, particularly in the case of extensive breeding areas. Moreover, taking into account that a first dose is able to prime the immune system, even in the presence of high levels of MDA, revaccination of animals before moving to fattening areas would be highly recommended in order to ensure a satisfactory immunity. This approach is mandatory in several countries in South America. In piglets born to FMD vaccinated sows, MDA had a suppressive effect on the early vaccination response, which may vary with the quality and

potency of the vaccine and with the titre of MDA present in the piglets at the time of vaccination. Early studies showed that this suppression was complete in 1-, 2- and 4-week-old piglets and partial in 8-week-old piglets (Francis and Black, 1986). Later studies demonstrated that MDA did not entirely suppress the antibody response induced by vaccination of 2-week-old piglets (Chenard et al., 2008). Additionally, vaccination at 10 days of age and revaccination at 60 days overcame the window of susceptibility to FMD derived from the drop in MDA, recording high levels of antibodies up to 6 months of age (Fondevila et al., 1996). Vaccination at 8 weeks induced protective levels of neutralizing antibodies that were maintained for the productive 24-week period, time at which protection against challenge was also registered (Liao et al., 2003). Vaccination of 30-day-old lambs or younger in the presence of MDA induced an antibody response that persisted at protective levels for at least up to 100–150 days of age (Cunliffe and Graves, 1970; Späth et al., 2008). Performance of FMD vaccine under field conditions The importance of routine mass vaccination programs to reduce and eradicate FMD is unquestionable but its success depends on a number of factors (Doel, 1999; McVey and Shi, 2010) related to: the quality, purity, potency and safety of the vaccine, and its match against the circulating viruses; immunity and health status of the host; the age of calves at first vaccination; intrinsic animal aspects, which may result in significant animal to animal variation; the operational aspects of the vaccination campaign including proper records of livestock to be vaccinated, vaccination regime and intervals between vaccination events; vaccine storage and distribution. Finally, the application of the vaccine should follow the recommendations of the manufacturer. In order to ensure the successful outcome of these multifaceted vaccination campaigns, the national services need to substantiate their effectiveness by demonstrating absence of viral activity (Bergmann et al., 1996, 2003c) and population immunity (León et al., 2014). In fact, these are part of the OIE requirements in order to apply for recognition of the FMD free status where vaccination

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is practised or to substantiate freedom from FMD infection after emergency vaccination (OIE, 2015t; Paton et al., 2006). Of major relevance for the confirmation of absence of viral activity has been the development and application over the past years of methods to distinguish infection regardless of vaccination (NCP or DIVA tests). The diagnostic strategy is based on the detection of antibodies against viral NCPs (wrongly named non structural) that take part during the replication process and that, in principle, are induced only during infection and not after immunization with conventional inactivated vaccines (Bergmann et al., 1993, 2000; Neitzert et al., 1991). Due to the conserved nature of these proteins, infection with any serotype of FMD virus can be detected with a single serological test. The NCP test system consists of a screening ELISA method that detects antibodies against the polyprotein 3ABC, followed by confirmation of suspect or positive samples by an enzyme linked immunoelectrotransfer blot assay that detects antibodies against five NCPs (Bergmann et al., 2000; Malirat et al., 1998). In Argentina, using this system, the testing of more than 23,000 serum samples/year of 12- to 24-month-old cattle under vaccination programme, during the period 2006–2011, revealed a very low percentage of reactive animals (Smitsaart et al., 2015), which fell within the specificity of the ELISA 3ABC/EITB diagnostic test system applied (Bergmann et al., 2003a). These data demonstrated and confirmed previous studies that the use of conventional purified vaccines did not interfere with the interpretation of the results of serological surveys, thus supporting the evaluation of viral circulation in the zone or country (Bergmann et al., 1996, 2000, 2003c). In terms of herd immunity, the goal is to confer immunity on a sufficient number of animals and farms to prevent the transmission of the virus if it is introduced in the population to be protected. Consequently, monitoring the levels of protection achieved after vaccination, in terms of both individual animals and farms, is essential to identify eventual failures of the campaign. Although it would be difficult to be prescriptive about the level of vaccination coverage required, it is generally accepted that reaching 80% herd immunity would be quite appropriate for field protection (OIE, 2015t).

The estimation of the proportion of protected animals and protected farms in vaccinated populations is based on the evaluation of antibody levels against specific structural proteins measured by a single dilution liquid phase competitive blocking ELISA (slpELISA) (Robiolo et al., 2010). As mentioned before, predetermined correlation tables associating probability of protection (referred to as EPP) and slpELISA titres are used to classify the samples as belonging to protected or unprotected animals, which will depend on a cut-off titre value specific for each FMD virus strain. Annually, the Argentine National Animal Health and Agri-food Quality Service (SENASA) evaluates the effectiveness of the mass vaccination campaign through the analysis of serum samples obtained using a stratified random design, usually collected before the next vaccination cycle (180 days after vaccination). Approximately 30,000 samples of 2250 herds were collected between 2005 and 2009, and analysed initially by the ELISA/EITB system to exclude animals with antibodies induced by eventual infection, so that only the antibodies induced by vaccination were measured. Subsequently, the negative ELISA/EITB samples were tested by slpELISA to detect antibodies against structural proteins of Argentina 2001, O1 Campos, and C3 Indaial strains. The results showed that vaccination coverage reached during consecutive vaccination campaigns is consistent with a level of protection necessary to sustain the status of FMD-free with vaccination (Cosentino et al., 2013). Safety performance During vaccine development, and in order to gain regulatory approval, safety tests should be carried out in the target species in order to record atypical local or systemic adverse reactions. For the case, such as in South America, where all vaccine batches are independently controlled in cattle by the official veterinary service, verification of absence of such reactions is performed in the same animals used for the vaccine potency and purity control tests. Also, as part of the evaluation of the vaccination programme, monitoring of post-marketing adverse reactions should be considered by the veterinary services. The apparent high frequency of systemic adverse effects after FMD vaccination seems more a myth than a reality. This paradigm could be explained

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considering that FMD vaccine is one of the most widely used biologicals in the veterinary market, so that in raw terms the frequency of adverse reactions seems to be high (Black, 1979). However, the rate of reported adverse reactions considering the number of vaccinations applied worldwide is negligible (Casas Olascoaga et al., 1999). In the 1970s, the application of aqueous vaccines produced a low incidence of postvaccinal allergic reactions of the immediate and delayed type (0.12 per 1000 doses administered in the Federal Republic of Germany during 1967 to 1970) (Lorenz and Straub, 1973) cited by (Black and Pay, 1975). More recently, the rate of anaphylactic reactions for W/O vaccines was even lower (1 per 5,000,000 doses administered in Argentina during 2007–2009) (A. Ham, unpublished). The vaccine components responsible of allergenic reactions remain uncertain. Nevertheless, it is speculated that they may be associated with antibiotics, BHK21 cell components of non-purified vaccines (Casas Olascoaga et al., 1999), viral proteins (Black, 1979) and bovine serum (Black and Francis, 1988). The unusually high anaphylactic reactions reported in 1984–1985 were detected mostly in multivaccinated dairy cattle and in black or white breeds (Casas Olascoaga et al., 1999). Regarding local reactions at the site of inoculation, oil vaccines are expected to produce a non pyogenic granuloma with presence of oil vacuolas (Rivenson et al., 1979). Important factors to minimize these effects are related to the quality of the mineral oil and surfactants, the antigen purity and concentration and the volume applied per dose (Aucouturier et al., 2001). In large scale studies in which cattle were vaccinated by different routes with W/O vaccines, a few animals presented small local reactions at the site of inoculation shortly after vaccination, which disappeared in the following 3–4 months (Martino et al., 2007; Rivenson et al., 1979). Regarding prevention of side effects, the use of appropriate vaccination practices together with the incorporation of new technological developments, particularly concerning adjuvants and downstream processing, are essential to ensure maximal vaccine performance with minimum side effects (Aguilar et al., 2012; Cai et al., 2014; Casas Olascoaga et al., 1999; Martinod, 1995). The loss of meat from the neck region in slaughterhouses has been associated

with injections that do not meet essential hygiene requirements and aseptic practices (Rebagliati et al., 2005). In pigs, DOE and W/O vaccines commercialized mostly in Asian countries have reported rare and insignificant side effects. In controlled studies, high quality of DOE and W/O vaccines proved to be safe in pigs with little local reactivity after primary intramuscularly vaccination (Barnett et al., 1996; Smitsaart et al., 2004). Vaccine matching The genetic/antigenic diversity of FMD viruses is well recognized (Alonso et al., 1987; Domingo et al., 1980; Hyslop and Fagg, 1965; Pereira, 1976; Rowlands et al., 1983; Sobrino et al., 1983) and often generates debates on the selection and efficacy of inactivated vaccines containing specific viral strains (Doel, 2003; Mattion et al., 2004). Infection or vaccination with one of the seven serotypes known worldwide (Arrowsmith, 1977; Pereira, 1981) does not confer protection against the others. Within each serotype new antigenic variants arise continuously, due to mutation and recombination (Arrowsmith, 1977; Brooksby, 1982; Domingo et al., 1990; Kitching, 2005; Knowles and Samuel, 2003; Mittal et al., 2005; Pereira, 1981) which may affect immune responses, and thus the ability of vaccines to effectively protect against heterologous strains of the same serotype (Brooksby, 1982; Cartwright et al., 1982; Mattion et al., 2004; Nagendrakumar et al., 2011; Upadhyaya et al., 2014). The emergence of variants deserves special attention under subneutralizing field conditions in which antigenically distinct viruses resistant to neutralization has been previously reported (Maradei et al., 2014; Maradei et al., 2011; Tami et al., 2003). Such situations usually occur in endemic settings, either as a consequence of poor vaccination coverage, or deficient vaccine efficacy which could be due to a poor match of the vaccine strain with the field virus. Although the significance of FMD virus diversity to escape protective immunity is not quite clear, the relevance of having an appropriate strain in the vaccines can be inferred from field evidence. For example, during the epidemic waves of serotype C in 1984 and up to 1986 that were registered in Argentina, the incorporation in the vaccine of the

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C85 strain greatly reduced the number of outbreaks (Bergmann et al., 1988). Another illustration can be drawn during the 2000–2001 emergencies of the Southern Cone of South America, which affected large cattle populations in already free regions which had stopped vaccination 14 months before. A rapid control of the disease was attained in Argentina through inclusion of the field virus (strain Argentina 2001) in the vaccines (Mattion et al., 2004), while Uruguay, having a considerable smaller cattle population, managed to compensate the antigenic differences between the vaccine strain and the field virus by revaccinating 30 days after first vaccination with the prototype vaccine strain A24 Cruzeiro (Sutmoller et al., 2003). Within the Middle East, a high rate of evolution in FMD virus and emergence of new sublineages of serotype A viruses during 1996–2011 has required the regular development of new vaccine strains typically every 5–10 years, highlighting the inadequacy of the serotype A vaccines used in the region (Doel, 2003; Upadhyaya et al., 2014). Epidemic waves of serotype O were registered in Ecuador up to the year 2011, despite having reported in the previous two years the greatest historical availability of vaccines containing the O1 Campos vaccine strain for the vaccination program, and over 90% coverage during the 6-month vaccination cycles. Characterization of the acting viruses indicated poor protection of the vaccine to the field viruses which could explain the rapid appearance of new strains and the co-circulation of various variants (Maradei et al., 2011, 2014). Another interesting experience which shows how a pre-existing vaccine strain can help to control an extensive outbreak was the use of the O1 Campos South American vaccine strain to aid in the control of a devastating epidemic in pigs of serotype O in Taiwan in 1997, after 68 years of being FMD free without vaccination. Vaccine matching tests revealed a good match between the O1 Campos and the acting virus O Taiwan 97, which were reflected in the effective control of the epidemic (Chen et al., 1999; Yang et al., 1999). Such examples clearly indicate the significance of optimizing the vaccine program by matching the vaccine strain to the field viruses as closely as possible and preferably including a strain covering a broad antigenic spectrum, so that vaccines are capable of inducing high levels of protection after

a single dose. This is pertinent not only for a rapid and effective response in case of an incursion in free regions, especially when large cattle populations are involved, but also in endemic settings. In the latter case often a relatively high percentage of young livestock, the most susceptible animals, are being vaccinated for the first time. Additionally, there may be isolated pockets of virus with the possibility of antigenic drift and selection of new variants resistant to neutralization under conditions of insufficient protection conferred by the vaccine strain, reinforced by the rapid waning of immunity which could occur when a vaccine strain matches poorly with the field virus (Elnekave et al., 2013). In this context, one of the main challenges for the successful application of the ‘vaccination to live’ policy is to establish when to develop a new vaccine strain. To this aim it is crucial to understand the unresolved, yet central issue, of the significance of FMD virus variability for heterologous protection, both pointing to routine prophylactic vaccination as well as to emergency use, scenarios for which matching requirements are not necessarily the same. A more broadly reactive strain may be more appropriate for systematic vaccination, whereas for emergency use a more precise match could be more suitable. This understanding is rather complex because of the many variables that may be involved in intratypic (viruses of the same serotype) protection. The different vaccine strains (i.e. serotype and variants) and the various vaccine formulations, two main determinants of vaccine efficacy, can alter the outcome (Doel, 2003; Paton et al., 2005). In fact, vaccine potency can, in part, compensate for relevant differences between a field and a vaccine strain (Brehm et al., 2008; Doel, 2003). In addition, the final result of a vaccination process is certainly affected by previous vaccinations, previous infections with the same serotype or with a different serotype. Effectiveness can vary depending on how successful vaccination programs are implemented and on how well a vaccinated animal responds to the vaccine in terms of making protective antibodies. Cattle under certain conditions may have weakened immune systems and may have a lower antibody response. Also, responses may differ for the different susceptible species.

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Vaccine matching tests Considering that the effectiveness of a vaccine depends largely on the suitability of the chosen vaccine strain, in terms of how closely it matches those viruses circulating in the field and also on its capacity to protect a wide range of variants (Doel, 2003; Paton et al., 2005), permanent awareness of the strains prevailing in the field, their genetic distribution/evolution and particularly, assessment of the probable efficacy of the vaccine strain in use to control the disease is of utmost importance. To this aim, samples collected from different episodes, regions and at various times of exposure need to be characterized. A first group of assays are generally oriented to determine the genetic and antigenic relatedness between the field samples with the vaccine viruses available, through sequencing of the VP1 or P1 region and monoclonal antibody profiling, respectively. Although genetic studies are relevant to detect quantitative/qualitative changes of the field viruses when compared to other epidemiological relevant regional and extraregional viruses and with the vaccine strain, it is not possible at present to predict the impact of specific amino acid changes on antigenicity. Consequently, the genetic results should be taken with caution when extrapolating between nucleotide or deduced amino acid differences and antigenic homology (Ludi and Rodriguez, 2013; Paton et al., 2005). In fact, it has been reported that quite distantly related isolates may have similar immunogenic characteristics (Barnett et al., 2001; Hernandez et al., 1992; Samuel et al., 1988). Conversely, very close sequence homology may mask large antigenic differences (Crowther, 1993; Maradei et al., 2014). Recent studies indicated that when structural information on the location of the amino acid sequence in the virus is added to the sequence data, a better prediction of antigenic relationships has been obtained (Reeve et al., 2010). Particularly molecular dynamics may be an important asset for these predictions (V. Malirat, unpublished). Regarding antigenic relatedness, profiling ELISA using a panel of MAbs has been reported as a rapid and sensitive way to monitor the emergence of antigenically different strains, assessing also relevant differences with the vaccine virus regarding neutralization sites (Alonso et al., 1993; Mahapatra et al., 2008; Samuel et al., 1991; Seki et al., 2009;

Yang et al., 2014), but the impact of the changes for protection was mostly not determined. When the results of the genetic/antigenic characterization of the field strains reveal the emergence of new variants with potential changes in immunogenic sites, additional studies need to be oriented to further assess antigenic/immunogenic relatedness with the vaccine viruses (Maradei et al., 2011; Mattion et al., 2004). These second group of assays can be performed with representative samples of field isolates which can be selected, for example from a genetic analysis and if pertinent, include samples from different species and locations or at different times. Humoral immunity is known to be the most influential factor in preventing FMD (Ahl et al., 1990; Barnett et al., 2003b; Pay and Hingley, 1986; Pay and Parker, 1977; Saiz et al., 2002; Smitsaart et al., 1998; Van Maanen and Terpstra, 1989). In fact, many studies have shown that there is a strong correlation between homologous protection from virus challenge and FMD virus antibody response of primo-vaccinated cattle, either measured by VN test (Ahl et al., 1990; Pay and Hingley, 1986, 1992; Sutmoller et al., 1984; Sutmoller and Vieira, 1980) or liquid phase blocking sandwich ELISA (LPBE), which, in principle, is considered to be at least as reliable and precise as the VN test (Ahl et al., 1990; Amadori et al., 1991; Goris et al., 2008b; Periolo et al., 1993; Van Maanen and Terpstra, 1989). Thus, in vitro assays are widely used as relatively good predictors of homologous protection for vaccine batch QC and to establish population immunity (León et al., 2014; Maradei et al., 2008). Regarding heterologous protection, in vitro serological methods can also be used to quantify antigenic/immunogenic differences and thereby, in principle, estimate the likely cross-protection between a vaccine strain and a field isolate. A first step is to establish antigenic/immunogenic relatedness by comparing the antibody titres of serum samples collected from vaccinated animals against both the vaccine strain and field virus (Brehm et al., 2008; Paton et al., 2005). Pools of reference antisera are prepared against each vaccine strain to be matched, so called bovine vaccinal serum (BVS). An indirect relationship value (r1 value) is calculated for each reference BVS (Alonso et al., 1987; OIE, 2015a; Paton et al., 2005), which is the ratio of

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the reciprocal heterologous (field virus) to homologous (vaccine strain) serum titres quantified by LPBE or VN assays (Brehm et al., 2008; Jangra et al., 2005; Mattion et al., 2009; OIE, 2015a). For either VN or LPBE tests, BVS are derived from cattle 21–30 days after inoculation with the vaccine to be matched. There have been controversies regarding the interpretation of r1 data in view of the many variables that can affect the outcome of r1 results. For example, information on the vaccine strains is not always available, since in some cases it is considered an important proprietary asset. Different manufacturers may have different versions of the same vaccine strain. Indeed, differences have been observed between the cross-reactivity of vaccine strains with the same name (Paton et al., 2005). In addition, there is no standardization concerning the preparation of BVS whose cross reactivity is to be measured. Different BVS preparations against the same vaccine strain can lead to markedly different r1 results (Kitching et al., 1988; Paton et al., 2005). In fact, BVS preparations can vary for example, in the number of vaccinated animals used, the dose and purity of the antigen given, the adjuvant, time after vaccination at which BVS was collected, the titre range against the homologous virus, etc. (Mattion et al., 2009; Paton et al., 2005). It has been shown that results derived from pooled sera were more consistent than when calculated based on the mean reciprocal serum titres (Brehm et al., 2008; Mattion et al., 2009). The OIE prescribes the pooling of at least five different serum samples (OIE, 2015a). In addition, low-titre sera are less suited for r1 value determinations and, in principle, an appropriate reference BVS might be a medium to high VN or LPBE titre serum (Mattion et al., 2009). Another source of inconsistencies for r1 value determination is that some tests have intrinsic variability so that various repetitions may be needed for full reliability (Rweyemamu, 1984). In addition, different laboratories may use different cell culture systems and/or various protocols with results that cannot be directly compared (Ahl et al., 1990; Barnett et al., 2003b). Consequently, results obtained by different laboratories may not be equivalent. In fact, cross-validation studies on r1 value determination between laboratories (FMD-DISCONVAC, 2013; OIE/FAO, 2012) showed huge variation between the laboratories and techniques used.

The mentioned limitations can be largely overcome by using distinctive and well-characterized cell culture systems, particularly to perform the VN tests, as well as consistent and controlled viral strains and BVS. In South America, a regional network of National Laboratories selects and characterizes the official vaccine strains used in the region. These strains are sent to the National Reference Laboratories for further distribution to vaccine manufacturers and for use in official batch control. In addition BVS, rabbit and guinea pig antisera are produced with unified protocols. In general, values of r1 > 0.4 or > 0.3 for LPBE assay (Ferris and Donaldson, 1992) and VN test (Rweyemamu, 1984), respectively, have been suggested as indicative of significant relatedness between tested strains, and thus that the vaccine is likely to protect the field virus (OIE, 2015a). While the mechanisms behind immune protection are certainly more complex than just humoral antibody responses, as already mentioned, a good correlation has been shown between in vivo homologous protection and antibody titres measured by VN or LPBE assays (Maradei et al., 2008; Pay and Hingley, 1987; Van Maanen and Terpstra, 1989). However, relatively few studies assessing the correlation between heterologous protection and r1 values are available (Aggarwal and Barnett, 2002; Barteling and Swam, 1996; Brehm et al., 2008; FMD-DISCONVAC, 2013; Maradei et al., 2011, 2014; Mattion et al., 2004; Nagendrakumar et al., 2011) and in overall such studies gave varying results. There are documented cases where cross-protection was found in spite of low r1 values (Brehm et al., 2008) and vice versa (Nagendrakumar et al., 2011). These controversies are expected considering the many variables that can affect r1 determinations added to the inherent variability of the r1 value, which were not taken into account in the mentioned studies. Moreover, it is known that the degree of the titre that relates with protection is not the same for different strains, so that using r1 determinations with fixed values to predict protection for the various serotypes would not necessarily be quite appropriate. Nevertheless, when r1 values were determined with standardized reagents and methods, such as is the case in South America, they were quite valuable to assess the immunological relatedness between vaccine and field strains and results were quite in

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line with the observed protection. Thus, r1 values when determined under consistent conditions and with standardized methods are quite relevant and give an important input together with antigenic and genetic characteristics on the need or not to perform studies to further determine cross-protection (Maradei et al., 2011, 2013, 2014; Mattion et al., 2004). A more direct and comprehensive estimation of the protective capacity of a vaccine against a field virus, widely used in South America, establishes the suitability of a vaccine strain based on the serum titres of samples derived from animals vaccinated with a particular vaccine, against the field viruses (Alonso et al., 1987). This method, known as EPP, directly relates the serological titres obtained by VN or LPBE assays to the likelihood that cattle would be protected against a challenge of 10,000 infective doses after vaccination, based on predetermined correlation tables associating antibody titres with homologous clinical protection against the vaccine strain. The higher the titre, the better suited the vaccine. Although the degree of the titre that relates with protection is not the same for different strains, in general titres over 2.1 and 1.6 for LPBE and VN assays, respectively, can be considered as protective for most vaccine strains, which in overall results in an EPP close to 75% which, when a group of 16 vaccinated animals are used, is an indication that the vaccines will protect against the field strain (Maradei et al., 2008; PANAFTOSA, 2001). This correlation may not be strictly valid under heterologous conditions. However, generally a curve for the new emerging strain would not be available and up to date heterologous EPP estimations seem to be in line with homologous in vivo protection (Maradei et al., 2011, 2013, 2014). At present no conclusive results are available regarding which test should be used to test crossprotection through serology. Although neutralizing antibodies are considered to better correlate with protection, non-neutralizing antibodies may also be protective (Dunn et al., 1998; McCullough et al., 1992). It has been suggested that other factors than neutralizing antibodies might have an impact on protection (Cox et al., 2003; Oh et al., 2006; Parida et al., 2006) and might be even more relevant in case of heterologous challenge (Brehm et al., 2008). Immunogenic analysis of field isolates in relation to vaccine strains, based on VN or LPBE tests,

suggested that for serotype A viruses, the VN assay seemed to be the preferred test for vaccine matching purposes (Brehm et al., 2008; Jangra et al., 2005; Mattion et al., 2009; Paton et al., 2005). A more recent study also for serotype A, showed that when compared with the VN test, not only were the LPBE results more capable to distinguish between the suitability of various vaccines to protect field viruses, they were also more reproducible, as they are not influenced by variations in tissue culture susceptibility (Tekleghiorghis et al., 2014a). Significant variation has been reported by using VN test (Rweyemamu, 1984; Rweyemamu et al., 1978), while lower variation was registered in the LPBE (Amadori et al., 1991; Van Maanen and Terpstra, 1989). In addition, LPBE can be used with inactivated antigens outside high security laboratories, the data can be obtained earlier and they are friendlier for validation and automation. In addition, LPBE can use smaller volumes of BVS which is usually available in limited amounts. A disadvantage of the LPBE method is that it is harder to standardize the virus antigen concentration used in the test. When the results of the genetic/antigenic/ immunogenic characterization of the field strains reveal a loss in the effectiveness of the vaccine virus to protect the field isolates, the final evaluation needs to perform the gold standard test, which is the most direct method to measure cross-protection. It consists in vaccinating the relevant target species and then to challenge them by exposure to the virus isolate against which protection is assessed (OIE, 2015a). This will take account of both potency and cross-reactivity. There are two direct methods commonly in use, the PD50 which uses groups of at least five cattle inoculated with different dose volumes of vaccines (Bolwell et al., 1992; Brehm et al., 2008; Goris et al., 2007; Willems et al., 2012) and the PGP assay which employs groups of 16 animals inoculated with undiluted vaccines, the latter approach being mainly used in South America (Goris et al., 2008a; Maradei et al., 2008; Periolo et al., 1993; Vianna Filho et al., 1993). Although these in vivo methods are closely linked to the FMD in-field situation, observation of PD50 results indicated a lack of dose–response relationship in a large number of tests, which complicated the interpretation of the results (Goris et al., 2007; Pay and Parker, 1977; Stellmann et al., 1968). In

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overall the PGP method, where the vaccine is used undiluted, proved to be more reliable (Vianna Filho et al., 1993) and a good indicator of the protection observed in the field (Goris et al., 2008a). In vivo cross protection approaches have many disadvantages from the standpoint of animal welfare and biosafety, since they require the use of live FMD virus and appropriate biosecurity procedures and practices. In addition, they are expensive, the number of challenge viruses that can be assessed is limited and results can only be obtained after more than a month which is a major limitation considering that the decision to vaccinate often will have to be made within days. Consequently, in most countries, official animal health services as well as the OIE experts have supported the use of alternative in vitro testing methods. In this context, the results of the indirect in vitro assays which in overall are in line with the ones observed in the in vivo challenge test (Maradei et al., 2011; Mattion et al., 2009; Robiolo et al., 2010) merit further validation and acceptance. As stated above, another important determinant of the protection that a vaccine will afford is related to potency. A highly potent vaccine that stimulates a strong immune response may give greater protection against a heterologous virus than a vaccine that stimulates a weaker immune response (Brehm et al., 2008). Furthermore, booster doses of vaccine can increase potency and the subsequent breadth of antigenic cover provided by a given vaccine, although the onset of full protection may be delayed. It has been shown that there is no significant effect of adjuvant on the range of the antibody response, neither for mixing of antigens nor for the route of administration (subcutaneous versus intradermal) (Tekleghiorghis et al., 2014b). In contrast, the breadth of the antibody response depends mainly on the vaccine strain. A 10-fold higher antigen dose resulted in approximately four times higher titres against all tested strains. Vaccine strain selection As mentioned before, the importance of having vaccine strains that are as immunogenic and cross reactive as possible and with proven protection against circulating viruses is a key issue not only for systematic vaccination programs but also for the incorporation to strategic FMD virus inactivated

frozen antigens for rapid formulation into vaccines for use in case of an emergency (i.e. antigen banks). In South America, vaccines are formulated with selected strains harmonized for use in the region (Allende et al., 2003), choosing those of broad antigenic spectrum, high stability and good adaptability to replicate in cell culture at an industrial scale. These strains are: O1 Campos, A24 Cruzeiro and most of the Southern Cone countries comprise also virus C3 Indaial. The variant Argentina 2001 is also included in vaccine formulations in Argentina (Mattion et al., 2004). The production strains are characterized and distributed by the official control laboratory at a national level. In other regions there is variable harmonization of vaccine strain use at national and regional level. Moreover, there is a lack of consistent information on availability and use of different vaccine strains mainly because of eventual conflicts of interest. The decision whether changes in the vaccine strains are pertinent is rather multifaceted and is an issue which involves multiple focuses to work together and effectively in order to attain a successful outcome. The input of the surveillance system is very important. Epidemiologists need to have a rather effective surveillance system in order to capture any viral activity and particularly an FMD incursion. In addition, they should have a rather good understanding whether the vaccine is being properly applied. Field veterinarians need to investigate outbreaks and collect proper and representative samples. As mentioned before, the laboratory needs to determine the characteristics of the variant involved and perform an algorithm of tests to infer to what extent a vaccine may protect a new field strain for the various objectives that the vaccine will be used. In addition, it should assess which strains elicit antibodies capable of neutralizing a broad range of field viruses which, in general, will be preferred as vaccine strains to those that induce responses of narrow specificity (it should characterize and identify new vaccine strains to cover major antigenic variants). Vaccine producers need to produce and supply the vaccine and, if pertinent, make available the vaccine strain as well as the corresponding panel of BVS in order to be able to perform the assays to evaluate heterologous immunity. In addition, once the laboratory identified a candidate field virus for use as a vaccine strain, the manufacturer should

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determine whether it meets the characteristics required for an effective production: ability to grow in BHK21 suspension cultures in adequate yield, virus stability before and after inactivation, immunogenicity/protection induced by the antigen once is formulated into vaccine, inactivation conditions, among others. Many times these characteristics are quite difficult to meet, demanding a lot of time, or even not achievable (Barteling, 2004; Doel, 2003), in which case it may be advantageous to use known vaccine viruses even when they do not quite match. Veterinary authorities should encourage the establishment of international cooperation as well as active collaborations among reference laboratories in order to be aware of the various vaccine producers, when possible, and should determine the vaccination policy and purchase vaccine for use or for stock in vaccine banks. The final assessment depends largely on the different scenarios in which the vaccines need to be applied, for example, large or small cattle populations, young or older animals, and on the objective of this evaluation meaning systematic vaccination or to stock in strategic antigen banks. Moreover, the choice of vaccine strain to be used will depend very much on circumstances. In an emergency situation it will not be feasible to immediately develop a vaccine strain from a field isolate but it may be possible to supply a closely matched strain if required. Moreover, as mentioned, many field strains are difficult to adapt to cell cultures or they may not be very stable. Also, after adaptations, their antigenicity may be changed and impaired. Another concern when selecting field strains for use as a vaccine strain is that it may not induce broad protection, so that field viruses have a possibility to escape the protection by vaccination and develop new variants. Therefore, the process of selection of a vaccine strain should also consider its capacity to neutralize a broad range of viruses. Higher potency vaccines and two vaccinations can compensate for moderate antigenic differences between field and vaccine viruses (Mattion et al., 2004; Sutmoller et al., 2003), but revaccinations will not compensate against very significant intraserotype variation (Dubourget et al., 1987). In such cases, it is advisable to make all possible efforts to develop a new vaccine strain as soon as possible.

Antigen and vaccine banks Vaccination is an important strategy that may be implemented to control FMD emergency situations. In this context, strategic reserves of ready-to-use vaccines and/or inactivated antigens, registered or licensed according to the finished vaccine, known as antigen and vaccine banks (A/V banks), are essential instruments for contingency plans. During recent decades A/V banks acquired a key strategic and tactical role mainly due to the worldwide expansion of FMD-free areas (with or without vaccination) and to the intensification of international trade as a result of globalization, which reinforced the need to implement prevention and contingency plans. Moreover, the augmented acceptance of vaccination to respond to incursions in free regions, known as ‘vaccination to live’ policy, clearly strengthened the requirement for a rapid availability of effective vaccines. The benefits of applying this vaccination policy as an alternative to large scale culling of animals is being widely recognized, partly because of the widespread opposition generated by the latter, due to animal welfare concerns. The criteria that determine the approaches for the implementation of A/V banks are usually defined by governmental entities. Generally, contracts are established with vaccine manufacturers to provide ready-to-use vaccines and/or services such as production and storage of concentrated antigens of relevant vaccine strains and final formulation of vaccines. An important feature of A/V banks is its flexibility to rapidly increase its production rate and to comply with all the steps to finish and deliver vaccines on time. These attributes can be easily met if the bank is installed in an operational plant, which ensures the availability of well-prepared staff, appropriate equipment, and updated and validated procedures, all frequently subjected to internal and external audits in terms of GMP and biosafety. Moreover, when high amounts of doses are rapidly required, the accessibility to already approved stocks of reagents, raw materials and packaging supplies is essential. This ease of access allows finishing the vaccine without delays derived from purchasing, delivering and QC. A successful example of this approach was the use of the Argentinian A/V bank, which was essential to control the widespread FMD epidemics in Argentina in 2001. This case illustrates

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how a single bank assisted an emergency involving a herd of 60 million head of cattle and how it contributed to control the outbreaks in less than a year (Mattion et al., 2004). Recently, the concept of an international diagnostic bank to cover the demand for diagnostic reagents that may be rapidly needed in case of an emergency has been considered. The idea, conceptually similar to that of vaccine banks, proposes that several countries combine their diagnostic resources which would then be available to members in the case of an emergency (EU, 2010). Valuable information to design the requirements for A/V banks and eventually of diagnostic stocks is provided by risk assessment studies of the country or region considering herd at risk, epidemiological situation, and disease status of neighbouring areas. To this aim, FMD A/V banks are expected to have a dynamic and active cooperation between different international, regional and national veterinary services, regulatory agencies, reference laboratories, vaccine manufacturers, academic institutes and with other related stakeholders. Among other benefits, these collaborations allow becoming aware of emerging strains that need to be incorporated in the banks, and facilitate the harmonization among national regulatory agencies, of the requirements for vaccine authorization for use during FMD emergency situations, which should be established at least regionally. It should be noted that in the context of the risk of bio-terrorism, disease control authorities may consider it pertinent to restrict the release of information related to the storage of specific stockpiles of antigens and/or vaccines. Financial resources are needed to support A/V banks and strategic programs must be maintained and renewed by solid and long-lasting mechanisms. The OIE Manual (OIE, 2015a) describes the international standards for A/V banks along with the guidelines for storage and monitoring of concentrated antigen. A/V Banks are being increasingly required. Therefore, many countries have access to them either through a single contract with a vaccine manufacturer or as a member country of an international bank. Thus, they can have immediate availability of vaccines, regardless of world demand. The North American Vaccine Bank (NAFMDVB) was installed in 1982 for the USA, Canada and Mexico (Forman and Garland, 2002) and the

European Union (EU) established the EU vaccine Bank in 1992 (Barnett et al., 2010; Lombard and Fussel, 2007). In South America, Argentina was the first country to create a National A/V Bank through signing a contract with a vaccine manufacturer (Smitsaart et al., 2002). Later this manufacturer was awarded as a supplier of the NAFMDVB and, additionally in 2011, the USDA/APHIS issued a permit for importation and distribution of the Argentine local FMD vaccine (Bioaftogen®) to be used in case of emergency in the United States (Roth and Spickler, 2014). In addition, and within the framework of the South American Commission for the Fight against Foot-and-mouth disease (COSALFA), there has been progress towards the participative establishment of a regional A/V bank (COSALFA, 2015). The OIE World Fund has worldwide experience in the management of vaccine banks and the delivery of vaccines including vaccines for FMD. In this way, the OIE supports the provision of vaccines free of charge to developing countries destined to vaccinate target animal populations at risk and also to progressively achieve eradication (OIE, 2015s). An important progress has been the creation of a network of FMD vaccine banks (International Vaccine Strategic Reserves Network -IVSRN-(Barnett et al., 2010; Palma, 2004), which was endorsed by the OIE and FAO. The goal of the Network is to achieve, through mutually acceptable mechanisms, exchange of information and materials relevant to vaccine banks. Chief Veterinary Officers of QUADs (Australia, Canada, USA, New Zealand) are committed to support the network (Hickey, 2015). Ready-to-use vaccines Ready-to-use vaccines have the advantage of their rapid availability for immediate use during its lifetime. In this context, and as part of the contingency plan, veterinary services may choose to use already formulated polyvalent or monovalent vaccines as a primary barrier to prevent the spread of the disease. Contracts with companies that already have an on-going production and sales in countries that practise vaccination are highly desirable (Roth and Spickler, 2014). The application of ready-to-use vaccines was an important adjunct for the initial control of the emergency outbreaks which occurred in already free regions of the Southern Cone of South America

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during 2000–2001 (Mattion et al., 2004; Sutmoller et al., 2003). Storage of concentrated inactivated antigen As an alternative to ready-to-use vaccines, a more commonly adopted approach consists of stockpiles of concentrated inactivated antigens which, in general, can be stored over liquid nitrogen for a very long time for subsequent formulation into vaccine (Barnett and Statham, 1998). This strategy has economic benefits and avoids continually replacing vaccines that go beyond their shelf life. In addition, it has the advantage of allowing the adjustment of antigen payload according to the potency and amount of doses required (Lombard and Fussel, 2007). Quantification of entire virus particles, antigen integrity, safety and sterility controls must be performed before freezing and some of them are repeated on thawed aliquots at regular time intervals for stability testing. For the approval of the concentrated antigens, a vaccine formulated at a laboratory scale with a representative fraction of the antigen batch must pass final product testing. It is recommended to prepare a new laboratory scale vaccine to verify potency every 5 years after freezing. The mentioned controls are quite relevant because in special situations when the vaccine is required within a short period of time, sanitary authorities may agree to release the vaccine provided that it is manufactured with the same antigen content and formulae as the vaccine previously produced at a laboratory scale, prepared for the approval of the antigen concentrate (EMEA, 2004). In order to gain extra confidence, it may be desirable to verify the quality of the final finished vaccine through rapid in vitro tests, such as those described in ‘In process and final product control’. Regarding stability, oil vaccines formulated with concentrated antigens show similar shelf life as conventional vaccines prepared with freshly manufactured antigen (18–24 months) (Barnett et al., 2003a; A.M. Espinoza, unpublished). Viral components of A/V banks As for the strains to be included in the banks, it has been recommended to group the vaccine strains in three levels of priority. The classification was mainly based on the antigenic spectrum covered by the

vaccine strains available, assessed by in vitro matching tests between them and recent field isolates (OIE/FAO, 2013). In any case, as mentioned, each A/V reserve defines which vaccine strains should be stockpiled as concentrated inactivated antigen on the basis of risk assessment. Master seed-stock collections constitute an important support for the inactivated antigen banks. These collections allow for more alternatives of vaccine strains to be rapidly available for use in the event of new emerging viruses, without the delays resulting from the adaptation of new isolates to tissue culture (EU, 2010). In addition, they provide financial benefits. Nevertheless, regulatory and legal issues need to be addressed to ensure that vaccines derived from master-seed stocks are rapidly authorized in order to allow its application in case of a FMD event. In the case of an emerging strain, with relevant immunogenic differences from existing vaccine viruses, the development of a new vaccine virus from a representative field isolate could be required (see ‘Vaccine strain selection’). In this regard, the A/V bank should also have the technical capacity to adapt field strains to cell cultures, and generate master seeds according to international standards. In order to avoid delays derived from the approval of vaccines produced with the new antigens, the preparation of the vaccine using an intermediate antigen batch has been considered. This preparation can be used for potency testing while the whole antigen batch is being manufactured (EMEA, 2004). In this context, the use of in vitro methods for in process and final vaccine control can save considerable time and provide a good approximation on vaccine effectiveness (see ‘In process and final product controls’). Regarding the approval of vaccines produced with these emergent variants, the EU has proposed that in order to facilitate the authorization process, the concept of ‘mock up authorization’, introduced by human flu vaccines could be applied. This approach consists on the approval of the vaccine formulation with a specific strain along with limited clinical data (EU, 2010). In case of an epidemic with a different strain, regulators can assess data with the new strain, resulting in a brief authorization process. In summary, A/V banks should fulfil at least the following conditions:

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• high operational capacity with procedures and facilities that meet regulatory requirements; • high capacity for storing frozen concentrated antigens and continuous evaluation of its stability; • high capacity to formulate vaccines as well as flexibility to deliver in short time the finished products produced either from stored antigens or from freshly manufactured antigens; • supply of tools for diagnosis and for postoutbreak serosurveillance (viral activity and herd immunity assessment) • technical capacity to monitor FMD virus strains and vaccine strain selection; • cooperation and collaboration with reference laboratories, international A/V banks, regulatory agencies and academic sector. Concluding remarks There is no doubt that vaccination with conventional high-quality FMD vaccines, properly and extensively applied, can be used both to eradicate endemic FMD and to contain and to eliminate outbreaks that occur in free countries or zones. This is a result of the technical evolution achieved regarding equipment, quality of raw materials, new adjuvants, new purification processes and the use of reliable methods, which allowed manufacturing inactivated vaccines with high level of confidence in safety, purity and potency. An important contribution was also the introduction of improved methods for vaccine QC, along with increasing regulatory requirements, application of GMP rules, independent control by the veterinary services and systematic audits of vaccine plants. Another significant input was the optimization of the management of the vaccination campaigns controlled by the veterinary services, being the farmer’s participation of great value for the success of the control programs. In line with the 3R concept, there were also significant progresses to replace in vivo tests by in vitro methods. The establishment of A/V banks became quite relevant for FMD free countries with or without vaccination. The establishment of a shared diagnostic kit bank should be considered. The role of regional reference laboratories needs to be reinforced, particularly in regions with weak

veterinary services. This would be extremely valuable to provide an updated and reliable knowledge of the circulating strains and to ensure the quality of the vaccines in use and their performance. Post-marketing monitoring to confirm the vaccine safety and effectiveness, together with serosurveillance to assess herd immunity and viral activity, is highly desirable to give public confidence. Acknowledgements We are grateful to M. Spitteler, R Bellinzoni, A. Suárez, F. Barroumeres and A.M. Espinoza for helpful suggestions on the manuscript. References Abaracón, D., Alonso Fernández, A., Magallanes, N., Charles, E.G., and Durini, L.A. (1980). Protection of cattle following vaccination with oil-adjuvanted foot-mouth disease vaccine. Bol Cent Pan Fiebre Aftosa 37–38, 45–47. Abaracón, D., and Giacometti, H. (1976). Vaccines against foot-and-mouth disease in virus produced in cell cultures with bovine serum treated with polyethyleneglycol (PEG). Bol Cent Pan Fiebre Aftosa 21–22, 44–53. Abaracón, D., Mesquita, J.A., Giacometti, H., Sallúa, S., and Pérez Rama, R. (1982). Formulation of oil-adjuvanted foot-and-mouth disease vaccines containing antigen adsorbed to aluminum hydroxide. Bol Cent Pan Fiebre Aftosa 45–46, 43–50. Aggarwal, N., and Barnett, P.V. (2002). Antigenic sites of foot-and-mouth disease virus (FMDV): an analysis of the specificities of anti-FMDV antibodies after vaccination of naturally susceptible host species. J. Gen. Virol. 83, 775–782. Aguilar, N.M., Rossner, M.V., and Balbuena, O. (2012). Manual práctico de bienestar animal: recomendaciones para su implementación en el manejo de bovinos de producción, 1a edn (Argentina: Instituto Nacional de Tecnología Agropecuaria). Ahl, R., Haas, B., Lorenz, R.J., and Wittmann, G. (1990). Alternative potency test of FMD vaccines and results of comparative antibody assays in different cell systems and ELISA. Rep Res Gr Eur Com Contr FMD Lindholm (Denmark), 51–60. Alonso, A., Casas Olascoaga, R., Astudillo, V., Söndahl, M.S., Gomes, I., and Vianna Filho, Y.L. (1987). Updating of foot-and-mouth disease virus strains of epidemiological importance in South America. Bol Cent Pan Fiebre Aftosa 53, 11–18. Alonso, A., Gomes, M.D., Ramalho, A.K., Allende, R., Barahona, H., Söndahl, M.S., and Osorio, F.A. (1993). Characterization of foot-and-mouth disease virus by monoclonal antibodies. Viral Immunol. 6, 219–228. Allende, R.M., Mendes da Silva, A.J., and Comparsi Darsie, G. (2003). South American Standards for foot and mouth disease vaccine quality In Foot-and-mouth disease: control strategies, B. Dodet and M. Vicari, eds. (Paris, France: Elsevier SAS), pp. 331–336.

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Amadori, M., Archetti, I.L., Tollis, M., Buonavoglia, C., and Panina, G.F. (1991). Potency assessment of foot-and-mouth disease vaccines in cattle by means of antibody assays. Biologicals 19, 191–196. Arrowsmith, A.E.M. (1977). A survey of foot-and-mouth disease type O strains from the Far East. Dev. Biol. Stand. 35, 221–230. Aucouturier, J., Dupuis, L., and Ganne, V. (2001). Adjuvants designed for veterinary and human vaccines. Vaccine 19, 2666–2672. Augé de Mello, P., Astudillo, V., Gomes, I., and Campos García, J.T. (1975). Field application of inactivated oil adjuvanted foot-and-mouth disease virus vaccine: vaccination and revaccination of young cattle. Bol Cent Pan Fiebre Aftosa 19–20, 39–47. Augé de Mello, P., Costa, K.F., Alonso, A., Sutmöller, P., Pollak, A., and Millán, A. (1980a). Influence of the degree of dispersion in the aqueous phase on the immunogenicity of oil-adjuvanted foot-and-mouth disease vaccine. Bol Cent Pan Fiebre Aftosa 37–38, 11–15. Augé de Mello, P., and Gomes, I. (1977). Anamnestic response in cattle after revaccination with oil adjuvanted foot-and-mouth disease vaccines. Bol Cent Pan Fiebre Aftosa 27–28., 55–60. Augé de Mello, P., Sutmöller, P., Costa, K.F., and Millán, A. (1980b). Persistence of antibody response after revaccination with oil-adjuvanted foot-and-mouth disease vaccine: short communication. Bol Cent Pan Fiebre Aftosa 37–38, 39–40. Aznar, M.N., León, E.A., Garro, C.J., Robiolo, B., Filippi, J., Osacar, G., Walsh, M., and Duffy, S.J. (2011). FMD vaccination response on calves with colostral antibodies. In XV ISAH International Congress on Animal Hygiene, J. Köfer, and H. Schobesberger, eds. (Vienna, Austria), pp. 491–493. Bahnemann, H.G. (1972). The inactivation of foot-and-mouth disease virus by ethylenimine and propylenimine. Zentralblatt Veterinarmedizin Reihe B 20, 356–360. Bahnemann, H.G. (1990). Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine. Vaccine 8, 299–303. Bahnemann, H.G., and Mesquita, J.A. (1987). Oil adjuvanted vaccine against foot-and-mouth disease. Bol Centr Panam Fiebre Aftosa 53, 25–30. Barnett, P., and Statham, R.J. (1998). Long term stability and potency antigen concentrates held by the International Vaccine Banks. Rep Res Gr Eur Com Contr FMD Aldershot (United Kingdom) Appendix 38, 272–275. Barnett, P.V., Bashiruddin, J.B., Hammond, J.M., Geale, D.W., and Paton, D.J. (2010). Toward a global foot and mouth disease vaccine bank network. Rev. Off. Int. Epizoot. 29, 593–602. Barnett, P.V., Cox, S.J., Statham, R.J., and Aggarwall, N. (2003a). Progress on the use of high potency emergency vaccines. In Foot-and-Mouth Disease: Control Strategies, B. Dodet and M. Vicari, eds. (Paris, France: Elsevier SAS), pp. 273–286. Barnett, P.V., Pullen, L., Williams, L., and Doel, T.R. (1996). International bank for foot-and-mouth disease vaccine: assessment of Montanide ISA 25 and ISA 206,

two commercially available oil adjuvants. Vaccine 14, 1187–1198. Barnett, P.V., Samuel, A.R., and Statham, R.J. (2001). The suitability of the ‘emergency’ foot-and-mouth disease antigens held by the International Vaccine Bank within a global context. Vaccine 19, 2107–2117. Barnett, P.V., Statham, R.J., Vosloo, W., and Haydon, D.T. (2003b). Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach. Vaccine 21, 3240– 3248. Barteling, S.J. (1976). Use of polyethyleneglycol-treated serum for animal cell cultures. Dev. Biol. Stand. 37, 91–95. Barteling, S.J. (2002). Development and performance of inactivated vaccines against foot and mouth disease. Rev. Off. Int. Epizoot. 21, 577–588. Barteling, S.J. (2004). Modern Inactivated Foot-and-Mouth Disease (FMD) Vaccines: Historical Background and Key Elements in Production and Use. In Foot and Mouth Disease: Current Perspectives, F. Sobrino, Domingo, E., ed. (Great Britain: Horizon Bioscience), pp. 305–333. Barteling, S.J., and Meloen, R.H. (1974). A simple method for the quantification of 140S particles of foot-and-mouth disease virus (FMDV). Arch. Gesamte. Virusforsch. 45, 362–364. Barteling, S.J., and Swam, H. (1996). The potent aqueous and double oil emulsion foot-and-mouth disease type O1 vaccines from European Vaccine Banks probably protect against all other O1 strains. Rep Res Gr Eur Com Contr FMD Kibbutz Ma’ale Hachamisha (Israel), 90–96. Beck, E., and Strohmaier, K. (1987). Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. J. Virol. 61, 1621–1629. Bellinzoni, R., Magi, N., Régulier, E.G., Romo, A., and Spitteler, M.A. (2015). High Throughput Quantification and Characterization of Foot and Mouth Disease Virus and Products thereof (International application No. PCT/IB2015/054280, International filing date 05 June 2015). Bellinzoni, R.C., Levy, M.S., Régulier, E.G., Romo, A., Smitsaart, E., and Spitteler, M.A. (2012). Cuantificación exacta de partículas enteras de VFA mediante un método cromatográfico de exclusión molecular. (Argentine Patent Application AR085877A1. 24–05–2012). Bergmann, I.E., Astudillo, V., Malirat, V., and Neitzert, E. (1998). Serodiagnostic strategy for estimation of foot-and-mouth disease viral activity through highly sensitive immunoassays using bioengineered nonstructural proteins. Vet. Q. 20 Suppl 2, S6-9. Bergmann, I.E., de Mello, P.A., Neitzert, E., Beck, E., and Gomes, I. (1993). Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens. Am. J. Vet. Res. 54, 825– 831. Bergmann, I.E., Malirat, V., Dias, L.E., and Dilandro, R. (1996). Identification of foot-and-mouth disease virus-free regions by use of a standardized enzyme-linked immunoelectrotransfer blot assay. Am. J. Vet. Res. 57, 972–974.

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Peptide Vaccines Against Foot-and-mouth Disease Esther Blanco, David Andreu and Francisco Sobrino

Abstract Foot-and-mouth disease virus (FMDV) has been one of the pioneering systems in the development of synthetic peptide vaccines. Protection against FMDV infection is associated with the induction of neutralizing antibodies in the host species. The presence of a continuous B-cell epitope in a loop of capsid protein VP1 prompted its use in peptide constructions that elicited high levels of neutralizing antibodies in laboratory species. Nevertheless, this first generation of linear peptides conferred limited protection in natural hosts, reflecting the difficulties inherent to reproducing the immunogenicity of an entire virus by a simplified replica, such difficulties including lack of adequate T-cell epitopes to address MHC class II polymorphism, or the inefficient presentation of B-cell epitopes to the immune system. In this chapter we show how these challenges can be quite successfully overcome by a new generation of peptide vaccines that integrate B- and T-cell epitopes –the former in multimeric presentation– into a single molecular platform conferring solid protection against FMDV infection. Limitations of classical inactivated vaccines prompted the search for new immunogens Current vaccines are made of chemically inactivated whole virus preparations that are emulsified with adjuvants prior to intramuscular administration. These conventional vaccines have allowed the control of the disease in developed countries provided they are properly produced, stored and administered and that field-matched vaccine strains

13

are used for their formulations (see Chapter 12). Despite its wide use, immunization with chemically inactivated vaccines has disadvantages (Fig. 13.1) such as the need of a cold chain to preserve virus stability, the risk of virus release during vaccine production, the requirement of virus passage in tissue culture, not efficient for all field viruses and liable to select for antigenic variants, and the problems for serological distinction between infected and vaccinated animals (Barteling, 2004; Rodriguez and Gay, 2011). These drawbacks led the EU to adopt a non-vaccination policy for disease outbreaks in FMDV-free countries that relies on slaughtering of infected and contact herds and

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Inactivated whole virus vaccines + Good immunogenicity - Risk of virus escape - Low stability (cold chain required) - Limited immunogenicity (annual re-vaccination required) -

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- Antigenic variability (need for vaccine strain matching) - Polemicin the distinction infected/vaccinated animals

Figure 13.1  Advantages and disadvantages of chemically inactivated conventional current vaccines.

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strict limitations on animal movements and trading in case of viral outbreaks. As commented in Chapters 1 and 12, such FMDV re-emergences have caused massive and controversial culling of affected and suspected farm animals (Kitching et al., 2007; Sobrino and Domingo, 2001) that finally led the World Organization for Animal Health (OIE) to adopt a new ‘vaccination to live’ policy that considers the use of vaccines in response to FMD emergence in previously disease-free countries, as a way to reduce large-scale animal culling for disease control. Related to this scenario, much effort has been invested over recent decades in the search of safe and effective alternatives to conventional vaccines (Barteling and Vreeswijk, 1991; Brown, 1988; Grubman, 2005; and Chapter 14), peptide-based vaccines being one of the main approaches in this regard (Barteling, 1988; Cao et al., 2016; Purcell et al., 2007; Sobrino et al., 1999). In this chapter, we review early work leading to the use of a main FMDV B-cell linear epitope as a vaccine candidate, the limitations of this firstgeneration peptides as immunogens, as well as the much improved results obtained with multimeric (branched) peptides and their potential as new FMDV vaccines.

General features of the FMDV-specific immune response Protection against FMDV has been related to antibody-mediated compartments in both animal models and natural hosts (McCullough and Sobrino, 2004), as extensively addressed in Chapter 10. Inducing high titres of virus-specific antibody can relate to protection against challenge infection, although this relationship is not absolute since animals displaying similar titres of specific antibody can differ in resistance to FMDV infection. This is consistent with effector humoral immunity involving more than antibody and with the phagocytic system being necessary for removing antibody/virus complexes and destroying the virus (McCullough et al., 1992). The main antigenic sites recognized by B lymphocytes correspond to defined structural motifs exposed on the capsid surface, with amino acid sequences that accumulate variations among different serotypes and, within serotypes, among different viral isolates (see Chapter 4 and Acharya et al., 1989; Mateu, 1995). Among these motifs, the continuous immunodominant B-cell site located in the GH loop, around positions 140–160 of capsid protein VP1 (Fig. 13.2), has been extensively used

VP1 G-H loop (site A)

Site C Site D Site A

Continuous B cell site A

Integrin-Binding Motif (RGD)

Figure 13.2  Representation of the B-cell antigenic sites on the capsid of FMDV type C. Sites are as in (Mateu et al., 1994). On the right: a detail of the VP1 G-H loop in which the RGD triplet that mediates virus binding to molecules of integrins – which act as cell receptors – is depicted.

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to elicit neutralizing antibodies by various immunization approaches such as synthetic peptides (see below), recombinant proteins enclosing tandem copies of this sequence produced in different expression systems (reviewed in Brown, 1992; Domingo et al., 1990), or inclusion at permissive locations within heterologous virus-like particles such as those of the hepatitis B core (Clarke et al., 1987). For these and other reasons FMDV is regarded as an attractive, challenging model to study the requirements for B and T-cell stimulation resulting in efficient protective responses against such a highly variable virus (see Chapter 7). Induction of specific immune response involves recognition of B-cell epitopes inducing specific antibody production following antigen processing and presentation in the context of MHC class II molecules ensures stimulation of helper (Th)-lymphocytes to produce growth and differentiation factors necessary for development of the immune response (Fig. 13.3). Protection against FMDV in natural hosts has been achieved upon immunization with different vaccine strategies that did not elicit consistent levels of neutralizing antibodies (Borrego et al., 2006;

Diaz-San Segundo et al., 2014; Garcia-Briones et al., 2004; Perez-Martin et al., 2014; Sanz-Parra et al., 1999). Specific CD8+ T-cells – stimulated upon antigen processing and presentation of viral peptide epitopes by antigen presenting cells (APCs), mainly dendritic cells (DCs), in the context of MHC class I molecules (Fig. 13.3) – have been reported in host animals following infection or vaccination with inactivated virus (Guzman et al., 2010; Saiz et al., 1992). Despite these findings, the role of CTL responses in FMDV protection remains poorly understood. Relevant for mounting efficient T-cell responses is the interaction of FMDV with APCs, in particular with DCs that are key controllers of immune defence development and responsiveness, providing essential antigen presentation to T-lymphocytes and antigen delivery to B-lymphocytes (Fig. 13.3). Although FMDV can interact with APCs and DCs –in an enhanced manner when complexed to specific antibodies– producing a sort of abortive infection (see Chapter 10), a clear picture is not available on whether and how this interaction influences the outcome of the protective response against this virus.

IFN

IL-2

IFN

Proliferation and cytokines

Antigen Presenting Cell (APC)

IL-1 IL-2, IL-4, IL-5, IL-6

Live FMDV or inactivated FMDV vaccine

Macropinosome or endosome carrying FMDV

MIIC: MHC class II –containing late endosomal-like structure

TH lymphocyte CD4

Co-stimulation

TCR complex

MHC class IIMHC class-II mediated antigen restricted peptide presentation recognition

B lymphocyte Proliferation, differentiation, and Ig synthesis

Antigen processing Endosomes/lysosomes

Non-internalized or exocytosed antigen

Antigen recognition by B-cell receptors (surface Ig)

Antigen (intact) carried on APC surface Specific anti-FMDV antibody

Figure 13.3 Overview of main events on the interaction of antigen-presenting cells (APCs) with B and T-lymphocytes, leading to antibody production. Adapted from McCullough and Sobrino (2004).

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Identification of FMDV protein fragments containing B-cell neutralizing epitopes The use of peptides as FMDV immunogens stems from early observations made by Fred Brown and colleagues on the ability of viral protein fragments to elicit antibodies that recognized the intact virus [for an in-depth review, see (Rowlands, 2004)]. Early work revealed that FMDV antigenic and immunogenic properties were altered by trypsin treatment, yet without significantly modifying particle structure (Brown and Smale, 1970; Rowlands et al., 1971), and with VP1 as the only cleaved (at a single site) capsid protein (Burroughs et al., 1971). Interestingly too, limited trypsin digestion resulted in the generation of non-infectious virions, unable to bind to cells in culture (Baxt and Bachrach, 1982; Cavanagh et al., 1978), and pointing to a dual role of the VP1 region cleaved by trypsin, in both FMDV immunogenicity and cell attachment. Mapping of VP1 antigenic determinants (Strohmaier et al., 1982), using a series of VP1 fragments whose immunogenicity was tested in mice, allowed to predict the location of immunodominant antigenic sites, which fitted well with the earlier observations of trypsin-treated virus properties and with increasing data on the locations of highly variable regions, obtained by sequencing of capsid protein coding regions of different viral isolates (Beck et al., 1983; Dopazo et al., 1988; Knowles and Samuel, 2003). Successful FMDV particle crystallization and elucidation of its 3D structure revealed the presence of a protruding motif, exhibiting multiple conformations, located at the GH loop, around positions 140–160 of capsid protein VP1 (Acharya et al., 1989). This protruding, highly mobile loop was included in one of the previously identified immunogenic fragments that contained the single trypsin cleavage site in VP1. The loop also contained an RGD triplet, the binding motif to the cell receptors (integrins) (Fig. 13.2) (see Chapters 4 and 5 for details). This dual function — hosting an inmunodominant B-cell epitope and mediating the binding to the cell receptor — makes possible that changes in antigenicity may result in modulation of the interaction between the virus and their cell receptors (Nuñez et al., 2007; Tami et al., 2003). The identification of VP1 as holder of the GH loop and other antigenic sites involved in

eliciting neutralizing antibodies (see Chapter 4) led to exploration of the immunogenicity of recombinant versions of this capsid protein. Despite early experiments describing protection of pigs with bacterially produced VP1 (Kleid et al., 1981), the immunogenicity of microorganism-produced VP1 was shown to be quite low, discouraging the initial expectations. This lower immunogenicity of VP1 may be due to non-native folding in solution, hence inefficient exposure of the immunogenic sites displayed by the virus particle to B-cells (Brown, 1992; Sobrino et al., 2001). Peptides can induce neutralizing antibodies and protection The considerable amount of information gathered by late 1970s on FMDV antigenic structure prompted its use as a model system to explore the potential of peptides as vaccine candidates, a promising emerging approach (Lerner et al., 1981). As commented above, most of the work was conducted using the continuous, immunodominant B-cell site located in the GH loop of VP1. It was shown that uncoupled linear peptides containing the VP1 GH loop from viruses of different serotypes induced neutralizing antibodies in guinea pigs and mice (Pfaff et al., 1982) and protection in swine (Bittle et al., 1982). In 1986 (DiMarchi et al., 1986) protection to viral challenge was reported in cattle immunized with a chimeric peptide in which the sequences of the GH loop and of the C-terminus (residues 200–213) of VP1 were linearly juxtaposed, the latter segment also selected for its ability to elicit neutralizing antibodies (Strohmaier et al., 1982). These peptide vaccines induced significant levels of anti-FMDV neutralizing antibodies and protected guinea pigs against homologous virus challenge (Doel et al., 1992). Nevertheless, it became soon evident that the immunogenicity of these linear constructs in a number of host species was substantially lower than that of conventional vaccines (Doel et al., 1988), and that the correlation between serum neutralizing activity and host protection was poorer in animals immunized with peptide vaccines than in those immunized with conventional vaccines (Barteling, 1988; Collen, 1994; Sobrino et al., 2001).

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Advantages and limitations of FMDV peptide vaccines Peptide-based vaccines utilize minimal components as specific immunogens to elicit effective immune responses. Despite the potential advantages of this approach, development of successful FMD peptide vaccines has been hampered by the difficulties associated with the identification and mimicking of viral epitopes involved in efficient induction of protection. An additional problem when using a limited number of viral epitopes as immunogens stems from the antigenic heterogeneity of FMDV in the field (Barteling and Woortmeyer, 1987; Domingo et al., 1992). The main advantages and challenges of peptide vaccines are presented in SWOT analysis form in Fig. 13.4. The adaptive immune system comprises two arms, one responsible for the humoral response (B-cells) and one for the cytotoxic immune response (CTLs), both dependent on T-helper cells (Th cells). A peptide vaccine must therefore incorporate epitopes recognized by both B and T-cells, ideally with the latter being widely recognized and presented by MHC alleles frequently occurring in

S •  •  •  •  • 

STRENGTHS safety: absence of infectious material è no risk of reversion to virulence, genetic integration or recombination total differentiation between infected and vaccinated animals (DIVA capability) fast, affordable large scale production by robust synthetic methods chemical stability (lyophilized form) simple handling, storage and transport (no cold chain required)

O •  •  •  •  • 

OPPORTUNITIES

flexibility: irrelevant or potentially troublesome sequences can be excluded easy structural fine-tuning to modulate immunogenicity, stability, solubility adaptability: fast response to new strains, emergency situations multivalency: various epitopes combined on a single molecular platform registered as a pharmaceutical (simpler than a biological)

individuals from natural host populations (Collen, 1994; Sobrino et al., 1999). Sequences recognized as B and T-cell epitopes can overlap substantially or be located at different discrete regions in the proteins of pathogens. Despite initial evidences that the immunogenic GH loop in VP1 could be also recognized as a helper T-cell epitope (Francis et al., 1987a), an inefficient priming for virus-specific T-cells was found in cattle (Glass and Millar, 1995; Van Lierop et al., 1995). These results highlighted that the lack of efficiently recognized T-cell epitopes was an important limitation for eliciting solid protective immunity within outbred populations as those of FMDV natural hosts. In a further attempt to advance towards an FMDV vaccine, a linear peptide reproducing the VP1 GH loop epitope, either alone (peptide A), or juxtaposed to the C-terminal secondary site C (peptide AC), or further elongation of the latter with a VP1 T-cell epitope identified in vaccinated cattle (Collen et al., 1991) (peptide ACT) were evaluated. In a large-scale vaccination trial in cattle, the highest protection (above 40%) was afforded by peptide ACT (Taboga et al., 1997). Protection

W •  •  • 

T •  • 

WEAKNESSES

B epitope definition not always simple: linear vs. conformational (continuous/discontinuous) T epitopes often insufficiently specified generally low immunogenicity of peptides è adjuvants, multimerization required

THREATS vaccine trial policies increasingly restrictive in some parts of the world vaccine trials (prototype validation) are slow processes

Figure 13.4 A SWOT (strengths, weaknesses, opportunities, threats) analysis of peptide vaccines against FMDV at the time of this writing.

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showed a limited correlation with specific serum neutralizing activity and a higher correlation with the induction of T-cell responses. Consistently, associations were observed between certain DRB alleles (DRB3.2 *1, 3 and 7) and increased levels of protection and the presence of others (DRB3.2 *12 and 18) and lack of protection, supporting that the polymorphisms in genes of bovine class I and class II MHC affected recognition of the individual epitopes, resulting in the animal to animal variation observed in both humoral and cellular immune responses (Garcia-Briones et al., 2000). These results reinforced the need of adequate T-cell activation for efficient peptide-based protection. The study also underlined the limitation of vaccines based on a single linear peptide in achieving protection against a highly variable RNA virus as FMDV, as escape mutants with amino acid substitutions at the GH loop could be isolated from non-protected challenged cattle (Taboga et al., 1997; Tami et al., 2003). An additional challenge, that of substantial antigenic variability, particularly at the main B-cell site located on the GH loop of VP1, has also to be reckoned with. In an effort to address the limitation posed by this problem in the design of peptide vaccines against HIV, the group of Tartar produced combinatorial peptide libraries, named mixotopes, where the different antigenic variants were proportionally represented, with the assumption that an immunogen with a high level of sequence variation could improve the broadness of antibody response (Gras-Masse et al., 1992). In the case of FMDV, mixotopes corresponding to the GH loop and containing either ca. 103 or ca. 105 peptides each, were used to immunized guinea pigs, but limited immunogenicity was observed (Oliveira et al., 2005). Induction of strong immune responses requires a sustained presentation of antigen in a stimulatory context; hence an important goal of peptide vaccine design is overcoming degradation by extracellular proteases. This goal has been addressed by peptides made up of non-native, protease-resistant D-amino acids. Peptides made with D-enantiomers will adopt the mirror image conformation of the canonic, l-amino acid structures. To overcome this problem, the D-peptides are assembled in the reverse order from the natural sequence, generating ‘retro-inverso’ peptides that adopt conformations resembling the natural antigen (Van

Regenmortel et al., 1998). A single inoculation of retro-inverso peptide corresponding to FMDV residues VP1[141–159] induced longer-lasting and higher antibody titres in immunized animals than the corresponding l-peptide. The antibodies cross-reacted strongly with virus particles and with L-peptides and conferred substantial protection in guinea pigs challenged with the cognate virus (Briand et al., 1997). Characteristics of the regions mimicked by FMDV peptides: continuous and discontinuous B-cell epitopes Whereas most peptide-based vaccine candidates so far reported, for either FMDV or other targets, are designed to reproduce continuous B-cell epitopes, i.e. uninterrupted stretches of a protein sequence recognized by antibodies, there is ample evidence that most antibody binding sites are discontinuous, i.e. made up from residues distant in sequence –often located in different subunits of a structurally complex antigen, e.g. a viral particle– but brought spatially close by the folding of the native structure. Reproduction of such discontinuous –also named conformational– epitopes by chemical means is a largely unaddressed task in molecular engineering. Much of its challenge lies in, first, defining which structural components of the antigen – i.e. residues or parts thereof – are relevant for antibody interaction and, second, spatially arranging them so that a native-like orientation of the critically interacting moieties can be accomplished. The daunting complexity of this task explains the low success rate of such efforts, judged by the rather few examples in the literature (Chamorro et al., 2009; Eichler, 2004; Franke et al., 2004; Howie et al., 1998, 1999). In the specific field of FMDV, only two efforts in this direction have been reported. A first study (Borràs et al., 1999) aimed to mimic the FMDV discontinuous antigenic site D, defined by mutational analysis (Mateu et al., 1998) as consisting of five residues situated on three external loops of capsid proteins VP1 (T193), VP2 (S72, N74, H79) and VP3 (E58) (see Chapter 4). The three loops were spliced together by means of two interconnecting units (a disulfide bridge and a poly-proline segment) into a single peptide construction (Fig. 13.5) that was able to elicit modest levels of neutralizing antibodies.

Advantages, Challenges and Future of Peptide Vaccines |  323 A

B

VP2 (132-137)

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S72 Pro8

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VP3Y63(58-61)

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P P P P T G

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Figure 13.5  (A) Front view of antigenic discontinuous site D of FMDV, showing the five residues defining the site (displayed in CPK) and adjoining regions. Surface most-exposed fragments are coloured white. Also included is VP2[132–137] loop, with a highly exposed Arg (also in CPK) presumed to interact with antibodies. (B) Elements defined in (A) are visualized as defining a cycle (purple arrow). (C) Incorporation of viral surface elements into a 26 residue cyclic peptide (Villen et al., 2006) reproducing VP1 (193–195, TGD), VP3 (58–60, ENV; Y63 and K84) and VP2 (72–80, PSQNFGHMHK and 133–137, SEKDR, reorganized from native SDREK for structural reasons). (D–F) Cross-sectional view of site D showing the five key residues and other elements included in a previous, first-generation replica of the site (Borràs et al., 1999). Antigenically relevant fragments were connected via a poly-proline module and a disulfide bond (shown in white), all involving residues at the inner side of the capsid.

In a second, more successful attempt (Villen et al., 2006), the approach taken was to generate a mimic of the contact surface, by structure-guided rational assembly of the native fragments adjoining the five key residues defining site D. The result of this design was a medium-size (24-residue) quasi-circular array that could be rather accurately mimicked by a disulfide-linked cyclic peptide. That a reasonably accurate replication of site D was achieved is shown by the fact that the cyclic peptide was able to bind FMDV-derived monoclonal antibodies, and that amino acid residues assumed – in above-mentioned mutational studies – to be involved in antibody recognition were confirmed by NMR to be spatially close to antibody paratopes. In addition, the cyclic peptide construct was successfully evaluated as a vaccine candidate on a guinea pig model. The cyclic peptide, unconjugated to a carrier protein, generated an antibody response comparable to that of virus-immunized animals in terms of FMDV neutralization in vitro, but unfortunately did not elicit full protection of the animals against FMDV

challenge. No further efforts to replicate this discontinuous epitope have been reported, to the best of our knowledge. However, one can hypothesize, in view of the success of vaccines based on multimeric (branched) presentation of the GH loop continuous epitope (see below), that combining (i.e. non-covalent admixture) the dendrimeric construct with the surface contact mimic of discontinuous site D might afford a vaccine of even superior performance. This hypothesis remains for the moment untested. Characteristics of the regions mimicked by the FMDV peptides: T-cell epitopes For the design of novel subunit vaccines, particularly peptide vaccines, a detailed knowledge of the pathogen (viral) epitopes eliciting cytolytic (CD8+) and helper (CD4+) T-cells relevant for protection in the natural hosts is crucial. As mentioned above, T-cells recognize specific antigens

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in the context of MHC molecules, and the genes coding for these molecules are highly polymorphic within an outbred population of a particular species (see Chapter 10). Therefore, the recognition of T-cell epitopes depends on the MHC alleles present in a particular animal, a phenomenon termed MHC restriction. The strength of T-cell epitope binding to MHC molecules is one of the critical determinants of immunogenicity (Lazarski et al., 2005). Peptides binding with higher affinity to MHC molecules are more likely to be displayed on the cell surface where they can be recognized by their corresponding T-cell receptors (TCRs) (Weber et al., 2009). Among T-cell epitopes with high affinity (immunodominant) and recognized by different MHC allelic forms (promiscuous), those conserved among different viral strains (heterotypic) are preferred for vaccine application, as they can be widely recognized by host T-cells and induce heterotypic responses. These features are particularly relevant for FMDV, a virus that exhibits high sequence variability (see Chapter 7) and affect different domestic and wild species (see Chapters 8 and 9). T helper epitopes Identification and characterization of FMDVspecific T-cell epitopes was initially addressed by using outbred animals and overlapping peptides spanning VP1 capsid protein. These studies allowed identification of T-helper epitopes recognized by cattle (Collen et al., 1991) and pig (Rodriguez et al., 1994) lymphocytes. A disadvantage for vaccine improvement of the T-cell epitopes identified in these experiments was the high sequence variability of VP1 among different FMDV isolates and serotypes (Carrillo et al., 2005). Interestingly, in VP4, another structural protein highly conserved among FMDV serotypes, MHC-promiscuous T-cell epitopes were identified for cattle (Van Lierop et al., 1995); one of them, VP4[20–34], was shown to bind at least four different cattle MHC haplotypes and to be presented by MHC class II DQ molecules (Gerner et al., 2009). Furthermore, VP4[20–34] was also recognized as a porcine T-cell epitope in FMDV-stimulated lymphocytes from vaccinated outbred pigs, providing further evidence of the promiscuous nature of this region (Blanco et al., 2000). Upon T-cell epitope recognition, effector (inflammatory) or regulatory (suppressive) T-cells can be

activated, depending on the co-stimulatory signals expressed (Weber et al., 2009). Unfortunately, incorporation of the nucleotide sequence corresponding to the VP4[20–34] epitope into a DNA vaccine turned out to be detrimental, promoting exacerbation of clinical signs after FMDV challenge (Ganges et al., 2011), and a fusion protein corresponding to amino acid positions VP1[133–158] and VP4[20–34] did not afford complete protection in the guinea pig (Zhang et al., 2002). Search for FMDV heterotypic T-cell epitopes has also been focused on non-structural proteins (NSP), since most of these proteins exhibit low sequence variability among viral isolates and serotypes, and therefore are expected to be recognized in the context of heterotypic FMDV infections. In addition, expanding T-cell epitope search to NSPs allows broadening the repertoire of viral epitopes recognized by host immune defences. The T-cell antigenicity of FMDV NSP was first analysed using recombinant fusion proteins expressed in E. coli, leading to the identification of 3AB and 3C polypeptides as main inducers of specific and consistent lymphoproliferative responses in all the FMDVinfected pigs analysed (Blanco et al., 2001). Fine epitope mapping of 3AB and 3C by means of overlapping peptides allowed the identification of 11 heterotypically recognized epitopes. One of them, 3A[21–35], was shown to induce lymphoproliferation and FMDV neutralizing antibodies in vitro when displayed in linear tandem with the VP1 GH loop peptide. The relevance and potential of this 3A[21–35] T-cell epitope as a key element in novel, fully protective peptide vaccines, has been subsequently confirmed (Blanco et al., 2016; Cubillos et al., 2008, 2012; Monso et al., 2013) and is described in more detail in the next section. The presence of T-cell epitopes within NSP 3D, the viral RNA polymerase, has been demonstrated in both cattle (Collen et al., 1998) and swine (Foster et al., 1998). Indeed, the contribution of this protein to the protective immune response to FMDV was confirmed by the observation that pigs immunized with recombinant vaccinia virus expressing 3D protein were partially protected against viral challenge (Garcia-Briones et al., 2004). In this study, 3D overlapping peptides permitted identification of different T-cell epitopes that were efficiently recognized by pig lymphocytes, upon both primary and secondary (heterotypic) FMDV

Advantages, Challenges and Future of Peptide Vaccines |  325

infection. Interestingly, one of these peptides, 3D[346–370] was also shown to be recognized by inbred pigs (Gerner et al., 2006). In conclusion, a fairly broad set of CD4+ T-cell epitopes identified in cattle and swine is available as candidates for subunit and peptide vaccines improvement. CTL epitopes Characterization of CTL epitopes in outbred populations is complicated by the limited availability of inbred animals among FMDV natural hosts, and has therefore been mostly done in inbred mice. However, the biological significance for natural host species of murine-defined CTL epitopes cannot be taken for granted and requires experimental confirmation. In addition, the cytopathic nature of FMDV has hindered the use of FMDV-infected cells as targets. All these limitations underscore the scarce reports on FMDV CTL epitopes (Barfoed et al., 2006). So far it has been described a solely BoLA class I-restricted CD8+ T-cell epitope in the structural protein VP1 (Guzman et al., 2010). In an attempt to circumvent the above limitations, predictive approaches have been recently described to identify FMDV peptides presented by swine (SLA) and bovine (BoLa) MHC class I molecules. Based on in silico predictions and in vitro verifications, FMDV peptides were found to bind SLA-2*0401 and SLA-1*0401 MHC alleles commonly expressed in commercial pig breeds (Pedersen et al., 2013). Analyses of antigenspecific, MHC class I-restricted T-cells using MHC tetramers made with the peptides identified, showed that at least one of them reacts with FMDVspecific T-cells (Patch et al., 2011). A similar in silico approach predicted the binding of several peptides from FMDV structural proteins to common Bola class I molecules (Pandya et al., 2015). The CD8+ response to such peptides has not yet been evaluated in vaccinated/infected cattle. Non FMDV-specific immunodominant T-cell epitopes haven also been included in the design of peptide vaccines to provide T-cell help (Francis et al., 1987b). In this approach, activated T-cells would not be re-stimulated upon subsequent virus encounter, which can limit the potential stimulatory effect. Using this strategy, a linear peptide based on the VP1 GH sequence combined with that of a combinatorial library used as a source of T-cell epitopes was reported to confer protection

in pigs (Wang et al., 2002). However, this vaccine failed to afford any protection in cattle (Rodriguez et al., 2003). Current perspectives for improved FDMV peptide vaccines Despite the caveats already mentioned earlier in this chapter (Fig. 13.4), peptide-based vaccines are arguably one of the most advantageous alternatives to classical vaccines (i.e. dead or attenuated virus) against FMDV, not only for the complete safety achieved by the exclusion of the infectious agent but also by additional advantages such as their DIVA (differentiation between infected and vaccinated animals) capability, or their efficient production by chemical synthesis that facilitates characterization as pharmaceuticals, or their uncomplicated, cold chain-free transport and storage (Brun et al., 2011; Purcell et al., 2007). While these advantages are partially offset by the recognized low immunogenicity of peptides, this limitation can be addressed in various ways (Rueckert and Guzman, 2012), particularly by multimerization, whereby several epitopes can be displayed on a single scaffold. Different varieties of scaffolds have been described, including template-assisted synthetic protein (TASP) platforms (Tuchscherer and Mutter, 1995), gold nanoparticles (Chen et al., 2010), aromatic hydrocarbons (Chang et al., 2012), or four-armed star polymers (Liu et al., 2013), in addition to the classic lysine tree-based, multiple antigenic peptide (MAP) design pioneered by Tam (Tam, 1988; Tam and Spetzler, 1997). Many of these approaches rely on the considerable advances made in chemoselective ligation since the 1990s (Lu et al., 1991; Rose et al., 1995; Tam and Spetzler, 1995). In 2008 solid protection of swine against C serotype FMDV challenge was described by means of a vaccine prototype consisting of four copies of the continuous GH loop B-cell epitope tethered through thioether bonds to a single copy of the heterotypic 3A[21–35] T-cell epitope elongated at its N-terminus by a branching core of Lys residues (Fig. 13.6). This dendrimer construct, hereafter named B4T, generated high titres of FMDV-neutralizing antibodies in both pigs and outbred mice (Swiss CD1 strain), activated T-cells and induced IFN-γ release and induced full protection in swine upon

326  | Blanco et al. B epitope B epitope

Lys

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(S)

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=

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(S)

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B2T(mal)

=

CH 2

O

O =

(S)

O N O

Figure 13.6  General structure of FMDV vaccines combining one T-cell and four/two B-cell epitopes in a branched arrangement (Blanco et al., 2016). In all cases, the T epitope reproduces residues 21–35 (AAIEFFEGMVHDSIK) of non-structural protein 3A, and the B epitope (acetyl-PVTNVRGDLQVLAQKAARTC) reproduces the G-H loop of VP1, residues 140–160, serotype O-UKG 11/01. The (S) corresponds to the thiol group of the C-terminal Cys residue. All peptides are in C-terminal carboxamide form.

infection with homologous FMDV. Most remarkably, the B4T candidate vaccine elicited high titres of mucosal IgAs, similar to those achieved upon FMDV infection, which prevented contact controls to become infected when vaccinated animals were challenged with the live virus (Blanco et al., 2013; Cubillos et al., 2008). Interestingly, multimeric display of B-cell epitopes in the dendrimer was necessary for solid protection, since linear peptides including the GH loop B-cell epitope and the 3A[21–35] T-cell epitope did not afford full protection in pigs (Cubillos et al., 2012). Partial protection with linear peptides containing B and T-cell epitopes has been also recently reported for cattle (Zhang et al., 2015). This promising lead has been since pursued with dendrimers displaying the GH loop B-cell epitope of more epidemiologically consequential O rather than C serotypes. In studies with Swiss CD1 mice (Monso et al., 2013), the conventional view that higher B epitope multiplicity enhances immunogenicity was challenged by comparing the response of constructs with two or four copies of the B

epitope, and showing that bivalent (B2T; Fig. 13.6) constructs not only improve over co-linear display of B- and T-cell epitopes but also outdo tetravalent ones in both neutralizing antibody (humoral) and IFN-γ (cellular) responses, particularly when maleimide linkages are used to interconnect the B- and T-cell epitopes. More recently, this unexpected superior performance of the bivalent (B2T) over the tetravalent (B4T) peptide vaccine candidates has been conclusively verified in the swine, an FMDV natural host (Blanco et al., 2016). Similar to the earlier results in mice, the bivalent maleimide–linked construct [B2T(mal), Fig. 13.6] has an edge over an also bivalent but thioether-linked version [B2T(thi); Fig. 13.6], highlighting the impact of minute structural details on complex biological events such as vaccination with peptides. A further unexpected bonus of the maleimide–based construct is its extremely expedient production, due to the highly efficient thiol-ene conjugation chemistry, in contrast with thioether and other ligation chemistries. All in all, the B2T(mal) emerges as an efficient FMDV immunogen in swine,

Advantages, Challenges and Future of Peptide Vaccines |  327

eliciting a robust immune response in both serum and mucosae, with full (100%, 6/6 animals) protection and with additional features (i.e. production) that make it a vaccine candidate with realistic perspectives of clinical applicability.

definition for register as pharmaceuticals. While issues such as dosage, cost efficiency, immunization schedule or duration of the protection remain to be adequately addressed, FMDV dendrimer peptide vaccines are likely to enter the real vaccine world in the next years.

Concluding remarks Peptide vaccines are attractive and timely alternatives to traditional vaccines (Purcell et al., 2007). Despite the need for strategies to improve their antigenicity, discussed in this chapter, peptides are becoming a real alternative. Indeed, several peptidebased vaccines against human diseases, including cancer, are in clinical trial stages (Li et al., 2014). For a rational design of an efficient peptide vaccine two main limitations have to be considered: (i) the frequently incomplete picture of the immune effector mechanisms against a given pathogen, mainly the role of CD4 and CD8 lymphocyte responses in protection, and (ii) the difficulties in reproducing conformation-dependent B-cell antigenic sites that, in turn, restrict to conformation-independent (continuous) epitopes those that can be included in the peptide vaccine construct. For FMDV, the sequence of the VP1 GH loop has been proven a good mimetic of the antigenic role this loop plays in the viral particle, particularly when presented in a multimeric manner such us in dendrimers. Also, several CD4+ T helper-cell peptides have been identified in swine and cattle. Nevertheless, much remains to be learnt about comparable approaches in other host species, as well as on the role of responses mediated by CD8+ lymphocytes in the protection against FMD infection. In any case, the possibility of peptide dendrimer vaccines including not only T-helper epitopes that cooperate in B-cell maturation and efficient antibody production, but also CTL epitopes extending the antigenic spectrum of protection to infected cells, has undeniable interest. Indeed, the modular nature of peptide dendrimer vaccines such as B2T discussed above, and/or the possibility of administering different epitopes in dendrimer fashion in a single immunization. This approach enables combination of different T- and B-cell epitopes into single platforms that can increase the spectrum of protection, reduce the potential selection of antigenic variants in non-protected, vaccinated animals, and permit production of vaccines with adequate molecular

Acknowledgements Work at the authors’ laboratories for this chapter was supported by the Spanish Ministry of Economy and Competitiveness (grants SAF2011– 24899, and AGL2014-52395-C2 to FS and DA; AGL2013-48923-C2 to EB), the European Community’s Seventh Framework Programme (FP7, 2007–2013), Research Infrastructures action (NADIR), under the grant agreement No. FP7-228394 (to FS, DA, EB and AD), Comunidad de Madrid (S2013/ABI-2906-PLATESA to FS and EB) and Generalitat de Catalunya (2009SGR492 to DA). Work at Centro de Biología Molecular ‘Severo Ochoa’ was supported by Fundación Ramón Areces. References

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Control of Foot-and-mouth Disease by Using Replication-defective Human Adenoviruses to Deliver Vaccines and Biotherapeutics

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Fayna Diaz-San Segundo, Gisselle N. Medina, Marvin J. Grubman and Teresa de los Santos

Abstract Foot-and-mouth disease (FMD) is one of the most important viral diseases that can affect livestock worldwide. Although the disease has been successfully controlled in many geographic regions, mainly due to the enforcement of surveillance and trading policies, and the use of an available inactivated whole virus vaccine formulation, challenges remain as outbreaks are constantly detected in most of the developing world. With the expansion of globalization and the exponential growth of population in today’s world, recent outbreaks have wreaked havoc in disease-free countries resulting in devastating economic consequences. Novel vaccination policies that could be massively produced anywhere, that could induce early protection after vaccination at a low risk and affordable cost are needed. A novel vaccine approach using a recombinant replication-defective human adenovirus type 5 (Ad5) vector has recently been developed and has been granted for the first time in the last 50 years, a provisional U.S. licence for production and use in the U.S. mainland in outbreak situations. The Ad5-FMD vaccine delivers only FMDV capsid and some non-structural (NS) viral coding regions required for capsid processing and improved expression. Animals vaccinated with Ad5-FMD can be readily differentiated from infected animals (DIVA) using diagnostic assays employing the NS proteins not present in the vaccine, and production of this vaccine does not require expensive highcontainment manufacturing facilities since it does not contain infectious FMDV. One inoculation of this Ad5-FMD subunit vaccine can induce rapid, within 7 days, and relatively long-lasting protection

in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, suggesting that the Ad5 vector approach may be useful in semiannual FMD vaccine campaigns. To stimulate protection prior to the vaccine-induced adaptive immune response we have explored the possibility of using biotherapeutics also delivered by recombinant Ad5. Delivery of type I, type II and type III interferon (IFN) can fully protect swine against challenge with multiple FMDV serotypes. Similarly, delivery of type III IFN can protect cattle against FMD. Interestingly a combined approach of Ad5-FMD vaccine and Ad5-IFN can protect animals as early as 1 day post vaccination and protection can be complete. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread, this strategy may reduce the number of animals that need to be slaughtered during an outbreak as well as allow for differentiation of vaccinated from infected animals. In this chapter we will review the development of these approaches and current efforts to improve the strategy to make it affordable and effective for global use. Introduction Foot-and-mouth disease (FMD) is one of the most highly contagious viral diseases of animals (Grubman and Baxt, 2004). The aetiological agent, FMD

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virus (FMDV), is the type species of the Aphthovirus genus of the Picornaviridae family. The virus is antigenically variable and includes seven serotypes, A, O, C, Asia-1, South African Territories 1–3 (SAT1–3) and multiple subtypes within each serotype. FMDV contains a positive-sense, singlestranded RNA genome which is encapsidated by four structural proteins, VP1–4 (Mason et al., 2003). The viral RNA also codes for a number of non-structural (NS) proteins involved in various phases of virus replication and host cell control including Lpro, a 16 amino acid peptide 2A, 2B, 2C, 3A, three 21–22 amino acid 3B proteins, 3Cpro, and the viral RNA dependent RNA polymerase 3Dpol. Numerous reviews have described in detail the role of many of these proteins in the virus replication cycle (Belsham, 2005; Mason et al., 2003) and some of the chapters in this book provide more recent information on virus molecular biology and virus structure. The focus of this chapter, as well as a number of other chapters in this book, is to describe the various approaches that have been examined to effectively and rapidly control FMD. We will discuss a two-pronged strategy that we have developed using a viral vector, human adenovirus type 5 (Ad5), to deliver an FMD empty capsid subunit vaccine alone or in combination with Ad5 vectored type I, II or III interferon (IFN) genes. Foot-and-mouth disease FMD affects numerous cloven-hoofed animals including economically important livestock animals such as cattle, swine, sheep, and goats as well as numerous species of wildlife (Grubman and Baxt, 2004). The disease results in a high morbidity but generally low mortality, except in young animals that can develop cardiac complications. Infected animals usually develop lesions on the tongue, mouth, feet and teats, and as a result, often cannot eat, produce less milk, and in developing countries are unable to perform vital utilitarian functions such as ploughing fields. The World Organisation for Animal Health (OIE) lists FMD as a reportable disease and therefore by law, member nations are required to inform the organization about FMD outbreaks. OIE member nations, in which the disease is present, are not permitted to engage in trading of FMD-susceptible animals or their products. Thus, the presence

of FMD in a country can have severe economic consequences. Currently, upon an FMD outbreak countries attempt to restrict susceptible animal movement, slaughter infected animals, and decontaminate infected premises. Vaccination is an option in countries in which FMD is enzootic, but diseasefree nations prefer not to vaccinate or vaccinate followed by slaughter of vaccinated animals, as occurred in the 2001 outbreaks in the UK and The Netherlands (Pluimers et al., 2002; Scudamore and Harris, 2002). The current approved vaccine is a chemically inactivated whole virus preparation which is combined with an adjuvant prior to administration (see Chapter 12). Production of this vaccine involves the growth of large amounts of live virus and thus requires expensive high-containment facilities (Doel, 2003). While the vaccine has been successfully used for many decades there are a number of concerns with its use including, • It requires passage of virus in tissue culture which could result in selection of antigenic variants. • The virus may not be properly inactivated. • Depending on manufacturer’s protocol, the virus may not be sufficiently purified and thus still be contaminated with viral NS proteins resulting in difficulty in differentiating infected from vaccinated animals (DIVA). • The vaccination does not induce long-lasting immunity and protection generally requires semiannual vaccination campaigns. • The vaccination does not induce rapid protection – usually requires about seven days to induce a protective response. This is a major concern since FMDV replicates and spreads very rapidly. • The vaccine is not broadly protective and each serotype and often subtypes within a serotype require a specific vaccine. For these and other reasons researchers have been attempting for the past 40 years to develop novel vaccines which address some or all of the above mentioned concerns (Rodriguez and Gay, 2011). One of the obstacles in this process is the economic reality that animal vaccines or biotherapeutics must be inexpensive. Our approach

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has been to develop a product that (1) does not require infectious virus and thus can be produced in the U.S. in a BSL2 facility rather than requiring an expensive high-containment manufacturing facility, (2) allows differentiation of infected from vaccinated animals (DIVA), (3) is in a DNA format so that generation of antigenic variants during vaccine production is limited in contrast to production of the current whole virus vaccine, and (4) can be used in combination with a biotherapeutic that induces rapid protection regardless of virus serotype causing the outbreak. Adenovirus as vaccine vectors Adenoviruses (Ads) are non-enveloped, icosahedral pathogens associated with most vertebrates (Davison et al., 2003). They have a linear doublestranded DNA of about 26–45 kb and belong to the family Adenoviridae, which is divided into five genera based on the host species and the DNA composition. Ads vectors have become a very popular vehicle for gene transfer into mammalian cells (Tatsis and Ertl, 2004). Human Ad are by far the best characterized, and the vast majority of gene transfer studies involving Ad, whether for therapeutic or vaccine purposes, have been carried out with vectors derived from serotype 5 (Ad5) (Bradley et al., 2012; Tatsis and Ertl, 2004). Different strategies have been employed in the construction of Ad vectors. The most prevalent strategy involves deletion of one or more of the early genes (e.g. E1 gene) required for virus replication rendering the vector replication-defective (Schaack, 2005). Ad viruses with multiple genomic deletions have been used as delivery vectors, but in this case special cell lines, that usually have proprietary rights, are required for growth and propagation thus limiting the availability of these vectors for research and development. Multiple features of the virus such as high packaging capacity for transgene insertion, efficient transduction of both quiescent and dividing cells, and ability to grow with high titre make it a useful vector for gene delivery (Luo et al., 2007). Adenovirus vectored FMD vaccines and biotherapeutics It has been shown that FMDV empty capsids, e.g. virus particles lacking genomic RNA, are as antigenic as infectious virions (Rowlands et al.,

1975). Vaccine studies using numerous other viral systems including hepatitis B, human papilloma virus etc. have demonstrated that viral empty capsids or virus-like particles produced by recombinant DNA techniques are effective vaccines and some of these products are in current use (Roldão et al., 2010). Based on these results and knowledge of the replication requirements of FMDV (Mason et al., 2003), we have used recombinant DNA techniques to construct a replication-defective adenoviral vector containing the portions of the FMDV genome coding for the structural proteins as well as NS protein 3Cpro, which is necessary for processing of the capsid protein precursor (Vakharia et al., 1987; Mayr et al., 1999). In the following sections of this chapter we will go into detail regarding the development and testing of Ad5 vectored FMDV constructs for types A and O in both swine and cattle (Moraes et al., 2002, 2011; Pacheco et al., 2005). We will also present several approaches we have used, including addition of adjuvants, different routes of vaccination, etc. in order to achieve vaccine dose-sparing for a more practical use in the field (Diaz-San Segundo et al., 2014; Grubman et al., 2012) Evidence from our lab as well as others, demonstrated that FMDV replication in cell culture is highly susceptible to IFN treatment and that all virus serotypes are affected (Ahl and Rump, 1976; Chinsangaram et al., 2001; Grubman et al., 2012). Furthermore, IFN treatment can rapidly inhibit virus replication. Based on this information we hypothesized that a combination of IFN treatment and vaccination could both rapidly protect against infection with any FMDV serotype and induce a longer lasting specific immunity utilizing a vaccine against the virus strain circulating in the field (Grubman, 2003). In the following sections we will also present experimental data in swine and cattle examining the effectiveness of Ad5 delivered IFNs as well as some combination of Ad5-FMD and Ad5-IFN. Development of Ad5 vaccines to deliver FMDV antigens Ad5-FMD vaccines The use of replication-defective human adenovirus (Ad5-) to deliver FMD vaccines has been reported

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by different groups (summarized in Box 14.1 and Table 14.1). The initial studies using this vector were performed by Sanz-Parra et al. (1999) who demonstrated that two doses of an Ad5 vector containing the P1 coding region of FMDV serotype C, but lacking the 3Cpro coding region, induced partial protection in both cattle and swine. Mayr et al. used the same vector and the P1–2A coding region of FMDV A12, but included the 3Cpro coding region since it had been demonstrated that 3Cpro is required for processing of the capsid precursor P1–2A, allowing for capsid assembly (Mayr et al., 1999; Vakharia et al., 1987). The vaccine also contained partial sequences of the FMDV NS proteins 2B and 3B. There are a number of advantages of this vaccine platform including:

• the absence of other genomic regions such as the 3D coding region, which codes for one of the most immunogenic viral proteins allowing for differentiation of infected from vaccinated animals (DIVA); • the Ad5 vaccine is produced as a DNA molecule which is genetically stable since it is not subject to the high error rate of replication of RNA polymerases, and • the vaccine does not contain infectious FMDV and therefore can be produced in a BSL2 laboratory even in FMD-free countries (Grubman et al., 2010). In their proof of concept studies Mayr et al. (1999, 2001) demonstrated that the Ad5-FMD vaccine induced significant levels of neutralizing

Box 14.1  Schematic of the construction of Ad5-FMD vector vaccine FMDV structural precursor P1–2A, non structural full length 2B, truncated 3B and full length 3Cpro coding regions were cloned by recombinant DNA techniques and inserted into the E1 region of the replicationdefective Ad5 vector. Expression of the FMDV coding regions is under the control of the CMV promoter. The Ad5-FMD vector was propagated in HEK 293 cells. Expression of capsid proteins was verified by western blotting using polyclonal antibodies against VP0, VP1 and VP3. Ad5-FMD was used to inoculate animals resulting in synthesis of FMDV empty capsids and induction of an FMDV-specific immune response. P1

VP0

VP3

2A2B3B 3C

VP1

E1 LITR Pac I

CMV

E3 Ad5 DNA

polyA

Promoter

Pac I

0

-VP0

RITR

1

3

FMDV empty capsid

-VP1 -VP3

LITR, RITR-left and right internal terminal repeats of the Ad5 genome; CMV- cytomegalovirus promoter; PacI- restriction enzyme sites in pAd5 vector; poly A- polyadenylic acid tract; ΔE1 and E3- deletions in Ad5 early regions 1 and 3.

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Table 14.1 Development of Ad5 vectored FMD vaccine Year

Milestone

Reference

1998

First construction of an Ad5-FMD vaccine, but lacking the 3Cpro coding region. Achieved partial protection in swine

Sanz-Parra et al. (1998)

1999

First publication of proof-of-concept study of Ad5-FMD vaccine containing the 3Cpro coding region. Expression and processing of FMDV capsid proteins, induction of a FMDV-specific neutralizing antibody response in mice and in swine

Mayr et al. (1999)

2001

Characterization of the immune response in pigs vaccinated with Ad5-A12 and demonstration of protection against challenge.

Mayr et al. (2001)

2002

Construction of an Ad5-FMD vector against a field strain of FMDV, A24 Cruzeiro. Demonstrated protection with a single dose at 7 days post vaccination

Moraes et al. (2002)

2003

Co-expression in vitro of capsids from two FMDV field strains in one Ad5 vector (A24 and O1 Campos) and induction of neutralizing antibody response against both FMDV serotypes in swine.

Wu et al. (2003)

2004

Agreement between U.S. Department of Homeland Security, U.S. Department of Agriculture, Agriculture Research Service, and Genvec, Inc. to develop a commercial Ad5-FMD vectored vaccine

2005

Protection of bovines vaccinated with one dose of Ad5-A24 at 7 days post vaccination. Pacheco et al. (2005)

2008

Addition of the complete 2B coding region to the Ad5-FMD vaccine. Vaccination of swine with this vector enhances protection

Pena et al. (2008)

Ad5-FMD vector expressing O/China/99 capsid and 3Cpro protects swine

Li et al. (2008)

Ad5-VP1 fused to IFN can protect swine against FMD

Du et al. (2008)

2010

Construction of Ad5-FMD vectors against multiple serotypes and subtypes induces protection in cattle

Grubman et al. (2010)

2011

Increased efficacy of an Ad5-O1 Campos vaccine containing the complete 2B coding region is associated with an enhanced cell-mediated immune response

Moraes et al. (2011)

2012

Route of infection, number of sites of inoculation significantly reduces the vaccine protective dose in swine

Grubman et al. (2012)

2013

Approval of a conditional license by USDA, APHIS, Center for Veterinary Biologics for inclusion of an Ad5-A24 vaccine in the U.S. Veterinary Vaccine Stockpile and use in emergency situations

Colby et al. (2013)

2013

Mucosal adjuvants delivered intranasally with Ad5 can improve efficacy of Ad5-FMD in mice

Alejo et al. (2013)

2014

Addition of molecular adjuvants, such as poly ICLC, improves efficacy and reduces the Diaz-San Segundo vaccine protective dose in swine. et al. (2014)

2015

Inclusion of an RGD in the fibre of Ad5-FMD does not improve vaccine efficacy in cattle Medina et al. (2016)

antibodies and protected swine against challenge with FMDV serotype A12. Further studies demonstrated that the approach was also effective against FMDV serotype A24, a field strain of FMDV (Moraes et al., 2002). One dose of an Ad5-A24 vector administered intramuscularly (IM) conferred protection in both swine and cattle against direct inoculation challenge with FMDV as early as seven days after vaccination and protection in swine lasted for at least 42 days (Moraes et al., 2002; Pacheco et al., 2005). Moreover a boost given to cattle at 9 weeks after the initial inoculation, significantly enhanced the FMDV-specific

neutralizing antibody response and the vaccinated animals, housed in the same room as the control animals, were protected when challenged 14 days later (Grubman, 2005). These later results suggest that the Ad5-FMD approach could also be useful in vaccination campaigns in enzootic areas where vaccinations are given twice annually. FMDV strains that are circulating and causing outbreaks in many parts of the world for the past number of years belong to serotype O. Outbreaks of serotype O have been recurrent in many parts of Asia including Taiwan, Japan, South Korea, China, Vietnam, Russia, etc. (Nishiura and Omori,

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2010; Paton et al., 2009) as well as in the Americas (Sutmoller et al., 2003). It has been demonstrated that inactivated serotype O vaccine induces a lower immune response compared with serotype A antigen (Doel et al., 1994; Pay and Hingley, 1986). As a result of its poorer immunogenicity commercial type O vaccines usually contain 4- to five-fold more antigen that A vaccines. An Ad5O1Campos (Ad5-O1C) vaccine was also evaluated by scientists at Plum Island Animal Disease Center (PIADC). Swine inoculated with 5 x 109 pfu of this vaccine developed lower levels of FMDV-specific neutralizing antibodies than swine inoculated with an equivalent dose of Ad5-A24. Furthermore, in efficacy studies this dose of the Ad5-O1C vaccine did not protect pigs against homologous challenge, although disease signs were delayed and considerably less severe than controls all of which died (Caron et al., 2005). Testing in cattle of a similar vaccine resulted in 50% protection (Moraes et al., 2011). However, it is important to note that in both experiments viraemia was eliminated or considerably reduced and virus shedding was reduced 1–3 logs in vaccinated animals compared with controls. These results are particularly important since pigs are major amplifiers of virus and a reduction in virus shedding can significantly reduce virus spread. Nevertheless, it is clear that further modifications are required to use the Ad5 approach against FMDV serotype O, at least the subtype O1 Campos. Ad5 biodistribution in a natural host Strategies to further enhance the efficacy and potency of Ad5-FMD vaccines require detailed examination of their biodistribution in the natural host. Most of what is known about the biodistribution of recombinant Ad5 (rAd5) vector vaccine has been performed in mouse models, which describe preferential sequestration of systemically administered Ad vectors to the liver posing serious adverse effects (Imperiale, 2008; Kalyuzhniy et al., 2008). However, recent studies that documented the biodistribution of Ad5-A24 administered IM in cattle (Montiel et al., 2012, 2013) showed that despite Ad5-A24 distribution in multiple visceral organs, vector DNA was not found in the liver. Specifically, elevated mRNA transcription at 24 and 48 hpi with peak occurrence of transgene expression at 48 hpi was observed. Abundant vector DNA was detected at the injected sites, in muscle and lymph nodes.

In addition, detection of histological changes after vaccination indicated the presence of extensive oedema and cellular infiltrates consisting of large mononuclear cells with fewer neutrophils and small mononuclear cells (Montiel et al., 2012). Notably, this study showed the presence of large quantities of cellular markers found in antigen-presenting (APC) and other immune cells in tissues where the Ad5 transgene expression was also detected – providing a clear indication of the host response to Ad5-FMD vectors (Montiel et al., 2013). Examination of Ad5-FMD biodistribution in swine or other FMDV host species as well as delivery by alternative routes should provide additional insights for critical events in Ad5-FMD vaccine function. Improvement of the efficacy of Ad5 vaccines To enhance the potency and efficacy of the Ad5FMD vector approach and develop a commercially viable FMD vaccine candidate against many circulating serotypes a number of strategies have been evaluated: Effect of inclusion of the complete FMDV 2B coding region in the Ad5-FMD vector After infection of cells with FMDV, as well as other picornaviruses, there is a drastic rearrangement of intracellular membranes which subsequently become the sites of viral replication (Aldabe and Carrasco, 1995; Buenz and Howe, 2006; Cho et al., 1994; de Jong et al., 2002; Monaghan et al., 2004; Suhy et al., 2000). A number of viral NS proteins have been implicated in the rearrangements including NS protein 2B. To attempt to enhance the synthesis of FMDV capsid proteins and the efficiency and/or stability of capsid assembly we constructed second generation Ad5-FMD vectors containing the fulllength 2B coding region (Ad5-FMD-2B) (Moraes et al., 2011; Pena et al., 2008). Infection of cells with this vector induced significant rearrangement of cytoplasmic membranes similar to what is observed in FMDV-infected cells (Pena et al., 2008). Swine inoculated with an Ad5-A24–2B vector developed an enhanced FMDV-specific neutralizing antibody response compared with the first generation vector which contained only a portion of the 2B coding region and these animals showed no clinical signs of disease after FMDV challenge (Pena et al., 2008).

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A similar approach was applied for the vaccine against FMDV O1Campos (Ad5-O1C-2B). Although complete protection was not achieved, a slightly higher percentage of cattle vaccinated with this vaccine and challenged 21 days later with FMDV O1 Campos were completely protected from clinical disease than cattle vaccinated with the first generation vaccine only containing a truncated version of NS viral protein 2B. Interestingly, addition of full-length 2B to Ad5-O1C resulted in an approximately 10-fold reduction in virus shedding and an enhanced specific vaccine-induced T-cell response compared with the group administered the first generation vaccine lacking the complete 2B coding region (Moraes et al., 2011). Effect of route and number of sites of inoculation for Ad5-FMD vaccines A single dose of 5 × 109 pfu of Ad5-A24 administered IM can protect swine against challenge at 7, 14, or 42 days post-vaccination (dpv). The effect of route, number and location of inoculation sites was further evaluated for their ability to enhance vaccine potency. By simply changing the route of inoculation from IM to subcutaneous (SC), increasing the number of inoculation sites from one to two or four and administering and vaccinating in the neck instead of the back limb of swine, it was demonstrated that the protective vaccine dose could be lowered by 25-fold (Grubman et al., 2012). These results indicated that the route, number and injection location of Ad5-FMD are important determinants to consider when evaluating potency and efficacy of the Ad5-FMD vaccine. Effect of broadening tropism of Ad5 by modifying the fibre protein Attachment of Ad5 to the cell occurs upon interaction between the fibre and the coxsackie and adenovirus receptor (CAR) (Bergelson et al., 1997; Tomko et al., 1997). This is followed by the interaction between the RGD (Arg-Gly-Asp) motif present within the penton base and the cell surface integrins which mediate the internalization of the virus into the cell (Bergelson et al., 1997; Greber et al., 1993; Wickham et al., 1993). Genetically engineered Ads that have an RGD motif inserted in the HI-loop of the fibre can use αvβ3 and αvβ5 integrins to attach to host cells (Coughlan et al., 2009; Sengupta et al., 2011) broadening their tropism

(Barnett, Crews and Douglas, 2002; Hidaka et al., 1999). This strategy has been particularly important for targeting dendritic cells (DCs), which are critical players for the initiation of immune responses but lack CAR receptors (Worgall et al., 2004). A comparison of the Ad-FMD vaccine with and without an extra RGD motif in the fibre knob was evaluated against serotype O1Campos in cattle (Adt.O1C.2B.RGD versus Adt.O1C.2B) (Medina et al., 2016). Although enhanced transgene expression was demonstrated in vitro in epithelial cells infected with the Adt.O1C.2B.RGD vector, no significant increase in efficacy of the vaccine containing the fibre RGD compared with the vaccine without the fibre RGD was detected in cattle when challenged with FMDV O1Campos at 21 dpv. In contrast to mice and humans, addition of an RGD in the Ad5 fibre did not improve transduction of cattle immune cells enriched in APCs (Medina et al., 2016). Nonetheless, enhanced T-cell responses and presumably enhanced memory T-cell responses in animals vaccinated with the RGD construct may play a role in improving protection from challenge in the long term. Further studies with a larger statistical sample number and challenge at later times post-vaccination (e.g. six to eight weeks), as well as development of more sensitive techniques to identify specific subpopulations of immune cells, may contribute to a better understanding of the possible effects of RGD modification of the Ad5-FMDV fibre on humoral and cellular immunity in cattle. Evaluation of adjuvants for Ad5-FMD IFN Numerous studies indicate that in addition to antiviral activity IFN-α/β can act as an adjuvant and boost the immune response to antigens (Brassard et al., 2002; Cull et al., 2002; Le Bon et al., 2001; Proietti et al., 2002). The adjuvant effect of porcine interferon α (poIFNα), also delivered by an Ad5 vector (Ad5-poIFNα), was examined in swine vaccinated with Ad5-A24 and challenged with FMDV at 42 days post inoculation (de Avila Botton et al., 2006). Although strong protection was observed with the vaccine alone, addition of (Ad5-poIFNα) resulted in improved protection. Despite the limited knowledge of the swine immune system, the authors observed that protection correlated with increased levels of IgG1 and IgG2 as well as an

340  | Diaz-San Segundo et al.

increase in the IgG1/IgG2 ratio. These results were consistent with previously reported studies in cattle that suggest a role for a specific antibody isotype in promoting protection against FMD (Capozzo et al., 1997; Mulcahy et al., 1990). Thus, poIFN-α enhances the long-term level of protection induced by the Ad5-FMD vaccine. More recently Su et al (Su et al., 2013) have shown that IFN-α acts as an adjuvant in the generation of T follicular helper (Tfh) cells and antigen-specific antibody responses when it is co-expressed with FMDV VP1 proteins in an Ad5-FMD subunit vaccine (Ad5-VP12A-poIFNα). Inoculation of mice with Ad5-VP12A-poIFNα enhanced the generation of Tfh, the secretion of IL-21 protein and the expression of Bcl-6 mRNA, compared with Ad5 vectors solely expressing VP1 and substantially increased the number of germinal-centre (GC) B-cells and formation of GCs. Furthermore, IFN-α enhanced the antibody response as revealed by increased production of IgG and subclasses of IgG1 and IgG2a. Poly-ICLC We also examined the inclusion of an adjuvant in the vaccine formulation. Adjuvants can significantly enhance the maturation of APCs including DCs and the antigen specific cellular response (Kool et al., 2008; Pulendran and Ahmed, 2006). Poly ICLC is a synthetic, double-stranded polyriboinosinicpolyribocytidylic acid molecule stabilized with poly-L-lysine and carboxymethyl cellulose which has enhanced biostability in animals, compared with poly IC (Nordlund et al., 1970) and is a known toll-like receptor 3 (TLR3) and MDA-5 agonist that can activate multiple innate immune pathways (Meylan and Tschopp, 2006; Stahl-Hennig et al., 2009). In rodents and primates, poly ICLC is a strong IFN-α inducer and provides antiviral and adjuvant activity (Caskey et al., 2011; Harrington et al., 1979; Levy et al., 1975; Tenbusch et al., 2012). With respect to FMD, it has been shown that poly IC has an adjuvant effect when combined with an FMD multiepitope protein or inactivated FMD vaccine (Cao et al., 2012, 2013, 2014; Zhou et al., 2014). Combination of poly ICLC with Ad5A24–2B vaccine delivered SC at two sites in the neck, reduced the Ad5-A24–2B protective dose by approximately 80-fold compared with administration of vaccine alone. Interestingly, the enhanced

efficacy of the combination approach correlated with a stronger antigen specific cell-mediated immune response (Diaz-San Segundo et al., 2014). Therefore, at least in swine, polyICLC is a strong adjuvant of Ad5-FMD. Mucosal adjuvants The oral and respiratory mucosae are primary sites for FMDV replication in the host (Arzt et al., 2010). It is hypothesized that stimulation of local immunity in these tissues may help prevent initial infection and viral spread. Several bacterial proteins including V. cholera and E. coli heat-labile enterotoxin (LT) display adjuvant activity at mucosal surfaces (Rappuoli et al., 1999). Ad5 vectors encoding either of two LT-based mucosal adjuvants, LTB and LTR72 have been used together with Ad5-A24 vaccine to assess their ability to augment mucosal and systemic humoral immune responses and protection against challenge with FMDV (Alejo et al., 2013). Adult mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. This group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common entry pathway. Evaluation of Ad5-FMD empty capsid vaccines for different FMDV serotypes Outbreaks of serotype O have been recurring in many parts of Asia including Taiwan, Japan, South Korea, China, Vietnam, Russia, as well as the Middle East, Africa and the Americas (Nishiura and Omori, 2010; Paton et al., 2009). Inactivated serotype O vaccine induces a lower immune response compared with serotype A antigen and requires more antigen than A vaccines (Doel et al., 1994; Pay and Hingley, 1986). These results have been reproduced when evaluating an O1 Campos Ad5-O1C-2B vaccine (Caron et al., 2005; Medina et al., 2016; Moraes et al., 2011). However, recent studies have shown that a similar vaccine against serotype O1 Manisa (Ad5-O1M2B) is very effective. Swine vaccinated with as low as 4 × 107 pfu of Ad5-O1M-2B, a five-fold lower

Control of FMDV with Ad5 Vaccines and Biotherapeutics |  341

dose than that required for serotype A24, were fully protected against challenge as early as 7 dpv (de los Santos, unpublished data). Similar results were obtained with an Ad5-Asia vaccine but in a mouse model. C57BL/6 mice immunized with a single 107 pfu dose of Ad5Asia-05 were protected against challenge with 100 times the lethal dose of FMDV Asia1/YS/CHA/05 (Zhou et al., 2013). Additional studies with Ad5 vectors containing the P1–2A coding region from numerous other FMDV serotypes and strains have been tested in cattle (Grubman et al., 2010). These studies highlight the potential use of Ad5FMD vaccines against current circulating FMDV strains in Asia. Ad5-FMD VP1 subunit vaccines Given the immunogenicity of FMDV VP1, several studies have focused on utilizing VP1 as a peptide vaccine (Brown et al., 1992; Sobrino et al., 1999; Taboga et al., 1997). However, poor results and limited protection in the natural host were observed. Utilization of FMDV VP1 as a peptide vaccine in the context of rAd5 has been examined in combination with porcine IFN-α (Du et al., 2008a,b). In the latter study, the rAd5 expressing VP1 from FMDV O/LY/2000 strain fused with IFN-α enhanced both humoral and cell-mediated immune responses to FMDV in mice (Du et al., 2008b). Interestingly, addition of multiple VP1 epitopes in rAd5 VP1 (B and T-cell epitopes) fused with IFN-α enhanced immune responses in mice when compared to rAd5 VP1 or rAd5 IFN-α alone. Most importantly, this approach provided protection from virulent FMDV type O challenge in guinea pigs and swine (Du et al., 2008a). Recently, Su et al. (2013) provided evidence that combinatorial use of IFN-α and VP1 could enhance the generation of specific T-cells (Tfh), secretion of IL-21 and Bcl-6). These studies summarized the importance of VP1 and its use as a candidate vaccine when employed in combination with an adjuvant. Development of Ad5-delivered biotherapeutics/antivirals as a control strategy for FMD Although both commercial inactivated and newly developed Ad5 based FMD vaccines fully protect animals against the disease, they require about 7

days to induce complete protection from clinical disease (Moraes et al., 2002; Pacheco et al., 2005; Pandya et al., 2012). However, in the event of an FMD outbreak in a disease-free country, the induction of rapid protection, prior to the development of vaccine-stimulated adaptive immunity, is necessary to block or limit disease spread and thus potentially reduce the number of animals that have to be slaughtered. Early protection against viral infection is dependent on the induction and the sensitivity to IFNs and IFN-stimulated genes (ISGs) (Fensterl and Sen, 2009; Schoggins and Rice, 2011). IFNs are glycoproteins with strong antiviral activities that represent the first line of host defence against invading pathogens (Ank et al., 2006; Basler and Garcia-Sastre, 2002; Frese et al., 2002; Henke et al., 2001; Samuel, 2001; Shrestha et al., 2006). These proteins are classified into three groups, type I, II and III IFNs, based on the structure of their receptors on the cell surface (Fensterl and Sen, 2009). Type I IFNs (IFN-α and IFN-β) signal through a heterodimeric receptor complex formed by IFNAR1/IFNAR2, type II IFN (IFN-γ) signals through the complex IFN-γR1/IFN-γR2 and type III IFNs bind the receptor complex IL-28Rα/ IL-10Rβ. Despite the receptor differences the three IFN families transduce signals though the JAKSTAT pathways and type I and type III IFNs induce redundant responses which result in the inhibition of viral replication. Similar to many other viruses, FMDV is highly sensitive to the action of IFN (Ahl and Rump, 1976; Diaz-San Segundo et al., 2013). Thus, to be a successful pathogen, the virus counteracts the innate immune response by blocking the expression of IFN (Chinsangaram et al., 1999; de los Santos et al., 2006) through a combination of mechanisms including, degradation of translation initiation factor eIF-4G, which is required intact for translation of capped messages (Devaney et al., 1988; Kirchweger et al., 1994); degradation of nuclear factor-κappa B (NF-κB) (de los Santos et al., 2007); inhibition of the activation of interferon regulatory factors 3 and 7 (IRF-3; IRF-7) (de los Santos et al., 2007; Wang et al., 2010) affecting adaptor molecules including TRAF-6, TBK1 (Wang et al., 2011); impairment of cellular activity of the cytoplasmic pathogen recognition receptor (PRR) RIG-I (Wang et al., 2012). These results suggest that use of IFN could be a successful strategy to

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rapidly control FMDV in vivo. However, since IFN protein is rapidly cleared, its clinical use requires multiple inoculations of high doses for a prolonged time (Lukaszewski and Brooks, 2000; Qin et al., 1998; Santodonato et al., 2001) which can lead to adverse systemic effects (Qin et al., 1998). To overcome this problem, bovine and porcine IFN-α, IFN-β, IFN-γ and IFN-λ have been delivered with the same replication-defective Ad5-system used for the FMD vaccine, thus allowing animals to produce the protein endogenously and spread it systemically.

Lastly, an alternative anti-FMD strategy using Ad vectors to deliver siRNAs have been explored. The sections below summarize the knowledge thus far reported in these fields (Box 14.2 and Table 14.2). Type I IFN Initial studies in cell culture demonstrated that in supernatants from Ad5-poIFN-α infected IB-RS-2 cells, poIFN-α was detected as early as 4 hours post infection (hpi), its expression continued for at least 30 hpi and more importantly, the protein

Box 14.2  Construction and characterization of Ad5-IFNs IFN coding regions (bos Taurus/sus scrofa) were cloned by recombinant DNA techniques and inserted into the E1 region of the replication-defective Ad5 vector. Expression of recombinant IFN is under the control of the CMV promoter. The Ad5-IFN vectors were propagated in HEK 293 cells and their expression was verified by western blotting using polyclonal antibodies against bovine or porcine IFN α,β,γ, and λ. Antiviral activity of IFN-α was assessed by a plaque reduction assay against all 7 FMDV serotypes (A, O, C, Asia, SAT-1, SAT-2 and SAT-3).

LITR, RITR, left and right internal terminal repeats of the Ad5 genome; CMV, cytomegalovirus promoter; PacI- restriction enzyme sites in pAd5 vector; poly A, polyadenylic acid tract; ΔE1 and E3, deletions in Ad5 early regions 1 and 3.

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Table 14.2 Development of Ad5 vectored IFN and antivirals Year

Milestone

2001

Construction of an Ad5-poIFN-α vector. Proof-of-concept study showed that one dose Chinsangaram et of vector protects swine when challenged with FMDV A24 Cruzeiro 1 day later al. (2003)

2003

Swine inoculated with Ad5-poIFN-α are protected for 3–5 days

Moraes et al. (2003)

2003

Ad5-IFN-α partially protects cattle against FMD

Wu et al. (2003)

2006

Ad5-siRNA targeting structural and non-structural FMDV proteins blocks viral replication in a mouse model

Chen et al. (2006)

2007

A combination of Ad5-poIFN-α and Ad5-poIFN-γ protects swine at doses that when used individually were not protective

Moraes et al. (2007)

2010

IFN-induced protection against FMDV infection correlates with enhanced tissuespecific innate immune cell infiltration and interferon-stimulated gene expression.

Diaz-San Segundo et al. (2010)

2011

Ad5-poIFN-α inoculated swine are protected against multiple FMDV serotypes using different challenge methods. Ad5-poIFN-α dose can be reduced by altering route and site of inoculation. Ad5-poIFN-β also protects swine challenged with FMDV 1 day later

Dias et al. (2011)

2012

Molecular adjuvants, such as poly ICLC, can protect swine against FMD or improves potency of Ad5-IFN-α

Dias et al., 2012

Identification of bovine type III IFN as an antiviral against FMD in cattle

Diaz-San Segundo et al. (2012)

Ad5-poIFN-α antiviral activity is enhanced when used in combination with Ad5-siRNA against FMDV proteins in a mouse model

Kim et al. (2012)

2013

An Ad5 vector containing type III bovine IFN significantly delayed and reduced severity of disease in cattle challenged with FMDV one day later.

Perez-Martin et al. (2013)

2014

Swine administered Ad5-poIFN-λ and challenged by contact were completely protected from clinical disease, viraemia, and viral RNA and had no virus shedding

Perez-Martin et al. (2014)

An Ad5 vector containing a constitutively active porcine IRF7/IRF3 synthetic construct has antiviral activity and protects mice against FMD

Ramirez-Carvajal et al. (2014)

Bicistronic Ad5-poIFN-α/γ displays enhanced activity against FMD in swine

Kim et al. (2014)

2015

Ad5-poIFN-α/γ antiviral activity is enhanced when used in combination with Ad4siRNAs against FMDV proteins in swine

Kim et al. (2015)

2016

An Ad5-poIRF7/IRF3(5D)protects swine against FMD at doses lower than Ad5-poIFN-α Ramirez-Carvajal et al. (in press)

2016

Combination of Ad5-FMD and Ad5-boIFN- λ confers complete protection of cattle against FMD at 3 days post treatment

obtained had biological antiviral activity against FMDV (Chinsangaram et al., 2003). Furthermore, all serotypes of FMDV were very sensitive to the supernatants of these cells (Grubman et al., 2012). Studies in swine demonstrated that after one IM inoculation with 1 × 109 pfu/animal of Ad5-poIFN-α, relatively high levels of antiviral activity (~800 U), starting 16 hpi to 72 hpi, were detected in plasma and, importantly, animals were completely protected against FMDV A24 when challenged intradermally (ID) 24 hours after the Ad5-poIFN-α treatment (Chinsangaram et al., 2003). Furthermore, complete protection induced by Ad5-poIFN-α in swine lasted for 3–5 days, and there was a delay in disease onset, reduced severity

Reference

Diaz-San Segundo et al. (submitted)

of clinical signs, and a significant reduction in viraemia when the challenge was performed 7 days post inoculation (dpi) or one day prior to the treatment (Moraes et al., 2003). Similar results were obtained with an Ad5-vector expressing poIFN-β. In this case, although the antiviral activity was lower than that induced by Ad5-poIFN-α with the same doses of Ad5s, animals were also completely protected against ID challenge with FMDV A24 (Dias et al., 2011). FMDV is an antigenically variable virus and a control strategy using biotherapeutics could potentially rapidly protect susceptible animals against any FMDV strain and overcome the limited coverage of vaccines against heterologous strains

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(Domingo et al., 2003; Grubman and Baxt, 2004). Swine experiments in which animals were inoculated with the Ad5-poIFN-α vector and challenged ID 24 hours later with FMDV serotypes O1 Manisa or Asia1 showed complete protection, independent of the FMDV serotype used for challenge (Dias et al., 2011). Although these proof-of-concept studies demonstrated that Ad5-poIFN-α could rapidly protect swine against FMD, these experiments were performed by direct ID challenge. However, the natural route of FMDV infection is by aerosol (Alexandersen et al., 2003; Pacheco et al., 2010, 2012). Therefore, the efficacy of treatment of swine with Ad5-poIFN-α was also evaluated by contact exposure challenge with animals that had been previously inoculated with FMDV. At 24 hours post Ad5-poIFN-α or Ad5-control vaccination, swine were directly exposed for 18 hours to donor animals which had been previously inoculated with FMDV and displayed clear clinical signs of disease. In contrast to the Ad5-control inoculated animals, all Ad5-poIFN-α-treated animals were completely protected against disease, indicating that Ad5-poIFN-α treatment is effective against multiple routes of FMDV exposure (Dias et al., 2011). In studies to understand the mechanism by which IFN protects swine against FMD we examined the expression of ISGs in skin, peripheral blood mononuclear cells (PBMCs), and lymphoid tissues and also evaluated possible immune cell recruitment to the skin and lymph nodes. Protection of swine inoculated with Ad5-poIFN-α correlated with recruitment of skin DCs (DiazSan Segundo et al., 2010) that showed a partial maturation phenotype with increased expression of CD80/86, and decreased phagocytic activity (Diaz-San Segundo et al., 2013a). At the same time, an increase in the number of natural killer (NK) cells in draining lymph nodes was noticeable (DiazSan Segundo et al., 2010). These findings correlated with up-regulation of a number of ISGs with antiviral activity including PKR and OAS, which block FMDV replication in cell culture (Chinsangaram et al., 2001; de los Santos et al., 2006) as well as cytokines and chemokines, including monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, and IFN inducible protein 10 (IP-10) which are involved in chemoattraction of DCs and NK cells (Diaz-San

Segundo et al., 2010, 2013a) and in epidermal DC maturation (Fujita et al., 2005). Furthermore, using a mouse model for FMDV developed by Salguero et al. (2005), IP-10 C57Bl/6 knockout mice treated with murine IFN-α (muIFN-α) prior to challenge were not protected against disease whereas WT C57Bl/6 mice were completely protected, indicating that IP-10 is directly involved in protection induced by IFN against FMDV (Diaz-San Segundo et al., 2013b). Preventative therapy with Ad5-type I IFN only had limited efficacy in cattle. Inoculation of bovines with 1x1010 pfu/animal of Ad5-poIFN-α or Ad5bovine IFN-α (Ad5-boIFN-α) induced a relatively low level of systemic antiviral activity (100–200 U/ ml) and challenge of these animals with FMDV A24 by ID inoculation in the tongue only resulted in a short delay and reduced severity of disease compared with control animals (Wu et al., 2003). Type II IFN Approximately a decade after the discovery of type I IFN, another molecule with antiviral activity was described (Wheelock, 1965). In the early 1970s the active principle was recognized as being distinct from classical virus-induced interferons, leading to its designation as immune IFN or type II IFN (Falcoff et al., 1973), and eventually IFN-gamma (IFN- γ). This protein is a multifunctional cytokine produced by T-helper 1 (Th1) and NK cells, and its biological functions include immunoregulatory, anti-neoplastic, and antiviral properties (Biron and Brossay, 2001). The signal transduction pathways elicited by type II IFNs are different than those induced by type I IFN (Levy and Darnell, 1990). Interestingly, the combination of type I and type II IFNs can synergistically induce gene expression (Levy and Darnell, 1990; Thomas and Samuel, 1992). Following the previous work with IFN-α, and to examine the potential antiviral effect of IFN-γ on FMDV replication an Ad5 vector containing the porcine IFN-γ gene (Ad5-poIFN-γ) was constructed. Ad5-poIFN-γ was able to block FMDV replication in cell culture. Furthermore, an enhanced protective effect against FMDV in vitro when type II IFN was combined with type I IFN was observed (Moraes et al., 2007). Interestingly, the action of type II IFN in combination with type I IFN could synergistically block virus replication in vivo; swine inoculated with the combination of

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Ad5-poIFN-γ and Ad5-poIFN-α were completely protected against FMD challenge when used at doses that, individually, were not effective (Moraes et al., 2007). More recently, a similar approach in swine, using an Ad5 that expressed bicistronically porcine IFN-α and IFN-γ also showed an enhancement of the antiviral activity compared with Ad5 constructs that expressed either IFN alone in swine (Kim et al., 2014). In cattle, similarly to treatment with Ad5-type I IFN (Wu et al., 2003) treatment with a combination of type I and II IFNs (1 × 1010 pfu of each IFN/ animal) followed by challenge with FMDV A24 ID 1 day later only showed partial protection, with 4 days’ delay in the onset of the disease and lower levels of viraemia than control animals (Moraes et al., unpublished). Type III IFN A new family of IFNs, type III IFN or IFN lambda (IFN-λ), has been identified in several species including humans, mice, chicken, swine and cattle (Díaz-San Segundo et al., 2011; Kotenko et al., 2003; Sang et al., 2010; Sheppard et al., 2003). Furthermore, in vitro antiviral activity against FMDV has been demonstrated for both, bovine IFN-λ3 and porcine IFN-λ1 and –λ3 (Díaz-San Segundo et al., 2011; Wang et al., 2011). Although type I and type III IFNs induce similar innate antiviral responses, they signal through different receptors (Sheppard et al., 2003; Uzé et al., 2007). A relatively higher abundance of the type III IFN receptor in mucosal epithelial tissues makes IFN-λ an attractive candidate for control of infections that mainly initiate in these tissues (Pulverer et al., 2010; Sommereyns et al., 2008). In fact, the main natural route of FMDV infection is via aerosol through the upper respiratory tract (Alexandersen et al., 2003), with the nasopharynx region as the primary site of viral replication before subsequent spread to the lungs (Arzt et al., 2010; Pacheco et al., 2010), making this type of IFN a very appealing candidate to be used against FMDV. Inoculation of cattle with Ad5-boIFN-λ3 resulted in low systemic antiviral activity, but the induction of several ISGs in most tissues of the upper respiratory tract which are targets of FMDV infection (Díaz-San Segundo et al., 2011). Interestingly, there was an enhanced effect in the upregulated ISGs when the treatment included a

combination of Ad5 expressing type I and III IFNs. Inoculation of cattle with 1–1.5 × 1011 pfu/animal of Ad5-boIFN-λ3 followed by ID challenge in the tongue with FMDV 24 hpi, resulted in a significant delay (6 to 12 days) and reduced severity of disease (Perez-Martin et al., 2012). Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that best resembles the natural route of infection (Pacheco et al., 2010). In this experiment, no clinical signs of FMD, viraemia or viral shedding in nasal swabs were found in the Ad5-boIFN-λ3 treated animals, for at least 9 days post challenge and one of three animals remained free of disease during the entire experiment (Perez-Martin et al., 2012). These results indicated that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV, a species in which treatment with type I IFN had not been previously successful. To examine the efficacy of IFN-λ in swine, animals were inoculated with various doses of Ad5-poIFN-λ3 and one day later exposed by contact to swine that had clinical signs of FMD (Perez-Martin et al., 2014). Swine administered the highest dose of vector were completely protected from clinical disease, had no detectable viraemia or viral RNA, and no virus shedding. Two of three animals in each of the lower dose groups were also protected. This type of challenge resembles the natural route of infection in a commercial production facility or a small farm, where naive animals might be physically exposed to infected animals. Interestingly, protection was achieved even when no consistent levels of systemic antiviral activity or up-regulation in the expression of ISGs in PBMCs were detected (Perez-Martin et al., 2014), which is consistent with previous reports showing that the expression of the IFN-λ receptor and sensitivity to this type of IFN are highest in epithelial tissues and not in leucocytes (Pulverer et al., 2010; Sommereyns et al., 2008). Although more experiments collecting several tissue samples after IFN-λ3 treatment are needed to determine the role of specific ISGs in inducing protection in swine against FMDV at the primary sites of entry, replication, and dissemination, the use of type III IFN is a viable, effective, and safe strategy to control FMD early after infection in the field.

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Novel innate immune modulators The administration of Ad5-IFN, type I, II or III to cattle or swine up regulates a number of genes resulting in protection against FMDV (Diaz-San Segundo et al., 2014, 2013, 2011, 2010; Moraes et al., 2007). However, during the course of a viral

infection a broader response is initiated because of the interaction of unique viral molecules (‘pathogen associated molecular patterns’ [PAMPs]) with specific PRRs present in host cells (Honda and Taniguchi, 2006; Medzhitov and Janeway, 1998) (Box 14.3). The events involved in the

Box 14.3  Schematic of induction of antiviral pathways Ad, VEE, or synthetic RNAs such as poly IC can infect/enter cells and be recognized by different PRRs such as c-GAS, DAI, RIG-I, MDA5, TLRs, triggering many signal transduction pathways that culminate with the activation of transcription factors and the induction IFN/other molecules expression. Subsequently, IFN proteins are secreted, bind to their receptors on the same (autocrine) or other cells (paracrine) and activate of the JAK/STAT pathways inducing expression of numerous IFN stimulated genes with direct antiviral activity, e.g. OAS, Mx1, or involved in the induction of an inflammatory response, e.g. IRF-1, CXCL9. Alternative pathways may lead to secretion of other antiviral products involved in the innate response against viral infection.

VEE

Endosome

DAI

TRIF

MAVS

TLR3

TRAF3

MYD88

IRF3/7/? IRF3/7/?

IFN ER nucleus

IFN-R IRF-1,CXCL9 IFN-R

JAK1 TIK2

NF-κB AP-1

STING

STAT

STAT

TBK1 IKKε

JAK1 TIK2

TRAF6

Mitochondrion cGAMP

TLR7 TLR8 TLR9

Other antiviral genes

STAT

cGAS RIG-I/ NOD2 MDA5

PolyIC

STAT

Ad

IFNs Type I Type II Type III

Inflammatory response OAS,Mx1 Antiviral response

Unknown mechanism

Ad, adenovirus; AP-1, activator protein 1; cGAMP, cyclic di-GMP-AMP; cGAS, cytosolic GAMP synthase; CXCL9 (chemokine (C-X-C-motif) ligand 9; DAI, DNA-dependent activator of IRFs; ER, endoplasmic reticulum; GAS, γ‑activated sequence; IFN, interferon; IFN-R, IFN receptor; IRF, IFN regulatory factor; IKKε, IκB kinase‑ε; JAK1, janus kinase 1; MAVS, mitochondrial antiviral signalling protein; MDA5, melanoma differentiation-associated gene 5; Mx1, Myxoma resistance protein; MYD88, myeloid differentiation primary response protein 88; NF-κB, nuclear factor‑κB; NOD2, NOD-containing protein 2; OAS1, 2′,5′-oligoadenylate synthetase 1; PRRs, pattern recognition receptors; poly IC, polyinosinic:polycytidylic acid; STAT, signal transducer and activator of transcription, STING, stimulator of IFN genes; TBK1, TANK-binding kinase 1; TRAF, TNF receptor-associated factor; TRIF, TIR domain-containing adaptor protein inducing IFNβ; TYK2, tyrosine kinase 2; VEE, Venezuelan equine encephalitis virus.

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induction of the antiviral IFN response includes the activation of a series of transcription factors, i.e. IRFs, NF-κB, etc. Activated IRFs and NF-κB are required for IFN induction as well as upregulation of additional antiviral genes, some of which are induced by mechanisms independent of type I IFN (Kawai and Akira, 2011). By directly administering IFN, the molecular interactions that normally occur during viral infection are bypassed. It is hypothesized that treatment of animals with both IFN and various PAMPs may result in a broader, enhanced, and prolonged antiviral response than with Ad5-IFN treatment alone, including the activation of a number of signalling molecules that may potentially result in a positive feedback induction of additional IFN (Honda and Taniguchi, 2006; Marié et al., 1998). Three different strategies that could enhance the antiviral effects of IFN have been tested: (i) the use of double stranded RNA, poly IC, in combination with IFN treatment (Dias et al., 2012); (ii) the use of viral replicon particles (VRPs) derived from Venezuelan equine encephalitis (VEE) virus that could act as RNA factories (Diaz-San Segundo et al., 2013b); or (iii) expression of a constitutively active transcription factor, IRF7/3(5D) fusion protein, delivered with the Ad5 vectored platform (Ramirez-Carvajal et al., 2014). Poly-ICLC As mentioned above, poly-IC is a synthetic, double-stranded RNA that binds TLR3 and MDA-5 cellular receptors. Importantly, inclusion of poly-ICLC, a stabilized version of poly IC, acts as an adjuvant of the Ad5-FMD vaccine. Treatment of specific porcine and bovine cell lines with poly IC alone significantly reduces FMDV replication. Interestingly, in bovine cells, the combination of poly IC and boIFN-α had a synergistic effect as compared with each treatment alone, and there was up-regulation in the expression of a number of cytokines, chemokines, transcription factors, and genes with direct antiviral activity. To test the effect of this molecule in vivo in combination with type I IFN, swine were inoculated using 5- to 15-fold reduced Ad5-poIFN-α doses combined with different doses of poly ICLC. The results from this experiment indicated that 8 mg of poly ICLC is able to protect swine against FMDV A24 ID challenge when used alone or combined

with Ad5-poIFN-α (10-fold reduction compared with the dose of Ad5-poIFN-α necessary to protect by itself), demonstrating that the use of PRR agonists alone or in combination with IFNs may represent an effective and broad-spectrum antiviral strategy to combat FMDV infection and perhaps other viral diseases of livestock species (Dias et al., 2012). VRPs Pushko et al. (1997) have constructed VRPs consisting of VEE virus particles containing a defective VEE genome, in which the structural genes downstream of a subgenomic promoter are replaced by a heterologous gene/cassette of interest. Packaging of these recombinant genomes is performed in helper cell lines that provide VEE capsid and replicase activity in trans. VRPs can only undergo a single-cycle of infection when used as vaccines. VRPs have been used as vaccines to deliver various foreign genes (Hooper et al., 2009; Lee et al., 2003) including FMDV capsids (unpublished data). Konopka et al. (2007, 2009) demonstrated that null VRPs, VRPs lacking any foreign gene, induce an early innate immune response in mice within 1 to 3 hpi, resulting in the up-regulation of a number of ISGs and the production of type I IFN protein, and can also protect mice against VEE challenge (Konopka et al., 2009). In the case of FMDV, pretreatment of cells with VRPs containing green fluorescent protein (VRP-GFP) as well as porcine IFN-α (VRP-poIFN-α) significantly reduced FMDV replication in infected porcine or bovine cells, and inhibition lasted for at least 5 days. A number of genes were upregulated after treatment of swine cells with either VRP-GFP or VRP-poIFN-α (Diaz-San Segundo et al., 2013). VRPs were also very effective against FMDV in vivo in a smallanimal model. Adult C57BL/6 mice, which can be lethally infected with FMDV (Salguero et al., 2005), survived challenge when pretreated with VRP-GFP (Diaz-San Segundo et al., 2013b), indicating that VRP treatment is an effective approach in vivo to stimulate a strong innate IFN dependent immune response. Using the VRPs to express IFN in the natural host is a very promising strategy to initiate the IFN pathway at various levels, therefore inducing a robust innate immune response able to protect against FMD.

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IRFs IRF3 and IRF7 are key activators of the IFN α/β genes. A construct, IRF7/3(5D), containing 246 amino acids from human IRF7 (the DNA binding and constitutive activation domains) and 295 amino acids from human IRF3 (5D) (the transactivation and signal response domains) was described to induce the activation of IFN promoters in vitro (Lin et al., 2000). A constitutively active porcine IRF7/3(5D) [poIRF7/3(5D)] synthetic construct expressed using a replication-defective human Ad5 [Ad5-poIRF7/3(5D)] showed antiviral activity against FMDV in vitro and in vivo. Expression of poIRF7/3(5D) enhanced the antiviral activity of Ad5–poIFN-β against FMDV. Furthermore, mice inoculated with an Ad5-poIRF7/3(5D) developed no viraemia after FMDV serotype A24 challenge, and their sera had high levels of antiviral activity correlating with increased systemic levels of murine IFN-α/β (muIFN-α/β) (Ramirez-Carvajal et al., 2014). Interestingly, this Ad5-poIRF7/3(5D) has been proven to be very effective in swine, since low doses of this Ad5 (1 × 108 pfu/animal) can completely protect the animals against FMDV challenge 24 hours after the treatment (RamirezCarvajal et al., 2016). Although a mild systemic antiviral activity was observed in swine treated with Ad5-poIRF7/3(5D), additional analyses are needed to elucidate mechanisms of protection induced by poIRF7/3(5D). In any case, this antiviral strategy can contribute to the development of improved biotherapeutics to control FMDV infection in animals. Ad5-small RNAs as antivirals against FMD RNAi has been explored as an alternative control strategy against FMD. RNAi is a natural process by which double-stranded RNA directs sequence specific post-transcriptional gene silencing (Fire, 1999; Hammond et al., 2001; Sharp, 1999). Specific inhibition of endogenous or pathogen mRNA by RNAi can be triggered by the introduction of 21–23 nucleotide (nt) duplexes of RNA (siRNAs) or by transcription of DNA precursors into short hairpin RNAs (shRNAs) homologous to target sequences (Brummelkamp et al. 2002; Elbashir et al., 2002). Over the past years, several laboratories have used this technology to attenuate viral infection in cell culture (Coburn and Cullen, 2002; Gitlin et al.,

2002; Jacque et al., 2002; Phipps et al., 2004) and in animals (McCaffrey et al., 2003). In the case of FMD, this technology was initially tested in vitro against homologous strains of FMDV (Chen et al., 2004; Kahana et al., 2004). However, one of the advantages of an antiviral strategy is the possibility of targeting several serotypes. Therefore, the technology was later proven successful against four different FMDV serotypes in vitro (de los Santos et al., 2005). In order to deliver the RNAs in vivo, several groups have used the Ad5 platform. With this strategy swine were protected when two Ad5 constructs targeting structural and NS protein coding regions were combined (Chen et al., 2006). Interestingly, this strategy was useful even when animals were treated 3 days after the challenge (Kim et al., 2008). More recently, a slightly different strategy expressing shRNA against a structural and NS protein in tandem also induced protection in a guinea pig and in a suckling mouse model (Kim et al., 2010; Xu et al., 2012). However, FMDV possesses resistance mechanisms against antiviral agents, such as mutation against siRNA target sequences (Belsham and Normann, 2008; de la Torre et al., 1988; Pariente et al., 2003; Pfister and Wimmer, 1999). Kim et al. (2012, 2015) anticipated that the combination treatment of antiviral agents with different mechanisms might be more advantageous in overcoming their individual limitations. They initially demonstrated that the combination of Ad-poIFN-α and Ad-siRNA was a successful strategy against FMD in a mouse model (Kim et al., 2012). More recently, they have improved the strategy using a new Ad5 construct simultaneously expressing porcine IFN-α and -γ (Ad-poIFN-αγ), combined with an Ad4 expressing three siRNAs (Ad-3siRNA) targeting FMDV mRNAs for NS proteins. This combination treatment was effective against all serotypes of FMDV in swine (Kim et al., 2015). Thus, a combined treatment with Adporcine IFN-α/γ and Ad-3siRNA could work as a fast-acting antiviral treatment to induce protection prior to the induction of vaccine mediated adaptive immunity. Combination of Ad5-FMD vaccines and Ad5-IFN Thus far, different strategies to develop and improve the use of replication-defective Ad5-based vaccines

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and biotherapeutics to induce either, a robust long lasting adaptive and cellular immune response with strong neutralizing antibody levels or to induce a rapid innate immune response to control FMD have been discussed. However, a complete control strategy would ideally include both, a rapid control of disease spread as well as long-lasting protection that would ultimately cover livestock from being infected with FMDV. Therefore, it would be reasonable to combine both treatments as the next step in designing the best control strategy against FMD. In a proof of concept study in swine, Moraes et al. (2003) treated animals with a combination of Ad5-poIFN-α and Ad5-A24 subunit vaccine challenging at 5 dpi with FMDV A24. Treated animals were completely protected against FMD, and developed a significant adaptive immune response (Moraes et al., 2003). More recently, similar results were described in cattle treated with a combination of Ad5-boIFN-λ3 and Ad5-O1Manisa (Ad5-O1M) and challenged by aerosol exposure (Diaz-San Segundo et al., submitted). As expected, animals treated with Ad5-boIFN-λ3 alone or in combination with Ad5-O1M were protected when challenged at 24 hpi while animals treated with Ad5-O1M alone were not. Interestingly cattle treated with the combination of Ad5-boIFN-λ3 and Ad5-O1M were protected when challenged 3 dpi while animals treated with Ad5-boIFN-λ3 or Ad5-O1M alone showed either no or partial protection. Remarkably, protection of animals treated with the combination occurred despite the absence of detectable neutralizing antibodies or antiviral activity in serum at the time of challenge. These two studies demonstrated that with a careful design strategy, it is possible to control FMD in the event of outbreaks in disease-free or enzootic countries. If successful, this approach could significantly reduce disease spread and ultimately lead to eradication. Future directions Although significant progress has been made in the development of Ad5 delivered FMD vaccines, as demonstrated by the granting in 2012 of a conditional licence for inclusion of an Ad5-A24 vaccine in the US National Veterinary Vaccine Stockpile by the Center for Veterinary Biologics of the Animal Plant and Health Inspection Service of the USDA

(USDA Product Code 1FM1.R0; Colby et al., 2013), a number of improvements, as well as field testing, are still necessary for using this approach as a viable alternative to the current inactivated whole virus vaccine. Perhaps the major obstacle is the economics of Ad vaccine production based on the vaccine dose required to induce protection. As discussed in this article, we have shown that the dose can be considerably reduced by (a) including the coding region for the FMDV NS protein 2B in the construct; (b) altering the site and route of vaccination; and (c) including an adjuvant in the vaccine formulation. Approaches to further improve the efficacy of the vaccine by broadening vector tropism to target DCs in cattle, have not resulted thus far in enhanced protection (Medina et al., 2016). However, additional experiments are needed to determine if similar approaches such as substituting the fibre of Ad5 by the fibre of other Ad serotype could target bovine DCs or other APCs resulting in a beneficial effect. In addition, similar studies are needed in other livestock species of interest, including swine and ovine. Further studies are also required to determine the optimal adjuvant/s that should be included in the vaccine formulation. For example, adjuvants which act as PAMPs and can initiate an innate immune response resembling natural virus infection as well as initiating/enhancing antibody and cell-mediated immune responses, have shown advantages over some traditional adjuvants (Goulet et al., 2013). As mentioned in this chapter, poly ICLC is effective and results in Ad5 vaccine dose sparing, but only after 21 dpv (Diaz-San Segundo et al., 2014). Additionally, studies on duration of immunity conferred by Ad5-FMD should be investigated. In a preliminary study we demonstrated that cattle given an Ad5-A24 boost at 9 weeks developed a significant increase in FMDV-specific neutralizing antibodies and were protected when challenged 2 weeks post-boost (Grubman, 2005). This result is of particular importance since it demonstrated that the Ad5-FMD vaccine approach could be used in vaccination campaigns without concern that antibodies against the Ad5 vector would block vector delivery and transgene expression. Development of more broadly protective FMD vaccines is also an area that requires more research. Chimeric viruses containing epitopes from more

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than one FMDV serotype can protect swine against challenge with either serotype (Rieder et al., 1994), while an Ad5 vector containing more than one capsid precursor can induce an immune response to each capsid (Wu et al., 2003). Designing Ad5 vectors with epitopes conserved across FMDV subtypes are approaches that also need to be examined. The combination of an Ad5 delivered IFN or transcription factor vector and Ad5-FMD vaccine can induce both rapid protection and hopefully long lasting vaccine specific protection, but more research is required to understand which approach is appropriate for cattle and swine. Likewise studies examining vaccine efficacy as well as the combination approach should be performed in other economically important species including sheep and goats. Another topic of concern in the use of replication-defective Ad5 derived vectors is the emergence of replication-competent adenovirus (RCA) (Xiang et al., 1996; Kumar et al., 2015) due to the possibility of homologous recombination in packaging HEK 293 cell lines required for production of recombinant Ad5. Our Ad5-FMD vectors have approximately 1% RCA (Mayr et al., 1999). Utilization of vectors with additional deletions or modification in overlapping sequences should also be considered (Mizuguchi and Kay, 1999). In fact, the licensed Ad5-A24 vaccine was constructed with an Ad5 partially lacking regions of E1, E3 and E4 and produced in proprietary cell lines (GenVec, Inc). However, a fine tuning will be required balancing the usually reduced yield in large scale production of such vectors. Undoubtedly, the use of the adenoviral vector platform to control FMD in cattle and swine has resulted in a reliable and versatile vehicle for antigen delivery, however many challenges remain and are the subject of current research. References Ahl, R., and Rump, A. (1976). Assay of bovine interferons in cultures of the porcine cell line IB-RS-2. Infect. Immun. 14, 603–606. Aldabe, R., and Carrasco, L. (1995). Induction of membrane proliferation by poliovirus proteins 2C and 2BC. Biochem. Biophys. Res. Commun. 206, 64–76. Alejo, D.M., Moraes, M.P., Liao, X., Dias, C.C., Tulman, E.R., Diaz-San Segundo, F., Rood, D., Grubman, M.J., and Silbart, L.K. (2013). An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored

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Antiviral Therapies for Foot-and-mouth Disease

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Annebel R. De Vleeschauwer, David J. Lefebvre and Kris De Clercq

Abstract Prevention and control of foot-and-mouth disease (FMD) is traditionally based on zoosanitary measures and vaccination. The genetic and antigenic diversity of the FMD virus (FMDV), its highly infectious nature and enormous dynamic potential illustrate that pan-serotype targeted control measures that inhibit immediately upon administration viral replication would be a valuable support tool or alternative to vaccination to control FMD outbreaks in an early stage. In this respect, antiviral drugs against FMDV come into focus. Many research groups have pursued various approaches in search of anti-FMD therapies. Although the research topic is rather young some encouraging achievements have been reported, but for all approaches several issues still need to be addressed before reliable practical application can be reached. In this chapter we review the history and the state of the art of antiviral drugs against FMDV. Nucleoside analogues, small chemical molecules, oligonucleotides, new molecular biological techniques such as RNA interference, single-domain antibodies and derivatives from natural products are covered. Overall challenges and strengths and weaknesses specific to the different approaches are discussed. Background Traditional preventative measures for foot-andmouth disease (FMD) at farm level include basic knowledge of infectious diseases, hygienic husbandry and veterinary practices and quarantine of purchased animals. At the level of markets and slaughterhouses preventive measures include staff education, adequate hygiene management including measures for vehicles and veterinary inspection

of animals and animal products. At a regional or country level effective prophylaxis includes efficient veterinary services and authorities, surveillance and monitoring of animal diseases, animal identification, surveillance and control of animal movements including fencing and trade restrictions and traceability of animals and animal products. Effective prophylaxis may also comprise voluntarily or compulsory vaccination and post-vaccinal serological monitoring. Early detection of FMD based on prompt notification of suspected cases and effective clinical and laboratory diagnosis is of primary importance to keep the size of the outbreak within well manageable limits (Gibbens et al., 2001; Gibbens and Wilesmith, 2002; Muroga et al., 2012). In case of an outbreak of FMD traditional control measures include movement restrictions in particular for but not limited to animals and animal products, the establishment of protection and surveillance zones, culling of infected herds and carcass disposal, sanitary measures including cleaning and disinfection of infected premises and of visiting people and vehicles and pre-emptive culling and/or emergency vaccination of herds at risk (EU, 2003; OIE, 2015). Although the strategy to apply pre-emptive culling for the containment of an outbreak of FMD may be highly efficient from sanitary and export trade perspectives, this strategy is becoming more and more controversial, particularly from an ethical point of view. Recent cases have shown that pre-emptive culling (whether or not preceded by emergency vaccination) may provoke the death of tens or hundreds of thousands or even millions of healthy animals (Bouma et al., 2003; Kitching et al., 2006; Mansley et al. 2011; Muroga et al., 2012; Wee et al., 2004). This is not only unacceptable

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with regards to animal welfare but is also a huge waste of resources. This is in sheer contrast with the twenty-first century vision of a highly productive yet sustainable and animal-friendly agriculture (EU, 2013; FAO, 2014). Despite the disadvantages of pre-emptive culling and despite the fact that vaccination is a very powerful tool to prevent the spread of FMD, vaccination does not offer the Holy Grail. The successful eradication of smallpox (1980) and rinderpest (2011) can at least partially be attributed to the fact that these viruses existed in a single serotype and that vaccination offered protection for at least several decades in humans (smallpox) (Taub et al., 2008) or life-long in cattle (rinderpest) (Taylor et al., 2006). Foot-and-mouth disease virus (FMDV), however, exists in seven different serotypes and a variety of antigenic subtypes. Vaccine strains need to be antigenically ‘matched’ to the circulating field strains and protective immune responses induced by vaccination usually do not last longer than 6 months (see Chapter 12 and Domenech et al., 2010). After formulation – for emergency vaccines this is usually from strategic frozen antigen stocks – FMD vaccines easily degrade if the stringent requirements for maintenance of the cold chain are not respected. As FMD vaccines usually do not contain preservatives, it is also essential to vaccinate in a hygienic manner, according to good veterinary practices. Once a vaccine flask has been punctured, the vaccine should be administered without any delay (Philippe Dubourget, personal communication). Highly potent FMD vaccines rapidly induce a strong immune response but animals can still develop clinical disease if the infection with FMDV takes place within the first 4–7 days following vaccination (Golde et al., 2005). Emergency vaccination is also ineffective if the animals are newly infected with FMDV before the time of vaccination. Antiviral drugs are available for a number of human diseases such as HIV infection, hepatitis B and C, influenza and various types of herpes and provide not only a very efficient but also a very attractive way of medicinal treatment. There is a significant interest in the commercial development of antiviral drugs for animals, but in Europe only recombinant feline interferon omega is commercially available. Up-to-date companion animals are increasingly treated with antiviral drugs that were originally developed for humans. Therapy is

mostly empiric and dosage, route and interval of administration are often extrapolated from data in humans and laboratory animals. As these antiviral drugs were not specifically developed for a certain viral infection in a certain animal species, these treatments often lack effectivity due to poor bioavailability and in some cases even can be toxic at therapeutic doses. Treatment regimens and success rates of antiviral therapy in companion animals are poorly documented in scientific literature (Dal Pozzo and Thiry, 2014; Hartmann, 2012). The commercial availability of antiviral drugs for large animals would have numerous advantages in particular for viral diseases for which a good vaccine is not yet available, such as African swine fever, or diseases for which vaccine strategies need to be extensively tailored, such as FMD. In the case of FMD manufacturers envisage an antiviral drug that is active against several serotypes, preferably all seven, and that exerts strong inhibition of the viral replication from the first administration onwards. This drug must be active at low compound concentrations and have a favourable Absorption, Distribution, Metabolism, Excretion – Toxicity (ADME-Tox) profile, a high therapeutic index and a high barrier towards the selection of resistant viral mutants. This drug should be stable at ambient temperatures, have a long shelf life and should be easily palatable, soluble in drinking water or formulated into a bolus. With regards to food safety and the selection of resistant viral mutants in humans this drug should preferably not be active against human viruses related to FMD, such as human enteroviruses or rhinoviruses, or have at least a short withdrawal period. With regards to environmental safety it is preferred that this drug is not toxic for animal species after accidental uptake of medicated feed or drinking water. A persistent or ecotoxic effect of the drug or its metabolites excreted by treated animals in the environment is undesired. In FMD-enzootic settings where vaccination is not routinely applied ‒ usually in developing countries ‒ antiviral drugs might not only prevent livestock to become infected with FMDV it might also relieve disease and accelerate recovery in infected livestock and reduce mortality in young animals. In this way antiviral drugs would reduce losses of milk, meat and draught power and reduce decreased fertility, enhancing the food security and contributing to an improved household income

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and livelihood of smallholders (Bayissa et al., 2011; Jemberu et al., 2014). We are however well aware that food safety is a major concern when antiviral drugs would be administered to smallholders’ livestock of which the products are intended for daily consumption (milk) or have a high value (meat). In settings where vaccination against FMD is routinely applied, antiviral drugs might be a complementary tool. High potency vaccines that match the FMD outbreak strain are not always timely available in sufficient numbers or vaccination might be difficult to deploy in more remote areas. In such cases the administration of antiviral drugs might immediately reduce viral replication, excretion and transmission, irrespective of the serotype of the FMD outbreak strain and independent of technical requirements such as maintenance of the cold chain. In zones or countries that were previously free from FMD, antiviral drugs might as well bridge the time gap between the moment of vaccination and the moment that vaccinated animals are effectively protected (‘immunity gap’). Moreover, simulation exercises with classical swine fever in densely populated pig areas have shown that antiviral drugs might constitute a cost-effective, stand-alone alternative for emergency vaccination (Ribbens et al., 2012; Backer et al., 2013). Ideally, antiviral drugs can be quickly and safely administered in the feed or drinking water or as a bolus and are an easy and efficient way to treat large herds. In case of a large outbreak, this requires that antiviral drugs are sufficiently available from stockpiles. In small herds ( 50 µM (Osiceanu et al., 2014). Data look promising but much more research needs to be done. None of the presently reported small chemical molecules is sufficiently potent for practical application to prevent or treat FMDV infections in the field. Compounds need to be optimized and EC50 values in the low nanomolar region are desirable. In addition, the evaluation of the anti-FMDV activity is mainly based on in vitro

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experiments and experiments in rodents and the question remains whether optimized molecules will be effective to block FMDV replication in cloven-hooved animals. Other issues that remain to be explored include the pharmacological and toxicological profile, the stability and solubility, the genetic barrier to resistance of FMDV towards these compounds and the feasibility of large scale production and administration to livestock. Nucleic acid-based strategies The study area of antiviral approaches against FMDV of which the majority of reports originate is the one using nucleic acid-based strategies. The study of sense and antisense oligonucleotides to block FMD viral replication started in the early nineties. Studies on RNA interference (RNAi) against FMDV first emerged at the beginning of this century. Antisense oligonucleotides and RNAi approaches act on the same principle: the binding of an oligonucleotide to a target RNA through Watson–Crick base pairing. Sense sequences, on the other hand, may interact indirectly through competition with viral sequences for cellular cofactors required for viral RNA replication or more likely through functional distortion of a structural viral genome motif required for RNA replication (Bigeriero et al., 1999). Sense and antisense oligonucleotides Antisense oligonucleotides (ASO) are singlestranded molecules of about 20 nucleotides designed to be complementary to target mRNA. Base-pairing between the ASO and its target results in RNase H-dependent degradation of the target RNA or direct inhibition of translation (reviewed in Cohen, 1991; Spurgers et al., 2008). The group of Professor Dr. Sobrino was the first that described the anti-FMDV effect of ASO. Six antisense oligodeoxyribonucleotide (ODN) sequences corresponding to the IRES (IRES1 and IRES2), the functional translation initiation regions (AUG1 and AUG 2), the 2A gene and the beginning of the 3′ NCR sequence of FMDV O/ Kaufbeuren were designed and micro-injected either alone either as a mixture of two in BHK-21 cells that were immediately thereafter infected with O/Kaufbeuren at a moi of 2. The expression of the

VP1 protein was transiently inhibited (35–52% inhibition at 5 hpi) by the ODN targeted at the AUG2 sequence at concentrations of 125–250 µM (not 20 µM). However, no inhibitory effect on virus yield was detected on BHK-21 covered with ‘AUG2’ ODN containing medium 2 hours prior to and after infection with O/Kaufbeuren at a moi of 0.001. The optimized form of the ‘AUG2’ ODN which contained a phosphorothioate linkage reduced the virus yield by ≥ 40% up to 19 hpi. Combined administration of the ‘AUG2’ ODN with other ODN did not increase the viral inhibition (Gutiérrez et al., 1993). In addition, this group tested sense (s) and antisense (as) RNA molecules that target the 3′ terminal region of FMDV serotype C (C-S8) (p-3′D s and as, p-3′1 s and as, p-3′2 s and as) and the 5′ non-coding region including the proximal element of the IRES site and the two functional initiation AUGs (p-5′1 s and as) for their inhibitory effect on the translation of purified FMDV RNA in an in vitro rabbit reticulocyte lysate system. Of all molecules tested, only the p-5′1 as molecule induced a significant inhibition (68%) of the translation and protein synthesis compared to the untreated control. When co-transfected with infectious homologous FMDV RNA all but the p-5′1 s molecule resulted in marked reduction of the VP1 expression (~ 35–75% inhibition, > 60% for p-5′1as) at 5 hpi and inhibition of plaque formation (~ 25–85% inhibition, > 60% for p-5′1as) at 36 hpi on BHK-21 cells. The degree of inhibition was inversely related to the length of the molecules. Injection of the p-5′1 as molecule in BHK-21 1 hour prior to infection with C-S8 (moi of 0.01) inhibited plaque formation by 30–40% (Gutiérrez et al., 1994). Combination of p-5′1 as with p-3′2 s or p-3′2 as increased the inhibitory effect compared to the single molecules (Bigeriero et al., 1999). Inhibition percentages of heterologous FMDV serotypes O (O/Kaufbeuren) and A (A5Ww), with 79–91% sequence identity to the homologous C-S8 strain in the target regions, were similar as those of C-S8 (Bigeriero et al., 1999). The inhibition was specific for FMDV as the plaque formation of EMCV or SVDV was not affected. The observed effects were dose-dependent and an excess (200- to 20,000-fold) of RNA molecules compared to FMDV RNA was needed to obtain the reported antiviral effects. Efficient hybridization between viral RNA and the transcripts was crucial for the inhibition of the PFU as PFU inhibition

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percentages were markedly higher (up to 90% when p-5′1 as and p-3′2 s or p-3′2 as were combined) when viral RNA and the oligonucleotides were allowed to anneal under renaturing conditions prior to transfection into cells (Gutiérrez et al., 1994; Bigeriego et al., 1999). In further research, Rosas et al. (2003) demonstrated that constitutive expression of p-3′1 as, p-5′1 as separately or together in BHK-21 cells reduced the plaque formation (37–90%) and virus yield (37–99%) at 24 hpi and delayed CPE formation by ≥ 4 days after infection with 50–100 PFU of the homologous C-S8 strain, as well as with the heterologous O/Kaufbeuren strain. C-S8 virus recovered from p3′1 as and p-5′1 expressing cells did not show a decrease in infectivity upon six further passages on these cells, nor were mutations in the target sequences found in the virus recovered after the sixth passage (Rosas et al., 2003). Morpholino oligomers Phosphorodiamidate morpholino oligomers (PMOs) are ASO in which the ribose ring of the nucleotide (nt) backbone is replaced with a morpholine ring leading to an increased stability and solubility and decreased off-target toxicity. Unlike ASO, PMO do not mediate their inhibitory effect via RNase-mediated degradation of their RNA target but by interference with the mRNA processing and translation since they are typically designed against IRES or AUG start codons (Spurgers et al., 2008). Six peptide-conjugated PMO (PPMO) complementary to sequences in the 5′ and 3′ untranslated regions of the FMDV strain A24 Cruzeiro genome were designed by Vagnozzi et al. (2007). Peptide conjugation increases cell-uptake and persistence of the PMO. The PPMOs targeting the 3′ side of the IRES (5D) and the AUG translation initiation regions (AUG1 and AUG 2) induced a reduction in viral RNA production, viral protein expression and viral titres up to 6 log10 in BHK-21 cells treated with 5 µM PPMO before and after infection with A24 Cruzeiro at a moi of 0.5, whereas the activity of the PPMO directed against the 5′ side of the S-fragment, the 5′ side of the IRES and the 3′UTR region was less outspoken with reductions in virus titres up to 2 log10. Cell viability staining indicated 5 µM as the maximum tolerable concentration of PPMO without apparent cytotoxic effects. In accordance with the number of sequence mismatches between the designed PPMO and other FMDV serotypes, the

PPMO targeting AUG1 reduced viral replication of FMDV strain A12 to a similar extent as the A24 but FMDV serotypes Asia1 and C to a lesser extent (~ 2 log10), and the PPMO targeting 5D resulted in titre reductions from 2 log10 to 7 log10 of FMDV serotypes O, Asia1, C, SAT1 and SAT2 at a PPMO concentration of 25 µM. Serial passage of FMDV A24 Cruzeiro in the presence of concentrations of ‘AUG1’ PPMO increasing from 0.5 µM to 1 µM resulted after three passages in the absence of CPE, undetectable viral RNA and the inability to isolate infectious virus upon three blind passages. On the other hand, five serial passages of FMDV A24 Cruzeiro in the presence of concentrations of ‘5D’ or ‘AUG2’ PPMO increasing from 0.5 µM to 2.5 µM resulted in variant viruses that had advantages in growth in the presence of the PPMO compared with the wild type virus. Comparative sequence analysis of the ‘5D’ and the ‘AUG2’ PPMO variant viruses, the wild type virus and the virus passaged five times in the absence of PPMO revealed nonsynonymous nt substitutions in the PPMO target site (Vagnozzi et al., 2007). RNA interference RNA interference (RNAi) is a cellular pathway that plays a role in cellular gene regulation and may act as a cellular antiviral mechanism especially in plants and invertebrates but the RNA silencing machinery is conserved in vertebrates (reviewed in Gitlin and Andino, 2003; van Rij and Andino, 2006; Arbuthnot, 2011). Although no direct evidence of the function of RNAi as a natural antiviral mechanism in mammals has been reported (Cullen, 2014), Elbashir et al. demonstrated in 2001 that introduction small interfering (si) RNA into mammalian cells could silence target genes by RNAi. Since then, this mechanism has been widely exploited as the subject of research aiming at the development gene-specific therapeutics for viral diseases, including FMDV. Several RNAi approaches including plasmid- or vector-encoded short hairpin (sh) RNA and endogenous micro (mi) RNA complementary to the FMDV genome have been studied for their antiviral effect against FMDV in vitro and some in vivo and are discussed below (overview of in vivo studies in Table 15.1). Some studies even aimed at the development of transgenic animals naturally resistant to FMDV infection. Some promising data have been

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achieved, but generally these techniques, like s and as oligonucleotides, have to overcome some obstacles before practical and widespread use in the field of livestock diseases, and especially FMDV, may be envisaged (Paroo and Corey, 2004; Grubman and de los Santos, 2005; Bayry and Tough, 2005). Ideally for an effective and broad panserotype antiviral activity against FMDV the target region in the viral genome is essential during the replication cycle of the virus and is highly conserved between different serotypes of FMDV. Moreover, the use of siRNA as an antiviral agent could lead to a selective pressure on the siRNA target that might result in the appearance of escape variants due to changes in the target sequence, therefore high sequence identity between the interfering RNA and the target sequence is warranted and simultaneous targeting multiple genomic regions may be useful. SiRNA and shRNA Introduction of exogenous double-stranded (ds) RNA into cells results in the sequence specific post-transcriptional silencing of complementary mRNA. Small interfering (si) RNAs are generally 21 to 23 nucleotide ds RNA molecules that mediate

cleavage of target transcripts through the activation of the RNAi pathway. Short hairpin (sh) RNA are plasmid- or vector-expressed and are intracellularly processed into siRNA. Much research on this topic was led by Professor Dr Zheng at Fudan University in China. His group was the first to show a 80–90% sequence specific reduction of the plasmid expressed VP1 region of the HKN/2002 FMDV serotype O genome (pVP-EGFP-N1) in BHK-21 cells at 24 hours after simultaneous transfection of these cells with plasmids expressing the inverted repeat of either a 21 or 63 nt sequence (shRNA) of this VP1(pNT21, pNT63). Reduction was determined by immunofluorescence microscopy and by fluorescence activated cell sorting based on the expression of the enhanced green fluorescent protein p-EGFPN1 by the reporter plasmid containing the VP1 target sequences and co-transfected with the shRNA-expressing plasmid and RT-PCR analysis. In addition, transfection of BHK-21 cells with either the pNT21 or pNT63 plasmid 24 hours prior to infection with 100 TCID50 of HKN/2002 delayed CPE formation up to 24 hpi, but not up to 48 hpi. The virus yield at 12 hpi in transfected

Table 15.1 Overview of in vivo studies on RNAi against FMDV

Reference

Target genomic region

siRNA sequences based on FMDV strain

Animal species

In vivo test challenge virus

Chen et al. (2004)

VP1

O/HKN/2002

Suckling mice

O/HKN/2002

Chen et al. (2006)

VP1, 3D

O/HKN/2002

Guinea pigs and O/HKN/2002 large white pigs

Kim et al. (2008)

2B, 3C

O/SKR/2000 and O/SKR/2002 Suckling mice

O/SKR/2002

Pengyan et al. (2008)

2B, 3D

Sequences available from GenBank

Suckling mice

O and Asia1

Joyappa et al. (2009)

IRES, 3D

Three serotypes of Indian origin

Guinea pigs

O/R2/75

Cong et al. (2010)

VP4, 2B, 3D

3D O/HKN/2002, VP4 Asia1/ YNBS/58 and 2B O/CHA/99

Suckling mice, guinea pigs and pigs

O/HKN/2002 and Asia1/ YNBS/58

Kim et al. (2010)

2B, 3C

O/SKR/2000 and O/SKR/2002 Suckling mice

O/SKR/2002

O, A and Asia1

Transgenic bovine embryo

Asia1/Ys/CHA/05

Wang et al. (2012) VP2, VP3, VP4, 2B Xu et al. (2012)

VP1, 2B

O/HK/2002

Guinea pigs

O/HK/2002

Jiao et al. (2013)

2B, 3D

O/HKN/2002

Transgenic suckling mice

O/HKN/2002

Chang et al. (2013)

IRES

O/HN/CHA/93

Suckling mice

O/HN/CHA/93, A/AF/72, Asia1/ Jiangsu, O/Tibet/China/1/99 and O/CHN/Mya98/33-P

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cells was reduced by 1000-fold compared to mocktransfected control cells, but this effect gradually waned and no difference was seen at 72 hpi. The inhibition of viral replication was demonstrated to be sequence specific as it was not seen when cells were infected with another serotype O FMDV strain (CHA/99) or a pseudorabies virus. In suckling mice, subcutaneous (SC) administration of either pNT21or pNT63 plasmid at 6 hours prior to challenge with 20 lethal dose (LD)50 of HKN/2002, resulted in a survival rate of 75% of the mice at 5 dpi. No or borderline VP1 mRNA could be detected in the internal organs of the surviving treated mice at 5 dpi, whereas mock-treated animals all died within 36 hpi with high amounts of visceral VP1 mRNA. The protective effect of the pNT21or pNT63 plasmids was less outspoken when administered simultaneously with virus challenge or when the viral challenge dose was higher (100 LD50). Simultaneous administration of the VP1 expression plasmid together with the VP1 specific shRNA 6 hours prior to challenge increased the survival rate of mice compared with that in mice challenged after only being given the VP1 specific shRNA (Chen et al., 2004). This research group further explored the use of siRNA targeted to various more conserved regions of the FMDV genome which were selected based on sequence similarity (85–98%) of FMDV strains of serotype O, A, C and Asia1 in the NCBI database with the HKN/2002 strain. The selected genome regions included the 5′ non-coding region (5′NCR), the 3′ non coding region (3′NCR), VPg, VP4 and 3D (Pol). ShRNA expressing plasmids and p-EGFP-N1 reporter plasmids containing the target sequences were constructed using similar techniques as Chen et al. (2004). In BHK-21 cells transfected with these shRNA plasmids, the expression of EGFP and the target RNA yield at 24 hours post transfection (hpt) was significantly reduced compared to mock controls, and this was similar for all target sequences. Infection of BHK-21 cells with 100 TCID50 of HKN/2002 5 hours after transfection and titration of the supernatant at 24 and 48 hpi revealed a 10- to 1000-fold reduction in virus titres compared with mock controls. The antiviral effect was extended to 6 dpi. When challenged with a heterologous serotype O (CHA/99) a similar reduction in virus yield was observed at 48 hpi with all siRNAs, whereas upon challenge with FMDV

serotype Asia1 (YNBS/58) 10-fold reduction was seen with the 5′NCR, Pol and 3′UTR siRNA only and the effect lasted up to 48 hours but not 60 hpi. Despite the significant reduction seen with siRNA treatment, homologous HKN/2002 virus titres still reached up to > 4 log10 TCID50 per 100 µl supernatant at 48 hpi. Infection of BHK-21 cells 1 hours before transfection resulted in an increased RNAi effect (delayed CPE formation and lower virus titres) and the suppression of the viral replication was prolonged until 198 hpi as compared with pretreated cells with all but the 3′NCR specific shRNA (150 hpi) (Liu et al., 2005). To address difficulties that might be associated with the in vitro and in vivo delivery of RNAi, this research group used similar plasmid construction techniques (Chen et al., 2004) to examine the efficacy of shRNA directed against the VP1 (pAd5-NT21) and 3D (pAd5-Pol) region of FMDV HKN/2002 delivered by a recombinant replication-defective human adenovirus type 5 (rAd5). Treatment of IBRS-2 cells at a moi of 5 and 10 with pAd5-NT21 or pAd5- Pol 12 hours before infection with 100 TCID50 of HKN/2002 inhibited the formation of virus-induced CPE until 72 hpi, reduced the viral RNA yield at 36 hpi and the virus yield in the supernatant at 72 hours by 100% compared with the untreated control. When challenged with CHA/99 only pAd5-Pol inhibited the CPE formation and the virus yield. Typical CPE, high viral RNA and virus yield were obtained in untreated FMDV-infected and treated PRV infected control cells from 24 hpi on. Intramuscular (IM) injection of 106 PFU of pAd5-Pol in guinea pigs 24 hours prior to intradermal challenge with 50 (infectious dose) ID50 of HKN/2002 mitigated clinical signs and protected three out of five animals completely from vesicular lesions and fever that were present in all untreated control animals. However, when challenged with 200 LD50 only one out of five treated animals was protected. Treatment results were also less favourable for pAd5-NT21. Treatment of guinea pigs with a mixture of pAd5-NT21 and pAd5-Pol and viral challenge 24 hours later, or twice with this mixture with a 24 hour interval and viral challenge at the time of the second treatment did not increase clinical protection. Large white pigs inoculated IM with a ‘low’ dose of 2 × 109 PFU for each of a mixture of pAd5-NT21 and pAd5- Pol and challenged 24 hours later with 100 ID50 of

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HKN/2002 were markedly protected against the development of virus induced vesicular lesions and fever that were present in all control pigs from 5 dpi on. Only one out of three treated pigs showed mild vesicles at 15 dpi. When pretreated with a double dose (4 x 109 PFU each) only one out of three animals was completely protected against clinical signs, but in the two other pigs disease was delayed (6 and 10 dpi) and milder compared with control-treated animals. Upon infection, control-treated animals showed high degree of viraemia at 6 dpi but not at 14 dpi and virus neutralizing antibodies at 14 and 21 dpi. None of the pigs treated with the low dose had detectable viral RNA in their serum at 6 dpi, and only the one pig showing delayed clinical signs was viraemic at 14 dpi, but cross-contamination from infected animals could not be excluded. Viraemia was not examined in the pigs treated with the double dose. All pigs that showed clinical disease developed significantly higher antibodies against 3ABC polyprotein of FMDV than animals that were protected from clinical symptoms (Chen et al., 2006). To further meet the challenges of the high genetic variability of FMDV and in search of an optimal vector for delivery of siRNA, the group of Professor Dr Zheng constructed plasmids expressing shRNA targeted at conserved genome regions that corresponded to 3D gene sequence fragments of O/HKN/2002 (P3D-NT19 and p3D-NT56), VP4 gene sequence fragments of Asia1/YNBS/58 (pVP4-NT19 and pVP4-NT65) or a 2B gene sequence fragments of O/CHA/99 (p2B-NT25) and introduced those in a recombinant attenuated Salmonella choleraesuis delivery vector (rS. cho). This vector was chosen because unlike the rAd5-vector the rS. cho vector could be more often recovered from the lymph node, tonsils, lung and alimentary tract of treated pigs, sites that correspond more with the infection routes of FMDV than the liver which was reported to be the main site of rAd5-vector recovery. The virus titres of FMDV serotype O strains HKN/2002, CHA/99 and serotype Asia1 strains YNBS/58 and Jiangsu/2005 was 30–300-fold reduced at 48 hpi with 100 TCID50 of BHK-21 cells transfected with the p3D-NT56 or the p2B-NT25 shRNA compared with the controls, whereas the other shRNA induced cross-serotype inhibition to a lesser extent. SC injection of suckling mice with p3D-NT56 or p2B-NT25 shRNA

resulted in a survival rate of 20% with 20 LD50 of HKN/2002 at 5 dpi and up to 40% with YNBS/58, whereas 100% of the mock-treated mice had died within 60  hpi. IM administration of 109 CFU of the recombinant rS. cho vector carrying the p3D-NT56 shRNA (p3D-NT56/S. cho) to guinea pigs 36 hours prior to intradermal challenge with 50 ID50 of HKN/2002 protected four out of five animals from vesicular lesions and fever that were seen in all mock-treated animals. However only one out of five animals was protected when pre-treated with 108 or 1010 CFU of this recombinant. Combined administration of the 106 CFU of pAd5-Pol (Chen et al., 2006) and 109 CFU of p3D-NT56/S. cho did not increase the level of protection as two out of five animals developed clinical FMDV signs. In swine IM injected in the neck with 5 × 109 CFU of p3D-NT56/S. cho and IM challenged in the neck with 100 ID50 of HKN/2002 24 hours later the disease onset was delayed to 10 dpi and disease severity was much lower than that of mock-treated control animals. The latter displayed severe disease from 3 dpi onwards. Pigs treated with 5 × 109 CFU of p3D-NT56/rS. cho also developed lower virus neutralizing and 3ABC antibodies suggesting the amount of virus in these animals was reduced compared with control animals. Increase of the dose of from 5 × 109 CFU to 5 × 1010 CFU, however, decreased the level of protection (Cong et al., 2010). Several other research groups used RNAi technologies to target structural and/or non-structural viral targets. A chronologic review of these studies is presented. Kahana et al. (2004) aligned all FMDV sequences available in GenBank at that time to identify three regions with sequences of at least 22 bp with 100% identity in all FMDV sequences, leading to the design and synthesis of one shRNA targeting the 2B region and two targeting the 3D region. In BHK-21 cells transfected with shRNA using an RNAi shuttle and immediately thereafter infected with 103 PFU of FMDV serotype O1 Geshur, viral replication at 24 hpi was inhibited by 80–97% as determined by viral RNA synthesis when transfection was done with each of the shRNA separately and > 98% when a mixture of all three shRNA was used. Virus yield at that time was inhibited > 90% and 100%, respectively. The amount of negativesense RNA, reported as an essential element of the viral replication, was inhibited in a similar order of

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magnitude. At 30 hpt the cellular mRNA amount in transfected cells was similar to that of untransfected cells suggesting no adverse effect of transfection on the cell-viability (Kahana et al., 2004). Mohapatra et al. (2005) infected BHK-21 cells transfected with a mixture of 12–30 base pair (bp) long siRNA generated by in vitro transcription against the genome regions VP3-VP1, 2A-2C or 3D-3′UTR of FMDV Asia1 (IND 63/72) with 100 TCID50 of this FMDV strain. The virus yield of IND 63/72 in the transfected cells was > 90% reduced in the cell supernatant at 24 hpi, compared with controls. The greatest effect (120-fold reduction in virus titre) was seen with the siRNA mixture directed against the 2A–2C region at 24 hpi. At 48 hpi the antiviral effect had nullified. In addition, these authors constructed 21 bp siRNAs against the L-5′UTR and the 2B–2C junction regions, as alignment of FMDV serotype O, A, C and Asia1 had revealed these regions as highly conserved between the serotypes. In BHK-21 cells transfected with these siRNA a > 99% inhibition of the replication of FMDV serotypes O (O/IND/R2/75), A (A/IND/17/77), C (C/BOM/64) and Asia1 (IND 63/72) was observed at 24 hpi. At 48 hpi, the inhibition was > 87% against all four serotypes with the most potent siRNA directed against the 2B–2C junction. Despite the high percentages of inhibition of the virus replication by these 2 siRNA, virus titres in the supernatant still reached up to 4.5 and 5.6 log 10 TCID50/ml at 24 hpi and 48 hpi, respectively. Serial passage of the supernatant collected at 24 hpi from siRNA transfected cells did not induce the emergence of mutant viruses at passage level 2 (Mohapatra et al., 2005). De los Santos et al. (2005) selected seven conserved target regions (CRE, IRES1, IRES2, 2B1, 2B2, 2C and 3D) from FMDV A12-IC based on the alignment of sequences of the seven FMDV serotypes to construct shRNA containing plasmids and corresponding firefly luciferase reporter plasmids. Upon transfection of human embryonic kidney (HEK)-293T-cells specific silencing of luciferase activity of 90% was obtained with the 2B1 shRNA and 50% with the IRES1, IRES2 and 3D shRNA at 24 hpt, but not with the other shRNA. Similar findings were done when IBRS-2 cells were transfected with the shRNA and infected 48 hours later with 100 PFU of FMDV A12-IC, i.e. 2B1 shRNA and IRES1 shRNA inhibited the virus replication at

48 hpi a 100- and a 50-fold, respectively, but further experiments with the 2B1 shRNA showed that virus titres at 72 hpi reached similar levels as controls (> 106 PFU/ml). The inhibitory effect of 2B1 was shown not to be mediated by the activation of the IFN pathway. Double transfection of IBRS-2 cells with 2B1 shRNA with a 20 hour interval and challenge 24 hours after the second transfection resulted at 6 hpi in a decrease in total viral RNA production of 70% and a >80% decrease in viral proteins 2B, VP0, VP1 and VP3 as determined by Western and Northern Blot analyses compared to mock-transfected control cells. In addition, infection with different FMDV serotypes of 2B1 shRNA transfected IBRS-2 cells revealed reduction of virus titres at 24 hpi compared to controls of 97% for the homologous A12-IC strain and 92% for the heterologous strain O1 Campos, between 74 and 88% for Asia1, O/Taiwan/2/99 and SAT2, and only 2% for C3 Resende, indicating the 2B region – being one of the most conserved sequences across all FMDV serotypes – as another potential target for silencing of multiple FMDV serotypes (de los Santos et al., 2005). Transient siRNA expression in BHK-21 cells transfected with plasmids bearing 21–23 nt of the 3D (pSiFMD2) or 2B1 (pSiFMD3) sequence of FMDV 24 hours prior to infection with FMDV serotype O, A and Asia1 (5 × 10³ TCID50/cell) barely reduced the replication of FMDV as indicated by the formation of CPE at 24 hpi and the virus yield in the supernatants at 18 to 48 hpi, which was only slightly lower than that observed in mock-transfected and untransfected controls. SC administration of 5–100 µg plasmids to suckling mice 6 hours before challenge with 5 LD50 FMDV serotype O and Asia1 resulted in a survival rate of 30 to 40% at 5 dpi whereas mock-treated mice all had died within 3 dpi. When challenged with 20 LD50, the survival rate at 5 dpi decreased to 10% (Pengyan et al., 2008). Joyappa et al. (2009) identified three highly conserved sequences in the 3D genomic region and one in the IRES region by sequence alignment of strains of three serotypes of Indian origin. Corresponding shRNA were tested for their inhibitory effect of viral replication in BHK-21 cells transfected with one of these shRNAs and infected with 100 TCID50 of FMDV O/R2/75 18 hours later. The shRNA targeting the IRES region did not exhibit any

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inhibitory effect on viral replication in vitro. Two shRNA targeting 3D decreased viral RNA yield and virus titres in supernatant at 24 hpi ~ 10,000-fold compared with the control. Upon IM administration of either of these two shRNA targeting the 3D region ≥ 80% of guinea pigs was protected from vesicular lesion development when challenged with 103 ID50 of O/R2/75 24 hours later ( Joyappa et al., 2009). The VP1 gene of FMDV was also the target for shRNAs developed by Lv et al. (2009). Five out of 24 designed shRNAs silenced ≥ 60% of the expression of EGFP from reporter plasmids containing the VP1 gene of FMDV O (O/NY00) in a sequence specific manner in HEK-293T-cells. A reduction of 76–97.5% in viral RNA yield was obtained with four shRNAs in BHK-cells and three of these delayed CPE development by 6–12 hours and reduced the level of viral progeny ~ 100-fold 24 hours after infection with O/NY00, but the difference with mock-transfected control cells gradually waned at 36–48 hpi. Mixing the shRNAs did not increase the inhibitory efficacy. One extra passage of virus recovered from shRNA treated cells on cells expressing the same shRNA, did not induce reduced susceptibility of the virus to the shRNA. On the other hand, a single point mutation in the target region of a shRNA, which was naturally present in the O/NY00 virus stock, reduced the silencing effect of this shRNA by 3.7-fold (Lv et al., 2009). The majority of results have been achieved by transfection of siRNA into cells, and this may be problematic for practical applicability of RNAi. In attempts to get around in vitro and in vivo problems associated with the delivery of RNAi different promoter systems and delivery vectors have been studied. Recombinant adenovirus constructs expressing shRNA targeting the 3C (Ad-3C1) or 2B (Ad-2B) region of the FMDV serotype O (O/ SKR/2000 or O/SKR/2002) were used to infect IBRS-2 cells prior to and at various time points after infection with 100 TCID50 of FMDV strain O/ SKR/2002. Pre-treatment of cells with the Ad-3C1 and a combination of Ad-3C1 and Ad–2B construct prior to virus challenge resulted in a > 90% reduction of the RNA yield, a respective ~ 10,000- or ~ 1,000-fold reduction in virus titre and a reduction of viral protein as determined by antigen ELISA of ~ 90% and ~ 65%, respectively. The inhibition was

lower using the Ad–2B construct alone with 70% reduction in viral RNA, ~ 100-fold reduction in virus yield and ~ 45% in viral protein load. Cells pre-treated, pre- and post-treated at 6 hpi or posttreated at 6 hpi with a combination of Ad-3C1 and Ad–2B construct showed a similar ~ 1,000-fold inhibition of viral RNA at 48 hpi. At 72 hpi a similar high degree of inhibition was only seen in the pre- and post-treated cells and the inhibitory effect had declined most in the pre-treated cells. Posttreatment with the Ad-3C1 and Ad–2B constructs at 1 hpi or 12 hpi inhibited the RNA yield most effectively (~ 1000-fold at 72 hpi), whereas this effect was abolished when cells were post-treated at 24 hpi. Intraperitoneal (IP) infection of mocktreated suckling mice with 125 LD50 of FMDV O/SKR/2002 resulted in a survival rate of 20% at 3 dpi and 0% at 6 dpi. When suckling mice were administered Ad-3C1, Ad-2B or a combination IP at both 24 hours and 6 hours prior to viral challenge the survival rate increased to 58% at 7 dpi. Furthermore, an additional injection with Ad-construct at 3 dpi enhanced the survival rate up to ~ 80% at 7 dpi (Kim et al., 2008). Having in mind the variable nature of the FMDV and the low fidelity of the viral RNA polymerase, it is not inconceivable that a selective pressure is exerted on the viral target by siRNA. This might give rise to the emergence of variant viruses with reduced sensitivity to this siRNA. The approach of simultaneously targeting several FMDV genomic regions to counteract the viral mechanism to escape the RNAi effect was examined. Kim et al. (2010) studied the effect of co-expression of multiple shRNAs from a single vector against FMDV. Therefore, additional recombinant adenovirus constructs simultaneously expressing shRNA targeting the 2B and 1 or 2 3C sequences of the FMDV serotype O (O/SKR/2000 and O/SKR/2002) under control of three U6 promotors (Ad-U63B-U63C1and Ad-U63B-U63C1-U63C2) or U6 and CMV promotors (Ad-U62B-U63C1-CMV3C2) were generated. The inhibitory effect of these shRNA on FMDV O/SKR/2002 was determined by RT-pPCR and virus titration at 24 and 48 hpi. To this end, IBRS-2 cells were treated 12 hours before infection with 100 TCID50 of FMDV O/SKR/2002. The inhibitory effect on RNA and virus yield of the Ad–U63B-U63C1 construct was ~100-fold higher than that of the single Ad-3C1 and Ad-2B

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constructs at 48 hpi. The antiviral effect of single constructs Ad-U63C2 or the Ad-CMV3C2 was in the same order of magnitude as that of Ad-3C1 and Ad-2B, but the combined administration of Ad-U62B-U63C1 together with one of the AdU63C2 or the Ad–CMV3C2 constructs decreased the effect of the former. In contrast, when the three different shRNAs were simultaneously expressed in one construct (Ad-U63B-U63C1-U63C2 and Ad-U62B-U63C1-CMV3C2) the antiviral effect was ~ 100-fold enhanced at 54 hpi with Ad-U63B-U63C1-U63C2 being most effective. The effect seemed to be dose dependent, as increasing the Ad-construct dose from 3 x 106 TCID50 to 1 x 107 TCID50 increased the observed antiviral effect. Treatment of the cells with Ad-U63B-U63C1-U63C2 showed toxic to IBRS-2 cells as the cell viability was reduced by ~  47% compared with untreated control cells. This toxic effect was not seen with Ad-U63B-U63C1-CMV3C2. Cross-inhibition of FMDV serotype O, A, Asia1 and C occurred in IBRS-2 cells with Ad-U63B-U63C1-U63C2 or Ad-U62B-U63C1-CMV3C2, but was lower against FMDV serotype SAT1, SAT2 and SAT3. These observations corresponded to a complete sequence identity of the 2B target region between serotype O and A, and of the 3C1 and 3C2 regions between serotype O, A, Asia1 and C, whereas the SAT serotypes each showed seven differences in the 2B region and two to four differences in the 3C1 and 3C2 regions. Intraperitoneal infection of mocktreated suckling mice with 125 LD50 of FMDV O/ SKR/2002 resulted in survival rate of 20% at 4 dpi and 0% at 6 dpi. When suckling mice were administered Ad-U63C2 or Ad-CMV3C2 IP, survival rates were similar to those reported earlier with Ad-3C1, Ad-2B and Ad-U62B-U63C1 (Kim et al., 2008). Treatment with a mixture of Ad-U62B-U63C1 and Ad-U63C2 slightly enhanced survival rate but this difference was not statistically significant. However, mice treated with Ad-U62B-U63C1-U63C2 or Ad-U62B-U63C1-CMV3C2 had a survival rate of 90% at 7 dpi, which was higher than obtained with any other Ad-construct treatment. Ninety per cent of Ad-U62B-U63C1-U63C2-treated mice also survived challenge with A22/IRQ 24/64 or Asia1/MOG/05 until 7 dpi, whereas all control mice had succumbed by that time. The effect of Ad-U62B-U63C1-CMV3C2 against challenge

with A22/IRQ 24/64 or Asia1/MOG/05, was less pronounced, with survival rates of ~ 50–70% at 7 dpi (Kim et al., 2010). Targeting more than one viral gene was also studied by Xu et al. (2012) through the development of an Ad-5 vector carrying VP1 and 2B shRNAs against the genome of FMDV O/ HK/2002 expressed alone or in tandem (Ad-VP1, Ad-2B and Ad-VP1–2B). In IBRS-2 cells pretreated with Ad-VP1–2B at a moi 10 and infected with 100 PFU 12 hours later, the titres of O/HK/2002 were reduced by a ~1000-fold at 48 hpi compared with the controls, and this reduction was superior to that of treatment with the single shRNAs or a combination of them. Marked reduction of viral RNA production (>99%) was obtained when IBRS-2 cells were treated with Ad-VP1–2B 12 hours before or at the time of viral infection with O/HK/2001 but reduction decreased or absent when treated at 12 or 24 hpi. Pre-and post-treatment resulted in a complete absence of viral RNA until 48 hpi and a > 99% reduction at 72 hpi. IM injection of guinea pigs with a high dose (108 PFU) of Ad-VP1–2B and intradermal challenge with 100 ID50 of O/ HK/2002 24 hours later resulted in a protection against virus induced lesions and mortality of 60% of the animals until 7 dpi which was higher than the protection conferred by the single or combined administration of the Ad-shRNAs. Treatment at the time of viral challenge or double treatment with the second treatment at the time of challenge did not increase the protection rate. However, triple treatment before, at the time of challenge and 48 hours thereafter increased the protection rate up to 80%. Viral challenge 48 or 72 hours after administration of Ad-VP1–2B decreased the rate of protection to 40% and 20% respectively at 7 dpi (Xu et al., 2012). Another approach addressing the issue of the highly genetic variability and high mutation rate of FMDV is to target a host cell sequences instead of a virus sequence. Using a lenti-viral vector expressed RNAi targeted to the porcine integrin αv subunit of PK-15 cells, Luo et al. (2011) established a stable iαv-PK-15 cell line in which the expression of the integrin αv subunit of the FMDV receptor was 89.5% downregulated as determined by qRT-PCR analysis of the αv mRNA. Infection of these cells with 100 TCID50 of FMDV O/CHA/99 resulted in a > 1000-fold reduction of virus titre in the

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supernatant at 48 hpi compared with controls (Luo et al., 2011). miRNA Unlike siRNA or shRNA which are of exogenous nature, micro (mi)RNA are dsRNA molecules encoded in endogenous genes. Generally, miRNA does not require full complementarity to bind with target mRNA, e.g. one type of miRNA may regulate many genes, as well as one gene can be regulated by several miRNAs. Transgene expressed shRNA in transgenic cell lines or animals may in some way be considered as artificial miRNA (amiRNA). Pengyan et al. (2010) used the SIFMD2 and pSiFMD3 plasmids (Penyan et al., 2008) to generate transgenic mice through microinjection of the siRNA in the male pronucleus of the zygote and embryo transplantation. Upon IP infection with 100 LD50 of FMDV Asia1 and euthanasia at 72 hpi, non-transgenic mice had detectable FMDV antigen in their liver, kidneys and spleen as demonstrated by immunohistochemistry, whereas FMDV antigen was only detected in the spleen of the transgenic mice, and the number of positive cells in their spleen was reduced compared with the non-transgenic controls. Pathological examination revealed necrosis in the cardiac muscle and congestion in the liver of non-transfected infected control mice, but not in the transfected infected mice and a significant increase of splenic corpuscles and macrophage epitheloid cell nodes in the junction of the white pulp and the red pulp in the transgenic mice compared with non-transgenic mice (Pengyan et al., 2010). Unfortunately, no additional virological examinations were performed in this study. Co-transfection of HEK-293T-cells with one of three lentiviral shRNAs targeting the VP2, VP3 or VP4 region of FMDV and their respective FLAGtagged viral gene sequence induced a significant inhibition of viral gene expression determined by Western Blot analysis as compared with mocktransfected cells. Viral replication as expressed by TCID50 of FMDV Asia1 (Asia1/Ys/CHA/05) in BHK-21 cells stably expressing one of the three shRNAs infected with 100 TCID50 of this strain was inhibited for 91–98% relative to the controls. Transgenic bovine embryos were generated by transfer of transgenic bovine fetal fibroblasts expressing VP4 shRNA into enucleated oocytes. The resulting embryos were transferred into cows.

At 4 months of pregnancy fetuses were surgically recovered and after confirmation of the integration and expression of the VP4 shRNA, tongue epithelium was collected. Both primary VP4 shRNA transgenic and non-transgenic tongue epithelium cells were infected with 100 TCID50 of FMDV. At 24 and 48 hpi an inhibition in viral RNA yield and virus yield of > 91% was obtained in the transgenic cells compared to the non-transgenic cells (Wang et al., 2012). Jiao et al. (2013) constructed plasmids containing shRNAs targeting the 3D and 2B genome region of FMDV HKN/2002 (PB-EN3D2B) that induced a reduction of 77.7% in EGFP expression in BHK cells co-transfected with an EGFP reporter plasmid, following similar methodology as Chen et al. (2004).These PB-EN3D2B plasmids were used to establish a transgenic IBRS-2 cell lines and transgenic mice. Infection of six lines of transfected cells with 20 TCID50 of FMDV O/HKN/2002 resulted in complete abrogation of viral replication in two cell lines (no CPE and no detectable virus in the cell supernatants at 72 hpi) and a delay of CPE formation until 48 hpi and a 94% reduction in virus titre at 48hpi in another cell line, compared with controls. In the three remaining cell lines, the replication of FMDV was not inhibited. Similar findings were done upon challenge of these cell lines with the FMDV serotype Asia1 ( Jiansu/2005). The shRNA copy number integrated in the cellular genome was shown not to be correlated with the magnitude of the observed antiviral effect. Transgenic suckling mice containing 1 or 2 shRNA integrations in their genome did not exhibit significant resistance to FMDV infection upon challenge with 10 LD50 of HKN/2002 and all died. When the challenge dose was lowered to 3 LD50, 27–41% of the transgenic mice survived, but also 22% of control mice did ( Jiao et al., 2013). Four miRNA expression plasmids targeting conserved regions of the 3D gene sequence of different FMDV isolates significantly silenced the EGFP reporter plasmid containing the O/CHA/99 3D sequence in BHK-21 cells. In BHK-21 cells transfected with these miRNAs a 6.3- to 400-fold reduction in virus titre and a 41.5–82.1% reduction in viral RNA load compared with mock-transfected control cells was observed at 24 hpi, but the effect had waned at 48 hpi (Du et al., 2011). Co-transfection of one of four miRNAs targeting the IRES of

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FMDV O/HN/CHA/93 and corresponding homologous EGFP reporter plasmids in BHK-21 cells resulted in 44.3–81.4% EGFP silencing at 48 hours compared with mock-transfected controls. When co-transfected with EGFP reporter plasmids containing IRES sequences of heterologous FMDV strains A/AF/72 or Asia1/Jiangsu/China/2005, the miRNA resulted in 38.7–71.4% EGFP silencing at 48 hours compared to mock-transfected controls. Transfection with a mixture of the two most potent miRNAs or with a plasmid expressing these two miRNA structures (dual miRNA) enhanced the homologous EGFP silencing up to 84.7% and 95% respectively and the heterologous EGFP silencing 88.3 and 96.6% respectively. Transgenic BHK-21 cells lines with stable integration of the dual miRNA in their genome were established and infected with one of the FMDV strains O/HN/ CHA/93, A/AF/72, Asia1/Jiangsu/China/2005, O/Tibet/China/1/99 or O/CHN/Mya98/33-P at a moi of 5–50. Up to 72 hpi the virus replication of all but the O/CHN/Mya98/33-P was markedly reduced as determined by qRT-PCR. Simultaneous SC administration of the dual miRNA and 50 to 100 LD50 of each of the viruses mentioned above in the neck of suckling mice resulted in 100% mortality with FMDV O/HN/CHA/93, AF/72 and Asia1/Jiangsu/China/2005 but the time of death was delayed by 6 hours compared with control mice that all had died at 42 hpi, whereas 75–100% of the mice infected with O/Tibet/China/1/99 or O/ CHN/Mya98/33-P survived up to 7 dpi (Chang et al., 2013). Gismondi and co-workers (2014) established transgenic BHK-21 cell lines constitutively expressing amiRNA targeted against one of two sites in the 3D region (3D1 and 3D2) of the FMDV genome, based on sequence homology between FMDV strains A/Arg/01, O1 Campos and C3 Indaial. Similarly, BHK-21 transgenic cells containing a transgene directed against the 3′UTR region of the FMDV genome were established, but no expression of 3′UTR amiRNA was detected. Silencing activity of the transgenic cells was evaluated by co-transfection with reporter plasmids encoding the Renilla luciferase (RLuc) gene fused to FMDV fragments enclosing the amiRNA target sequences of A/Arg/01 and a control plasmid encoding the firefly luciferase. The RLuc activity, as determined by quantification of the total RNA by RT-PCR, at

24 hpt was abolished in both tested 3D1 transgenic cell lines, but was less outspoken in 3D2 transgenic cells (silencing of about 50% in only one out of two examined 3D2 transgenic cell lines). No crosssilencing was observed when reporter plasmids encoding the sequence of O1 Campos were used. Degradation of the reporter RLuc mRNA was shown to be involved in the observed silencing of A/Arg/01 reporter plasmids. However, upon infection of the 3D1 transgenic cell lines with FMDV A/Arg/01 (moi of 0.01 and 5) the virus replication as evaluated by viral titres and viral RNA was not impaired. Sequence analysis of the 3D1 target region in virus that replicated in these cells did not reveal any mutation that could explain reduced susceptibility to the amiRNA. In addition, comparison of the predicted structural organization of the amiRNA target sequence in the reporter plasmid and that of the full-length FMDV sequence, hybridization reactions with a fluorescent DNA oligonucleotide resembling the 3D1 amiRNA and transfection with nude viral RNA showed that the lack of silencing of the viral replication in cell culture as opposed to the strong silencing of reporter gene expression in the 3D1 transgenic cells could not entirely be explained by structural differences in the target sequence between the plasmid and the virus sequence (Gismondi et al., 2014). Hu et al. (2015) aligned FMDV strains of serotype O, A and Asia1 to select conserved sequences of the VP1 gene for the design of shRNA expression vectors. Upon infection with 100 TCID50 of FMDV serotype O strain OS/99 of shRNA-transfected BHK-21 cells, the most potent shRNA reduced the viral RNA yield by 96.8% in comparison to untransfected control cells. This shRNA was selected to generate transgenic pigs through somatic cell nuclear transfer using shRNA transfected primary porcine fibroblasts. Based on the expression level of the amiRNA in their fibroblasts, the transgenic pigs could be divided into a ‘high’ and a ‘low’ expression group. When fibroblasts isolated from the transgenic pigs were infected with 100 TCID50 of OS/99 the viral RNA yield was reduced up to 30-fold at 36 hpi in the ‘high’ expression group compared with untransfected controls and the correlation between the ami RNA expression and the inhibition of viral RNA expression was positive. Challenge of pigs with 100 LD50 of OS/99, revealed a marked protection against FMDV induced fever

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and vesicular lesions in transgenic pigs compared to non-transgenic pigs. Whereas the latter all showed severe disease from 3 dpi on, fever was absent in the ‘high’ expression transgenic pigs and only one small vesicular lesion was present at 9 dpi. Lesion development was also mild and delayed until 7 dpi in the ‘low’ expression transgenic pigs. Consistent with the clinical protection, the viral RNA load in the serum of transgenic pigs was strongly reduced at 1, 3, 5, 7 and 10 dpi and different internal organs at 10 dpi compared with non-transgenic pigs (Hu et al., 2015). The seemingly unlimited target range for nucleic acid based methods implies a promising broad therapeutic potential. In human medicine several therapeutic candidates for metabolic, hereditary, degenerative, neurological or tumoral diseases are in clinical development. Antiviral RNAi-based drugs against infections with hepatitis B and C viruses, respiratory syncytial virus, the human immunodeficiency virus or the Ebola virus are in the developmental pipeline and some have entered phase I or II clinical studies. The present overview shows that a lot of effort has been done to study the potential of RNAi-based strategies to inhibit the replication of FMDV. Depending on the construct design, RNAi seems an efficient method to inhibit the replication of FMDV in vitro but in vivo experiments yield more varying degrees of success and effects are usually short-lived. RNA-based strategies hold some inherent challenges and additional research is needed to address these, especially when bearing the highly contagious and genetically diverse nature of the FMDV and the treatment of livestock in mind. A first challenge is the seemingly suboptimal efficacy when it comes to reduction of the infectious viral load. Although virus titres in infected cells are often significantly reduced when expressed as a percentage, the actual virus titres of infectious virus that can be recovered from treatedinfected cells are often quite high. In extension, the transition from in vitro activity to in vivo shows that he latter is often inferior to what would be expected based on the in vitro findings. Target site accessibility during viral replication and the conformation of the target RNA are important to consider when developing an RNAi strategy (Gismondi et al., 2014). Another challenge is the in vivo delivery, systemic spread and in vivo stability of the siRNA. In in vitro studies transfection is used to introduce the

siRNA in the cell and an overdose of siRNA relative to target RNA is needed to obtain an inhibitory effect. This is difficult to achieve in vivo. Progress in this field has been made by vector-based delivery like replication-defective human adenovirus (Chen et al., 2006) or recombinant S. cho (Cong et al., 2010). Also, the emergence of FMDV genome resistant to the siRNA seems to be easily induced in siRNA treated cells. Combined administration of siRNA targeting different sequences of the FMDV has been reported as an approach to overcome this hurdle. Off-target effects due to recognition of other genes with a similar sequence should be avoided, taking into account the different natural host species of FMDV. The use of RNAi methods to generate genetically engineered transgenic cell lines and animals with reduced susceptibility to FMDV is challenging. Discussion about the potential use of amiRNA to generate animals with reduced susceptibility to FMDV falls beyond the scope of this chapter and for the advances in farm animal transgenesis the reader is referred to review articles like Kues and Niemann (2011) and other. Passive immunization with llama antibodies (Nanobodies®) Passive transfer of (hyper)-immune serum may offer rapid protection against FMDV infection. In the beginning of the twentieth century it was a frequent practice in Europe to administer immune serum or blood from animals recovering from FMDV infection to other animals to protect or cure them from the disease. However, this method was quickly abandoned because of the risk for iatrogenous transmission of FMDV and of inadequate or too short protection (Blancou, 2002). Unlike in other mammalian species, 45–75% of the antibodies of the members of the family of the Camelidae only contain heavy chains. The isolated variable antigen-binding single domains of these antibodies (VHH fragment), known and patented as Nanobodies®, have been explored and developed as therapeutic agents (Reviewed in Vanlandschoot et al., 2011). The passive immunization with recombinant llama single-domain antibody fragments against FMDV was mainly described at the Central Institute for Animal Disease Control in The Netherlands. Upon immunization of llamas with a mixture of four FMDV strains O1 Manisa,

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A22 Turkey/98, A24 Cruzeiro and Asia1 Shamir 24 VHH that neutralized O1 Manisa in vitro were selected. Mapping of the antigenic sites to which the VHH bind by competition ELISA revealed that the VHH recognized four (I, II, III and IV) functionally independent antigenic sites more or less corresponding to the described antigenic sites of the VP1 protein of FMDV serotype O. In ELISA, all VHH recognizing antigenic sites I, II and IV bound to homologous and heterologous FMDV strains of serotype O (O1 Manisa, O Taiwan/97, O1 British Field Strain 1860 (BFS)/67A), A (A22 Turkey/98, A22 Iraq 24/64, A24 Cruzeiro/Brazil/55), Asia1 (Asia1 Shamir) and C (C1 Detmold), although for some high concentrations (up to 10 mg/ml) of VHH were required. VHH recognizing antigenic site III on the other hand only reacted with the FMDV serotype O strains. Fifteen VHH were able to neutralize O1 Manisa in vitro at concentrations of 0.15–3.3 mg/ml, one at a concentration of 10 mg/ml and five not at concentrations below 10 mg/ml. Some combinations of VHH resulted in a synergistic effect lowering the total needed VHH concentration by 3- to 8-fold. The four most potent VHH were PEGylated to increase their serum halflife and administered IM at a dose of 4 mg/kg to guinea pigs 24 hours prior to intradermal challenge with 10³ TCID50 of O1 Manisa. Despite the presence of a virus neutralizing titre in their serum at 0 and 3 dpi, the guinea pigs that received single VHH were not protected and all developed generalized FMDV lesions at 3 dpi. Combination of two VHH resulted in a 1 day delay of lesion development and protected three out of six animals against lesions up to 5 dpi when the experiment was stopped. Guinea pigs that received immune serum of guinea pigs previously immunized with FMDV O1 Manisa did not develop generalized FMDV until 5 dpi, despite the fact that this serum neutralized less efficiently in vitro and these guinea pigs had lower neutralizing titres in their serum at 0 and 3 dpi compared to the VHH immunized guinea pigs. These findings illustrate the crucial role antibody-mediated opsonophagocytosis plays in in vivo protection against FMDV which cannot be achieved by VHH since they lack the Fc receptor (Harmsen et al., 2007). In search to overcome this hurdle Harmsen et al. (2008) generated three VHH2 – that consist of a VHH binding to FMDV genetically fused to a VHH binding with high affinity to porcine

immunoglobulin (pIg) (Harmsen et al., 2005) – using three of the above described VHH against O1 Manisa. This fusion resulted in a 100-fold increase in serum half-life compared to monovalent VHH. The in vitro affinity and FMDV neutralizing capacity of the VHH2s was comparable to that of the FMDV VHH. In vivo, all but three of the 16 large white pigs administered with one of the VHH2 or a mixture of them at a dose of 3 mg/kg and challenged 24 hours later with 10³ PFU of O1 Manisa developed generalized FMD at 2 to 3 dpi comparable to mock-treated control pigs. Three pigs did not develop FMD and no infectious virus could be detected in their blood or oropharyngeal fluids (OPF) up until 14 dpi when the experiment ended. The transmission of FMDV from VHH2 treated and FMDV-infected pigs to untreated control pigs at 24 hpi was not prevented, despite the lower viral load in the serum and OPF of the treated pigs compared with infected control pigs (Harmsen et al., 2008). To improve the in vivo activity, other FMDV binding VHHs with higher neutralizing capacity were selected, two FMDV VHHs were fused into a bivalent FMDV VHH2. This VHH2 was in its turn fused with a pIg VHH resulting in a so-called VHH3. Three out of five pigs administered intravenously with 50mg/kg of an equimolar mixture of 2 VHH3s and infected intradermally 24 hours later with 104 TCID50 of O1 Manisa did not develop FMD and had reduced and variable amounts of viral RNA in their serum and OPF between 1 and 17 dpi compared with control pigs. These three treated pigs did not transmit FMDV to untreated control pigs. In the other two treated pigs both the development of FMDV (5–6 dpi) as the transmission to control pigs was delayed (6 dpi) compared with untreated control pigs (2 dpi). IM administration in the hind leg of 1g of the above VHH3 mixture at the time of IM injection in the neck with a full dose of conventional FMDV O1 Manisa double-oil emulsion vaccine in pigs resulted in a significant decrease in FMDV virus neutralizing antibodies compared with pigs that were solely vaccinated, the ELISA antibody response on the other hand was not affected (Harmsen et al., 2009). Similarly, two VHH with high affinity to FMDV serotype O were isolated from the immune library from two camels immunized with an inactivated FMDV serotype O vaccine. These VHH showed high affinity to FMDV serotype O but did not

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cross-react with FMDV serotype A and Asia1 limiting their practical applicability against FMDV in the field. Conjugation with fluorescent semi-conductor nanocrystals turned these VHH into an interesting tool for tracing FMDV type O virions in laboratory studies with FMDV (Wang et al., 2015). The present data provide an interesting proof-of concept for the use of passive FMDV immuno-prophylaxis using VHH but there is a need for improvement with regard to potency, dosage, route of administration (individual intravenous) and panserotype activity before practical applicability against FMDV in the field would be possible. It also remains to be determined whether FMDV variant viruses with reduced sensitivity to VHH would be readily selected on, especially since the VHH are directed towards the highly variable surface proteins of FMDV. Antiviral properties of natural products In earlier times several treatments based on natural products like herbs and plants (pine tar, pomegranate, garlic, turnip, hyssop, root of mallow) have been proposed to treat lesions associated with FMDV infections (Reviewed by Blancou, 2002), however the majority if not all of them without demonstrated efficacy nor scientific background. Nowadays traditional medicine including herbal treatment is still often empirically applied to soothe FMDV lesion and promote their healing in countries endemic with FMDV. Medicinal plants, their derivatives or animal products have been and are more and more explored as alternative sources of novel drug discovery however few have been examined for their activity against FMDV. Meliacine (MA) is a cyclic peptide isolated from the leaves of the plant Melia azedarach L that contains aliphatic amino acids and a single glucose unit. It was reported to inhibit the replication of FMDV strains of serotype O (O1 Campos, O 69) and A (A24, A87) with 52–90% in BHK-21 cells infected at a moi of 1 and over 99% when infected at a moi of 0.001, whereas the strains O1 Caseros and C3 Resende were not sensitive (Wachsman et al., 1995). In BHK-21 cells covered with medium containing 0.5, 5, 50, 100 or 500 µg/ml of purified MA after 1 hour virus adsorption of FMDV serotype O (O1 Campos), the virus replication was inhibited in

a dose dependent way as determined by a plaque reduction assay on the supernatant collected at 18 hpi. The EC50 – the concentration that reduced plaque counts by 50% in treated cultures compared to untreated ones – was determined at 0.5 µg/ml with no signs of cytotoxicity up to 100 µg/ml. However although the virus yield was reduced by 2 logs in cells treated with 50 µg/ml, the virus yield still reached up to 7 log PFU/ml at 18 hpi. Addition of MA before, during or maximum 1 hour after virus adsorption was necessary for a strong inhibition of the viral replication (PFU). Pre-incubation of high concentrations of MA with 106 PFU of virus for 1 hour before incubation on BHK-21 cells did not result in a decrease in virus titre compared to untreated virus, suggesting MA did not have a virucidal effect. The reducing effect of MA on the production of photoresistant neutral-red labelled FMDV and on the staining of intracellular acidic compartments suggested that MA inhibits the process of uncoating of FMDV in BHK-21 cells through reduction of the acidification of intracellular acidic vesicles (Wachsman et al., 1998). Flavonoids are ubiquitous in photosynthesizing cells and therefore present in most plants. The molecular structure of the flavonoid compounds backbone is 2-phenyl-1,4-benzopyrone, which consists of two phenyl rings and a heterocyclic ring. The flavonoid apigenin with three –OH substitutions at carbon positions 5, 7 and 4′ is present in many fruits and vegetables (e.g. parsley, celery, celeriac) and has been reported to have inhibiting effects on HIV activation and to enhance the antiviral activity of acyclovir against human and veterinary herpes viruses (reviewed in Cushnie and Lamb, 2005). A dose-dependent effect of the flavonoid apigenin (4′,5,7-trihydroxyflavone) on the replication of FMDV strain O/ES/2001 in BHK-21 cells was reported, whereas six other flavonoids did not exhibit anti-FMDV activity (Qiang et al., 2015). Addition of cell-culture medium containing 20 µg/ ml apigenin after 1 hour adsorption of the FMDV strain O/ES/2001 at a moi of 0.1 on BHK-21 cells strongly reduced the VP1 expression at 20 hpi and the CPE formation and virus titre obtained in the supernatant at 24 hpi, respectively when compared to mock-treated control. The EC50 in BHK cells was determined at 8.593 µg/mL and the cytotoxic concentration (CC)50 at 31.43  µg/mL. Timeof-drug-addition studies revealed that apigenin

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(20 µg/ml) exerted its anti- FMDV effect after virus entry in the cell. The viral replication was not inhibited when apigenin was added before or during adsorption of the virus, nor when the virus and the apigenin were pre-incubated before addition to the cells. Intracellular viral RNA yield was nearly undetectable in treated cells up until 22 hpi. Transfection of BHK-21 cells with a plasmid containing the IRES of FMDV fused to EGFP and treatment of these cells with apigenin revealed an inhibition of the EGFP expression in a dose dependent-manner. These data indicated IRES as a target of apigenin and the suppression of the IRES-mediated translational activity of FMDV as the mechanism of action (Qiang et al., 2015). Chitosan is a linear polysaccharide composed of randomly distributed β-(1–4)-linked d-glucosamine and N-acetyl-d-glucosamine. It is produced from chitin derived from the cell walls of fungi and shells of insects and crustaceans like shrimp. Administration of 0.5 mg chitosan granules and O/CHN/99 to suckling mice resulted in an average delay of death time of 0.2–2.1 hours in the chitosan-treated compared to untreated animals and little difference was seen when the chitosan was administered simultaneously with, or 3 or 7 days prior to viral challenge. Similar results were seen with the Asia1/JS/05 FMDV strain (Li et al., 2010). Although at present the above-described products are far from ready to be used for the practical control of FMDV, futher investigations into this kind of compound may allow the development of efficient and pharmacologically appropriate drugs against FMDV. Summary While to date the use of antiviral drugs in veterinary medicine remains limited to companion animals, there is an increasing interest for their therapeutic potential to control of livestock diseases including FMDV. The development of antiviral drugs for livestock is still at an early stage and requires a very specific approach. Drug administration to foodproducing animals implies specific requirements to safeguard food safety including among others the determination of maximum residue levels in products of treated animals. This requires a thorough knowledge of the pharmacokinetic profile of

the antiviral product in all target species and the pharmacological and possible toxicological effects in humans. Second, the scale of treatment in the event of an outbreak of an epizootic viral disease like FMD in livestock exceeds many times that in companion animals. The massive administration of antiviral drugs yields certain risks including the possibly rapid generation of virus variants resistant to the antiviral drug. To manage this risk, a thorough, fundamental scientific knowledge of the barrier to resistance, the fitness and transmission efficiency of any resistant variants is needed. Another important factor is the cost of treatment of livestock which is in first instance determined by the product and the required dosage. Therefore, a simple and inexpensive synthetic process which is suitable for production of large quantities and a low effective dose is desirable. The cost of veterinary interventions, withdrawal periods and residue analyses should also be considered. For the treatment of large numbers of animals on short time notice under field circumstances, administration through the feed seems a practical approach. However, any method for in vivo administration poses specific demands and challenges including bio-availability, systemic spread and stability. These factors are among others important to warrant administration of the optimal dose of the antiviral agent and by doing so to limit to a minimum the probability that variant viruses with reduced susceptibility to the antiviral agent emerge. Given the highly infectious nature of FMDV, the short duration of the replication cycle, the large number of virus particles excreted by infected animals and the small number of virus particles required for infection FMDV is readily transmitted to susceptible animals often resulting in an explosive epidemic. To be able to nip an FMDV outbreak in the bud, an antiviral agent should be very effective at potently inhibiting the virus replication shortly after administration. Various approaches and many research efforts have been and are pursued in search of an adequate antiviral agent to combat FMDV and although the majority of studies remain limited to in vitro experiments and/or preliminary efficacy studies in laboratory animal species, some encouraging achievements have been reported. It remains to be answered whether the antiviral activity observed in these models can be translated into FMDV target species as very few studies have been performed in these

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species. Every approach meets specific challenges, strengths and weaknesses and these have been summarized at the end of the corresponding sections. So far, the majority of agents encounter a lack in one or more aspects that are vital for appropriate use in the control of FMD outbreaks. For each approach more research is needed to improve the antiviral potency, to gain insight into mechanisms of action and the barriers to resistance and to explore the efficacy, pharmacokinetic behaviour and safety in FMDV target species and to examine practical synthesis and applicability. The large number of recent studies on this topic illustrate that the field of research on specific antivirals against FMDV is rather young and in constant evolution which creates space and a positive outlook for future progress. References

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Mathematical Models of the Epidemiology and Control of Foot-and-mouth Disease

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Michael J. Tildesley, William J.M. Probert and Mark E.J. Woolhouse

Abstract This review considers how epidemiological models are constructed, how they deal with real-life complexities such as spatial heterogeneity, how they can be applied to specific foot-and-mouth disease (FMD) outbreaks or epidemics, and how they can be used to explore the impact of control measures. A detailed description is provided of the application of a particular model, the ‘Keeling’ model, of the spread of FMD between farms in the UK during the 2001 epidemic. The review concludes with a discussion of how modelling has developed since the 2001 outbreak and is likely to develop in the future. The emphasis throughout is on ‘good practice’, especially how theoretical models relate to biological data and how models can sensibly be used to inform decisions about disease control strategies. Introduction Mathematical models of the spread of infectious diseases were first developed early in the twentieth century and have made important contributions to improving epidemiological understanding and designing control programmes for many human diseases, including malaria, measles, tuberculosis, HIV/AIDS and more recently Ebola (Anderson and May, 1991; Daley and Gani, 1999; Keeling and Rohani, 2008; Kucharski et al. 2015; Drake et al., 2015). There have also been numerous applications of mathematical models to animal diseases, including BSE (Anderson et al., 1996), classical swine fever (Stegeman et al., 1999), scrapie (Matthews et al., 2001), bluetongue virus (Gubbins et al. 2008) and various wildlife diseases (e.g. Hudson et al., 2002). Mathematical models are potentially powerful

tools for infectious disease epidemiologists because infectious diseases have inherently complex dynamics. For example, as explained later on, the relationship between the rate at which individuals infect one another, the transmission rate, and the final size of an epidemic is non-linear, i.e. doubling the transmission rate does not simply double the final number of cases. These complex relationships make it very difficult, on the basis of practical experience alone, to develop a quantitative understanding of the epidemiological process, or to generalize from one situation to others, or to predict the impact of control measures. Tackling these kinds of problems requires the kind of precise, formal, quantitative framework that mathematical models can offer, with the obvious proviso that the models must have a sound biological basis to be of practical value. The potential importance of mathematical models was underlined in the Royal Society of London’s report on the 2001 epidemic of FMD in the UK, which stated that: ‘Quantitative modelling is one of the essential tools both for developing strategies in preparation for an outbreak and for predicting and evaluating the effectiveness of control strategies during an outbreak’ (Royal Society, 2002). The Royal Society’s report should encourage the more widespread use of mathematical models in veterinary medicine. Since 2001, models have played a much more central role in epidemiological analyses, designing control programmes, contingency planning and policy making, not just for FMD but for other infectious diseases of animals (e.g. Savill et al., 2006; Gilbert et al., 2008; Brooks-Pollock et al., 2014). Successive sections of this chapter will consider how models are constructed, how they deal with real-life complexities such as spatial

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heterogeneity, how they can be applied to specific FMD outbreaks or epidemics, and how they can be used to explore the impact of control measures, proceeding to a detailed description of the application of a particular model, the ‘Keeling’ model, of the spread of FMD between farms in the UK, and concluding with a discussion of how mathematical modelling of FMD has developed since the 2001

Box 16.1  Glossary Adaptive management. A framework for structured decision making in the face of uncertainty, with the aim of reducing uncertainty through time so as to improve management outcomes (see, for example, Shea et al., 2014). Basic reproduction ratio. The average number of new cases of infection directly generated by a single case introduced into a previously unexposed host population. Also known as R0. Bias. A systematic tendency for the value of a parameter to be over-estimated or underestimated. Bootstrapping. A method of generating confidence intervals around a parameter estimate by repeatedly sampling with replacement from the source data set to create ‘new’ datasets and reestimating the parameter value each time. Case reproduction ratio. The average number of new cases generated by a single case. Also known as R or Rt. It can be estimated at any stage of an epidemic and is generally expected to be less than R 0. Compartments. Discrete subsets of the host population defined according to their infection status. Commonly used compartments are susceptible, exposed (or latently infected), infectious and recovered/removed. These occur in SEIR models of infection dynamics. Deterministic. The output is fully determined by the inputs. Chance is not involved. Difference equations. Equations using discrete time steps, e.g. 1 day. Differential equations. Equations using calculus, i.e. infinitely small time steps.

outbreak and how modelling of livestock disease is likely to develop in the future. The emphasis throughout is on ‘good practice’ (Woolhouse et al. 2011), especially how theoretical models relate to biological data and how models can sensibly be used to inform decisions about disease control. A glossary provides explanations of technical terms (Box 16.1).

Ensemble modelling. A range of techniques for combining outputs from multiple epidemiological models so as to improve predictive projections (see, for example, Lindström et al., 2015). Estimation. The procedure by which the value of a parameter is estimated from data. There are many different statistical approaches to parameter estimation, including least squares fitting, maximum likelihood fitting, martingale estimators and Bayesian methods. Parameters will almost always be estimated with some degree of uncertainty (giving rise to confidence intervals) and may be subject to bias, noise or non-independence. Generation time. The average interval between the time of infection of a case and the time of infection of new cases generated from it. Latin hypercube sampling. A technique for sensitivity analysis which can explore the effects of variations in many different inputs at the same time, but which is much more efficient than comparing outputs for every possible combination of inputs. Microparasites. Pathogens which cause infections which can usefully be represented in terms of compartments, e.g. susceptible or infected. The term is generally applicable to infections by viruses, bacteria or protozoa. Microsimulation. Stochastic mathematical models in which each individual in the population is represented explicity, as opposed to tracking the number of individuals in each of a set of compartments. Model. Here, a mathematical representation of a dynamic process, such as the spread of an infection through a population.

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Network. In this context, the numbers and distribution of links between hosts, where ‘link’ implies an opportunity to transmit infection. An example is the network of farms linked by movements of livestock. Noise. Inherent variability and/or imprecision in data which makes it difficult to obtain precise parameter estimates. Non-independence. Correlation between two or more parameter estimates such that there is a range of combinations of parameter values which are consistent with the data. Non-linear. Here, used in the mathematical sense that the rates of change between different compartments in a model (e.g. susceptible to infected) are not simply proportional to the value of a single variable. This can lead to complex relationships between the inputs and outputs of a model. Non-stationary. Meaning that parameter values are not constant through time. Parameter. A constant (i.e. a fixed number) in a mathematical term which determines how the value of a variable in a model changes through time. Sensitivity analysis. A generic term for various methods of exploring how model outputs are related to model inputs in order to determine which of the model’s assumptions and/or parameter values are most important in determining its behaviour. One method for carrying out sensitivity analysis is Latin hypercube sampling.

Model structure Types of model There are various ways in which mathematical models can be used in epidemiological studies. One useful distinction is between retrospective and predictive models. Retrospective modelling involves fitting mathematical equations to epidemiological data and is used as a technique for the quantitative interpretation of those data (e.g. Haydon et al., 1997; Howard and Donnelly, 2000; Ferguson et al., 2001b; Tildesley et al., 2008). Predictive models that have been fitted to epidemiological data can be used to examine alternative scenarios, such as the

SEIR. An abbreviation for a commonly used mathematical model with four compartments: susceptible, exposed (latently infected), infectious and recovered/removed. Stochastic. The output is not fully determined by the inputs. Chance is incorporated in the process and every realization of that process can produce a different outcome. Stochastic effects are particularly important where the numbers involved are small, e.g. at the start or during the ‘tail’ of an epidemic when there are few infectious individuals. Transmission kernel. A mathematical function describing the relationship between transmission rate and distance. Transmission rate. The average rate at which a single infectious individual infects susceptible individuals. Validation. The process where the outputs of a model are compared with a fully independent data set, i.e. one which was not used to provide estimates of any of the model’s parameters. Variable. A quantity whose value is tracked in a mathematical model. An example is the number (or fraction) of individuals in one of the compartments of a SEIR model. Well-mixed. This implies that every individual in a population is equally likely to infect every other individual. Although clearly a simplification the assumption that a population is well-mixed is often used as a starting point in developing a model of the spread of infection.

implementation of different control measures (e.g. Keeling et al., 2001; Morris et al., 2001; Tildesley et al., 2006; Shea et al., 2014). Predictive models are used in two ways. Firstly, current data can be used as the basis for predicting the course of an ongoing epidemic (e.g. Ferguson et al., 2001a). The other is exploratory, modelling a range of possible epidemiological scenarios rather than focusing on a particular event; such models are often used to aid contingency planning (e.g. Garner and Lack, 1995; Durand and Mahul, 2000; Keeling et al., 2003; Buhnerkempe et al., 2014). Models may be deterministic or stochastic. Deterministic models (e.g. Haydon et al., 1997;

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Ferguson et al., 2001a,b) generate a fixed output for a given set of inputs. Stochastic models (e.g. Garner and Lack, 1995; Keeling et al., 2001; Morris et al., 2001; Chis Ster et al., 2009; Buhnerkempe et al., 2014) generate variable outputs for a given set of inputs; these models incorporate chance in the epidemiological process and each model run can produce a different result. Deterministic models are typically formulated as a set of coupled differential equations representing the dynamics of subsets of the host population corresponding to different infection states (see below). Stochastic models fall into two main categories: a set of coupled stochastic difference equations analogous to differential equations; and microsimulation or state-transition models, where the current state of each host in the population is represented individually. Stochastic models are often (though not always) much more difficult to compute, to fit to data, and to interpret than comparable deterministic models, but they do better reflect the intrinsic uncertainty in any epidemiological process. Enormous increases in computer processing power in recent years have made stochastic models, particularly microsimulations, a much more practical option, and are being increasingly widely used. Each mathematical approach has its advantages and disadvantages and it is important to recognize that no one approach is ‘right’ and that there can be no single model for the epidemiology of FMD or any other infectious disease. Rather, the merits of different approaches have to be judged against the purpose of the modelling exercise. Indeed, there may be advantages to modelling work being carried out in parallel: for example, four different modelling approaches were used to inform policymakers during the course of the UK 2001 FMD epidemic and the close agreement between these greatly increased confidence in the robustness of the outputs (Kao, 2002). More recent innovations have included the use of adaptive management and ensemble modelling, such that multiple models can be used in the event of novel outbreaks of disease to reduce the risks associated with making control decisions in the early stages of outbreaks, when there is significant uncertainty regarding how the disease is spreading through the population (Shea et al., 2014; Lindström et al., 2015; Probert et al., 2016).

Compartments The standard approach for modelling the epidemiology of microparasite (virus, bacteria or protozoa) infections is to divide the host population into different compartments: susceptible, denoted S; exposed, i.e. infected but not yet infectious, E; infected and infectious, I; recovered (or removed), R. The dynamics of infection are then represented by the movement of hosts from one compartment to another (Fig. 16.1A), as is described below. Such a model is usually referred to as an SEIR model or a SLIR model (the L standing for ‘latent’) (Anderson and May, 1991). If vaccination is involved there may also be a compartment, denoted V, representing vaccinated individuals or premises (Hutber and Kitching, 1996), and in order to differentiate between the contribution to infectious pressure from notified infectious and infectious but undetected individuals a notified compartment may also be introduced ( Jewell et al., 2009b). The SEIR structure is widely used and has the advantage of simplicity, but some care is needed in its application to foot-and-mouth disease virus (FMDV) infections. The first problem is that the compartments susceptible, exposed, infectious and recovered correspond only imperfectly to the states that can be defined in the field (Fig. 16.1B). FMDV infection can be demonstrated by the detection of virus, the detection of antibodies to the virus, or the appearance of clinical signs (Hughes et al., 2002a). There is, however, no simple correspondence between these assays and whether or not the host is exposed, infectious or recovered; for example, hosts may transmit infection before clinical signs appear (and in some hosts, notably sheep, clinical signs may not be detected at all) (Charleston et al., 2011). A second problem is that the SEIR compartments are discrete; that is, a host is either susceptible or exposed or infectious or removed, and all hosts are equally susceptible when classed as susceptible and equally infectious when classed as infectious. This is clearly a major simplification: viraemias may vary by orders of magnitude over the time course of a single infection and between infections of different hosts (Hughes et al., 2002b); and antibody levels similarly vary greatly with time and between hosts (Woolhouse et al., 1996b). One factor affecting the course of a single infection, and how infectious an infected host will be, is the virus

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(A)

Susceptible

S

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f (•) 1

E

Infected

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Figure 16.1  Compartments, in theory and practice. (A) A diagrammatic representation of a simple SEIR model showing the flow of hosts between susceptible, latently infected, infectious and removed compartments. The number of hosts (or fraction or density) in these compartments is represented by the variables S, E, I and R respectively. The rate of flow is specified by three expressions: f1(·), the rate at which susceptible hosts become infected; f2(·), the rate at which exposed hosts become infectious; and f3(·), the rate at which infectious hosts are removed. Different models use different mathematical expressions, representing different levels of detail and incorporating different numbers of parameters. (B) A diagrammatic representation of the course of an FMD infection in a single host (or single farm). The top panel illustrates that the transition between exposed and infectious does not correspond to the appearance of clinical signs: animals may be infectious before clinical signs appear. In practice, there is inevitably a further delay before clinical signs are observed and reported. The bottom panel, however, illustrates how these states can be represented as distinct compartments in a mathematical model.

dose to which it is exposed (Hughes et al., 2002c), which itself may be highly variable in practice. Mathematical models attempt to take account of this variability within compartments in various ways. One solution is to define more compartments (e.g. subclinically infected or partially immune) (Hutber and Kitching, 1996). A more sophisticated but computationally demanding alternative is to represent the ‘state’ of a host quantitatively rather than qualitatively (e.g. levels of viraemia rather than infected or not infected; antibody titre rather than infected or recovered) (Woolhouse et al., 1996b; White and Medley, 1998; Stringer et al., 1998). This requires functional relationships, e.g. between antibody titre and susceptibility, to be defined. These

are useful refinements which merit further development in future, linking mathematical analyses to experimental studies of the dynamics of infections within individual hosts (Chis Ster et al., 2012). A third problem is that the unit of epidemiological interest is not necessarily an individual host; many applications of mathematical models consider populations of livestock farms rather than of individual animals (e.g. Haydon et al., 1997; Durand and Mahul, 2000; Howard and Donnelly, 2000; Ferguson et al., 2001a; Keeling et al., 2001; Tildesley et al., 2006). There is no conceptual difficulty with classifying individual farms as susceptible, exposed, infectious or removed, but these compartments are unlikely to be equivalent

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to those for individual hosts. Considerable care must be exercised in ‘scaling up’ from the animal level to the farm level – the time scales and nature of the epidemiological processes involved are likely to be distinctly different. As an example, consider how a farm makes the transition from exposed to infectious. If this occurs when the first infected animal becomes infectious then the farmlevel latent period will be of the same order as the individual-level latent period. But if considerably more virus is required for transmission between farms than for transmission between individual animals, as might be expected given that different transmission routes may be involved, then the farm-level latent period may be longer, possibly much longer. The situation is further complicated in that the dynamics of FMDV outbreaks on individual farms are likely to be highly variable (Woolhouse et al., 1996b), just as are the dynamics of infections of individual animals. This is particularly evident in countries such as the USA, where there is a significant variation in farming practices in different parts of the country. For example, in states such as California, large dairy farms can have herds in excess of 15,000 animals and therefore it may not be reasonable to assume that the disease progresses through all animals on the farm at the same rate (Carpenter et al., 2011). Whilst its limitations must be borne in mind, it should be recognized that the SEIR approach has proved extremely useful for developing a quantitative understanding of the epidemiology and control of a wide variety of microparasite infections (Anderson and May, 1991; Keeling and Rohani, 2008) and is likely to remain a standard format for epidemiological models for some time to come. Parameters and variables An SEIR model describes infection dynamics in terms of changes in the numbers (or densities or fractions) of hosts in the compartments susceptible, exposed, infectious and removed. These numbers are represented by the variables S, E, I and R respectively. Movements between these compartments occur at rates specified by a set of mathematical terms (see Fig. 16.1A). In the simplest formulation, one term specifies the rate at which susceptibles become infected, another the rate at which exposed individuals become infectious, and another the rate at which infectious

individuals recover or are removed. There may also be terms specifying, for example, the rate at which susceptibles are vaccinated and the rate at which vaccinated individuals lose their protection. Each of these terms incorporates one or more parameters (mathematical constants which determine how the values of the variables change), such as the transmission rate (see below). The exact functional form depends on the underlying biology and the type of mathematical model being used. To take a simple example, the term σE is often used in differential equation models to represent the rate at which exposed and infected individuals become infectious. Expressed thus, the implication is that exposed and infected individuals become infectious at a constant per capita rate represented by the parameter σ, that the mean latent period is 1/σ, but that the modal latent period is extremely short. This last aspect is often regarded as unsatisfactory, and an alternative is to make the latent period itself a fixed constant. The implication, when expressed this way, is that all exposed and infected individuals become infectious at a fixed time after they were first infected, with no variation. The biological reality is likely to lie between these extremes. There are many potentially suitable functions to describe that situation, e.g. the gamma distribution, but these are inevitably more complex (i.e. they incorporate more parameters) and are more problematic to compute (Stringer et al., 1998). Consequently, constant rates or constant periods are frequently assumed in practice, although it is often straightforward to compare models of both types to see if there are significant differences in their outputs (Woolhouse et al., 1997b; Keeling and Grenfell, 2002). The number of parameters in a mathematical model is limited only by the level of biological detail which is to be represented (though an increase in the number of parameters will result in an increase in computational cost to carry out the simulations). For example, it is possible to represent different transmission routes separately and so have one or more parameters relating to each of airborne spread, direct contact, livestock movements, vehicle movements and so on. One microsimulation model for FMD which takes this approach has over 50 different parameters (Morris et al., 2001). This allows the model to be more realistic, but does create problems with parameter estimation and model validation.

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Quantifying transmission A key parameter for any epidemiological model is the basic reproduction ratio, R0, which is defined as the average number of secondary cases arising from the introduction of a single primary case into a previously unexposed population. If R0 > 1 then each case is more than capable of replacing itself and an epidemic can take off. On the other hand, if R0 1, so that each case produces, on average, more than one new case and a major epidemic is possible. The bottom diagram illustrates the situation where R01, the epidemic may fail to take off through chance alone, and that, even if R0  1 then the epidemic is growing and may be regarded as ‘out of control’ at time t, indicating that additional control measures may be warranted. On the other hand, if Rt