Advances in Virus Research, Volume 113 0323989926, 9780323989923

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VOLUME ONE HUNDRED AND THIRTEEN

ADVANCES IN VIRUS RESEARCH

Serial Editors MARGARET KIELIAN THOMAS C. METTENLEITER MARILYN J. ROOSSINCK

ADVISORY BOARD SHOUWEI DING JOHN FAZAKERLY KARLA KIRKEGAARD JULIE OVERBAUGH DAVID PRANGISHVILI  FELIX A. REY JUERGEN RICHT JOHN J. SKEHEL GEOFFREY SMITH MARC H.V. VAN REGENMORTEL VERONIKA VON MESSLING

VOLUME ONE HUNDRED AND THIRTEEN

ADVANCES IN VIRUS RESEARCH Edited by

MARGARET KIELIAN Albert Einstein College of Medicine, Bronx, New York, United States

THOMAS C. METTENLEITER Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald – Insel Riems, Germany

MARILYN J. ROOSSINCK Department of Plant Pathology and Environmental Microbiology, Center for Infectious Disease Dynamics, Penn State University, University Park, PA, United States

Academic Press is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States 525 B Street, Suite 1650, San Diego, CA 92101, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 125 London Wall, London, EC2Y 5AS, United Kingdom First edition 2022 Copyright © 2022 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-98992-3 ISSN: 0065-3527 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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Contents Contributors

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1. Diversity of viral RNA silencing suppressors and their involvement in virus-specific symptoms

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Vitantonio Pantaleo and Chikara Masuta 1. Introduction 2. Principles of RNA silencing pathways in plants 3. Multilayer strategies of viruses for suppressing RNA silencing at the cellular level 4. Concluding remarks Acknowledgments References

2. Animal models of alphavirus infection and human disease

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Cormac J. Lucas and Thomas E. Morrison 1. Introduction 2. Alphavirus transmission cycles 3. Human clinical disease 4. Animal models 5. Animal models of human disease 6. Animal models of unique aspects of alphavirus infection 7. Virus strains used in animal models 8. Conclusions Acknowledgments References

3. Enteroviruses: The role of receptors in viral pathogenesis

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Emma Heckenberg, Justin T. Steppe, and Carolyn B. Coyne 1. Introduction 2. Transmission and viral life cycle 3. Enterovirus structure 4. Enteroviruses and their receptors 5. Enterovirus disease modeling 6. Concluding remarks References

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Contributors Carolyn B. Coyne Department of Molecular Genetics and Microbiology; Department of Pathology, Duke University School of Medicine, Durham, NC, United States Emma Heckenberg Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, United States Cormac J. Lucas Department of Immunology and Microbiology; RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, United States Chikara Masuta Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan Thomas E. Morrison Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, United States Vitantonio Pantaleo Department of Biology, Agricultural and Food Sciences, Institute for Sustainable Plant Protection, Bari, Italy Justin T. Steppe Department of Pathology, Duke University School of Medicine, Durham, NC, United States

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CHAPTER ONE

Diversity of viral RNA silencing suppressors and their involvement in virus-specific symptoms Vitantonio Pantaleoa,∗ and Chikara Masutab,* a

Department of Biology, Agricultural and Food Sciences, Institute for Sustainable Plant Protection, Bari, Italy Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan *Corresponding authors: e-mail address: [email protected]; [email protected] b

Contents 1. Introduction 2. Principles of RNA silencing pathways in plants 2.1 Diversification of RNA silencing suppressors 2.2 Suppression strategies 3. Multilayer strategies of viruses for suppressing RNA silencing at the cellular level 3.1 Geminiviruses: A complete armamentarium of small and large antiviral defense suppressors 3.2 Potyviruses: Multitasking HCPro 3.3 Tombusviruses: Different effects of p19: siRNA-binding ability and beyond 3.4 Cucumoviruses: Further expansion of 2b protein functionality by post-translational modification 4. Concluding remarks Acknowledgments References

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Abstract RNA silencing is an evolutionarily conserved and homology-dependent gene inactivation system that regulates most biological processes at either the transcriptional or post-transcriptional level. In plants, insects and certain mammalian systems, RNA silencing constitutes the basis of the antiviral defense mechanism. To counteract RNA silencing-based antiviral responses viruses adopt strategies of replication and host invasion that include mechanisms of RNA silencing suppression. Indeed, viruses can express proteins known as RNA silencing suppressors (RSSs). Over the last two decades, silencing studies in plant virology have been largely devoted to the discovery and description of RSSs. The result has been exciting and these studies have revealed (i) an incredible diversity of proteins and mechanisms of RSSs

Advances in Virus Research, Volume 113 ISSN 0065-3527 https://doi.org/10.1016/bs.aivir.2022.06.001

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belonging to various viral taxonomic groups, (ii) the multifunctionality of RSSs: they can fulfill several functions during viral infection and target one or more key points in the RNA silencing machinery. Some RSSs of model viral systems have been the subject of exceptional in-depth studies; they have proven to be real molecular tools for studying plant physiology, plant biology and virus–plant interactions, even in some cases extending the knowledge of the response of plants to other biotic and abiotic stressors. RSS diversity in phylogenesis, in mechanism of action and the frequent presence of more than one RSS in a single viral genome all suggest that they are extremely plastic in evolving to overcome host defenses. In this chapter, we present and discuss the most recent findings related to the well-studied RSSs of four viral taxonomic groups: geminiviruses, potyviruses, tombusviruses and cucumoviruses.

1. Introduction Viruses are infectious entities that invade the cells of living organisms of every kingdom, including bacteria, protists, archea, fungi plants and animals. Viruses likely represent the most extensive genetic and biological diversity on the planet as revealed by genomic data. Viral diversity is defined by a wide array of virus types, such as those with double-stranded (ds) DNA, single-stranded (ss) DNA, as well as dsRNA and ssRNA genomes, and reverse transcribing genomes (ICTV, 2012). Viruses can also be pathogens, and plant viruses are among the most important pathogens, causal or co-causal agents of devastating crop diseases around the globe. As such they are capable of affecting entire agro-ecosystems and associated industrial systems and of generating relevant social impacts (Hull, 2002). The scientific community’s interest in studying viral replication mechanisms and plant defense strategies stems from the fact that such knowledge can help develop disease protection and crop improvement strategies. The difference in viral genome organization implies a difference in replication strategy. The genetic information held and orchestrated by the viral genome encodes a minimal number of proteins with indefinite functionality, which interact and interfere with, modify, redirect, inhibit, utilize, or hijack host proteins. Indeed, plant viruses invade the host plants following various steps such as intra- and intercellular movement, suppression of antiviral defenses, genome replication, viral gene expression, and acquisition and transmission by vectors and spread into the environment exploring novel hosts. As previously stated, viruses are obligate infectious entities. However, to ensure infection, key essential functions need to be provided

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by viral proteins; these can be facilitated by different proteins in different viruses and/or accomplished through distinct virulence strategies, but their functions cannot be lost during evolution. During host-virus coexistence a set of complex interactions involving virus attack and host defense is developed. These include mechanisms ascribed to pattern-triggered immunity, effector-triggered immunity, and the RNA silencing-based antiviral response (reviewed in Leonetti et al., 2021).

2. Principles of RNA silencing pathways in plants RNA silencing is a fundamental genetic regulatory mechanism conserved in eukaryotic organisms. It can act at the transcriptional (transcriptional gene silencing, TGS) or post-transcriptional level (posttranscriptional gene silencing, PTGS) and has many diverse roles including developmental regulation, stress response or defense against invading nucleic acids like transposons or retrotransposons. Antiviral short-interfering RNAs (siRNAs) are the main actors of the RNA silencing process. SiRNAs guide the targeting to nucleic acids of infecting viruses, resulting in the inhibition of viral replication, accumulation and spread. The RNA silencing pathway is initiated by the Dicer family of RNA endoribonuclease III (RNase III) enzymes, processing the full sequence dsRNA of the replicating viral genome into 20- to 25-nucleotide-long siRNAs. In plants, multiple Dicer-like (DCL) enzymes function redundantly or cooperatively to detect RNA and DNA viruses (Guo et al., 2019b). siRNAs are incorporated into the largely unknown multisubunit RNA-induced silencing complex (RISC) upon recruitment by members of the Argonaute (AGO) protein subfamily. Once incorporated into RISC, one of the two siRNA strands, the passenger strand, is removed (Iki et al., 2010). The other siRNA strand, the guide strand, pilots RISC toward viral nucleic acids in a sequencespecific manner, resulting in either RNA degradation or translational and transcriptional repression (Pantaleo et al., 2007). When this pathway results in transcriptional and post-transcriptional changes, it is known as TGS and PTGS, respectively (reviewed in Shimura and Pantaleo, 2011). Despite extensive studies carried out on antiviral PTGS, it remains unclear how AGO proteins (e.g., AGO1 and AGO2) target viral RNAs (Alvarado and Scholthof, 2012). Plant RNA viruses replicate within membrane invaginations, from outer membranes of cellular organelles (mitochondria, peroxisomes, chloroplasts, endoplasmic reticulum) that protect

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their genomes from silencing (Lalibert
e and Sanfac¸on, 2010). Otherwise, antiviral AGOs may target viral transcripts during their translation by either cleavage or translational repression, preventing genome replication and encapsidation (Brodersen et al., 2008; Miozzi et al., 2013; Pantaleo et al., 2007). One of the fascinating aspects of virus-induced RNA silencing in plants is the ability of siRNAs to travel from the site of infection to remote tissues and retain antiviral activity. This signal is used to activate RISC in remote tissues that are not yet infected. Such a remote and efficient signal points to the amplification and transport of the silencing signal (Sanan-Mishra et al., 2021). Indeed, plant RNA-dependent RNA polymerases (RDRs) are required for both the cytoplasmic and chromatin RNA silencing in plants (Dalmay et al., 2000; (Mourrain et al., 2000). In Arabidopsis thaliana, the RDR1 and RDR6 orthologues are required in the cytoplasmic antiviral RNA silencing of viruses with some specificity for different viral RNAs (Dalmay et al., 2001; Xie et al., 2001). A secondary set of dsRNA is amplified by the host’s RDRs from the primary ssRNA templates, essentially amplifying the siRNA, and are termed transitive siRNAs (De Felippes and Waterhouse, 2020). In transitive silencing, the siRNAs are used as primers by RDRs to produce dsRNA, which DCL2 or DCL4 then processes to produce 22- or 21-nucleotide transitive siRNAs that degrade complementary mRNAs (Fusaro et al., 2006; Vaistij et al., 2002). RNA silencing in plants can be achieved locally at the site of infection (local silencing), or in a remote tissue or organ (systemic silencing). Indeed, the amplified viral siRNAs then travel through either plasmodesma to neighboring cells or by phloem to remote tissues. The mechanisms are still not fully understood; however, recent studies carried out on miRNA would suggest the movement of siRNAs to be actively mediated by the host factors (Brioudes et al., 2021) and restricted by RISC use (Devers et al., 2020) rather than be passive.

2.1 Diversification of RNA silencing suppressors RSSs are viral proteins that actively inhibit the antiviral RNA silencing response, providing an evasion route for viruses and a reasonable co-existence of the virus and its host (Csorba et al., 2015). Viral RSSs target different steps in the silencing pathways with diverse mechanisms, implying convergent evolution, i.e., having analogous features but non-homologous motifs. Indeed, RSSs from plant viruses display high sequence diversity

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and are frequently gained and lost over evolutionary time scales. Conversely, plant antiviral RNA silencing genes show high rates of adaptive evolution and motif conservation. The relative rate of protein evolution is higher for RSSs than for other genes. In addition, specific studies carried out in several plant viruses have shown that strong RSS evolution/diversification is attributable to episodic selection rather than to pervasive positive selection (Murray et al., 2013). In other words, the diversification of plant virus RSSs is strongly boosted by frequent jumps between host species/ genotypes rather than a one-to-one coevolution.

2.2 Suppression strategies Antiviral silencing suppression mediated by RSSs was first discovered in potyviruses (Brigneti et al., 1998; Kasschau and Carrington, 1998). For example, suppression strategies by RSS proteins include siRNA sequestering and preventing their entry into the RISC (Anandalakshmi et al., 1998; Silhavy et al., 2002; Vargason et al., 2003; Ye et al., 2003); dsRNA binding to prevent them from being processed by the antiviral DCLs (Qu et al., 2003); decreasing the activity of antviral DCLs (Haas et al., 2008); AGO quenching; targeting for proteasomal degradation and selective autophagy (Baumberger et al., 2007; Bortolamiol et al., 2007; Csorba et al., 2010; Derrien et al., 2012; Zhang et al., 2006); suppression of systemic silencing by RDR6 inhibition of transitive siRNA synthesis (Guo et al., 2013) or interfering with the production of RDR1-dependent virus-derived small interfering RNAs (vsiRNAs) (Diaz-Pendon et al., 2007); and destabilization of vsiRNAs by viral RNase III (Kreuze et al., 2005). The last two decades, particularly the 12 years following the discovery of RSS, have witnessed unprecedented numbers of publications featuring viral RSSs, uncovering a plethora of functions and different properties of these viral proteins in different viruses. In most cases, however, these studies have only scratched the surface of the diversity of RSS, for example, through the identification of homologous RSSs in different species of a viral taxonomic group and the confirmation of RNA silencing function with the help of standard experiments. By considering, instead, that RSS evolution/diversification is attributable to episodic selection rather than pervasive positive selection (see previous paragraph), it is likely that RSS from individual viral species may have acquired previously unknown mechanisms. Indeed, when a systematic study is applied to RSSs then the full amplitude of their functional and mechanistic portfolio will be unveiled.

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3. Multilayer strategies of viruses for suppressing RNA silencing at the cellular level 3.1 Geminiviruses: A complete armamentarium of small and large antiviral defense suppressors Geminiviruses (Family: Geminiviridae) belong to the largest known family of plant viruses and possess a circular ssDNA genome (Bennett and Agbandje-McKenna, 2020). The virus causes devastating diseases in crops around the globe, compromising food security. The taxonomic structure of the family includes eight genera, based on the host range, insect vector, and genomic structures: Becurtovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, Turncurtovirus, and Begomovirus (Zerbini et al., 2017). Most species described to date belong to the genus Begomovirus, which has small genomes composed of one or two DNA molecules of less than 3 Kb each. The coding space, therefore, is packed and its expression is optimized by bidirectional and up to seven partially overlapping open reading frames (ORFs). The ORFs are organized in such a way as to provide the virus with the ability to initiate the replicative cycle, slow down, or suppress the antiviral defense at the cellular level and invade the entire host (Gupta et al., 2021). For example, tomato yellow leaf curl virus (TYLCV), one of the most studied geminiviruses, is a monopartite begomovirus expressing six known ORFs, encoding capsid protein (CP)/V1 and V2 in the virion strand, and replicase protein (Rep)/C1, C2, C3, and C4 in the complementary strand. The primary function of each protein derived from each of the TYLCV ORFs is in fact well known, and the need for the virus to have many counteracting factors to the plant’s antiviral defense, whether RNA silencing-based or hormone signaling, is already clear. For instance, V2 is the first identified geminivirus strong suppressor of PTGS (Zrachya et al., 2007), and many recent studies have confirmed V2 activity in suppressing methylation-mediated TGS by hampering host histone deacetylase HDA6 (Wang et al., 2014; Wang et al., 2018). More recently, V2 has been shown to interact with and inhibit AGO4, the main component of the RNA-dependent DNA methylation (RdDM) machinery, playing key roles in the defense against TYLCV. Indeed, the geminiviral genome forms mini-chromosomes (reviewed in Hanley-Bowdoin et al., 2013) and is subject to epigenetic modifications, including cytosine DNA methylation and histone modifications. Recent studies on the ability of V2 to suppress methylation of the TYLCV genome

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have also revealed the importance of Cajal bodies (i.e., spherical nuclear bodies largely consisting of proteins and RNAs) in gene regulation mechanisms, drawing a clear link between them and RdDM. Indeed, Wang et al. (2020) showed that the viral genome is subject to AGO4-mediated TGS in the presence of Cajal bodies and that this antiviral defense is reduced in the presence of V2 or altogether abolished in the absence of Cajal bodies (where V2 colocalizes with AGO4). TYLCV C2 and C4 suppress cellular PTGS and the cell-to-cell movement of PTGS, respectively; both alter phythormone signaling, such as jasmonic acid (JA) signaling and salicylic acid (SA)-dependent defenses (Lozano-Dura´n et al., 2011; Luna et al., 2012; Rosas-Diaz et al., 2018). It is worth pointing out that the notable multifunctionality of C4 has recently been discovered thanks to the identification of protein signals that mediate its association with the plasma membrane (including those structurally included in plasmodesmata) and chloroplast membrane (chloroplast transit peptides). Indeed, at the PM, C4 interacts with the receptor-like kinases (RLKs) BAM1 and BAM2 and hinders the intercellular diffusion of PTGS (either vsiRNA-mediated or miRNA-mediated, Fan et al., 2021; Rosas-Diaz et al., 2018). Following defense activation, which is triggered by the presence or activity of the virus-encoded Rep protein, plasma membrane-associated C4 (but not plasmodesmata-associated C4) is translocated to the chloroplast, where it interacts with calcium sensing receptor-dependent defense responses, including induced transcriptional reprogramming by pathogen associated molecular pattern triggered immunity, SA biosynthesis, callose deposition, and broad-spectrum anti-bacterial and anti-fungal resistance. Meanwhile, C4 is localized at plasmodesmata to perform the functions of the “guardian” to prevent the cell-to-cell movement of PTGS (Medina-Puche et al., 2020). The RSS repertoire of geminiviruses has been recently expanded. Neglected small ORFs, other than those previously described, are indeed expressed during infection. Some of these ORFs encode small proteins less than 10 kDa. They can harbor distinctive features, such as transmembrane signals, and localize in specific subcellular compartments that have not been previously described for any of the other viral proteins. The novel localizations include the Golgi apparatus and mitochondria (Gong et al., 2021). It should be noted that the recent report by Gong and collaborators (2021) identified up to 21 neglected transcription initiation sites within the TYLCV genome by taking advantage of cap-snatching by rice stripe virus (Lin et al., 2017). One of the additional ORFs of TYLCV is conserved

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among geminiviruses and encodes a 7.2-kDa Golgi-localized protein denoted as V3. V3 has biological functions upon expression in the context of viral infection. It is required for full viral infection in tomato and Nicotiana benthamiana and acts as an RSS either at the post-transcriptional or transcriptional level. For instance, V3 is able to suppress RNA silencing similarly to the tombusvirus p19 (Lakatos et al., 2006) in a transient expression assay or when expressed via a PVX viral vector. Another level of anti-viral RNA silencing response in plants is TGS, which acts through methylation of DNA at cytosine residues and is particularly relevant against geminiviruses (reviewed in Shimura and Pantaleo, 2011). V3 can also act as a TGS suppressor in N. benthamiana where the green fluorescent protein transgene is silenced due to methylation of the 35S promoter. At 10-day postinoculation (dpi), green fluorescence could be observed in systemic leaves where V3 is expressed from a PVX; as a consequence of V3-mediated TGS suppression this fluorescence persisted at 28 dpi. Interestingly, transgenic expression of V3 resulted in a lower level of genome-wide methylation (Gong et al., 2021). The authors propose a model for orchestrating the expression of V3 at the primary sites of infection by protecting the viral genome from PTGS and TGS during the early stages of formation of replication intermediates in the nucleus before expression of the potent and better known V2, which may promote efficient viral accumulation and invasion of host tissues (Gong et al., 2021). Another component of the RSS armament, among the most studied of the geminiviruses, is the AC2/C2 (begomovirus/curtovirus) protein. Both AC2 and C2 act as RSSs through an acid activation domain (AD) at the C-terminus of the protein, while C2 acts only in an AD-independent manner. For efficient silencing suppression, AC2 needs not only AD but also the nuclear localization signal (NLS) motif (at the N-terminus) as well as the Zn-finger motif. Suppression requires the presence of the protein in the nucleus, interaction with host DNA and induction of transcription. These conditions make AC2 a ’transcription-dependent silencing suppressor. Among the 30 plant genes that AC2 transactivates there is a calmodulinlike protein, a known host suppressor of PTGS (Anandalakshmi et al., 2000; Li et al., 2017; Nakahara et al., 2012) and there are proteins related to ABI3 and VP1, which are known transcriptional repressors of enzymes involved in chromatin methylation and TGS (Sun et al., 2015). Conversely, AC2 lacking the AD and C2 reversed PTGS and TGS in the vegetative phase with a “transcription-independent” mechanism (reviewed in Veluthambi and Sunitha, 2021).

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3.2 Potyviruses: Multitasking HCPro Members of the Potyviridae family, the most represented group of positive stranded RNA viruses in plants, adopt a polyprotein strategy to express several viral factors which play roles in the diverse infection-related pathways. Helper component proteinase (HCPro) is a papain-like cysteine proteinase responsible for self-cleavage from the polyprotein precursor with essential functions in polyprotein maturation. Moreover, HCPro has at least two independent additional functions: viral plant-to-plant transmission and RNA silencing suppression. Potyvirus HCPro is the first described and best characterized RSS (Anandalakshmi et al., 1998; Kasschau and Carrington, 1998). Burgya´n’s group (Lakatos et al., 2006) revealed the mechanism of HCPro RSS, i.e., to bind vsiRNAs and prevent their incorporation into anti-viral RISCs, analogously to the mechanism adopted by p19 of tombusviruses. The studies by Lakatos et al. (2006) were mainly based on transient protein expression tests and focused on the RSS interaction with vsiRNAs, the latter being the main players in antiviral RNA silencing. It should be noted that the action of HCPro as an RSS by vsiRNA sequestration has also been confirmed in vivo in turnip mosaic virus infection (Garcia-Ruiz et al., 2015). More recently, potyvirus HCPro has shown binding preference for vsiRNAs of 21–22 nt with 50 -terminal adenines over non-viral short (s)RNAs (i.e., of plant origin), regardless of the expression system (i.e., agrobacterium-mediated local transient expression vs virus-mediated systemic expression), consolidating the vsiRNA sequestration strategy to prevent their use by the silencing machinery (Toro et al., 2022). Further studies conducted in the context of potato virus A (PVA) infection have disclosed other functionalities of HCPro as an RSS. HCPro was shown to be co-involved in inhibiting both S-adenosyl-L-methionine synthase (SAMS) and S-adenosyl-L-homocysteine hydrolase (SAHH), inducing the accumulation of S-adenosyl-L-homocysteine (SAH). SAH has strong negative feedback on HEN1, so that HEN1 is unable to methylate sRNAs, which in turn leads to the suppression of RNA silencing (Ivanov et al., 2016). Accordingly, relative methylation levels of vsiRNAs were significantly less methylated in the presence of HCPro, regardless of its local or systemic expression (Toro et al., 2022). Moreover, in the same study it was shown that HCPro interacts with AGO1, the core component of RISC, and both are associated with ribosomes. Although it was already known that ribosome-associated AGO1 can inhibit gene expression of mRNAs via translation inhibition, the study

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by Ivanov et al. (2016) introduced the possibility that HCPro has a third mechanism of action as an RSS; i.e., that ribosome-associated multiprotein complexes containing HCPro relieve viral genomic RNA from translational repression (Ivanov et al., 2016). The same authors have recently refined the model by which HCPro associates with the translation machinery of the viral genome and rescues it from translation inhibition. That is, the “AELPR” motif of PVA HCPro ensures interaction with the VARICOSE group of regulatory proteins containing WD40 domains. Disruption of HCPro’s interaction with VARICOSE proteins also prevents silencing suppression (De et al., 2020).

3.3 Tombusviruses: Different effects of p19: siRNA-binding ability and beyond Tombusvirus p19 is one of the most extensively studied RSSs both structurally and biochemically, making p19 a model system for studying sRNA sequestration capacity both in vivo and in vitro. Similar to what has been observed for HCPro (Ivanov et al., 2016), tombusvirus p19 also prefers vsiRNAs to endogenous sRNAs, regardless of the mode of expression. Indeed, transgenic N. benthamiana expressing p19 of the cymbidium ringspot virus (CymRSV) supports the replication and invasion of the RSS-deficient CymRSV) (Cym19stop; Szittya et al., 2010), sequesters vsiRNAs but does not bind or significantly alter endogenous sRNAs. Accordingly, p19 expression during viral invasion specifically compromises vsiRNA loading into AGO1. In contrast to HCPro (Toro et al., 2022), p19 does not show any preference for a specific vsiRNA sequence or for the 50 -end nucleotide (Kontra et al., 2016). Recent affinity studies of p19 to plant miRNAs have nonetheless revealed that p19 can exhibit high preferences for endogenous sRNAs, such as miRNAs, and significant sequence-specific differences between conserved miRNAs (Fig. 1). More specifically, miRNAs directly involved in the regulation of certain key antiviral silencing proteins were analyzed: miR162 targeting the mRNA of DCL1, miR168 targeting the mRNA of AGO1, and miR403 targeting the mRNA of AGO2. In vitro analysis by electrophoretic mobility shift assays using purified p19 revealed considerable variations in binding strength; i.e., miR168 exhibited weaker binding to p19 and therefore had no influence on the competitive loading mechanism model for miR168’s action to control AGO1 (Dalmadi et al., 2021).

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AGO1-mRNA

miR168 DCL1-mRNA pre-miR168 miR162 p19

binding preference

miRNAs vsiRNAs

miR403 AGO2-mRNA

(@Plasmodesmata) BAM1/2

cell to cell movement of 21-nt vsiRNAs

Fig. 1 Multifunctional role of tombusvirus p19 in regulating functions of miRNAs and siRNAs by binding preferences and localization (see Section 3.3).

In contrast, miR162 and the miR403 isoforms displayed a high siRNAlike affinity (Fig. 1) (Pertermann et al., 2018; Pertermann et al., 2022). In vitro analyses with the aid of N. tabacum BY2 cell lysates have confirmed the ability of p19 to regulate the activity of AGO1-containing RISC in cleaving the targets of the three miRNAs under analysis. This is consistent with what has been repeatedly put forward, namely, that RSSs can alter the functionality of miRNA-mediated gene regulatory mechanisms in vivo. In fact, this is true but only perceivable when the CymRSV and Cym19stop virus replicate at moderate and comparable rates at the very early stages of infection (at 2 dpi). Under such conditions, the amount of viral RNAs has not yet been perceived to trigger the cascade processes of defense and where p19, yet unsaturated by vsiRNAs, has been produced to a sufficient level to start a soft but effective action to promote AGO1-miRNAs to act pro-virally. Indeed, at the early stage of tombusvirus infection, p19 acts as an efficient caliper of miR162 and miR403 but not of miR168; hence, the

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formation of functional AGO1/RISC containing miRNA 162 or miR403 (Pertermann et al., 2018) (Fig. 1). This is also possible because p19 induces AGO1 expression (Varallyay et al., 2010). In short, an exact and coordinated production in time and space of p19 allows p19 to overcome some cellular defense barriers by altering the functionality of miRNAs (Pertermann et al., 2018) and subsequently sequestering vsiRNAs, preventing their anti-viral action (Kontra et al., 2016). The mobile nature of RNA silencing allows for the information of a viral invasion to spread at the forefront of the infection, thus “immunizing” distant tissues. The functional relevance of mobile vsiRNA-mediated, non-cell-autonomous RNA silencing for plant recovery during a viral infection is well documented (Baucombe, 2004; Chellappan et al., 2004; Ghoshal and Sanfac¸on, 2015; Kørner et al., 2018; Ma et al., 2014; Medzihradszky et al., 2019). In tombusvirus infection, previous findings showed that the presence of 21-nt vsiRNAs correlated with the establishment of PTGS signaling. In addition, it has been proposed that these vsiRNAs act as PTGS signaling molecules for short distances, i.e., cell-to-cell (Havelda et al., 2005). Therefore, from the data gathered so far, it appears that the lonely RSS, p19, is not only capable of overcoming primary barriers by promoting the action of pro-viral miRNAs and defending the viral genome by preventing the incorporation of vsiRNAs into anti-viral RISCs, but is also able to promote cell-to-cell spread of the virus, a prerogative of long-distance spreading. Recently, p19 (from tomato bushy stunt virus) has been found associated with BAM1 and 2 particularly at the plasma membrane, preferentially in association with the plasmodesmata (Garnelo Gόmez et al., 2021) (Fig. 1). The study does not explain whether p19 bound to BAM1/1 is monodimeric or homodimeric (only the latter being capable of sequestering 21-long vsiRNAs) (Vargason et al., 2003; Ye et al., 2003), but is of great scientific relevance. In fact, the study further proves that there are significant links with “canonical” mechanisms of plant immunity (reviewed in Leonetti et al., 2021), reinforcing the notion that RLKs play a central role in antiviral defense. Furthermore, it clearly shows that unrelated RSSs can interact with RLKs, i.e., tombusvirus p19 (Garnelo Gόmez et al., 2021) and geminivirus C4 (Fan et al., 2021; Rosas-Diaz et al., 2018). Finally, but not of minor importance, the study suggests the possibility that p19 can suppress cell-to-cell movement RNA silencing independently of siRNA sequestration (Garnelo Gόmez et al., 2021) (Fig. 1).

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3.4 Cucumoviruses: Further expansion of 2b protein functionality by post-translational modification The cucumovirus 2b protein of cucumber mosaic virus (CMV), is another well studied RSSs. Early studies immediately highlighted the function of CMV 2b as a pathogenic determinant and a suppressor of transgenic PTGS (Lucy et al., 2000). CMV 2b contains NLSs, which are insufficient for enhancement of the 2b-mediated pathogenic response in host plants (Wang et al., 2004). Subsequently, the ability of 2b (constitutively expressed in A. thaliana) was discovered to block AGO1 cleavage activity, to inhibit miRNA pathways, attenuate RNA silencing and counteract host defense (Zhang et al., 2006). When expressed in the viral context, CMV 2b was able to reduce the accumulation of 21-, 22- and 24-nt vsiRNAs derived from DCL4, DCL2 and DCL3, respectively (Diaz-Pendon et al., 2007). At the same time, the ability of the 2b protein of CMV to bind vsiRNAs (Goto et al., 2007) allowed the sequestering of vsiRNAs, while CMV lacking 2b could confer discreet protection of the plant against viral overinfection or coinfection (Shams-Bakhsh et al., 2007; Ziebell et al., 2007; Ziebell and Carr, 2009). Moreover, similarly to the tombusvirus p19, tomato aspermy virus 2b, which is closely related to CMV 2b, exhibits affinity to antiviral and pro-viral miRNAs (Pertermann et al., 2018). Altogether, the evidence supports the notion that the ability of 2b protein to bind small RNAs is indispensable for its silencing suppressor functions. Indeed, its localization to the nucleus, capacity to self-interact and to interact with AGOs are not required for its RSS activity (Gonzalez et al., 2010). Nevertheless, nucleus/nucleolus localization of the 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. As already indicated for the other unrelated RSSs, the expression of the 2b protein in time and cell space determines its functionality; accordingly, the RSS activity of CMV 2b is predominantly exerted by the 2b protein residing in the cytoplasm (Du et al., 2014). The difference in the accumulation of 2b between the nucleus and cytoplasm is linked to 2b functions. In the nucleus/nucleolus, 2b accelerates symptom appearance, but cytoplasm-localized 2b has a primary role in suppressing RNA silencing. The very recent study by Kim et al. (2022) has clarified how 2b can shuttle between the nucleus and cytoplasm to maintain efficient suppression of host RNA silencing. The in vivo phosphorylation of 2b protein, both in infected N. benthamiana in the patches infiltrated with Agrobacterium containing 2b, had already been proven (Nemes et al., 2017). In addition, Kim and collaborators (2022) identified the serine residue at position 28 (S28) as

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an additional motif of phosphorylation. Importantly, S28 is located close to the NLS, and the study showed that phosphorylation at S28 is primarily required for nuclear localization by promoting the interaction between 2b and the carrier protein (importin [IMPα]). Du et al. (2014) found that phosphorylation and nuclear localization negatively regulated 2b RSS activity. Conversely, an elegant approach of nuclear fractionation and phosphoprotein enrichment showed that CMV 2b in the nucleus undergoes dephosphorylation and that the identified nuclear exporting signals promoted 2b export to the nucleus and the restoration of 2b RSS activity (Kim et al., 2022). Similar to the other unrelated RSSs, CMV 2b carries significant virulence activity in overcoming the barriers posed by the host plants, which are more largely attributable to canonical immunity. Such 2b activity is not strictly related to RNA silencing, but rather to other aspects of hormone-mediated signaling, since it is involved in counter-actions against SA- and JA-related defense responses. In addition, 2b can directly bind to a host factor that is efficient in scavenging cellular hydrogen peroxide and thus interferes specifically with the host factor, leading to the induction of necrosis (Inaba et al., 2011). The increase in plant reactive oxygen species levels has also been shown to be related to enhancing CMV acquisition and transmission by the vector insect, the aphid (Guo et al., 2019a). Likewise, the disruption of JA-mediated gene expression by the 2b protein influences CMV transmission by aphids (Lewsey et al., 2010; Wu et al., 2017). Both sRNA sequestration and host transcriptome reprogramming via hormone signaling has significant environmental impact, as the experiments using a CMV mutant lacking 2b and Arabidopsis silencing mutants implicated miRNAs in regulating the emission of pollinator-perceivable volatiles (Groen et al., 2016). Finally, the recent work by Shukla and collaborators (2021) has further disclosed the links between 2b RSS activity and its capacity to alter SA-signaling: (a) autophagy is induced by SA during CMV infection; it is dampened by CMV 2b, (b) in turn, autophagy promotes 2b turnover, and (c) autophagy provides resistance against CMV by reducing viral RNA accumulation in an RNA silencing-dependent manner (Shukla et al., 2021). These combined studies foster the idea that viral RSSs exhibit functional specificity in the context of the infected cell in terms of compartmental localization and temporal expression. They also show that macroautophagy/autophagy is the conserved intracellular degradation pathway which is emerging as an additional feedback mechanism of plant counter-response to viral RSSs.

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4. Concluding remarks Review articles on viral RSSs to date have focused on discovering and introducing the mechanism of action of viral RSSs. Even if the RSSs of a particular virus are described as having multiple action points, it has not been discussed why the virus evolves for multilayered RSS activity. In this review, we provide a detailed description regarding the fact that the RSSs of potyviruses, cucumoviruses, tombusviruses, and geminiviruses are multifunctional proteins that not only possess multi-layered RSS activity but also exhibit a variety of intracellular functions (resumed in Fig. 2). The multilayered RSSs activity is definitely advantageous for viral survival when the virus adapts to a new host, and subsequent episodic positive selection results in the acquisition of new intracellular functions that are suited to each successive viral infection. Because all of the viruses presented in this review have a wide host range, it is likely that RSSs have been the driving force behind viral host switching. It has been widely recognized that many RSSs, if not all, are involved in determining the disease phenotype, i.e., strong, mild or symptomless. Importantly, RSSs may determine the breakdown of resistance, which underscores the importance of gaining insight into their mechanisms of action and potential implications for disease control. The functionalities of RSS described so far suggest that plant viruses adopt very diverse strategies against plant’s RNA silencing, which are entrusted to one or more RSSs in well-coordinated spatio-temporal action. This implies that viral RSSs are tools at the service of viruses and that the advanced assumptions of their strong interference with gene regulation processes mediated by endogenous miRNAs or siRNAs are not entirely well supported (unless RSSs are transiently or transgenically overexpressed). Nevertheless, the notion that RSSs are symptom determinants remains firmly established. It is therefore likely that most viral-specific symptoms are due to the viral titre in tissues, which is indirectly influenced by the action of RSS, while specific symptoms are due to gene silencing mediated by vsiRNAs that escape RSS sequestration (Fig. 3). Specific and functional selectivity of RSSs for vsiRNAs remains to be established. Lastly, the full exploitation of the functional properties of viral RSSs, such as those described in this review, is deepening our understanding of the intricate plant–virus interaction. Consequently, more insight is acquired into both virus evolution and differentiation and plant gene expression and signal transduction.

RSS strategies RSS diversity in cellular functions

siRNA-binding AGO-binding DNA methylation

Load

Initial infection Host adaptation

Positive selection pressure

SA/JA-related genes Episodic Environmental changes selection

Multi-layer RSSs

Host jumping

Autophagy/Proteasome

Viral protein for RSS

Simple RSS

Load Translation inhibition

Change in subcellular localization

Modification in DNA methylation

Fig. 2 Schematic representation of the diversity of viral RSSs that may have gained new cellular functions in molecular evolution driven by episodic positive selection. The newly gained functions are important for viruses to survive when adapting to new hosts. Considering that viral RSSs should mainly work for host adaptation, the virus with multilayer RSS strategies is at an advantage. As for the newly acquired functions and multiple mechanisms of RSS activity, whether the two work in tandem will depend on a case by case basis.

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Viral suppressors of RNA silencing in plants

Viral invasion of the cell

Viral replication and host invasion High viral titre Severe sympthoms

affinity.-based Anti viral miRNA sequestration

Anti viral RNA silencing

vsiRNAs

Very Early infection

Viral protein for RSS Expression

Late infection and vsiRNA saturation

Escaping from RSS sequestration

Host gene silencing and Specific sympthoms

miRNA sequestration Isi mpaired

Viral enrtry into cell

Fig. 3 Viral RSSs as symptom determinants. RSSs with strong 21- to 24-nt short RNA sequestration activity, such as p19 of tombusviruses, express a different sequencespecific affinity for highly conserved “anti-viral” miRNAs and a low affinity for “pro-viral” miRNAs. This results in an immediate ability of the virus in the permissive host to overcome the antiviral RNA silencing barriers and trigger viral replication processes in the cells. This leads to an increase in viral titre and invasion of the host with specific symptoms and, at the extreme limit, death of the host plant. This “escalation” is assisted by the RSS that sequesters vsiRNAs preventing their incorporation into anti-viral gene silencing complexes. RSSs thus become saturated with vsiRNAs and fail to bind plant miRNAs in a significant manner causing the direct dysfunction of miRNA gene regulation. Rather, vsiRNAs can escape RSS sequestration (i.e., due also to RSS-vsiRNA affinities) and when incorporated into gene silencing complexes are able to silence host genes in a sequence-specific manner and cause virus-specific disease symptoms.

Acknowledgments This work is financially supported by the Prize 2021 to V.P. from the Department of Biology, Agricultural and Food Sciences of the National Research Council for the project “AmaVirALS.”

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Wu, D., Qi, T., Li, W.-X., Tian, H., Gao, H., Wang, J., Ge, J., Yao, R., Ren, C., Wang, X.-B., Liu, Y., Kang, L., Ding, S.-W., Xie, D., 2017. Viral effector protein manipulates host hormone signaling to attract insect vectors. Cell Res. 27, 402–415. Xie, Z., Fan, B., Chen, C., Chen, Z., 2001. An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense. Proc. Natl. Acad. Sci. U. S. A. 98, 6516–6521. Ye, K., Malinina, L., Patel, D.J., 2003. Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426, 874–878. Zerbini, F.M., Briddon, R.W., Idris, A., Martin, D.P., Moriones, E., Navas-Castillo, J., Rivera-Bustamante, R., Roumagnac, P., Varsani, A., ICTV Report Consortium, 2017. ICTV virus taxonomy profile: Geminiviridae. J. Gen. Virol. 98, 131–133. Zhang, X., Yuan, Y.R., Pei, Y., Lin, S.S., Tuschl, T., Patel, D.J., Chua, N.H., 2006. Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev. 20, 3255–3268. Ziebell, H., Carr, J.P., 2009. Effects of dicer-like endoribonucleases 2 and 4 on infection of Arabidopsis thaliana by cucumber mosaic virus and a mutant virus lacking the 2b counter-defence protein gene. J. Gen. Virol. 90, 2288–2292. Ziebell, H., Payne, T., Berry, J.O., Walsh, J.A., Carr, J.P., 2007. A cucumber mosaic virus mutant lacking the 2b counter-defence protein gene provides protection against wild-type strains. J. Gen. Virol. 88, 2862–2871. Zrachya, A., Glick, E., Levy, Y., Arazi, T., Citovsky, V., Gafni, Y., 2007. Suppressor of RNA silencing encoded by tomato yellow leaf curl virus-Israel. Virology 358, 159–165.

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CHAPTER TWO

Animal models of alphavirus infection and human disease Cormac J. Lucasa,b and Thomas E. Morrisona,* a

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, United States RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, United States *Corresponding author: e-mail address: [email protected] b

Contents 1. Introduction 2. Alphavirus transmission cycles 3. Human clinical disease 3.1 Arthritogenic alphaviruses 3.2 Encephalitic Alphaviruses 4. Animal models 4.1 Natural reservoir hosts 4.2 Vectors 5. Animal models of human disease 5.1 Mice 5.2 Other rodents 5.3 Non-human primates 6. Animal models of unique aspects of alphavirus infection 6.1 Transmission studies 6.2 Co-infection studies 7. Virus strains used in animal models 8. Conclusions Acknowledgments References

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Abstract Alphaviruses are a large group (>30 species) of enveloped, positive-strand RNA viruses. The re-emergence of mosquito-transmitted alphaviruses associated with human diseases ranging from severe and potentially fatal neurological disease to chronic arthritic disease highlights the need to understand the biology and pathogenesis of alphaviruses. Here, we review the development and use of animal models of alphavirus transmission and human disease, and discuss areas for continued refinement of these models including possible avenues for future investigation.

Advances in Virus Research, Volume 113 ISSN 0065-3527 https://doi.org/10.1016/bs.aivir.2022.07.001

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2022 Elsevier Inc. All rights reserved.

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Abbreviations BBB BFV CHIKV CNS CSF DENV ECSA EEEV IHC IOL MADV MAYV MBL NHP NSV ONNV PFU PPI RNAi RRV SESV SFV SG SGE SINV SPDV spp. VEEV WEEV ZIKV μCT

blood-brain barrier Barmah Forest virus chikungunya virus central nervous system cerebrospinal fluid dengue virus East Central South African Eastern equine encephalitis virus immunohistochemistry Indian Ocean lineage Madariaga virus Mayaro virus mannose-binding lectin non-human primate neuronal-adapted Sindbis virus o’nyong ‘nyong virus plaque-forming unit pre-pulse inhibition RNA interference Ross River virus Southern elephant seal virus Semliki Forest virus salivary gland salivary gland extract Sindbis virus salmon pancreatic disease virus species Venezuelan equine encephalitis virus Western equine encephalitis virus Zika virus microcomputed tomographic

1. Introduction The re-emergence and geographic expansion of mosquitotransmitted alphaviruses such as chikungunya virus (CHIKV), Mayaro virus (MAYV), and Eastern equine encephalitis virus (EEEV) highlights the need to better understand mechanisms of alphavirus transmission and replication, correlates of clinical disease outcomes, and protective and pathogenic innate and adaptive immune responses (Azar et al., 2020; Cunha et al., 2020). Ethical and practical considerations have limited clinical studies of

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human alphavirus infection and thus, the development and characterization of laboratory animal models of human disease, protective immunity, and viral transmission are necessary to understand pathogenic mechanisms and to test therapeutic interventions and preventative measures. In this review of animal models of alphavirus infection, we summarize the clinical disease signs of alphaviruses known to infect humans, discuss progress on animal models of natural reservoir species and vectors, provide an overview of different animal models of human disease, and discuss areas in need of future investigation.

2. Alphavirus transmission cycles For the development of animal models of alphavirus transmission it is critical to understand the natural invertebrate and vertebrate hosts of alphaviruses. Moreover, a better understanding of how different hosts respond to alphavirus infection could help elucidate mechanisms of pathogenesis and protective immunity. Alphaviruses most commonly associated with human disease are transmitted in cycles between a variety of mosquitoes and vertebrate hosts (Chen et al., 2018). However, it should be noted that the Alphavirus genus includes over 30 species, including viruses not known to cause human disease or to be transmitted via mosquitoes, such as Southern elephant seal virus (SESV), fish alphaviruses like salmon pancreatic disease virus (SPDV), and the insect-specific Eilat virus (Chen et al., 2018; Forrester et al., 2012; Powers et al., 2001). Chikungunya virus (CHIKV), which causes an acute and chronic musculoskeletal disease in infected humans (see Section 3), is transmitted in urban environments between Aedes (A.) aegypti mosquitoes and humans and in sylvatic cycles between arboreal Aedes spp. and non-human primates (NHPs) (Pezzi et al., 2020; Weaver et al., 2020). The 2004–2006 La R
eunion epidemic and numerous subsequent outbreaks demonstrated that CHIKV could be efficiently transmitted to humans by A. albopictus mosquitoes (Pezzi et al., 2020; Renault et al., 2007; Tsetsarkin et al., 2007; Vazeille et al., 2007), emphasizing the risk of global expansion of endemic CHIKV transmission since the range of these mosquitoes includes more temperate latitudes than that of A. aegypti. Ross River virus (RRV) and Barmah Forest virus (BFV) are endemic to Australia and Papua New Guinea (Harley et al., 2001; Michie et al., 2020). There is considerable debate regarding the role of different mosquito species and vertebrate hosts for RRV maintenance and during RRV outbreaks.

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Nevertheless, the current consensus is that RRV and BFV are predominantly transmitted between Aedes spp. or Culex annulirostris mosquitoes and marsupials (RRV) or birds (BFV), whereas A. vigilax and A. procax are important vectors for human-mosquito-human transmission (Claflin and Webb, 2015; Kain et al., 2021; Stephenson et al., 2018). The related arthritogenic alphaviruses o’nyong’nyong virus (ONNV) and Mayaro virus (MAYV) do not appear to have established consistent urban human-mosquito-human transmission cycles. Most studies indicate that MAYV is transmitted in Central and South America between Haemagogus spp. mosquitoes and NHPs, and possibly birds (Pezzi et al., 2020). In contrast to most arboviruses, ONNV is transmitted by anopheline mosquitoes, including Anopheles funestus and Anopheles gambiae (an important vector of malaria in Sub-Saharan Africa) (Pezzi et al., 2020). ONNV can be transmitted human-mosquito-human, but the vertebrate hosts that participate in enzootic transmission of ONNV remain unidentified (Celone et al., 2021; Pezzi et al., 2020). Sindbis virus (SINV) is naturally maintained between Culex unnivatus, Culex torrentium, and Culiseta morsistans mosquitoes and passerine birds in South Africa and Northern Europe, although incidental transmission to humans, which are dead end hosts, by Aedes and Ochlerotatus spp. mosquitoes does occur (Adouchief et al., 2016). The equine encephalitis viruses are endemic in the Americas. Eastern equine encephalitis virus (EEEV) is transmitted between Culiseta melanura mosquitoes and passerine birds (enzootic) or Aedes and Culex spp. and equines and humans (epizootic) (Armstrong and Andreadis, 2022; Burkett-Cadena et al., 2022). Venezuelan equine encephalitis virus (VEEV) cycles between spiny rats and cotton rats (Sigmodon spp.) and Aedes spp., Ochlerotatus taeniorhynchus, and Psorophora spp. mosquitoes for epizootic strains or Culex spp. mosquitoes for enzootic strains (Weaver et al., 2004). Western equine encephalitis (WEEV) epizootic transmission is associated with similar vectors as EEEV, but the primary enzootic vector for WEEV is Culex tarsalis (Weaver and Barrett, 2004). Importantly, horses serve as potent amplifying hosts for EEEV and VEEV, as outbreaks in equines characterized by high equine viremia are often associated with human cases (Gonzalez-Salazar et al., 2003). In addition, the encephalitic alphaviruses can be efficiently transmitted by aerosol, leading to their development as biowarfare agents (Croddy et al., 2002).

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3. Human clinical disease The development of informative animal models of human disease requires in-depth knowledge of alphavirus infection outcomes in human populations. Although the global spread of CHIKV since 2004 has greatly increased knowledge of human infection with CHIKV and related arthritogenic alphaviruses, our understanding of the general features of human alphavirus infections remains limited. In this section, we summarize key features of human alphavirus infections that can guide continued development of animal models that mimic human disease.

3.1 Arthritogenic alphaviruses 3.1.1 Disease signs and symptoms Acute infection with arthritogenic alphaviruses, including CHIKV, MAYV, ONNV, RRV, and SINV generally presents with a sudden onset of high fever, severe joint pain and inflammation, muscle pain, and rash (Fig. 1).

Fig. 1 Human clinical disease signs and symptoms during alphavirus infection. Shared and unique disease signs and symptoms of arthritogenic (CHIKV, MAYV, ONNV, RRV, SINV) and encephalitic (EEEV, VEEV, WEEV) alphavirus infection in patients. This figure was made using Biorender.com.

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In most cases, the arthritis and arthralgia are symmetrical and affect multiple joints (i.e., polyarthritis/polyarthralgia), especially joints in the fingers, wrists, ankles, and feet (Zaid et al., 2021). Notably, infection with any of the arthritogenic alphaviruses can progress to post-acute (3 weeks to 3 months post illness onset) and chronic stages (>3 months post illness onset) characterized by the persistence of musculoskeletal tissue pain and inflammation that can be debilitating. In addition, atypical presentations have been observed, including encephalitis in the young and the aged and cardiovascular symptoms (Bonifay et al., 2018; Cotella et al., 2021; G
erardin et al., 2016). Fatal CHIKV cases are rare and often associated with pre-existing comorbidities, such as diabetes, and atypical disease presentations including encephalitis (de Lima et al., 2021; G
erardin et al., 2016). 3.1.2 Pathology In general, there is a paucity of information regarding tissue pathology associated with acute arthritogenic alphavirus infection of humans. During RRV infection, the accumulation of monocytes, vacuolated macrophages, and natural killer cells in synovial fluid has been reported (Fraser et al., 1981; Hazelton et al., 1985). In acute CHIKV infection, T cell and macrophage cellular infiltrates were detected in skeletal muscle tissue (Ozden et al., 2007). In addition, in a post-mortem study of fatal CHIKV cases, mononuclear infiltrates were detected in connective tissue and the synovial sheath surrounding tendons in the hand (Sharp et al., 2021). The limited knowledge of tissue pathology associated with post-acute and chronic stages of alphavirus-induced musculoskeletal disease comes predominantly from studies of human CHIKV infection. Imaging analysis of affected joints (e.g., wrists, ankles, fingers) has revealed synovitis and tenosynovitis, joint effusion, and myositis (Manimunda et al., 2010; Mogami et al., 2017; Simon et al., 2007). In addition, joint degeneration and bone lesions, detectable by MRI and X-ray, have been observed ( Javelle et al., 2015; Manimunda et al., 2010). Analysis of synovial fluid collected from a single patient with chronic CHIKV disease identified the presence of vacuolated macrophages, CD14+ monocytes, and activated CD56+ NK cells, as well as CD4+ and CD8+ T cells (Hoarau et al., 2010). In addition, synovial lining hyperplasia and cellular infiltrates including macrophages and T cells were identified in synovial tissue biopsy material collected from the same individual (Hoarau et al., 2010). Similarly, synovial hyperplasia and mononuclear cell infiltrates were detected in knee biopsy tissue obtained from patients with post-acute (i.e., 5 weeks post symptom onset) RRV arthritis (Soden et al., 2000).

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3.1.3 Virology Arthritogenic alphavirus infection of humans often results in detectable viremia (Fig. 2). Viremia can range from 101 to 108 plaque-forming units (PFU)/mL of blood, and typically lasts 2–8 days (Appassakij et al., 2013; Chusri et al., 2014; Kain et al., 2021; Riswari et al., 2016). As such, most clinical isolates of arthritogenic alphaviruses have been obtained from human serum or plasma, although a small number have been obtained from skin lesions (Kurkela et al., 2004; Malherbe et al., 1963). Ross River virus antigen was detected by specific immunofluorescence in monocytes and macrophages present in synovial fluid collected from four acute

Fig. 2 Viral and pathologic features during acute and chronic arthritogenic alphavirus infection. Acute arthritogenic alphavirus infection is characterized by viremia. In multiple joints, typically symmetrical, synovitis and tenosynovitis with mononuclear cell infiltration and viral antigen and RNA are observed. In patients that do not resolve disease, viral RNA has been detected in synovial biopsies, and viral antigen has been reported in synovial macrophages. This figure was made using Biorender.com.

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cases, but intact virus was not identified by electron microscopy or cell culture (Fraser et al., 1981). In tissue biopsy samples from humans with acute CHIKV infection, CHIKV antigen was detected in skeletal muscle fascia, muscle satellite cells, fibroblasts of the joint capsule, and dermis (Couderc et al., 2008; Ozden et al., 2007). During the chronic phase of disease, immunostaining of synovial and muscle tissue biopsy material identified perivascular macrophages and muscle satellite cells, respectively, as positive for CHIKV antigen (Fig. 2) (Hoarau et al., 2010; Ozden et al., 2007). The synovial tissue also was positive for CHIKV nucleic acid by RT-PCR (Hoarau et al., 2010). Consistent with these findings, synovial biopsy tissue collected from the knees of patients with RRV arthritis 5 weeks after symptom onset was positive for RRV RNA (Soden et al., 2000).

3.2 Encephalitic Alphaviruses 3.2.1 Disease signs and symptoms Acute infection with encephalitic alphaviruses (EEEV, Madariaga virus (MADV), VEEV, WEEV) leads to a febrile illness following a 1–14 day incubation period (Curren et al., 2018, (CDC), 1995). Most infections with VEEV are symptomatic, whereas a large portion of EEEV and WEEV infections remain subclinical (Carrera et al., 2013; Weaver et al., 1996). Prominent manifestations in clinically apparent cases include fever, convulsions, headache, photophobia, myalgias, chills, vomiting, and diarrhea (Fig. 1) (Franck and Johnson, 1970; Soto et al., 2022; Zacks and Paessler, 2010). The overall case fatality rate varies greatly among the encephalitic alphaviruses, ranging from 107 PFU by intravenous inoculation, alters disease severity in this rhesus macaque model. Studies in this model have identified reduced B and T cell responses, higher viral loads for a longer duration, and altered expansion of myeloid cell populations in aged animals infected with CHIKV-LR (Messaoudi et al., 2013). These findings reflect diminished immune responsiveness in the elderly, providing a strong model for studying age-dependent clinical disease severity (Salminen, 2022). Despite the presence of viral RNA in lymphoid and joint-associated tissues in CHIKV 37997-infected pregnant macaques at

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21 dpi (after viremia and clinical disease signs subside), a lack of detectable viral RNA or germinal center development in fetal tissue indicated no transplacental transmission, suggesting CHIKV is not vertically transmitted (Chen et al., 2010). Both the rhesus macaque and cynomolgus macaque models of CHIKV infection have been used to investigate antiviral preand post-prophylactic treatments including vaccination and intravenous administration of human monoclonal antibodies against CHIKV during acute disease, emphasizing how the usefulness of this model at mimicking aspects of human disease supports the development of novel therapeutic strategies (Broeckel et al., 2017; Roy et al., 2014). The cynomolgus macaque model also provides strong evidence for persistent CHIKV infection. CHIKV RNA was detected in lymphoid organs, liver, synovial and muscle tissues for months after inoculation. Moreover, infectious virus was recovered from spleen, liver, and muscle tissue at 44 dpi and viral antigen and RNA were detected in CD68+ macrophages, implicating macrophages as a cellular site of viral persistence (Labadie et al., 2010). 5.3.2 Encephalitic alphaviruses in non-human primates While EEEV, VEEV, and WEEV are naturally transmitted by mosquito bite, which is usually mimicked in the laboratory using needle-based subcutaneous inoculation, studies in NHPs have primarily used aerosol-based inoculation as these viruses are highly infectious biohazard threats when in aerosolized form (Croddy et al., 2002). For more detailed description of aerosol exposure, we direct the reader to the Section 6.1.1. Infection of cynomolgus macaques with EEEV, VEEV, or WEEV by aerosol exposure results in fever, viremia, lymphopenia, and encephalitis, and mortality is dose-dependent for both EEEV and WEEV, although in one study comparing high and low dose EEEV infection, survival was inversely correlated with the development of apparent disease signs (Albe et al., 2021; Reed et al., 2004; Reed et al., 2005). WEEV has been less studied in NHPs than EEEV and VEEV, however, a small proportion of cynomolgus macaques infected with the virulent WEEV CBA-87 strain by aerosol exposure succumb to disease with evidence of viral antigen and lymphocytic infiltrate present in CNS tissue and a correlation between serum glucose levels and severity of clinical encephalitis (Reed et al., 2005). Studies with EEEV and VEEV using minimally invasive implantation of telemetry devices demonstrate significant disruption of macaque behavior, including disrupted circadian rhythm, cardiac abnormalities, disinterest in food and water, and increased intracranial pressure following aerosol exposure; these parameters

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and their measurement with telemetry are excellent quantitative ways of assessing encephalitic disease in a manner more easily translatable to observation of human patients by trained medical professionals (Ma et al., 2019; Ma et al., 2020b; Trefry et al., 2021). Characterization of the pathophysiology following EEEV-mediated disease revealed viremia preceded fever with early elevated levels of IP-10 in the plasma, an increase in traumatic brain injury genes and inflammatory cytokines and chemokines within the brain, and high amounts of infectious virus in macaques that succumbed to infection (Albe et al., 2021). The few macaques that survived infection with EEEV strain V105 had evidence of viral RNA but not infectious virus in their brain, suggesting survival may be associated with differential control of infection through restriction of production of infectious viral progeny (Albe et al., 2021). Given that EEEV is administered by aerosol exposure in this model, it is not surprising that viral RNA is not present in visceral organs at the time of death, and interestingly, active viral replication appears to be restricted to neurons and even more restricted to regions of the brain near the olfactory tract (Williams et al., 2022). This particular study concluded that due to this limited viral tropism and lack of observed microscopic lesions at the time of necropsy, EEEV-mediated disease is primarily associated with neuronal dysfunction instead of direct cell death (Williams et al., 2022). The exact mechanism of CNS disease during VEEV infection of cynomolgus macaques is also unclear, however, VEEV aerosol-exposed animals displayed early reduction in expression of the MOG glycoprotein, which has been associated with autoimmune encephalitis when overexpressed, and S100b, which may serve as a sensor for brain injury and can affect the cellular p53 response; viral downregulation of these factors may contribute to CNS pathology and subsequent encephalitic disease (Koterski et al., 2007). Older studies have used rhesus macaques to model VEEV-mediated disease, however, these animals develop a milder illness, and CNS pathology was not examined (Bowen et al., 1980). Recently, EEEV, VEEV, and WEEV infection via aerosol exposure or subcutaneous inoculation was compared in cynomolgus macaques to attempt to better mimic the natural route of human infection (Smith et al., 2020). All animals infected with WEEV or VEEV by subcutaneous inoculation survived infection, and only VEEV-infected animals displayed limited pathology consisting of inflammation in the meninges and gliosis in the cerebellum (Smith et al., 2020). In contrast, 75% of animals infected subcutaneously with EEEV succumbed to infection within 6–9 dpi, and viral RNA was detected in CNS tissue accompanied by acute inflammation of the

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meninges, hemorrhage in the cerebrum, and perivascular cuffing in multiple regions. However infectious virus in the brain/olfactory bulb was only detected in one animal, in contrast with the aerosol-exposed animals (Smith et al., 2020). Ultimately, the cynomolgus macaque model recapitulates several key aspects of human encephalitic alphavirus disease, including differences in severity between VEEV and EEEV upon natural exposure and the increased infectiousness of these viruses upon aerosol transmission. These systems can be used to examine viral factors associated with pathogenesis in a relevant animal model and to test novel vaccines and antivirals. For example, such a test of a post-exposure prophylactic monoclonal antibody treatment following VEEV aerosol exposure, suggested that it could be directly applied to treating possible laboratory aerosol exposures (Burke et al., 2019).

6. Animal models of unique aspects of alphavirus infection 6.1 Transmission studies Viral transmission is critical for the alphavirus life cycle and for the establishment of disease (Fig. 6). In order for mosquito-transmitted alphaviruses to spread, the virus must establish a viremia of sufficient magnitude and duration in the vertebrate host to infect blood-feeding mosquitoes. Thus, understanding the viral and host determinants of viremia is a key research area of interest (Ander et al., 2022). To do this, researchers have utilized a reductionist system to study alphavirus viremia in which a defined number of viral particles or infectious units are inoculated intravenously into mice, and the clearance rate of viral particles from the blood circulation is determined (Ander et al., 2022; Carpentier et al., 2019). This simplified system bypasses the effects of de novo viral replication and allows for the determination of the fate of alphaviral particles as well as the identification of host and viral factors that influence the rate of viral clearance, such as the scavenger receptor MARCO (Carpentier et al., 2019) and specific amino acid residues in virion surface glycoproteins (Bernard et al., 2000; Carpentier et al., 2019), respectively. 6.1.1 Aerosol transmission Studies in mice and NHPs have been used to evaluate infection and disease upon aerosol exposure, which is generally performed using head-only aerosol exposure in a class 3 biological safety cabinet with aerosol droplets generated using a nebulizer. For these studies, the respiratory capacity of the

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Fig. 6 Utility of alphavirus transmission models. Alphavirus transmission has been studied using specific models of experimental viremia in which the fate of viral particles in the blood circulation can be assessed following intravenous inoculation, following aerosol inoculation, and using mosquito and mosquito factor-based models to more closely mimic natural infection. This figure was made using Biorender.com.

animals is determined prior to exposure, and the actual inhaled dose is determined by plaque assay on samples collected by impaction on gelatin-filters (Reed et al., 2004; Taylor, 2007). These steps are essential as aerosol exposure can be a more highly variable method of inoculation than needle-based inoculation. In mice, aerosol exposure of 7–9-week-old female BALB/c mice to EEEV Pe-6 causes dose-dependent mortality and recapitulates the severe encephalitis observed in human patients infected following a mosquito bite (Phelps et al., 2019). Histological evaluation of these mice suggested that the brain supports productive EEEV infection, however, further study of EEEV replication in cells of the murine CNS in the context of aerosol infection is needed. In NHPs, aerosol exposure of adult cynomolgus macaques and common marmosets to EEEV (FL91-4679

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and FL93-939) also results in dose-dependent lethality and neurological disease similar to that observed in humans (Porter et al., 2017; Reed et al., 2007). These studies highlight the infectiousness of aerosol-transmitted EEEV, although as of yet, there have been no reported cases of aerosolized EEEV infection in humans for comparison. Enhanced infectivity and disease upon aerosol exposure of cynomolgus macaques to VEEV can be more directly correlated with human infection due to early reports of laboratory VEEV aerosol exposure and subsequent disease (Reed et al., 2004). In addition, while no natural CHIKV infections by aerosol exposure have been reported, CHIKV is capable of aerosol transmission in cynomolgus macaques, causing viremia and increased markers of liver damage although no overt clinical disease was observed (Cirimotich et al., 2017). 6.1.2 Mosquito and mosquito component-based infection models One important consideration for alphavirus animal models is that generally virus stocks are diluted in cell culture media or saline solution prior to inoculation into the animal, while in natural infection, virus is transmitted as part of mosquito saliva (Dudley et al., 2017; Thangamani et al., 2010). There is a large interest in understanding the impact of mosquito saliva and other arthropod feeding factors on the dynamics of alphavirus infection as mosquito and tick salivary factors have been shown to influence host susceptibility, tissue viral burden, viral dissemination, and immune responses for a variety of arboviruses (Cox et al., 2012; Edwards et al., 1998; Le Coupanec et al., 2013; Limesand et al., 2000; McCracken et al., 2014; Moser et al., 2016; Osorio et al., 1996; Schneider et al., 2006; Styer et al., 2011; Wanasen et al., 2004). One approach used is the co-inoculation of mice with both a defined amount of virus and sonicated salivary glands collected from virus-naı¨ve mosquitoes to mimic natural infection (Schneider et al., 2004). When this approach was used with SINV strain H55K70, which has similar mouse virulence phenotypes to the commonly used strain AR339, the presence of salivary gland extract (SGE) was associated with reduced type I and II IFN responses and an overall shift from protective TH1 to TH2 cytokine responses (Schneider et al., 2004). The most direct method to investigate natural mosquito transmission of alphaviruses is to allow mosquitoes to blood feed on laboratory animals. For example, infection of mosquitoes by blood feeding on EEEV-infected chicks followed by blood feeding on naı¨ve laboratory mice was used as an early method for estimating the amount of virus inoculated by mosquito bite (Chamberlain et al., 1954). Since then, cell culture systems and infectious virus derived from recombinant cDNA clones have allowed direct

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infection of mosquitoes instead of blood feeding on chicks, for example, in experiments with outbred CD-1 mice using A. aegypti mosquitoes infected with CHIKV LR2006 (Thangamani et al., 2010). However, the authors of this study did not report the efficiency of CHIKV infection between needle-inoculated and mosquito bite-transmitted CHIKV, as the amount of virus actually picked up and transmitted by a single mosquito is variable (Chamberlain et al., 1954; Miller et al., 2021; Styer et al., 2007). Recently, an alphavirus infection model has been described in which mice are bitten by up to 5 naı¨ve mosquitoes on a defined site and then immediately inoculated with a known virus titer at the same site; this model allows for consistent infection of a defined number of viral particles rather than the inconsistency observed by allowing infected mosquitoes to feed on laboratory mice (Fletcher et al., 2020; Pingen et al., 2016). This model has been used for study of the avirulent SFV4 and virulent SFV6 strains and clearly demonstrates that inoculation of virus at mosquito bite sites results in increased viral replication at the infection site and more rapid viral dissemination for both avirulent and virulent SFV (Pingen et al., 2016).

6.2 Co-infection studies Alphaviruses circulate in the same regions of the world as other important human pathogens, such as DENV, Zika (ZIKV), Plasmodium spp., Leishmania spp., and HIV, and in regions where people are more burdened with helminth infections (Brooker, 2010; Mala et al., 2021; Pasaribu et al., 2019). DENV and ZIKV are both transmitted by A. aegypti mosquitoes, which also transmit CHIKV, making it important to characterize how potential co-infection of both mosquito or mammalian hosts alters transmissibility, clinical disease, and development of protective immunity (Fig. 7); a neglected area of public health concern (Vogels et al., 2019). 6.2.1 Plasmodium spp. While Plasmodium species that cause malaria are primarily transmitted by Anopheles gambiae mosquitoes, co-infection of humans remains a strong possibility due to the significant geographic overlap in malaria and CHIKV endemic regions and the isolation of CHIKV from other Anopheles mosquito species known to transmit malaria (Mala et al., 2021). In addition, ONNV is transmitted by A. gambiae, making it vital to understand how ONNV and Plasmodium co-infections of both mosquitoes and mammals impact transmission and disease (Pezzi et al., 2020). In one study, researchers found that prior infection of WT C57BL/6 mice with rodent Plasmodium spp. (either the lethal PbA strain or the non-lethal Py17x strain) prior to inoculation

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Fig. 7 Utility of alphavirus co-infection models. Co-infection of hosts with alphaviruses and other infectious agents, such as Plasmodium parasites or dengue virus, is an understudied area of research. However, some studies have assessed the impact of co-infection on alphavirus pathogenesis and immunity in mammalian systems and fecundity, transmission, and vector competence in mosquito models. This figure was made using Biorender.com.

with CHIKV strain SGP11 reduced viremia and suppressed tissue viral load and joint inflammation, whereas concomitant co-infection had no effect on viremia but reduced peak joint inflammation (Teo et al., 2018). Similar findings were observed following inoculation of ONNV into Plasmodium-infected mice (Torres-Ruesta et al., 2022). Although these studies investigated both an experimental cerebral malaria (ECM) model and an uncomplicated malaria infection model that recapitulates liver stage disease, metabolic acidosis, parasitemia, and severe anemia, inoculation of CHIKV or ONNV occurred during the acute stage of Plasmodium infection when the innate immune response is activated. Thus, it could be of interest to modify these models and assess the outcome of CHIKV or ONNV infection following inoculation during the chronic stage of malaria infection (De Niz and Heussler, 2018; Teo et al., 2018). In addition, testing the effect of other Plasmodium species on CHIKV and ONNV disease, such as P. chaubaudi, which establishes chronic malaria in mice (Stephens et al., 2012), could enhance the utility and impact of these systems. 6.2.2 Dengue virus A few studies have experimentally co-infected mosquitoes with CHIKV and DENV to evaluate replication kinetics and transmission potential. Le Coupanec et al. infected 7-day old female mosquitoes with a blood meal containing the CHIKV 06.21 isolate from La R
eunion with the A226V mutation and a 1974 human serum DENV-2 isolate. Although CHIKV RNA levels in the salivary glands (SGs) were lower in co-infected mosquitoes at 2 dpi, CHIKV RNA levels were significantly higher in SGs of co-infected mosquitoes at 13 dpi; increased DENV-2 RNA was present

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in SGs of co-infected mosquitoes consistently throughout infection. These data highlight the concern that co-infection of CHIKV and DENV-2 could lead to increased transmission and larger outbreaks (Le Coupanec et al., 2017). Similarly, A. albopictus mosquitoes collected from La R
eunion island as eggs in 2006 can be infected with both DENV-1185/04 and CHIKV 06.21 either by simultaneous oral co-infection or an intrathoracic inoculation of DENV-1 followed later by a CHIKV-containing blood meal. The authors acknowledge this model has some limitations due to mosquito survival time and willingness to blood feed under the laboratory conditions (Vazeille et al., 2010). Nevertheless, the ability of two different vectors implicated in multiple DENV and CHIKV epidemics to be simultaneously infected with both viruses under laboratory conditions is promising for future investigation, as human DENV/CHIKV co-infection has increasingly been reported in places such as La R
eunion island and Madagascar (Furuya-Kanamori et al., 2016; Ratsitorahina et al., 2008).

7. Virus strains used in animal models As arthropod-borne viruses that cycle between vertebrate and invertebrate hosts, alphaviruses are constrained in the degree to which they can adapt to one host over the other (Coffey et al., 2008; Vasilakis et al., 2009). As viral genomic sequencing and development of robust recombinant cDNA clone systems have become widespread, the complications of passaging viral isolates in mammalian cell culture or laboratory animals prior to experimental use has become more appreciated, since adaptive mutations can confound studies of viral infection, pathogenesis, and immunity in animal models (Alcorn and Klimstra, 2022; Ciota and Kramer, 2010; DuPai et al., 2019; Gardner et al., 2012; Greene et al., 2005c). For example, comparison of the cell-culture adapted CHIKV strain 181/25 with WT CHIKV isolates in mice lacking the type I and II IFN receptors (AG129) or the STAT1 transcription factor involved in type I and type II IFN responses (STAT129) revealed that WT CHIKV infection was 100% lethal in both AG129 and STAT129 mice, whereas CHIKV 181/25 was 100% lethal in AG129 mice but only 50% lethal in STAT129 mice, emphasizing the dramatic effect cell culture adaptations can have on infection outcome (Gardner et al., 2012). This study, and many others, emphasizes the need to utilize low passage viral isolates and/or cDNA clone-derived virus based on direct viral genome sequencing of primary samples or low passage isolates. We provide a table summarizing the origin and passage history of alphaviruses commonly used in animal model systems (Table 1).

Table 1 Alphavirus strains commonly used in animal models Virus

Passage History

GenBank Accession No.

LR2006-OPY1 Human

2006 Vero-3, SM-1, Vero-1

DQ443544.2

Tsetsarkin et al. (2006), Tsetsarkin et al. (2007)

IOL

SL15649

Human

2006 Vero-3

GU189061.1

Hawman et al. (2013), Morrison et al. (2011)

ECSA

Ross

Human

1953 SM-16, Vero-2, C6/36–1 AF490259.3

Langsjoen et al. (2018), Powers et al. (2001)

WA

37,997

Aedes furcifer

1983 AP61 cells-1, Vero-1

AY726732.1

Tsetsarkin et al. (2007), Vanlandingham et al. (2005)

Asian

AF15561

Human

1962 Unk, Vero-2

EF452493.1

Gorchakov et al. (2012), Hawman et al. (2016)

Asian

181/25

AF15561 derivative

1986 MRC-5  18

MW473668.1 Gorchakov et al. (2012), Levitt et al. (1986)

I

T48

Aedes vigilax

1959 SM-10, Vero-2

GQ433359.1

Morrison et al. (2006)

III

SN11

Human

2009 C6/36-1, BHK-1

JF699395.1

Liu et al. (2011)

II

DC5692

Aedes 1995 C6/36-1, Vero-1, BHK-1 HM234643.1 Jupille et al. (2011) camptorhyncus

SG650

Human

1996 Vero-1

AF079456.1

Vanlandingham et al. (2006)

IMTSSA/5163 Human

2004 Vero-2

DQ399055.1

Chan et al. (2020)

Culex annulirostris

1974 SMB-2, Vero-2

U73745.1

Herrero et al. (2014)

Human

2001 Unk, Vero-1

DQ487410.1

Earnest et al. (2019), Weise et al. (2014)

Lineage

CHIKV IOL

RRV

ONNV

BFV

G2

Strain

BH2193

MAYV genotype D CH

Source

Year

Key References

SINV

genotype D TRVL4675

Human

1954 SM-13, BHK-1

AF398378.1

Figueiredo et al. (2019), Powers et al. (2001)

genotype D TRVL15537

Coquellitidia venezuelensis

1957 SM-5, Vero-2

MK573240.1

Chuong et al. (2019)

genotype L BeH407

Human

1955 Unk-2

MK573238.1

Earnest et al. (2019), Fox et al. (2015)

I

AR339

Culex pipiens 1952 SMB-9

JX682537.1

Labrada et al. (2002), Tucker et al. (1993)

I

TR339

I

NSV

I SFV

AR339 consensus sequence derivative AR339 derivative

Pierro et al. (2008)

1977 alternating SMB and WM-6

Lustig et al. (1988), Thach et al. (2000)

TE

chimeric Toto1101, NSV, and SV1A (SV1A is SMB-7, BHK-5, CEF-2 version of AR339)

Lustig et al. (1988), Pierro et al. (2008)

TE12

chimeric Toto1101 and NSV

Lustig et al. (1988)

S.A.AR86

Culex spp.

1954 Unk

U38305.1

Suthar et al. (2005)

SFV4

Aedes abnormalis

1942 Mouse-4

KP699763.1

Pingen et al. (2016)

SFV6

recombinant derivative of SFV4

A7 (74)

AR2066 derivative

N/A

1959 SMB-7

Pingen et al. (2016)

X74425.1

Michlmayr et al. (2014) Continued

Table 1 Alphavirus strains commonly used in animal models—cont’d Virus

EEEV

VEEV

WEEV

GenBank Accession No.

Key References

1942 Mouse-8, SMB-2

AY112987.1

Michlmayr et al. (2014)

Aedes albopictus

1992 Vero-3

AY722102.1

Gardner et al. (2011), Vogel et al. (2005)

FL93-939

Culiseta melanura

1993 Vero-1, SMB-1

EF151502.1

Arrigo et al. (2010a), Honnold et al. (2015a)

NA

V105-00210

Human

2005 Vero-3

KP282670.1

Yu et al. (2015)

IAB

TrD (Trinidad) donkey brain 1943 GP-1, Vero-6, BHK-1

L01442.2

Kinney et al. (1989)

IAB

TC-83

TrD derivative

1967 FGHC-83

L01443.1

Kinney et al. (1989)

IC

INH-9813

Human

1995 Vero-3

KP282671.1

Yu et al. (2015)

ID

ZPC738

sentinel hamster

1997 BHK-1

AF100566.1

Anishchenko et al. (2004)

Group A

McMillan

Human brain

1941 Mouse-2, SMB-1, Vero-2 GQ287640.1

Group B

Imperial 181

Culex tarsalis 2005 Vero-2

Group B

Montana-64

Horse brain

1967 DE-2

GQ287643.1

Group A

Fleming

Human

1938 SM-5, Vero-3

MN477208.1 Burke et al. (2020)

Lineage

Strain

Source

Year

L10

Aedes abnormalis

NA

FL91-4679

NA

Passage History

Logue et al. (2009) Logue et al. (2009) Logue et al. (2009)

SM ¼ suckling mouse, SMB ¼ suckling mouse brain, WM ¼ weanling mouse, DE ¼ duck embryo cells, MRC-5 ¼ human fibroblast cell line, C3/36 ¼ A. albopictus cell line, AP61 ¼ A. pseudocutellaris cell line, GP ¼ guinea pig, BHK ¼ baby hamster kidney cell line, FGHC ¼ fetal guinea pig heart cell cultures, Unk ¼ unknown, N/A ¼ no passage history, NA ¼ North American, ECSA ¼ East/Central/South African, IOL ¼ Indian Ocean lineage, WA ¼ West African * Bold text denotes strain with existing infectious clone system.

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As mentioned above, alphavirus reverse genetics systems were established more than 30 years age (Boyer and Haenni, 1994; Levis et al., 1986; Rice et al., 1987). In addition to permitting the role of specific amino acid or nucleotide mutations in replication and infection to be tested, researchers have combined this technology with animal models in many creative ways to advance our understanding of alphavirus infection, pathogenesis and immunity. For example, to investigate the antiviral activity of specific cytokines against SINV infection in the CNS, Binder and Griffin engineered the cDNA for TNF-α or IFN-γ into the SINV genome downstream of a second subgenomic promoter, revealing a role for both cytokines in promoting or hampering the clearance of SINV infection (Binder and Griffin, 2001). Other examples include the generation of a recombinant SINV library encoding a whole genome-targeting siRNA library to identify host factors that impact SINV replication in vivo (Varble et al., 2013), a recombinant CHIKV strain engineered to express the Cre recombinase to identify cellular targets of infection following inoculation of tdTomato flox-stop reporter mice (Young et al., 2019), numerous recombinant alphaviruses engineered to encode luciferase or fluorescent proteins to track viral infection and dissemination, including in live animals (Sun et al., 2014), and the use of recombinant CHIKV and RRV strains encoding wellcharacterized CD4+ and CD8+ T cell receptor epitopes to investigate T cell responses during infection (Burrack et al., 2015; McCarthy et al., 2018).

8. Conclusions As described above, there currently exist a variety of useful animal models of alphavirus infection in both mammals and mosquitoes, however, gaps remain in our ability to understand alphavirus infection using animal models. For example, a robust small animal model of chronic arthralgia induced by the arthritogenic alphaviruses remains elusive. Enhanced understanding of human clinical disease facilitated by patient cohort studies would aid the future development of such a model. Also, commonly used strains of laboratory mice often rapidly succumb to encephalitic alphavirus infection, hindering study of chronic neurological sequelae observed in a large proportion of symptomatic human patients. Use of chimeric SINV and EEE/ VEE/WEE viruses or generation of attenuated viruses and intracranial inoculation could potentially facilitate development of a murine model of chronic neurological disease following encephalitic alphavirus infection. Moreover, reflection of the vast genetic and microbiome diversity of the

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human population is significantly limited in inbred mice, and a link between alphavirus pathogenesis and microbiome-regulated responses has been reported (Winkler et al., 2020). To overcome these issues, future work could apply alphavirus infection to more population-representative collaborative cross (CC) mice and non-laboratory “dirty” mice (Masopust et al., 2017; Noll et al., 2020; Threadgill et al., 2011). As noted, while several studies have attempted to definitively characterize natural reservoir hosts of alphaviruses in the laboratory, more extensive mechanistic studies to assess why some animals develop disease or support viremia over others are needed to better understand the ecology and host tropism that may dictate future outbreaks and spillovers. Finally, other areas of under-investigation include coinfection and comorbidity models. Alphaviruses share vector and regional similarities with other arboviruses of public health concern, and with helminth and parasitic diseases such as malaria. Moreover, patients with comorbidities such as type I and II diabetes, other autoimmune disorders, obesity, malnutrition, and cancer comprise an increasing proportion of the human population. As mentioned, some studies have been done with Plasmodium coinfection in mice and DENV-CHIKV coinfection in mosquitoes, and recently, a study comparing CHIKV, RRV, and MAYV infection in lean, obese, and malnourished mice suggested significant differences in morbidity (Teo et al., 2018; Weger-Lucarelli et al., 2019). Future studies and models should incorporate coinfections and comorbidities to enhance our understanding of their effect on alphavirus infection, pathogenesis, and immunity.

Acknowledgments We thank members of the Morrison laboratory for critical evaluation of this manuscript. This work was supported by Public Health Service grants R01 AI141436 and R01 AI148144 from the National Institute of Allergy and Infectious Diseases to T.E.M. C.J.L. was supported by an RNA Biosciences Initiative Research Scholar Award.

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CHAPTER THREE

Enteroviruses: The role of receptors in viral pathogenesis Emma Heckenberga, Justin T. Steppeb, and Carolyn B. Coynea,b,* a

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, United States Department of Pathology, Duke University School of Medicine, Durham, NC, United States ⁎ Corresponding author: e-mail address: [email protected] b

Contents 1. 2. 3. 4.

Introduction Transmission and viral life cycle Enterovirus structure Enteroviruses and their receptors 4.1 Enterovirus A 4.2 Coxsackievirus A and EV71 4.3 Enterovirus B 4.4 Coxsackievirus A9 4.5 Coxsackievirus B 4.6 Echoviruses 4.7 Enterovirus D 4.8 Enterovirus D68 4.9 Enterovirus D70 5. Enterovirus disease modeling 6. Concluding remarks References

90 92 95 96 98 98 99 99 99 100 101 102 102 103 105 106

Abstract Enteroviruses are among the most common viral infectious agents of humans and cause a broad spectrum of illness, which can range from mild and self-limiting to severe. Severe outcomes of enteroviral infections can include aseptic meningitis, bronchitis, acute liver failure, hand-foot-mouth disease (HFMD), hemorrhagic conjunctivitis, or acute flaccid myelitis and other paralytic syndromes. Enteroviruses initiate their replicative life cycles by attaching to a broad range of cell surface receptors, which play direct roles in the clinical outcomes of enteroviral infections. In this chapter, we review the transmission and viral life cycle of enteroviruses and discuss the diverse cell surface receptors that facilitate enterovirus attachment, entry, or genome release.

Advances in Virus Research, Volume 113 ISSN 0065-3527 https://doi.org/10.1016/bs.aivir.2022.09.002

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2022 Elsevier Inc. All rights reserved.

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1. Introduction Enteroviruses are positive-sense, single stranded RNA viruses belonging to the Picornaviridae family. The Picornaviridae are small (30 nm) nonenveloped viruses that infect a range of vertebrate hosts, including humans and livestock (Zell et al., 2017). An important characteristic of these viruses is their highly restricted host tropism, with most members generally only infecting a single host species (THE ENTEROVIRUSES; Committee on the Enteroviruses, 1957). Other members of the Picornaviridae family include Parechoviruses, Hepatoviruses, Cardioviruses, and at least 24 other genera of viruses (Chapter 26 - Picornaviridae, in Fenner’s Veterinary Virology, 2017). The enteroviruses discussed in this chapter naturally infect humans, but their natural infections of other species is limited, which often complicates the development of models of viral pathogenesis. Enteroviruses have an icosahedral capsid structure that allows for unique and specific interactions with their receptors (Racaniello, 1990). In addition, immunity to enteroviruses is serotype-specific, meaning immunity to one does not confer immunity to all (Pons-Salort and Grassly, 2018), which likely explains the high numbers of enterovirus infections experienced throughout the lifetime of humans. Enteroviruses are highly prevalent throughout the world, with key regional differences between species (Brouwer et al., 2021). Within the enterovirus genus there are currently several subgenera which are designated based on homology of the viral capsid protein (VP1) coding region (Oberste et al., 1999): Enterovirus A-D (EV-A-D) and Rhinovirus A-C (RV-A-C) (Fig. 1). This chapter will focus specifically on the EV-A-D subgenera. Enterovirus 71 (EV71) and Coxsackie A (CV-A) are members of the EV-A clade and are associated with Hand-Foot-andMouth-Disease (HFMD) and acute flaccid myelitis (AFM) (Bessaud et al., 2014; Hughes and Roberts, 1972). AFM can cause life-long sequelae including paralysis. The EV-B members include Echoviruses and Coxsackie B (CV-B) viruses and are the most common causes of enterovirus infections in humans, with disease outcomes ranging from mild febrile illnesses to more severe manifestations including aseptic meningitis or myocarditis (Kopecka, 1999; Tariq, 2022). Poliovirus, the most well studied enterovirus, is a member of the EV-C clade and can cause paralysis, which required the lifelong use of an iron lung to facilitate breathing (Mehndiratta et al., 2014). The most recent branch of the enterovirus family is the EV-D group, which includes Enterovirus D68 (EV-D68), and which has been more recently

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Fig. 1 Enterovirus phylogenetic tree: Phylogenetic tree depicting the different species of viruses within the enterovirus genus based on viral capsid protein (VP1) sequence homology. The species include Enteroviruses A–D (EV-A, EV-B, EV-C, EV-D) and the three Rhinovirus groups (A–C). Virus sequences were acquired from the NCBI Virus repository and the tree was generated using Geneious.

associated with upper respiratory diseases and AFM (Smura et al., 2010). However, it should be noted that although enterovirus infections can be associated with severe disease such as AFM, this is considered an uncommon outcome of infection as the incidence of AFM is likely