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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
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First edition 2022
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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
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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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Acquisitions Editor: Leticia Lima
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Typeset by STRAIVE, India
Contents
Contributors vii
1. Diversity of viral RNA silencing suppressors and their

involvement in virus-specific symptoms 1
Vitantonio Pantaleo and Chikara Masuta
1. Introduction 2
2. Principles of RNA silencing pathways in plants 3
3. Multilayer strategies of viruses for suppressing RNA silencing at the cellular
level 6
4. Concluding remarks 15
Acknowledgments 17
References 17
2. Animal models of alphavirus infection and human disease 25

Cormac J. Lucas and Thomas E. Morrison
1. Introduction 26
2. Alphavirus transmission cycles 27
3. Human clinical disease 29
4. Animal models 34
5. Animal models of human disease 41
6. Animal models of unique aspects of alphavirus infection 58
7. Virus strains used in animal models 63
8. Conclusions 67
Acknowledgments 68
References 68
3. Enteroviruses: The role of receptors in viral pathogenesis 89

Emma Heckenberg, Justin T. Steppe, and Carolyn B. Coyne
1. Introduction 90
2. Transmission and viral life cycle 92
3. Enterovirus structure 95
4. Enteroviruses and their receptors 96
5. Enterovirus disease modeling 103
References 106
v
This page intentionally left blank
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
vii
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
b
Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan
*Corresponding authors: e-mail address: vitantonio.pantaleo@cnr.it; masuta@res.agr.hokudai.ac.jp
Contents
1. Introduction 2
2. Principles of RNA silencing pathways in plants 3
2.1 Diversification of RNA silencing suppressors 4
2.2 Suppression strategies 5
3. Multilayer strategies of viruses for suppressing RNA silencing at the cellular level 6
3.1 Geminiviruses: A complete armamentarium of small and large antiviral
defense suppressors 6
3.2 Potyviruses: Multitasking HCPro 9
3.3 Tombusviruses: Different effects of p19: siRNA-binding ability and beyond 10
3.4 Cucumoviruses: Further expansion of 2b protein functionality by
post-translational modification 13
Acknowledgments 17
References 17
Abstract
RNA silencing is an evolutionarily conserved and homology-dependent gene inactiva-
tion system that regulates most biological processes at either the transcriptional or
post-transcriptional level. In plants, insects and certain mammalian systems, RNA silenc-
ing 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 Copyright # 2022 Elsevier Inc. 1

ISSN 0065-3527 All rights reserved.
https://doi.org/10.1016/bs.aivir.2022.06.001
2 Vitantonio Pantaleo and Chikara Masuta
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 knowl-
edge of the response of plants to other biotic and abiotic stressors. RSS diversity in phyl-
ogenesis, 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 func-
tionality, 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 ant-
iviral 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
Viral suppressors of RNA silencing in plants 3
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 (post-
transcriptional 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 inhi-
bition 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 sequence-
specific 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 mem-
brane invaginations, from outer membranes of cellular organelles (mito-
chondria, peroxisomes, chloroplasts, endoplasmic reticulum) that protect
their genomes from silencing (Laliberte and Sanfaçon, 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 ampli-
fied 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 pro-
cesses 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
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 sequester-
ing 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/diversi-
fication 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.
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 mono-
partite 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 ham-
pering 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
have also revealed the importance of Cajal bodies (i.e., spherical nuclear
bodies largely consisting of proteins and RNAs) in gene regulation mech-
anisms, 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 move-
ment of PTGS, respectively; both alter phythormone signaling, such as
jasmonic acid (JA) signaling and salicylic acid (SA)-dependent defenses
(Lozano-Durán 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 structur-
ally 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 immu-
nity, 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 move-
ment 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 localiza-
tions 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
among geminiviruses and encodes a 7.2-kDa Golgi-localized protein den-

oted 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 expres-
sion 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 partic-
ularly 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 con-
sequence 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 suppres-
sor. Among the 30 plant genes that AC2 transactivates there is a calmodulin-
like 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).
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 path-
ways. 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 transmis-
sion and RNA silencing suppression. Potyvirus HCPro is the first described
and best characterized RSS (Anandalakshmi et al., 1998; Kasschau and
Carrington, 1998). Burgyán’s group (Lakatos et al., 2006) revealed the
mechanism of HCPro RSS, i.e., to bind vsiRNAs and prevent their incor-
poration 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 interac-
tion 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 expres-
sion 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) infec-
tion 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 ribo-
somes. Although it was already known that ribosome-associated AGO1
can inhibit gene expression of mRNAs via translation inhibition, the study
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 transla-
tional 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 structur-
ally 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 expres-
sion 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 rev-
ealed that p19 can exhibit high preferences for endogenous sRNAs, such
as miRNAs, and significant sequence-specific differences between con-
served 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 consid-
erable variations in binding strength; i.e., miR168 exhibited weaker binding
to p19 and therefore had no influence on the competitive loading mecha-
nism model for miR168’s action to control AGO1 (Dalmadi et al., 2021).
AGO1-mRNA
miR168
DCL1-mRNA
pre-miR168
miR162
binding miRNAs
p19 preference
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 siRNA-

like 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 effici-
ent caliper of miR162 and miR403 but not of miR168; hence, the
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 subsequen-
tly 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 Sanfaçon, 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 immu-
nity (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 inde-
pendently of siRNA sequestration (Garnelo Gόmez et al., 2021) (Fig. 1).
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 function-
ality; 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 con-
taining 2b, had already been proven (Nemes et al., 2017). In addition, Kim
and collaborators (2022) identified the serine residue at position 28 (S28) as
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 phos-
phoprotein enrichment showed that CMV 2b in the nucleus undergoes
dephosphorylation and that the identified nuclear exporting signals pro-
moted 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 viru-
lence 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 experi-
ments 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.
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 tran-
siently 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
siRNA-binding RSS diversity in cellular functions

AGO-binding
DNA methylation
Load
Positive
selection SA/JA-related genes
pressure Episodic
Initial infection
Environmental changes selection
Host
adaptation Multi-layer RSSs Host jumping
Autophagy/Proteasome
Viral protein
for RSS
Simple RSS
Load Change in subcellular
localization
Translation inhibition
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.
Viral invasion Viral replication and host invasion

of the cell High viral titre
Severe sympthoms
affinity.-based Anti viral RNA silencing

Anti viral miRNA
sequestration
vsiRNAs
Very Early infection Host gene silencing and
Escaping from Specific sympthoms
Late RSS
infection sequestration
and
vsiRNA
Viral protein saturation
for RSS
miRNA
Expression 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 sequence-
specific 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 over-
come 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 regula-
tion. 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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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
b
RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, United States
*Corresponding author: e-mail address: thomas.morrison@cuanschutz.edu
Contents
1. Introduction 26
2. Alphavirus transmission cycles 27
3. Human clinical disease 29
3.1 Arthritogenic alphaviruses 29
3.2 Encephalitic Alphaviruses 32
4. Animal models 34
4.1 Natural reservoir hosts 34
4.2 Vectors 38
5. Animal models of human disease 41
5.1 Mice 41
5.2 Other rodents 52
5.3 Non-human primates 55
6. Animal models of unique aspects of alphavirus infection 58
6.1 Transmission studies 58
6.2 Co-infection studies 61
7. Virus strains used in animal models 63
8. Conclusions 67
Acknowledgments 68
References 68
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 Copyright # 2022 Elsevier Inc. 25

ISSN 0065-3527 All rights reserved.
https://doi.org/10.1016/bs.aivir.2022.07.001
26 Cormac J. Lucas and Thomas E. Morrison
Abbreviations
BBB blood-brain barrier
BFV Barmah Forest virus
CHIKV chikungunya virus
CNS central nervous system
CSF cerebrospinal fluid
DENV dengue virus
ECSA East Central South African
EEEV Eastern equine encephalitis virus
IHC immunohistochemistry
IOL Indian Ocean lineage
MADV Madariaga virus
MAYV Mayaro virus
MBL mannose-binding lectin
NHP non-human primate
NSV neuronal-adapted Sindbis virus
ONNV o’nyong ‘nyong virus
PFU plaque-forming unit
PPI pre-pulse inhibition
RNAi RNA interference
RRV Ross River virus
SESV Southern elephant seal virus
SFV Semliki Forest virus
SG salivary gland
SGE salivary gland extract
SINV Sindbis virus
SPDV salmon pancreatic disease virus
spp. species
VEEV Venezuelan equine encephalitis virus
WEEV Western equine encephalitis virus
ZIKV Zika virus
μCT microcomputed tomographic
1. Introduction
The re-emergence and geographic expansion of mosquito-
transmitted 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 repli-
cation, 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
Animal models of alphavirus infection and human disease 27
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 patho-
genesis and protective immunity. Alphaviruses most commonly associated
with human disease are transmitted in cycles between a variety of mosqui-
toes 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 pancre-
atic 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 mus-
culoskeletal 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
Reunion epidemic and numerous subsequent outbreaks demonstrated that
CHIKV could be efficiently transmitted to humans by A. albopictus mosqui-
toes (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.
Nevertheless, the current consensus is that RRV and BFV are predominantly
transmitted between Aedes spp. or Culex annulirostris mosquitoes and marsu-
pials (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 partici-
pate 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 devel-
opment as biowarfare agents (Croddy et al., 2002).
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.
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 inflam-
mation that can be debilitating. In addition, atypical presentations have been
observed, including encephalitis in the young and the aged and cardiovas-
cular symptoms (Bonifay et al., 2018; Cotella et al., 2021; Gerardin 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; Gerardin et al., 2016).
3.1.2 Pathology
In general, there is a paucity of information regarding tissue pathology asso-
ciated 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, mononu-
clear infiltrates were detected in connective tissue and the synovial sheath
surrounding tendons in the hand (Sharp et al., 2021). The limited knowl-
edge 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, detect-
able 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 individ-
ual (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).
3.1.3 Virology
Arthritogenic alphavirus infection of humans often results in detectable vire-
mia (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 mono-
cytes 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 multi-
ple 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.
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 iden-
tified 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 diar-
rhea (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 <1% for VEEV, 3–7% for
WEEV, and 50–70% for EEEV (Weaver et al., 1996). In addition, it has been
reported that abortions and fetal deaths occurred in pregnant women
infected with VEEV, and the virus was recovered from the brains of aborted
or stillborn fetuses (Franck and Johnson, 1970). Notably, long-term neuro-
logical sequelae, including convulsions, seizures, somnolence, and visual
disturbances can occur in survivors (4–14% for VEEV; 15–30% for
WEEV; 50–90% for EEEV) of encephalitic alphavirus infection (Ronca
et al., 2016).
3.2.2 Pathology
Characterization of the pathology that occurs during human infection with
encephalitic alphaviruses remains limited. In some neurological cases of
EEEV and VEEV, pleocytosis in the cerebrospinal fluid has been detected
(Deresiewicz et al., 1997; Soto et al., 2022). Autopsies of severe, fatal cases
of VEEV infection revealed cerebral edema, intracerebral hemorrhage,

perivascular infiltrates (with some extension into the adjacent parenchyma)
in the brain (i.e., encephalitis), mild infiltration of the leptomeninges
(i.e., meningitis), and lymphoid depletion and follicular necrosis in the
spleen and lymph nodes (Bastian et al., 1975; Lury and Castillo, 2004).
Examination of the brain in acute fatal cases of EEEV revealed focal enceph-
alomyelitis, characterized by perivascular cuffing, polymorphonuclear and
lymphocytic cellular infiltrates in the brain parenchyma, and focal areas of
neuronal cell death in the hippocampus, basal ganglia, pons, and frontal
and occipital lobes (Fig. 3) (de la Monte et al., 1985; Reddy et al., 2008;
Winter, 1956). MRI analysis of patients with EEEV-induced neurological
symptoms revealed focal lesions in the basal ganglia and thalami, brain stem,
cortex, and periventricular white matter (Deresiewicz et al., 1997; Lury and
Castillo, 2004; Winter, 1956).
Fig. 3 Viral and pathologic features during encephalitic alphavirus infection. Cerebral
edema, neuronal necrosis, and CNS lesions are observed during acute infection with
EEEV, VEEV, and WEEV with polymorphonuclear and lymphocytic cellular infiltrates in
the brain parenchyma and spinal cord meninges. Viral RNA and antigen are detectable
in the brain while infectious virus has been recovered from cerebrospinal fluid. Viremia
also occur, especially with VEEV infection. Viral and pathologic features of chronic
infection are understudied. This figure was made using Biorender.com.
3.2.3 Virology
There are limited virological data from human infections with encephalitic
alphaviruses. During acute VEEV infection, viremia of up to 102–106
PFU/mL has been detected (Fig. 3), with peak virus titers occurring the
day after the onset of symptoms (Bowen and Calisher, 1976; Franck and
Johnson, 1970; Scherer et al., 1972). Infectious VEEV also has been detected
in throat swabs and the CSF of patients with acute VEE (Fig. 3) (Bastian
et al., 1975; Bowen and Calisher, 1976; Franck and Johnson, 1970;
Scherer et al., 1972; Soto et al., 2022). In general, EEEV and WEEV are
undetectable in the blood at the time of neurological symptom onset,
possibly due to a lengthy prodrome. However, EEEV has been isolated
on rare occasions from the blood of human patients (Clarke, 1961). In
the brain of a fatal case, EEEV antigen was detected in the cell body and den-
drites of neurons (Garen et al., 1999). In addition, EEEV RNA was detected
in the brain and CSF from a fatal case in a person receiving B cell depleting
rituximab therapy (Hughes et al., 2021).
4. Animal models
4.1 Natural reservoir hosts
4.1.1 Natural reservoir hosts
Understanding alphavirus infection of natural reservoir species can shed light
on the determinants of human clinical disease described above, how geo-
graphic differences in the biology of these species influence viral transmis-
sion, and the potential for enzootic alphaviruses to spillover and become
epizootic. Alphavirus vertebrate reservoirs are species from which virus
can be isolated, demonstrate high levels of virus-specific antibodies in
seroprevalence surveys, and support high viremia under experimental labo-
ratory infection. However, these species must also be sufficiently abundant
that their presence increases the incidence of human disease (Kuno and
Chang, 2005; Stephenson et al., 2018). These parameters should be
supported by additional ecological and behavioral data, as some species that
fulfill these criteria may have minor roles in transmission in certain regions.
For example, a recent investigation of the role of monkeys in the sylvatic
transmission cycle of CHIKV noted that infant monkeys were positive
for CHIKV exposure even when virus could not be detected in native mos-
quito populations and when monkey herd immunity was high, leading the
authors to suggest, that at least in Senegal, monkeys serve as amplification
Fig. 4 Utility of alphavirus natural reservoir host and vector models. Species of rodents
(cotton rats, spiny rats), birds (wading birds, passerine birds), and equines can be exper-
imentally infected with alphaviruses to assess disease, viremia, and transmission poten-
tial. Similarly, experimental infection of mosquito species (Aedes spp., Anopheles spp.,
Culex spp., Culiseta spp.) reveals how alphavirus infection affects fecundity, vector
competence, and insect antiviral responses. This figure was made using Biorender.com
hosts of CHIKV as opposed to true reservoir species (Althouse et al.,

2018). Several excellent reviews have been published that summarize
alphavirus vector and reservoir species (Claflin and Webb, 2015; Pezzi
et al., 2020; Stephenson et al., 2018). For the purposes of this review, we
focus on animal models directly studied as putative reservoir species
(Fig. 4). Because non-human primates have been used extensively as
models of alphavirus-induced human disease (see Section 5.3), this section
primarily discusses studies investigating non-primate reservoirs of alphavirus
maintenance and amplification.
4.1.2 Rodents
Historical evidence indicates that viruses in the VEEV complex are
maintained naturally in rodent populations native to Central and South
America, specifically species of spiny and cotton rats. This evidence includes
infection of these rodents in nature, high rates of immunity in wild-caught
rodents, and the ability of these rodents to support viremia following exper-
imental virus inoculation in laboratory studies (Carrara et al., 2005; Carrara
et al., 2007; Coffey et al., 2004; Johnson and Martin, 1974). Laboratory
infection of spiny rats (Proechimys spp.) with the enzootic VEEV strains
Co97-0054, a sympatric strain to the Colombian forest in which the spiny
rats of this study originated, and 66,637, an allopatric strain from Venezuela,
resulted in the development of viremia and later seroconversion despite a
lack of detectable disease (Carrara et al., 2005), supporting the idea that
these rodents are reservoir hosts. However, it was not evaluated whether
the viremia was of sufficient magnitude to infect a mosquito during a blood
meal. Additional studies in cotton rats with the enzootic VEEV subtypes
ID (strain Co97-0054) and IE (strain 68 U201) similarly observed detectable
viremia, seroconversion, and little to no signs of encephalitis, fever, or
malaise (Carrara et al., 2007; Coffey et al., 2004), although disease appears
to be dependent on VEEV subtype, as infection with a Texas epizootic IB
strain was associated with illness and death in cotton rats (Howard, 1974).
Studies in cotton rats also have emphasized the importance of geographical
differences in reservoir populations as infection of cotton rats from
VEEV-naı̈ve areas with both enzootic and epizootic strains of VEEV
resulted in variable development of viremia, inconsistent antibody produc-
tion, and VEEV strain-dependent mortality (Carrara et al., 2007; Coffey
et al., 2004; Deardorff et al., 2009). Remarkably, infection of VEEV-
naı̈ve cotton rats collected from VEEV-endemic areas resulted in little to
no disease despite the development of viremia (Coffey et al., 2004,
Deardorff et al., 2009), suggesting allopatric speciation of both virus strains
and rat populations over time. Expanding beyond cotton rats, Deardorff
et al. collected multiple rodent species from the Chiapas state of Mexico
consisting of two species of mice and three species of rats, including cotton
rats, and infected them with VEEV MX01-22, a VEE-IE strain isolated from
Chiapas in 2001 and genetically similar to the virulent strains isolated
from equines during the previous two Chiapas outbreaks (Deardorff
et al., 2009). Strikingly, only Baiomys musculus (southern pygmy mouse)
uniformly developed encephalitic disease and succumbed to infection while
the other four rodent species developed viremia at levels considered capable
of infecting Culex spp. mosquitoes without any signs of disease; the authors
suggested that these data support the hypothesis that circulating VEEV
selects for disease resistance in wild rodents (Deardorff et al., 2009). In addi-
tion to VEEV, it is thought that South American (SA) EEEV strains (now
known as MADV) exist in a natural cycle between Culex mosquitoes and
rodents, although a study in which cotton rats and house sparrows were
experimentally infected with a North American (NA) EEEV strain
(FL93-939), or two different MADV strains (PE70 and CO2) resulted in
productive infection in both species for all 3 strains, albeit reduced survival
of house sparrows upon infection with FL93-939 (Arrigo et al., 2010a;
Arrigo et al., 2010b). Slight differences in peak viremic titers between cotton
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vivere. Venivano dalle cose che erano vive e ritornavano a muoversi,
cose che erano state come morte, in letargo, durante i lunghi mesi di
gelo. La linfa saliva su per i pini. Dai salci e dalle tremule
sbocciavano giovani gemme; i cespugli e le viti selvatiche si
rivestivano di verde; i grilli cantavano, la notte; e esseri striscianti e
rampicanti, d’ogni genere, uscivano, con infiniti fruscii, al sole.
Pernici e picchi risuonavano e picchiettavano nella foresta; gli
scoiattoli, cianciavano, gli uccelli cantavano, e sopra il capo
s’udivano le anitre selvatiche che venivano dal Sud disposte in abili
stormi a cuneo, che tagliavano l’aria.
Da ogni pendice giungeva il mormorìo d’acque correnti, la musica
d’invisibili fontane. Tutte le cose sgelavano, si piegavano, s’aprivano.
Il Yukon si sforzava di rompere il ghiaccio che lo teneva fermo,
rodendolo di sotto; mentre il sole rodeva di sopra. Si formavano dei
fori d’aria, si aprivano e s’allargavano fessure, mentre sezioni sottili
di ghiaccio cadevano intere nel fiume. E in mezzo a tutto questo
aprirsi, sbocciare e palpitare di vita che si risvegliava, sotto il sole
fiammeggiante e al dolce sospiro delle brezze, come viandanti della
morte, barcollavano i due uomini, la donna e gli huskies.
Coi cani cadenti e Mercede che piangeva sulla slitta, e Rico che
bestemmiava, e gli occhi di Carlo terribilmente acquosi, essi
entrarono barcollando nell’accampamento di Giovanni Thornton, alla
foce del fiume Bianco. Quando si fermarono, i cani caddero come
fulminati. Mercede s’asciugò gli occhi e guardò Giovanni Thornton.
Carlo si sedette a riposare s’un tronco d’albero: sedette molto
lentamente e dolorando, a causa del suo grande indolenzimento.
Rico parlò anche per gli altri. Giovanni Thornton stava dando gli
ultimi colpi di coltello ad un manico d’ascia che aveva fatto con un
pezzo di betulla. Tagliava e ascoltava, e rispondeva con monosillabi,
dando, solo quando era richiesto, chiari e concisi consigli.
Conosceva quella razza di gente, e dava consiglio con la sicurezza
che non sarebbe stato seguito.
— Ci hanno detto lassù che il fondo avrebbe ceduto, e che la miglior
cosa per noi era attendere, — disse Rico, in risposta
all’ammonimento di Thornton di non tornare ad esporsi a pericoli sul
cattivo ghiaccio. — Ci hanno detto che non avremmo potuto
raggiungere il fiume Bianco, ed eccoci qui. — Quest’ultime parole le
pronunciò in tono di dileggio trionfante.
— Vi hanno detto la verità, — rispose Giovanni Thornton, — Il fondo
può cedere ad ogni momento. Soltanto dei pazzi, assistiti dalla cieca
fortuna dei pazzi, possono essere giunti fin qui. Vi dico, francamente,
che non rischierei la mia carcassa su quel ghiaccio, per tutto l’oro
dell’Alaska.
— Forse perchè non siete un pazzo, — disse Rico. — Ciò
nonostante, noi proseguiremo per Dawson. — E agitò la frusta. —
Su, là, Buck! Ih! ih! Su, là! Avanti!
Thornton continuò a levigare il suo manico. Era tempo perso, lo
sapeva, interporsi tra un pazzo e la sua pazzia; mentre due o tre
pazzi o stupidi di più o di meno non avrebbero mutato la faccia del
mondo.
Ma i cani non si alzarono al comando. Da lungo tempo ormai erano
ridotti in uno stato tale, che a smuoverli e a rialzarli erano necessari
dei colpi. La frusta fischiò qua e là, senza pietà. Giovanni Thornton
strinse le labbra. Sol-leks fu il primo a levarsi a fatica in piedi. Segui
Teek. Poi venne Joe, che ululava dalla pena. Pike fece degli sforzi
penosi. Due volte cadde, quand’era già mezzo in piedi, e al terzo
sforzo riuscì a levarsi. Buck non fece alcun sforzo. Giaceva tranquillo
dove era caduto. La frusta lo colpì ripetutamente, ma egli nè mugolò
nè si mosse. Parecchie volte Thornton fece l’atto di voler parlare, ma
poi mutò idea. Mentre gli occhi gli s’inumidivano e le sferzate
continuavano, egli si alzò e si mise a camminare in su e in giù,
irrisoluto.
Quella era la prima volta che Buck mancava, ragione sufficiente per
far diventare furioso Rico. Egli lasciò la frusta e prese la mazza.
Buck rifiutò di alzarsi anche sotto la scarica di pesanti colpi che ora
s’abbattevano su lui. Come i suoi compagni, egli aveva appena la
forza sufficiente per alzarsi, ma, contrariamente a loro, aveva deciso
di non alzarsi. Egli aveva la vaga sensazione del destino che gli
sovrastava. Questa sensazione era stata fortissima in lui dacchè era
entrato nel banco, e non l’aveva più lasciato. Il ghiaccio sottile e
cattivo che sentiva sotto le zampe, tutto il giorno, pareva che gli
annunciasse un disastro vicino, e fuori, sul ghiaccio dove il suo
padrone cercava di trascinarlo. Rifiutò di muoversi. Egli aveva
talmente sofferto, ed era in così cattive condizioni, che i colpi non gli
facevano ormai molto male. Mentre essi continuavano a cadere su di
lui, quella scintilla di vita che gli rimaneva, vacillò e si spense. Egli
era quasi finito: si sentiva stranamente intorpidito. Quei colpi gli
arrivavano come da una lunga distanza. Le ultime sensazioni di
pena erano scomparse. Egli non sentiva più nulla, tranne, come
attutito, l’urto della mazza sul suo corpo, anzi su quello che non
pareva più il suo corpo; tanto era lontano.
Ma, improvvisamente, in modo inatteso, emettendo un grido
inarticolato simile al grido di un animale, Giovanni Thornton balzò
sull’uomo che maneggiava la mazza, e Rico fu scaraventato indietro,
come colpito dalla caduta di un albero. E mentre Mercede strillava,
Carlo guardava pensosamente asciugandosi gli occhi pieni d’acqua,
immoto, a causa del suo indolenzimento.
Giovanni Thornton stette davanti a Buck, facendo sforzi per
dominarsi, troppo agitato dalla collera per parlare.
— Se voi colpite ancora una volta questo cane, vi uccido, — riuscì a
dire alla fine, con voce rauca.
— È il mio cane, — rispose Rico, asciugandosi il sangue dalla bocca
mentre ritornava sui suoi passi. — Levatevi d’innanzi, o vi accomodo
io. Vado a Dawson.
Thornton stava in piedi tra lui e Buck, e non mostrava alcuna
intenzione di togliersi di là. Rico tirò fuori il suo lungo coltello da
caccia, mentre Mercede strillava, piangeva, rideva, in preda a una
crisi d’isterismo. Thornton battè le nocche della mano di Rico col
manico dell’ascia, facendo cadere il coltello a terra; e quando l’altro
cercò di raccogliere l’arma, picchiò, nuovamente; poi s’abbassò,
prese il coltello, e con due colpi tagliò i tiranti di Buck.
Rico non aveva più modo di lottare, ma aveva le mani, o, piuttosto,
le braccia piene di sua sorella; mentre Buck era quasi finito e non
poteva servire pel tiro della slitta. Alcuni minuti dopo uscirono dal
banco, e scesero giù per il fiume. Buck li udì andarsene e alzò la
testa a guardare.
Pike conduceva, Sol-leks era al timone, e tra loro erano Joe e Teek.
Zoppicavano e barcollavano. Mercede era adagiata sul carico della
slitta. Rico guidava, al timone, e Carlo veniva dietro zoppicando.
Mentre Buck li guardava, Thornton s’inginocchiava accanto al cane e
con ruvide mani gentili, tastando, cercava di accertarsi se vi fosse
qualche osso spezzato. Quando ebbe accertato che il solo male,
erano le molte ammaccature e uno stato di terribile sfinimento per
fame, la slitta era già a un quarto di miglio lontana. Il cane e l’uomo
stettero a guardarla mentre strisciava sul ghiaccio.
Improvvisamente videro sprofondare l’estremità posteriore, come in
un alto solco, e la stanga, con Rico che la teneva afferrata, agitarsi
nell’aria, mentre giungeva ai loro orecchi uno strido di Mercede.
Videro Carlo voltarsi e fare un passo per correre indietro, ma in quel
momento un’intera sezione del ghiaccio cedette, e cani e creature
umane scomparvero. S’era spalancato come un baratro, sotto il
ghiaccio, che aveva ceduto al peso della slitta.
Giovanni Thornton e Buck si guardarono.
— Povero diavolo! — fece Giovanni Thornton, e Buck gli leccò la
mano.
CAPITOLO VI.
PER L’AMORE DI UN UOMO.
Quando Giovanni Thornton ebbe un piede gelato, nel precedente

dicembre, i soci l’accomodarono bene, e lasciatolo perchè guarisse,
andarono lungo il fiume a preparare una zattera di tronchi segati, per
Dawson. Egli zoppicava ancora un po’ quando salvò Buck, ma
poichè la buona stagione continuava, guarì e cessò di zoppicare. E
lì, accovacciato sulla sponda del fiume, durante i lunghi giorni di
primavera, guardando l’acqua corrente, ascoltando pigramente i
canti degli uccelli e il ronzìo della natura, Buck riguadagnò
lentamente le sue forze.
Un riposo è veramente salutare, quando si è viaggiato per tremila
miglia; infatti bisogna confessare che Buck diveniva pigro a mano a
mano che le ferite gli si guarivano, e i muscoli gli si gonfiavano e la
carne ricominciava a coprire le sue ossa. In verità, erano tutti
placidamente in ozio, Giovanni Thornton, e Skeet e Nig, in attesa
della zattera che li portasse sino a Dawson. Skeet, ch’era un piccolo
setter irlandese, fece subito amicizia con Buck, il quale, in condizioni
d’agonia, era incapace di risentire le prime offerte del cane. Quella
bestiola aveva capacità mediche, che alcuni cani possiedono; e
come una gatta fa con i micini, essa lavorava e puliva le ferite di
Buck. Regolarmente, ogni mattina dopo ch’egli aveva finito il primo
pasto, essa sbrigava quel compito prefissosi, tanto ch’egli finì per
attendere le cure di essa, come attendeva quelle di Thornton. Nig,
egualmente amichevole, benchè meno espansivo, era un immenso
cane nero, mezzo segugio e mezzo cerviero, con occhi che
ridevano, e illimitato buonumore.
Con sorpresa di Buck, quei cani non manifestavano alcuna gelosia
verso di lui. Sembravano condividere la bontà e generosità di
Giovanni Thornton. A mano a mano che Buck diveniva forte, essi
l’allettavano ad ogni sorta di giochi ridicoli, ai quali non poteva fare a
meno di partecipare anche Thornton; e in questa maniera Buck
passò dalla convalescenza ad una nuova esistenza. Per la prima
volta, egli aveva amore, amore genuino e appassionato. Questo
sentimento egli non l’aveva mai provato nella casa del giudice Miller,
giù nella soleggiata Valle di Santa Clara. Con i figli del giudice,
andando alla caccia o a camminare, egli aveva stretto una specie di
società; con i nipoti del giudice, esercitato una specie di pomposa
tutela; con lo stesso giudice aveva una salda e dignitosa amicizia.
Ma amore, che fosse febbre e bruciore, adorazione, pazzia, non
l’aveva provato: c’era voluto Giovanni Thornton a suscitarlo.
Quest’uomo gli aveva non solo salvata la vita, — il che aveva la sua
importanza, — ma, si dimostrava, inoltre un padrone ideale. Mentre
altri curavano il benessere dei loro cani per un senso di dovere e per
necessità degli affari, egli curava il benessere dei suoi come se
questi fossero suoi figlioli; perchè non poteva fare altrimenti. Ed
andava oltre. Egli non dimenticava mai una buona accoglienza o una
parola di incoraggiamento, e sedeva a conversare a lungo o a
«cianciare», — come egli diceva — con gran divertimento suo e dei
cani. Egli aveva un modo tutto suo di prendere rudemente la testa di
Buck tra le mani, e di posare la sua testa su quella di Buck,
scrollandolo in avanti e indietro, chiamandolo con cattivi nomi che
per Buck erano nomi d’amore. Buck non conosceva gioia più grande
di quel rude abbracciamento e di quelle finte male parole borbottate,
e a ciascuna scrollata in avanti e indietro, sembrava che il cuore gli
balzasse fuori del corpo, tanto era grande la sua estasi. E quando
egli, lasciato libero, balzava in piedi, con la bocca ridente, gli occhi
espressivi, la gola vibrante per suoni non pronunciati e rimaneva,
così, immobile, Giovanni Thornton esclamava con ammirazione:
— Dio! ti manca solo la parola!
Buck aveva una forma di esprimere l’amore, che commoveva sino a
far male al cuore. Egli afferrava spesso la mano di Thornton in bocca
e chiudeva i denti così furiosamente da lasciarne le tracce sin molto
tempo dopo. E come Buck capiva che le male parole erano parole
d’amore, così l’uomo capiva che quel finto morso era una carezza.
Di solito, però, l’amore di Buck era espresso con l’adorazione.
Quando Thornton lo toccava o gli parlava, egli impazziva di gioia, ma
non mendicava queste prove di affezione. Diversamente da Skeet,
che doveva fregare il naso sotto la mano di Thornton e spingere e
spingere per essere accarezzato, o da Nig, che avanzava a grandi
passi e posava la sua grossa testa sul ginocchio di Thornton, Buck
era contento di adorare a distanza. S’adagiava per delle ore ai piedi
di Thornton, ansioso e attento, guardandolo in volto, esaminandolo,
studiandolo, seguendo col più vivo interesse la più fuggevole
espressione, ogni movimento di lineamento. O, come voleva il caso,
giaceva lontano, di fianco o accosciato, osservando il contorno della
figura dell’uomo o i movimenti del suo corpo. E spesso, tale era la
comunione in cui essi vivevano, che lo sguardo di Buck faceva
volgere il capo a Thornton, il quale contraccambiava lo sguardo,
senza parlare, col cuore che gli luceva negli occhi, come luceva il
cuore negli occhi di Buck.
Dopo che era stato salvato, Buck non perdeva mai Thornton di vista.
Dal momento che questi lasciava la tenda al momento in cui vi
rientrava, Buck gli era alle calcagna. Poichè i suoi transitorii padroni
gli avevano messo nel cuore il timore che nessun padrone fosse
durevole, aveva paura che Thornton svanisse dalla sua vita come
Perrault e François e il meticcio scozzese. Persino di notte nei suoi
sogni, era perseguitato da questa paura. Allora si scuoteva dal
sonno e andava, nel freddo, davanti la tenda, e si fermava ed
ascoltava il suono del respiro del suo padrone.
Ma nonostante questo suo grande amore per Giovanni Thornton,
che sembrava ricordare il morbido influsso della civiltà, la natura
primitiva che la terra nordica aveva ridestata in lui, rimaneva viva ed
attiva. Con la fedeltà e la devozione, sentimenti nati dal focolare e
dal tetto, egli conservava anche la selvatichezza e l’astuzia. Era una
creatura di natura selvaggia, venuta dalla selva ad accovacciarsi al
fuoco di Giovanni Thornton, anzichè un cane della mite terra del
Sud, con i segni di generazioni civili. Il suo grande amore gli
impediva di rubare a quell’uomo, ma di fronte a qualunque altro
uomo in qualsiasi altro accampamento, egli non avrebbe esitato un
momento; e con la furberìa e destrezza che usava, avrebbe evitato
d’essere preso.
Poichè la sua faccia e il suo corpo erano segnati dai denti di molti
cani, ora avrebbe combattuto più furiosamente che mai, ma con
maggior astuzia. Skeet e Nig erano troppo di buonumore per litigare,
eppoi, appartenevano a Giovanni Thornton; ma i cani estranei, di
qualunque razza e per quanto valorosi fossero, avrebbero
riconosciuto rapidamente la superiorità di Buck e si sarebbero trovati
a lottare per la vita con un terribile antagonista. E Buck era senza
pietà. Aveva imparato bene la legge della mazza e del dente, e mai
si lasciava fuggire un vantaggio o indietreggiava di fronte a un
nemico col quale avesse iniziato un combattimento mortale.
Aveva avuta la sua lezione da Spitz e dai principali cani nella lotta di
supremazia per la disciplina e pel corriere, e sapeva che non vi era
via di mezzo. Doveva o dominare o essere dominato; mostrare pietà
era una debolezza. La pietà non esisteva nella vita primordiale: era
considerata paura; e tale malinteso conduceva alla morte.
Uccidere o essere uccisi, mangiare o essere mangiati, era la legge;
e a questo comandamento, sorto dalle profondità del Tempo, egli
obbediva.
Egli era più vecchio dei giorni che aveva visti e dei respiri che aveva
emessi: ricongiungeva il passato al presente, e il senso dell’eternità
gli palpitava dentro con un possente ritmo al quale egli obbediva
come ubbidivano le maree e le stagioni. Egli stava accovacciato
accanto al fuoco di Giovanni Thornton, col suo petto largo, di cane
dai denti bianchi e dal pelo lungo; ma sapeva che dietro di lui erano
le ombre d’ogni genere di cani mezzi lupi e lupi selvaggi, che lo
insidiavano e spingevano, che gustavano il sapore della carne
ch’egli mangiava, assetati dall’acqua che egli beveva, fiutando con
lui il vento, ascoltando con lui e svelandogli i suoni della vita
selvaggia della foresta, dettandogli i suoi umori, dirigendo le sue
azioni, adagiandosi a dormire con lui e sognando con lui e oltre lui,
divenendo essi stessi la materia dei suoi sogni.
Imperativamente, quelle ombre lo chiamavano, ogni giorno più, a
mano a mano che il ricordo del genere umano e dei diritti del genere
umano s’allontanava da lui. Ogni volta che risuonava profondo dalla
foresta un appello, ed egli, udiva quell’appello, misteriosamente
attraente e vibrante, si sentiva obbligato a volgere le spalle al fuoco
e alla terra battuta intorno a lui, e a immergersi nel profondo della
foresta, procedendo senza sapere dove e perchè; senza
domandarselo neppure, giacchè l’appello risuonava con tono
imperativo e profondo nella foresta. Ma quando era pervenuto alla
morbida terra non tocca e all’ombra verde, l’amore per Giovanni
Thornton lo riconduceva al fuoco.
Soltanto Thornton lo teneva; il resto del genere umano non esisteva
agli occhi suoi. Viaggiatori passanti per caso potevano lodarlo o
accarezzarlo; ma egli rimaneva freddo ad ogni premura; e se
qualcuno si mostrava troppo espansivo con lui, egli si alzava e se ne
andava. Quando i soci di Thornton, Hans e Piero, arrivarono sulla
zattera tanto a lungo attesa, Buck non volle accorgersi di loro, finchè
non capì ch’erano cari a Thornton: dopo, li tollerava in una maniera
passiva, accettando favori da essi come se fosse egli a favorirli
accettandoli. Ed essi ch’erano semplici e generosi come Thornton,
perchè vivevano vicini alla terra, e pensavano semplicemente e
vedevano chiaramente, prima che spingessero la zattera nel largo
turbine vicino alla segheria di Dawson, avevano già imparato a
capire Buck e i suoi modi, e non insistevano per avere un’intimità
quale avevano ottenuta da Skeet e Nig.
Ma l’amore di Buck per Thornton, pareva crescere sempre più.
Questi solo, fra gli uomini, poteva porre un fardello sul dorso di Buck,
nel viaggio estivo. Nulla era troppo gravoso per Buck quando
Thornton comandava. Un giorno, (s’erano vettovagliati con quello
che avevano ricavato dalla zattera ed avevano lasciato Dawson per
le sorgenti del Tanana) gli uomini e i cani si trovavano a riposare
sulla cresta di una roccia che scendeva a picco su un letto di nude
rocce giacenti trecento piedi sotto. Giovanni Thornton sedeva vicino
al margine della roccia, Buck alle sue spalle. Thornton fu preso da
uno sconsiderato capriccio, e invitò Hans e Piero ad un esperimento
che aveva in mente.
— Salta, Buck!, — comandò egli, agitando il braccio sul precipizio.
Un istante dopo, egli era alle prese con Buck sull’estremità dell’orlo,
mentre Hans e Piero li tiravano indietro a salvamento.
— È straordinario, — disse Piero, dopo che il pericolo fu passato ed
ebbero ricuperato il respiro.
Thornton scrollò il capo.
— No, — disse, — è magnifico; ed è terribile pure. Sapete che
talvolta, mi fa paura?
Non vorrei essere l’uomo che vi mettesse le mani addosso,
quand’egli è vicino, — disse Piero, accennando col capo a Buck.
— Per Giove! — aggiunse Hans. — Neppur io.
A Circle City, alla fine dell’anno, le apprensioni di Piero divennero
realtà. Burton, il «nero», un uomo cattivo e falso, aveva preso a
litigare con un ingenuo al bar, e Thornton s’interpose bonariamente.
Buck, stava come soleva, accovacciato in un angolo, la testa sulle
zampe, osservando tutte le azioni del suo padrone. Burton, senza
che l’altro se l’aspettasse, diede un gran pugno a Thornton.
Thornton girò su se stesso ed evitò di cadere a terra aggrappandosi
alla barra del bar.
I presenti alla scena udirono non un abbaiamento o un ululato, ma
un ringhio che parve un vero e proprio ruggito, e videro il corpo di
Buck volare in aria, dal pavimento alla gola di Burton.
L’uomo si salvò avanzando istintivamente le braccia, ma fu
rovesciato per terra, con Buck sopra. Buck liberò i denti dalla carne
del braccio e cercò di afferrare nuovamente alla gola l’uomo, il quale
riuscì a coprirsi solo in parte, ed ebbe la gola squarciata. Allora la
folla ai gettò su Buck, che fu trascinato via; ma mentre un chirurgo
cercava di arrestare il sangue, Buck girava, su e in giù, ringhiando
furiosamente, e tentando di lanciarsi dentro, ricacciato solo da una
schiera di mazze ostili. Un’«assemblea di minatori» chiamata sul
luogo, giudicò che il cane aveva avuto una sufficiente provocazione,
e Buck fu liberato. Ma la sua reputazione era fatta; da quel giorno, il
suo nome si sparse per tutti gli accampamenti dell’Alaska.
In seguito, alla fine dell’anno, egli salvò la vita di Giovanni Thornton
in maniera del tutto differente. I tre soci tiravano una lunga e stretta
barca, con la pertica, lungo un cattivo tratto di corrente, sul Forty-
Mile Creek. Hans e Piero camminavano lungo la riva, trattenendo la
barca con una sottile corda manìla che avvolgevano di albero in
albero, mentre Thornton, rimasto sulla barca ne aiutava la discesa
per mezzo di una pertica, e gridando ordini alla riva. Buck, sulla riva,
preoccupato e ansioso, si teneva alla stessa altezza della barca,
senza mai distogliere gli occhi dal padrone.
In un punto, particolarmente cattivo, dove una catena di nudi scogli
appena sommersi sporgeva sul fiume, Hans passò la corda sopra,
rallentandola, e, mentre Thornton spingeva con la pertica la barca
fuori nella corrente, corse giù lungo la riva col capo della corda in
mano per trattenere la barca dopo sorpassati gli scogli. La barca
passò gli scogli, trascinata precipitosamente dalla corrente rapida
come una gora, allorchè Hans tirò la corda troppo bruscamente. La
barca ondeggiò, sobbalzò e si capovolse contro la sponda, mentre
Thornton lanciato dalla barca, era trasportato dalla corrente verso la
parte peggiore, un tratto di corrente furiosa nella quale nessun
nuotatore avrebbe potuto salvarsi.
Buck, che s’era gettato in acqua subito, a trecento metri raggiunse
Thornton. Quando sentì che il padrone gli s’era afferrato alla coda,
Buck si diresse alla riva, nuotando con tutto il suo meraviglioso
vigore. Ma il progresso verso la riva era lento; il progresso verso la
corrente incredibilmente rapido. Da sotto giungeva il fatale rombare
del punto dove la corrente diveniva più furiosa, rotta in contorti
stracci spumosi dalle rocce che tagliavano il fiume come i denti di un
enorme pettine. Il risucchio dell’acqua, là, dove incominciava l’ultima
pendenza, era terribile, e Thornton capì che sarebbe stato
impossibile raggiungere la riva. Passò furiosamente sopra una
roccia, si ferì contro la seconda punta e sbattè con terribile violenza
contro una terza. Allora s’afferrò a una punta sdrucciolevole, con
tutte due le mani, e liberando Buck, sopra il frastuono delle acque
agitate, gridò:
— Va’, Buck! Va’!
Buck non riuscì a fermarsi: trasportato dalla corrente, lottava
disperatamente, incapace di ritornare indietro. Quando udì il
comando di Thornton, ripetuto, si sollevò in parte fuori dell’acqua,
come per dare un ultimo sguardo, poi si volse, obbediente, verso la
riva, e nuotò poderosamente, sinchè non fu tratto in salvo da Piero e
da Hans, proprio al punto dove nuotare diveniva impossibile e la
distruzione era certa.
Essi sapevano che un uomo può tenersi afferrato ad una roccia
sdrucciolevole nel mezzo di una simile corrente, solo per pochi
minuti, e corsero quanto più rapidamente poterono su per la riva ad
un punto molto più su di quello dove si teneva afferrato Thornton.
Attaccarono la corda, con la quale avevano trattenuto la barca, al
collo e alle spalle di Buck, curando che non lo strangolasse nè
gl’impedisse di nuotare, e lo lanciarono nella corrente. Egli si mise a
nuotare poderosamente, ma non abbastanza diritto nella corrente.
S’accorse dello sbaglio troppo tardi, quando Thornton gli era quasi di
fronte, soltanto a cinque o sei colpi di distanza, mentre egli era
trasportato senza speranza oltre.
Hans, prontamente, tirò la corda, come se Buck fosse una barca. La
corda gli si strinse addosso, nel punto più forte della corrente, e il
cane fu sommerso, e sommerso rimase finchè il suo corpo non battè
contro la riva e fu tirato su. Era mezzo annegato, e Hans e Piero si
gettarono su lui facendolo respirare artificialmente, e facendogli
ributtare l’acqua. Il cane barcollò per rialzarsi, ma ricadde; ma il
debole suono della voce di Thornton giunse sino a loro, e benchè
essi non potessero intendere le parole, compresero che egli era agli
estremi. La voce del padrone agì su Buck come una scossa elettrica.
Il cane balzò in piedi e corse su per la riva precedendo gli uomini
sino al punto dov’era partito prima. Nuovamente fu attaccata la
corda e lanciato, e nuovamente egli nuotò, ma questa volta diritto
nella corrente. Aveva mal calcolato la prima volta, ma non avrebbe
sbagliato la seconda.
Hans mollava la corda, ma senza permettere allentamenti, mentre
Piero la teneva libera da nodi. Buck continuò a nuotare finchè fu in
linea retta sopra Thornton; poi si volse, e con la velocità di un treno
espresso piombò su lui. Thornton lo vide arrivare, e mentre Buck lo
colpiva come un montone che desse di cozzo, con tutta la forza della
corrente dietro, si sollevò sulla roccia e si afferrò con tutt’e due le
braccia al collo irsuto. Hans attorcigliò la corda ad un albero, e Buck
e Thornton furono sbattuti sott’acqua. Quasi soffocati, l’uno talvolta
sopra, talvolta sotto l’altro, trascinati sul fondo roccioso e ineguale,
sbattuti contro rocce e tronchi d’albero sommersi, raggiunsero la
riva.
Thornton ritornò in sè con la pancia in giù, violentemente spinto
innanzi e indietro, su un tronco portato dalla corrente, da Hans e
Piero. Il suo primo sguardo fu per Buck, sul cui corpo floscio e
apparentemente senza vita Nig urlava, mentre Skeet leccava il muso
bagnato e gli occhi chiusi del cane. Thornton, che pure era ferito e
ammaccato, esaminò accuratamente il corpo di Buck richiamato in
vita, e trovò tre costole spezzate.
— Questo fatto decide, — annunciò egli. — Ci accamperemo qui
dove siamo. — E s’accamparono, finchè le costole di Buck non
furono salde ed egli potè viaggiare.
Quell’inverno, a Dawson, Buck compì un’altra prodezza, non così
eroica, forse, ma tale da porre il suo nome di molte tacche sul palo
della fama, in Alaska. Questa prodezza fu particolarmente
vantaggiosa per i tre uomini; poichè essi avevano bisogno
dell’equipaggiamento ch’essa fornì, e poterono così fare un viaggio
da lungo tempo desiderato, nel lontano vergine oriente, dove non
erano ancora apparsi dei minatori. Il fatto ebbe origine da una
conversazione nella Birreria Eldorado, nella quale gli uomini
vantavano con orgoglio i loro cani favoriti. Buck, a causa della sua
fama, era la mira di quegli uomini, e Thornton era spinto
gagliardamente a difenderlo. Dopo una mezz’ora, un uomo affermò
che il suo cane poteva smuovere una slitta con un peso di
cinquecento libbre sopra, e tirarla; un secondo vantò che il proprio
cane ne poteva tirare seicento; e un terzo, settecento.
— Puf! puf! — fece Giovanni Thornton; — Buck può smuovere mille
libbre.
— E trarle dal ghiaccio? e tirarle per cento metri? — domandò
Matthewson, Re di Bonanza, un riccone, quello che aveva vantato le
settecento libbre come prodezza del suo cane.
— E rompere il ghiaccio, intorno, e tirarle per cento metri, — ripetè
Thornton, freddamente.
— Ebbene, — disse Matthewson, lentamente e deliberatamente, in
modo che lutti potessero udire, — scommetto mille dollari che non
può farlo. Ed eccoli qui. — Così dicendo, sbattè sul banco un
sacchetto di polvere d’oro delle dimensioni di una mortadella di
Bologna.
Nessuno parlava. Il bluff di Thornton, se era un bluff, era posto alla
prova. Egli sentì un’ondata di sangue caldo salirgli al volto. La lingua
l’aveva compromesso: giacchè non sapeva se Buck potesse tirare
mille libbre: mezza tonnellata! L’enormità della cosa lo spaventava.
Egli aveva una grande fiducia nella forza di Buck, ed aveva spesso
pensato che il cane fosse capace di tirare un simile carico; ma mai,
come ora, egli ne aveva considerato la possibilità, con gli occhi di
una dozzina di uomini fissi su lui, in attesa silenziosa. Inoltre, egli
non aveva mille dollari; nè li aveva Hans o Piero.
— Ho una slitta qui fuori, ora, con venti sacchi da cinquanta libbre di
farina, — continuò Matthewson con brutale sfida: — perciò non vi
preoccupate delle difficoltà.
Thornton non rispose: non sapeva che cosa dire. Guardava ora una
faccia ora un’altra, come un uomo distratto che abbia perduto la
forza di pensare, e cerchi in qualche luogo un oggetto che gli
richiami il pensiero. La faccia di Nino O’ Brien, un Re dei Mastodonti,
altro riccone, fermò i suoi occhi. Fu per lui come un lampo, che
sembrava spingerlo a fare quello che non avrebbe mai sognato di
fare.
— Puoi prestarmi mille dollari? — domandò, quasi mormorando.
— Certo, — rispose O’ Brien, gettando un sacchetto rigonfio accanto
a quello di Matthewson. — Benchè abbia pochissima fiducia,
Giovanni, che il cane possa compiere una tal prodezza.
Tutti quelli che si trovavano nell’Eldorado uscirono sulla strada per
vedere la prova. I tavoli divennero deserti, perchè i giocatori e quelli
che tenevano il banco uscirono a vedere il risultato della sfida e a far
scommesse. Parecchie centinaia di uomini impellicciati e con
manopole circondarono la slitta, tenendosi a poca distanza da essa.
La slitta di Matthewson, carica di mille libbre di farina era rimasta lì
ferma per un paio d’ore, e nel freddo intenso, (erano sessanta gradi
sotto zero) gli strisci s’erano gelati sulla neve battuta. Degli uomini
scommettevano, offrendo il doppio della posta, che Buck non
sarebbe riuscito a smuovere la slitta. Sorse un cavillo sul significato
della frase «liberare». O’ Brien asseriva che spettava a Thornton
liberare gli strisci dal ghiaccio, lasciando a Buck il compito di
trascinare la slitta dal peso morto; Matthewson insistette sostenendo
che la parola comprendeva anche il compito del cane di liberare gli
strisci dalla presa della neve gelata. La maggioranza di quelli che
avevano assistito alla scommessa decisero in suo favore, e allora le
scommesse salirono da tre ad uno contro Buck. Non vi era nessuno
che scommettesse in favore di Buck. Nessuno lo credeva capace di
quella prodezza. Thornton, ch’era stato spinto a scommettere, pieno
di dubbi, ed ora vedeva la slitta, il fatto concreto, con il tiro regolare
di dieci cani arrotolati nella neve davanti ad essa, sentiva ancora più
impossibile quel compito.
Matthewson si pavoneggiava, giubilante.
— Tre contro uno, — proclamò. — Metto giù altri mille dollari, a tre
contro uno, Thornton. Che ne dite?
Il dubbio pareva scritto sul volto di Thornton, ma lo spirito di lotta era
ormai desto, — lo spirito combattivo che s’eleva al disopra delle
scommesse, non riconosce l’impossibile, ed è sordo a tutto, tranne
al clamore della battaglia. Egli chiamò a sè Piero e Hans. Ma i loro
sacchi erano smilzi: col suo, i tre soci non potevano mettere insieme,
più di duecento dollari. Nella bassa marea delle loro fortune, quella
somma era tutto il loro capitale; tuttavia essi lo arrischiarono, senza
esitare, contro i seicento dollari di Matthewson.
Fu tolto l’attacco dei dieci cani, e Buck, col suo finimento e i suoi
tiranti, fu posto alla slitta. Egli aveva preso il contagio
dell’eccitamento generale, e sentiva di dovere rendere un gran
servizio a Giovanni Thornton. Si levarono mormorii di ammirazione,
per lo splendido aspetto dell’animale. Era in perfette condizioni,
senza un’oncia di carne superflua; formando le centocinquanta libbre
ch’egli pesava, altrettante libbre di risoluta energia. Il suo pelo luceva
come seta. Giù per il collo e attraverso le spalle, il suo manto, in
riposo com’egli era, mezzo irsuto, pareva sollevarsi ad ogni
movimento, come se l’eccesso di vigore rendesse vivo ed attivo ogni
pelo. Il largo petto e le pesanti gambe davanti erano proporzionate al
rimanente del corpo, dove i muscoli apparivano come saldi rotoli
sotto la pelle. Qualcuno palpò quei muscoli e li proclamò duri quanto
il ferro, e le scommesse scesero a due contro uno.
— Perdio, signore! Perdio, signore! — balbettò un membro
dell’ultima dinastia, un re delle Skookum Benches. — Vi offro
ottocento dollari per il cane, prima della prova, signore; ottocento
com’è.
Thornton scrollò il capo e andò al fianco di Buck.
— Dovete stare lontano dal cane, — protestò Matthewson. — Gioco
onesto e spazio in abbondanza.
La folla divenne silenziosa: soltanto si potevano udire le voci dei
giocatori che offrivano in vano, due contro uno. Tutti riconoscevano
in Buck un magnifico animale, ma venti sacchi da cinquanta libbre di
farina apparivano troppo grossi, ai loro occhi, per aprire i cordoni
della borsa.
Thornton s’inginocchiò accanto a Buck. Gli prese la testa fra le mani
e appoggiò la guancia alla guancia del cane. Non lo scosse
scherzosamente, come faceva volentieri; nè mormorò dolci male
parole d’amore; ma gli mormorò all’orecchio: — Come tu mi ami,
Buck. Come tu mi ami, — e Buck gemette con frenata ansia.
La folla guardava incuriosita. La faccenda si faceva misteriosa.
Sembrava come una congiura. Mentre Thornton s’alzava in piedi,
Buck afferrò la mano ricoperta dalla manopola tra le mascelle,
stringendola tra i denti e lasciandola andare lentamente, mezzo
riluttante. Era la risposta, non con parole, ma con segni d’amore.
Thornton si tirò bene indietro.
— A te, Buck, — diss’egli.
Buck tese i tiranti, poi li rallentò, per alcuni pollici; come aveva
imparato.
— Va! — risuonò la voce di Thornton, tagliente, nel silenzio perfetto.
Buck girò a destra, con un movimento che finì con un balzo che tese
i tiranti, e fermò, dopo una forte scossa, le centocinquanta libbre del
cane. Il carico tremò, e dagli strisci s’alzò un leggero crepitìo.
— A sinistra! — comandò Thornton.
Buck duplicò la manovra, questa volta a sinistra. Lo scricchiolìo si
mutò in un brusco frangersi del ghiaccio, la slitta girò leggermente su
se stessa, e gli strisci scivolarono graffiando la neve. La slitta era
liberata. Tutti trattenevano il respiro, intensamente, inconsci del fatto.
— Ora, avanti!
Il comando di Thornton risuonò come un colpo di pistola. Buck si
gettò in avanti, tendendo i tiranti, con sbalzi a scosse. L’intero corpo
era raccolto strettamente in sè nel tremendo sforzo, i muscoli si
gonfiavano e contorcevano come delle cose vive sotto il pelame di
seta. Il suo largo petto era proteso e abbassato sino a terra, la testa
in avanti, mentre le zampe gli si muovevano furiose, e gli unghioni
scavavano la neve battuta, in solchi paralleli. La slitta oscillava e
tremava, quasi smossa. Uno dei piedi del cane sdrucciolò, e un
uomo gemette rumorosamente. Poi la slitta si mosse, con un
succedersi rapido di scosse, benchè, in realtà non si fosse fermata
più... Mezzo pollice... un pollice... due pollici... Le scosse diminuirono
percettibilmente; a mano a mano che la slitta avanzava, Buck
cessava le scosse, finchè alla fine la slitta filò, senza oscillare.
Gli uomini mandarono un gran sospiro e ricominciarono a respirare;
chè senz’accorgersene, avevano cessato per un momento di
respirare. Thornton correva dietro la slitta incoraggiando Buck con
brevi parole liete. La distanza era stata misurata prima, e mentre il
cane s’avvicinava alla pila di legna da ardere, che segnava il termine
dei cento metri, incominciava a levarsi un plauso sempre più alto,
che divenne clamore di urli, allorchè, oltrepassata la legna da
ardere, il cane si fermò, ad un comando. Tutti gli uomini esultavano
pazzamente; persino Matthewson. Gettavano in aria cappelli e
manopole; scambiavano strette di mano col più vicino, chiunque
fosse, vociando, come in una confusionaria babele.
Ma Thornton cadde in ginocchio accanto a Buck. Con la testa centro
la testa, lo scrollava in avanti e in dietro. Quelli ch’erano corsi dietro
a lui l’udirono che malediceva Buck, e lo malediceva a lungo e con
fervore, ma dolcemente e amorosamente.
— Perdio, signore! Perdio, signore! — balbettò rauco il re di
Skookum Bench — Vi dò mille dollari per lui, mille, signore, mille e
duecento, signore.
Thornton s’alzò in piedi: aveva gli occhi bagnati. Le lagrime gli
rigavano liberamente e copiosamente le gote.
— Signore, — disse al re di Skookum Bench, — no, signore. Potete
andare al diavolo, signore. Non ho altro da dirvi e da fare.
Buck afferrò tra i denti la mano di Thornton, che lo scrollò avanti e
indietro. Come animati da comune impulso, gli spettatori si ritirarono
a rispettosa distanza; abbastanza discreti per interromperli
nuovamente.
CAPITOLO VII.
IL RICHIAMO DELLA VOCE.
Buck, facendo guadagnare mille seicento dollari in cinque minuti a

Giovanni Thornton, rese possibile al suo padrone di pagare alcuni
debiti che aveva e di partire con i suoi soci verso oriente, alla ricerca
di una favolosa miniera, la cui storia era vecchia quanto la storia del
paese. Molti uomini l’avevano cercata; pochi l’avevano trovata; e
molti non erano più ritornati dalla ricerca. Questa perduta miniera era
immersa nella tragedia e avvolta nel mistero. Nessuno sapeva chi
fosse stato il primo a scoprirla. La più antica tradizione finiva prima
della scoperta di essa. Si diceva che a principio era un’antica e
diroccata capanna.
Uomini sul punto di morire avevano giurato affermando l’esistenza
della capanna, e della miniera della quale la capanna segnava il
luogo, ribadendo la loro testimonianza con mostrar pepite ch’erano
diverse, per qualità, da qualsiasi tipo d’oro conosciuto nel Nord.
Ma nessun essere vivente aveva potuto saccheggiare quel tesoro, e
i morti erano morti; perciò Giovanni Thornton e Piero e Hans, con
Buck e una mezza dozzina d’altri cani, s’avventurarono nell’oriente
lungo un cammino sconosciuto, per ottenere ciò che uomini e cani
capaci come loro non avevano ottenuto. Essi risalirono in slitta, per
sessanta miglia, lo Yukon, volsero a destra nello Stewart River,
passarono il Mayo e il Mac Question, e continuarono, finchè lo
stesso Stewart non divenne un ruscello, varcando gli alti picchi che
segnano la spina dorsale del continente.

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1. Diversity of viral RNA silencing suppressors and their

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3. Enteroviruses: The role of receptors in viral pathogenesis 89

Diversity of viral RNA silencing

Advances in Virus Research, Volume 113 Copyright # 2022 Elsevier Inc. 1

by viral proteins; these can be facilitated by different proteins in different

2. Principles of RNA silencing pathways in plants

their genomes from silencing (Laliberte and Sanfaçon, 2010). Otherwise,

2.1 Diversification of RNA silencing suppressors

2.2 Suppression strategies

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among geminiviruses and encodes a 7.2-kDa Golgi-localized protein den-

3.2 Potyviruses: Multitasking HCPro

3.3 Tombusviruses: Different effects of p19: siRNA-binding

cell to cell movement of 21-nt vsiRNAs

In contrast, miR162 and the miR403 isoforms displayed a high siRNA-

formation of functional AGO1/RISC containing miRNA 162 or miR403

3.4 Cucumoviruses: Further expansion of 2b protein

an additional motif of phosphorylation. Importantly, S28 is located close to

siRNA-binding RSS diversity in cellular functions

Viral invasion Viral replication and host invasion

affinity.-based Anti viral RNA silencing

Animal models of alphavirus

Advances in Virus Research, Volume 113 Copyright # 2022 Elsevier Inc. 25

human alphavirus infection and thus, the development and characterization

2. Alphavirus transmission cycles

3. Human clinical disease

3.1 Arthritogenic alphaviruses

3.2 Encephalitic Alphaviruses

of VEEV infection revealed cerebral edema, intracerebral hemorrhage,

hosts of CHIKV as opposed to true reservoir species (Althouse et al.,

Quando Giovanni Thornton ebbe un piede gelato, nel precedente

Buck, facendo guadagnare mille seicento dollari in cinque minuti a

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