1 s2 - 0 S0065352722000197 Main

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
rats and house sparrows during MADV infection and the absence of disease
in mature cotton rats support the idea that cotton rats may be a MADV
reservoir host. However, the capacity of both species to support viremia
at a level above the minimum infectious dose for Culex spp. mosquitoes
has implications for future epizootic outbreaks (Arrigo et al., 2010a).
These experimental virus infections of putative reservoir species are critical
for the interpretation of existing seroprevalence studies and identification of
natural reservoirs for alphaviruses. Additionally, further examination of
the viral life cycle and host response in these models could provide insight
into the differences both in host response and viral replication during infec-
tions that are or are not associated with the development of disease.
4.1.3 Birds
Based on seroprevalence studies, EEEV is maintained in cycles between
Culiseta spp. mosquitoes and both aquatic and passerine birds (Armstrong
and Andreadis, 2022; Burkett-Cadena et al., 2022). Two studies in the
1990s captured EEEV-naı̈ve snowy egrets and glossy ibises and infected
them subcutaneously with EEEV to assess viremia and disease development.
The investigators found that the majority of birds survived infection but
developed both viremia and a strong virus-specific antibody response
(McLean et al., 1995; Mitchell et al., 1993). Further supporting the capabil-
ity of snowy egrets to function as a EEEV reservoir host and providing
insight into transmission dynamics, subcutaneous infection of egrets with
EEEV produced a sufficient viremia for transmission to A. albopictus mosqui-
toes during a blood meal (Mitchell et al., 1993). As A. albopictus mosquitoes
are highly anthropophilic, their potential competence for EEEV transmis-
sion is a cause for concern (Gomes Ade et al., 2005).
4.1.4 Horses
As amplifying hosts for EEEV, VEEV, and WEEV, equines are a useful
model for assessing pathogenicity and identifying viral and ecological deter-
minants of human disease outbreaks, however, due to their large size and
cost, equines are limited in their use as an animal model. Subcutaneous
inoculation of horses, aged 1–12 years, with VEEV isolated from equine
blood and passaged 13 times in guinea pigs and 2 times in chick embryos
caused fatal disease. The disease was characterized by viremia, a febrile phase,
destruction of lymphoid tissue architecture, severe disruption of the vascu-
lature in the brain, liver, and pancreas, and in some cases, encephalitis
(Kissling et al., 1956). Virological analysis highlighted a distinction between
VEEV and the other equine encephalitic viruses EEEV and WEEV, as
VEEV reached titers in the blood that were sufficient to infect A. triseriatus
mosquitoes during a blood meal (Kissling et al., 1956). Subcutaneous
infection of horses with the epidemic (epizootic) VEEV strains 71-180
(American) or Trinidad (TrD) also consistently caused fever and leukopenia,
with all VEEV 71-180-infected horses and a majority of VEEV TrD-
infected horses succumbing to infection. Moreover, liquefactive necrosis
and hemorrhage were described in the cerebral cortex, findings the authors
postulate as features that could be used to differentiate VEE from other
encephalitic disease in horses (Monlux and Luedke, 1973). Epizootic strains
of VEEV are highly pathogenic in horses whereas enzootic strains are gen-
erally avirulent, and infection with enzootic subtype ID strains protected
horses from challenge with epizootic strain P676 (Oberste et al., 1998;
Wang et al., 2001). However, infection of ponies with the enzootic
VEEV-IE NVSL 93-42124 strain (Chiapas, Mexico) did cause significant
neurologic disease (Sahu et al., 2003). These findings were consistent with
the two equine outbreaks in Oaxaca and Chiapas, Mexico during the 1990s
attributed to a VEEV-IE strain (Oberste et al., 1998). The high titer viremia
observed in VEEV-infected horses is associated with outbreaks of human
disease as equines are thought to serve as amplification rather than dead
end hosts (Gonzalez-Salazar et al., 2003). Experimental studies have empha-
sized this idea as (1) infection of horses with VEEV strains from the 1992
and 1996 Mexican outbreaks resulted in little to no disease and undetectable
viremia, suggesting the limited duration of those outbreaks was due to
lack of equine amplification, and (2) mutations in the viral envelope glyco-
proteins that differ between epizootic (IAB and IC) and enzootic (ID) strains
are sufficient to confer virulence and high viremia in horses (Anishchenko
et al., 2006; Gonzalez-Salazar et al., 2003; Greene et al., 2005b; Wang et al.,
2001). Ultimately, equine models of VEEV disease can be highly informa-
tive in understanding viral and ecological factors leading to outbreaks of
human disease.
4.2 Vectors
Mosquitoes. As described above, alphaviruses are transmitted by mosquito
vectors, including A. aegypti, A. albopictus, A. vigilax, A. procax, C. annulirostris,
Anopheles gambiae, A. funestus, and Ochlerotatus taeniorhynchus, and study of
viral infection in these different mosquito species is vital to elucidate trans-
mission mechanisms and for the development of vector control measures
(Kain et al., 2021; Pezzi et al., 2020; Weaver and Barrett, 2004). Experimental
infection of mosquitoes has been used to investigate vector competence,
virus-vector interactions, vector antiviral immunity, and horizontal and
vertical transmission (Fig. 4) (Huang et al., 2019). Here, we present a
few examples of studies that use vector models to answer these questions
and highlight some key issues associated with experimental mosquito
research.
Vector competence studies examine the capacity of a female mosquito to
support viral replication, viral dissemination and infection of the salivary
glands, and viral transmission to vertebrate hosts upon feeding. For example,
while Aedes spp. are not generally considered primary vectors of EEEV, the
isolation of EEEV from A. albopictus and its opportunistic feeding behavior
led one group to investigate vector competence of A. albopictus mosquitoes
for EEEV. Chicks were infected with EEEV FL91-4679 prior to being fed
upon by four different strains of A. albopictus mosquitoes (Turell et al., 1994).
All four mosquito strains were successfully infected and at least 40% trans-
mitted EEEV to naı̈ve chicks, suggesting that A. albopictus mosquitoes could
serve as a vector for EEEV FL91-4679.
When a mosquito ingests a blood meal containing an alphavirus, the
virus replicates in the midgut epithelial cells before disseminating through-
out the mosquito via the hemolymph. It then infects and replicates in the
salivary glands, from which the virus can be transmitted during a subsequent
blood meal (Cheng et al., 2016; Franz et al., 2015). To model this process,
female mosquitoes are fed a viremic blood meal, usually through membra-
nous material to simulate the vertebrate skin barrier. Midgut tissue, salivary
glands, and whole mosquitoes can then be analyzed for gene expression
and viral replication via a variety of methods (Liu and Cheng, 2016).
Viral tropism for midgut epithelial cells is evident, as ingestion of a blood-
meal containing a GFP-expressing SINV by A. aegypti mosquitoes resulted
in virus infection of enteroendocrine cells. These cells have important roles
in the transit of vesicles from the apical to basolateral side of the intestinal
epithelium, suggesting a connection between this tropism and viral dissem-
ination (Ahearn et al., 2020). Multiple studies have identified a critical role
for the insect antiviral pathway, RNA interference (RNAi), in restricting
infection of these intestinal midgut cells and replication in the salivary glands
of the mosquito. For example, abrogation of the RNAi pathway via knock-
down of the Aa-dcr2 gene enhances SINV dissemination, and expression of
RNAi suppressor factors from recombinant SINV and ONNV reduces
mosquito survival upon infection (Blair and Olson, 2014; Cheng et al.,
2016; Khoo et al., 2010; Myles et al., 2008). The role of RNAi in restricting
mosquito infection is consistent across alphaviruses, with CHIKV, RRV,
and MAYV all inducing expression of piwi-RNA and miRINAs in
A. aegypti, and knockdown of the Ago2 gene enhancing viral replication
in both midgut and head tissues as well as viral dissemination (Alto et al.,
2020; McFarlane et al., 2014; Sinclair and Asgari, 2020). Importantly, these
studies suggest RNAi is the primary antiviral defense against alphaviruses in
A. aegypti mosquitoes (Alto et al., 2020; McFarlane et al., 2014). Viral infec-
tion also is not benign in mosquitoes and comes with a fitness cost, as
A. aegypti fecundity was reduced by 30–50% upon infection with MAYV
regardless of the geographic origin of the mosquito strain (Sucupira et al.,
2020). However, despite A. aegypti being susceptible to MAYV and com-
petent for transmission under laboratory conditions, knowledge of the
immune response to MAYV infection in its enzootic vector Haemogogus
janthinomys remains lacking.
Several virological tools have been developed to elucidate viral determi-
nants of fitness in the mosquito. The TE/30 2 J double subgenomic SINV
virus is a useful way of introducing stably expressed genes into mosquito cell
lines but cannot efficiently infect midgut epithelial cells in the mosquito
(Frolov et al., 1996; Hahn et al., 1992; Higgs et al., 1993). Seabaugh
et al. developed a chimeric cDNA clone, MRE1001, containing sequences
from both the TE/30 2 J virus and the MRE16 SINV isolate that efficiently
infects mosquitoes, and they showed that MRE1001 virus infects midgut
cells and disseminates in at least 90% of mosquitoes, providing a useful tool
for identifying viral determinants of midgut infection and dissemination
(Seabaugh et al., 1998). Subsequently, substitution of E2 residues 95 and
96 or 116 to 119 from MRE16 to TE/30 2 J significantly enhanced infection
of midgut epithelial cells in A. aegypti mosquitoes, emphasizing the utility of
this mosquito infection model for understanding viral determinants of
infection (Pierro et al., 2008). Currently, there are alphavirus infection
models for A. aegypti and A. albopictus, yet there remains a critical need to
develop mosquito models using enzootic vectors such as Haemogogus spp.,
Culiseta melanura, other Aedes spp., and Culex spp. given their roles in both
maintenance and spillover of alphaviruses into urban cycles (Celone et al.,
2021; Pezzi et al., 2020; Stephenson et al., 2018). In addition, while
ONNV is the only alphavirus to infect and transmit via Anopheles mosqui-
toes, further study of viral determinants of infection of Anopheles gambiae
across different alphaviruses is essential given regional overlap between
malaria and alphaviruses like CHIKV and MAYV (Pezzi et al., 2020).
For example, two studies reported contradictory findings regarding the

molecular determinants of vector specificity for ONNV and CHIKV; the
latter normally does not infect Anopheles gambiae mosquitoes. In one study,
replacement of the CHIKV structural ORF with the ONNV structural
ORF was required to confer infection of Anopheles gambiae. In contrast, a
separate study found that substitution of the CHIKV nsP3 gene with
the ONNV nsP3 gene was sufficient (Saxton-Shaw et al., 2013;
Vanlandingham et al., 2005; Vanlandingham et al., 2006).
Another example of the utility of experimental mosquito infections is elu-
cidating mechanisms of vertical (transovarial) transmission of alphaviruses.
Transovarial transmission of dengue virus, a flavivirus, has been demonstrated
in A. aegypti through at least the F5 generation, and the alphaviruses CHIKV
and RRV have been isolated from wild caught male Aedes mosquitoes,
suggesting that vertical transmission of alphaviruses occurs in nature (Hardy
et al., 1983; Le Goff et al., 2011; Ratsitorahina et al., 2008; Rohani et al.,
2008; Rosen et al., 1983; Thavara et al., 2009). Infection of CHIKV-naı̈ve
female A. albopictus and A. aegypti mosquitoes originally caught in Thailand
with an Indian Ocean Lineage (IOL) strain of CHIKV revealed increasing
levels of infectious CHIKV in F2 to F4 generations that then declined in
the F5 and F6 generations, presenting experimental evidence of transovarial
transmission of CHIKV (Chompoosri et al., 2016). These data were in con-
trast to a previous study that failed to detect transovarial CHIKV transmission
in A. albopictus mosquitoes (Vazeille et al., 2009).
Finally, there are key issues associated with research in mosquito models,
including use of laboratory reared strains versus field collected experimental
subjects, the choice of species, and even regional population differences
within a species. Laboratory propagated strains can develop adaptations to
certain conditions that differentiate them from endemic mosquito
populations, propagation can lead to loss of genetic markers, and as noted
earlier, it is critical to evaluate virus-vector interactions in the mosquito spe-
cies involved in transmission. Gallichotte et al. present an excellent summary
on the potential use of cryopreservation to circumvent these limitations
(Gallichotte et al., 2021).
5. Animal models of human disease

5.1 Mice
The inbred laboratory mouse, Mus musculus, has been highly useful for inves-
tigating alphavirus pathogenesis (Fig. 5) and we point the readers to several
Fig. 5 Utility of alphavirus human disease models. Infection of laboratory mice (inbred
and outbred), rats, guinea pigs, hamsters, and non-human primates is used to model
alphavirus-induced human disease. Studies in these models enhance an understanding
of viral and immunological factors associated with viral replication, viremia, morbidity,
mortality, inflammation, clearance of infection, and long-term protection. Subsequently,
these models support testing of novel antiviral and immunomodulatory therapeutics
and vaccine strategies. This figure was made using Biorender.com
excellent recent reviews (Atkins and Sheahan, 2016; Haese et al., 2016;
Kafai et al., 2022; Ng, 2017; Steele and Twenhafel, 2010; Taylor et al.,
2015). In this section, we summarize existing inbred laboratory mouse
models of arthritogenic alphavirus infection as well as newer models for
re-emerging alphaviruses such as MAYV, introduce a humanized mouse
model of mosquito-transmitted CHIKV infection, and discuss common
murine models of encephalitic alphavirus infection. We note that many
studies, too numerous to review here, have exploited the genetic tractability
(e.g., gene knockouts, conditional gene knockouts, transgenics, etc.) of
inbred mouse models to define the role of specific cell types, pathways,
and genes in alphavirus infection, pathogenesis, and immunity, such as
the type I IFN system (Couderc et al., 2008; Grieder and Vogel, 1999;
Hwang et al., 1995; Rudd et al., 2012; Schilte et al., 2012; Winkler
et al., 2020). Additionally, pre-treatment of immunocompetent WT mice
prior to virus inoculation with a monoclonal antibody against IFNAR1,
the receptor for type I IFN, can be used as a model of lethal alphavirus
infection, particularly for the arthritogenic alphaviruses that are generally
less virulent in mice compared with the encephalitic alphaviruses.
Although this immunosuppression-based approach has limitations for path-
ogenesis studies, this system can be employed to rigorously test the protec-
tive capacity of antiviral therapeutics and vaccines against a lethal virus
challenge (Earnest et al., 2019; Earnest et al., 2021). Finally, it also is impor-
tant to note that the outcomes of both arthritogenic and encephalitic
alphavirus infections of laboratory mice are highly age-dependent, as youn-
ger mice are more susceptible to infection, exhibit more efficient viral
dissemination, and develop more severe signs of disease (Labrada et al.,
2002; Ryman et al., 2007; Taylor et al., 2015).
CHIKV. Traditional laboratory mouse models have been used for the
investigation of virological and immunological mechanisms of acute
CHIKV arthritic disease, providing key insight into risk factors and potential
therapeutic targets for human infection that otherwise are difficult to deter-
mine given the rarity of human joint tissue sampling and the sporadic nature
of CHIKV outbreaks. Subcutaneous inoculation of CHIKV-LR2006, an
IOL strain (Tsetsarkin et al., 2006), into the foot of 6-week-old WT
C57BL/6 mice results in the development of viremia, swelling of the inoc-
ulated foot, and inflammation in joint and muscle tissue (Gardner et al.,
2010). Histopathological analysis identified subcutaneous edema in the
footpad, arthritis (including mononuclear cell infiltrates and disruption of
the synovial membrane), tenosynovitis (including inflammatory cell infil-
trates surrounding connective tissue), and severe muscle necrosis (Gardner
et al., 2010). These findings were consistent with those observed following
subcutaneous inoculation of 14-day old WT C57BL/6 mice with 100 PFU
of the IOL strain CHIKV SL15649. This study performed virological and
histopathological analysis through 21 days post infection and found that viral
RNA persisted in the left and right ankle tissue and skeletal muscle tissue
displayed signs of both regeneration and a fibrous response (Morrison
et al., 2011). These models provide tools to evaluate signs of human
disease in immunocompetent adolescent to adult mice during arthritogenic
alphavirus infection. However, there remains a need for an immunocompe-
tent adult mouse model that recapitulates the persistent arthralgia associated
with chronic CHIKV disease in humans (Cunha et al., 2020; Hoarau et al.,
2010; McCarthy et al., 2019; Taylor et al., 2015). Further evaluation and
modification of the model described by Morrison et al., including the use
of 4 week-old WT C57BL/6 mice, resulted in the detection of significant
levels of CHIKV RNA and mild synovitis in murine joint tissue through
16 weeks post inoculation, suggesting this model may be useful for evalu-
ating some aspects of chronic CHIKV disease (Hawman et al., 2013).
Furthermore, infection of Rag1/ C57BL/6 mice, which lack mature B
and T cells (Mombaerts et al., 1992), resulted in more severe synovitis
and tenosynovitis through at least 6 weeks post infection, consistent with
increased levels of viral RNA in tissues compared with WT mice
(Hawman et al., 2013). Studies in humans have linked the early appearance
of neutralizing IgG3 antibodies to less severe CHIKV infection and a lower
incidence of chronic symptoms (Kam et al., 2012), emphasizing that this
genetically tractable C57BL/6 mouse model may be informative for eluci-
dating factors that lead to chronic disease. Although immunocompetent WT
C57BL/6 mice develop mild chronic synovitis, they do not develop the
long term clinical signs of disease in multiple joints that are characteristic
of chronic CHIKV disease in humans. Nevertheless, microcomputed tomo-
graphic (μCT) analysis of CHIKV-infected mice revealed there is reduced
bone volume in the joints and lesions characterized by periostitis, periosteal
bone proliferation around the metatarsal bones, and cartilage necrosis (Chen
et al., 2015; Goupil et al., 2016). The reduced bone volume is associated
with a high ratio of RANKL/OPG in joint tissue, which also is observed
in human patients, and can be alleviated if mice are pretreated with bindarit,
a small molecule inhibitor of monocyte chemoattractant protein synthesis
(MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7) (Chen et al.,
2015). CHIKV SL15649 infection of aged (18 months) C57BL/6 mice
causes an increased duration of viremia, enhanced early footpad swelling,
and joint tissue pathology at later timepoints compared with 12-week-old
adult mice (Uhrlaub et al., 2016). This study in aged mice also presented
evidence of persistence of replication competent virus in joint tissue
(Uhrlaub et al., 2016). Infection of adult BALB/c mice with CHIKV is
similar to C57BL/6, with mice developing musculoskeletal tissue pathology
including necrotic muscle degeneration and edema (Weger-Lucarelli et al.,
2014). Outbred CD-1 and ICR mice infected with CHIKV develop leth-
argy, weight loss, and hindlimb dysfunction but only in mice <7 days old
(Ziegler et al., 2008). Regardless of mouse strain, CHIKV infection of
neonatal mice causes lethal encephalitis and is generally used as a challenge

model for testing monoclonal antibody and other antiviral therapeutics
(Chen et al., 2020; Couderc et al., 2009).
In addition to traditional laboratory mouse models, human CHIKV dis-
ease has recently been modeled using humanized mice, which may provide
insight into how human immune cells respond to CHIKV infection. Hibl
et al. used the humanized NSG mouse strain to assess infection with
CHIKV 37997 (West African lineage) either intradermally or via direct
mosquito bite. Mice infected via mosquito bite developed erythema, which
is variable during human disease but can be concomitant with high fever, lost
weight, and exhibited reduced physical activity. Histological analysis rev-
ealed that through day 25 post infection, the majority of mosquito-bitten
mice had lesions characterized by bone marrow necrosis, myositis, and ten-
donitis in the skeletal muscle and hind limb joints (Hibl et al., 2021).
Although bone marrow necrosis is likely underreported in human disease
due to the rarity of sampling, bone marrow necrosis can occur following
edema and virus-associated vasculitis, which have been seen in both human
patients and other mouse models (Chen et al., 2014; Hibl et al., 2021). These
clinical signs were only observed in mice infected with CHIKV by mosquito
bite, yet it should be noted that mice used in this study were 8 weeks old due
to the requirement for 20–80% of circulating blood to be human CD45+
cells prior to experimental use (Haese et al., 2016; Hibl et al., 2021). As
mosquito saliva has known immunomodulatory effects and alphavirus
infection of mice is highly age-dependent, as will be discussed later in this
section, the difference in clinical disease observed between mice infected
by needle and infected by mosquito bite may be the result of interplay
between age-restriction on murine disease and the immunomodulatory
effects of mosquito salivary factors (Chan et al., 2015). Nevertheless, the
development of a humanized mouse model for CHIKV infection should
spur novel discoveries of protective and pathogenic virus-host interactions.
5.1.1 Ross river virus

A murine model of the related arthritogenic alphavirus, Ross River virus
(RRV), has been well established following translation of methods used
previously in outbred and CBA/H inbred mice to inbred C57BL/6 mice
(Lidbury et al., 2000; Morrison et al., 2006). Infection of 4-week-old
WT C57BL/6 mice with 103 PFU of RRV-T48 via subcutaneous inocu-
lation of the foot results in the failure to gain weight and in hindlimb
dysfunction characterized by the loss of gripping ability, altered gait, and

hindlimb weakness that largely resolves by 30 days post infection
(Morrison et al., 2006). These mice develop severe musculoskeletal tissue
inflammation (myositis, arthritis, and tenosynovitis) characterized by mono-
nuclear cellular infiltrates. Notably, B and T cell deficient Rag1/ and B
cell deficient μMT (i.e., Ighm/) C57BL/6 mice exhibit similar tissue
inflammation and injury as WT mice (Morrison et al., 2006), suggesting
an important role for cells of the innate immune system, such as macro-
phages, in pathology. In fact, depletion of macrophages from mice prior
to RRV infection results in less severe disease signs (Lidbury et al.,
2000). Further investigation of RRV-induced myositis and arthritis
identified a pathogenic role for mannose-binding lectin (MBL)-mediated
complement activation and deposition on virus-infected cells mediated
by interaction with viral N-linked glycans (Gunn et al., 2012; Gunn
et al., 2018; Morrison et al., 2007; Morrison et al., 2008). Additionally,
RRV-infected WT C57BL/6 mice exhibit reduced bone volume associated
with a disrupted RANKL/OPG ratio similar to that observed during
CHIKV infection (Chen et al., 2014), illustrating how this model allows
identification of inflammatory mediators with potential for therapeutic
targeting in human disease.
5.1.2 Barmah forest virus

A limited number of studies have characterized murine models of BFV infec-
tion. However, comparison of RRV-T48 with BFV (strain BFV2193)
infection in 4-week-old WT C57BL/6 mice suggested that BFV is less vir-
ulent than RRV in mice, which is consistent with observations in humans
(Herrero et al., 2014). BFV-infected WT C57BL/6 mice exhibited more
moderate disease signs compared with RRV-infected mice, including del-
ayed onset of tissue inflammation, more rapid clearance of infection, less
weight loss, and lower levels of pro-inflammatory cytokines (Herrero
et al., 2014). The lower expression of MCP-1/CCL2 and IL-6 detected
in BFV-infected mice suggests a mechanism for the absence of severe mus-
culoskeletal disease compared with RRV infection, as previous studies
indicated myeloid cell recruitment and IL-6 expression are critical for
RRV-induced bone loss and joint pathology (see Section 5.1.1) (Chen
et al., 2014; Herrero et al., 2014). For evaluation of clinical disease signs,
both the RRV and BFV models have relied on investigator scoring of
hindlimb dysfunction, which can be subject to bias despite blinding and
inclusion of controls. Recently, the DigiGait system, which provides
objective video-based assessment of movement and behavior in small

animal models, was successfully used to characterize disease in RRV and
BFV-infected mice. Consistent with prior studies (Herrero et al., 2014;
Morrison et al., 2006), significant differences in mouse gait were observed
for RRV-infected mice during peak disease compared with uninfected
control mice, whereas minor to no differences in gait were detected in
BFV-infected mice (Abeyratne et al., 2021).
5.1.3 Mayaro and o’nyong nyong viruses

MAYV, endemic in Central and South America, causes similar disease signs
as CHIKV and RRV following infection of humans (Pezzi et al., 2019).
Recently, infection of 4-week-old WT C57BL/6 mice with 103 PFU of
the genotype L MAYV strain BeH407 (via subcutaneous inoculation of
virus in the footpad) was reported to result in swelling in the ipsilateral ankle
similar to that observed in CHIKV-infected mice (Fox et al., 2015).
Notably, in contrast with CHIKV infection, swelling also was observed
in the contralateral ankle of MAYV BeH407-infected mice (Earnest
et al., 2019), suggesting that MAYV infection of mice may provide a better
model of the symmetrical polyarthritis observed in humans infected with
either MAYV or CHIKV. Additional mouse strains also can be used for
MAYV studies as infection of 11-day old WT SV129 mice with
106 PFU MAYV TR 4675 in the left footpad resulted in necrosis, edema,
and cellular infiltration in hind limb muscle tissue that was associated with
elevated expression of pro-inflammatory mediators including MCP-1,
RANTES, and IL-6. (Figueiredo et al., 2019; Santiago et al., 2015). In
contrast with CHIKV, MAYV, and RRV, ONNV has been reported to
replicate inefficiently in WT mice (Seymour et al., 2013). Thus, researchers
have used immunodeficient mice, such as Ifnar1/ mice, to study ONNV
arthritic disease (Fox et al., 2015; Seymour et al., 2013). However, recently
an immunocompetent mouse model of ONNV infection and joint disease
(e.g., necrosis, synovitis, tenosynovitis) based on the use of a clinical viral
isolate from Chad (IMTSSA/5163) and a higher viral dose (106 vs
103 PFU) was reported (Chan et al., 2020). Uniquely, this study also utilized
administration of labeled IL-2 and PET-CT imaging to evaluate accumula-
tion of CD4+ T cells in the infected joints of live animals (Chan et al., 2020).
5.1.4 Eastern equine encephalitis virus

The encephalitic alphaviruses EEEV and VEEV are lethal in mice regardless
of immunocompetency, which limits the utility of mouse models for study
of longer-term sequelae. Nevertheless, these models have been invaluable

for studies of acute pathogenesis (Steele and Twenhafel, 2010). Infection
of adult WT C57BL/6 mice with EEEV strain FL91–4679 by subcutaneous
inoculation caused 100% mortality and widely dispersed viral replication
throughout the CNS as early as 1-day post-infection (dpi). Viral replication
also was observed via immunohistochemistry (IHC) in metaphyseal osteo-
blasts, possibly suggesting a contributing mechanism to a higher incidence of
neuroinvasion, fulminant encephalitis, and high titer viremia seen in
EEEV-infected human children (Vogel et al., 2005). The widespread viral
replication in the brain was concluded to be the result of viral invasion of the
CNS via the vascular system rather than through the olfactory peripheral
nerves; yet other studies suggest both routes may be involved, and intranasal
and aerosol inoculation studies demonstrate rapid CNS invasion via the
olfactory sensory neurons using IHC for viral antigen over the first 5 dpi
(Honnold et al., 2015a; Honnold et al., 2015b). Subcutaneous inoculation
of 12-week-old BALB/c mice with EEEV FL93-939 produced inconsistent
weight loss, variable viral titers and antigen detected in the CNS, and none
of the mice succumbed during the study time frame (Honnold et al., 2015a).
Eight-week-old outbred CD-1 mice develop seizures and rapidly succumb
to encephalitis after 6 dpi following subcutaneous inoculation of EEEV
FL91-4679 (Gardner et al., 2011). EEEV FL91-4679 has strong affinity
for heparan sulfate (HS), promoting increased replication in the CNS and
decreased acute prodromal febrile disease, possibly explaining the rapid
mortality in CD-1 mice (Gardner et al., 2011). These variable mouse models
illustrate the need to standardize a model for assessing mortality and clinical
disease by peripheral inoculation, as all human EEE cases to this date have
been associated with mosquito transmission (Morens et al., 2019).
5.1.5 Venezuelan equine encephalitis virus

While VEEV is generally fatal in less than 1% of human cases, infection of
adult immunocompetent mice is 100% fatal within 7–10 days post subcuta-
neous virus inoculation ( Jackson et al., 1991; Steele and Twenhafel, 2010;
Weaver et al., 1996). Five-eight week old CB17 mice infected with the
VEEV TrD strain (derived from the recombinant V3000 clone) succumb
to fulminant encephalitis despite clearance of viremia and production of
VEEV-specific IgM as early as 3 dpi, and viral invasion of the CNS is
associated with replication in olfactory sensory neurons following high titer
viremia (Charles et al., 1995; Charles et al., 2001). Disease is exacerbated by
a pathogenic immune response involving T cells, and though some studies
have found a critical role for αβ T cells in controlling infection of the

CNS, infection of immunodeficient mice only marginally increases the
median time to death following severe spongiform encephalopathy from
VEEV-induced cytolysis of infected neurons (Brooke et al., 2010;
Charles et al., 2001; Paessler et al., 2007; Wang et al., 2001). This extreme
mortality in mice hinders the ability to study chronic manifestations of
VEEV disease, as long-term neurological sequelae are seen in 4–14% of
symptomatic human cases (Ronca et al., 2016). However, a study in immu-
nocompetent C57BL/6 mice using low dose (103 PFU) intranasal adminis-
tration of the attenuated VEEV vaccine strain TC-83 segregated the animals
based on the presence of apparent (e.g., ruffled fur, hunched posture, and
weight loss) or inapparent disease signs. In animals with apparent disease
signs, the authors identified both chronic impairment of pre-pulse inhibition
(PPI) and the acoustic startle response as well as histological damage to the
thalamus, providing a potential model for chronic human VEEV disease as
the acoustic startle response and PPI are common tools used to evaluate psy-
chiatric disorders and neurological decay in mice (Davis, 1980; Leung and
Jia, 2016; Ronca et al., 2017). Other studies have developed non-lethal
models of VEEV infection using subcutaneous inoculation of both outbred
CD-1 and inbred C57BL/6 mice with recombinant mutants of the virulent
TrD strain identified by direct mutation and in vivo revertant analysis,
including the V3533 and V3534 viruses that have significantly attenuated
capacity for replication in the CNS. Using these viral variants, researchers
determined that the early steps of VEEV infection involve transit to and
replication within the draining lymph node followed by dissemination
and invasion of the CNS. Within the CNS, the complement factor C3
plays an important role in control of disease pathology and the
pro-inflammatory genes Ccl2, Ccl5, Ccl6, and Ly6 are upregulated during
WT, but not attenuated, VEEV infection (Aronson et al., 2000; Brooke
et al., 2012; Grieder et al., 1995; Gupta et al., 2017). Finally, one challenge
to studying the pathogenesis of WT VEEV epizootic strains (i.e., subtypes
IAB and IC) are their status as HHS and USDA select agents. However
intranasal inoculation of C3H/HeN mice with the attenuated VEEV strain
TC-83 (a non-select agent that can be studied at BSL2) has been useful for
investigating determinants of neuroinvasion and pathology, as these mice
succumb to disease due to TLR4-mediated opening of the blood-brain
barrier (BBB) and NK cell-mediated pathology (Hollidge et al., 2021;
Steele et al., 1998; Taylor et al., 2012). Moreover, other non-select agent
VEEV strains, such as the subtype ID strain ZPC738, cause severe
encephalitis in mice similar to epizootic VEEV strains and can be studied at

BSL3 (Anishchenko et al., 2004; Ma et al., 2020a; Wang et al., 2001).
5.1.6 Western equine encephalitis virus

Similar to VEEV and EEEV, epizootic strains of WEEV induce rapid
lethal infection in immunocompetent mice, making investigation of the
long-term neurological damage associated with a subset of human cases
difficult (Bianchi et al., 1993; Logue et al., 2009; Nagata et al., 2006;
Ronca et al., 2016). Intranasal administration of WEEV in adult outbred
CD-1 mice results in viral entry into the CNS via the axons of olfactory sen-
sory neurons, similar to what has been observed for VEEV, while studies
using peripheral inoculation in the footpad revealed that CNS entry
occurs in the hypothalamus, anterioventral third ventricle region, and pineal
body, areas in which the BBB is absent, followed by viral trafficking along
neural pathways to other regions of the CNS (Phillips et al., 2013; Phillips
et al., 2016; Ryzhikov et al., 1995). These findings illustrate the importance
of inoculation route in understanding pathogenesis as clinical signs may
be similar between intranasal and peripheral inoculation, but the mechanistic
basis may be different, which could alter treatment strategies. Recently,
passive immunotherapy using polyclonal antibodies against the WEEV E1
glycoprotein to control infection was used to prevent lethal infection of
CD-1 mice following intranasal inoculation of the WEEV-McMillan
strain and subsequently assess the pathologic features associated with neu-
rodegeneration following ultimate clearance of infection at 8 weeks post
inoculation (Bantle et al., 2019). Pathologic features observed included loss
of dopaminergic neurons, persistent glial activation, and proteinase
K-resistant α-synuclein aggregates, suggesting that this model can replicate
neurodegeneration seen in human patients and provide a system to investi-
gate the underlying causes (Bantle et al., 2019). This model could be trans-
lated to studies of long-term neurological sequelae during VEEV and EEEV
infection, however, the requirement for passive immunotherapy to limit
mortality complicates interpretation of events during acute infection that
may contribute to the development of chronic disease manifestations.
While WEEV is generally lethal in mice, there exists considerable variation
in virulence between isolates, as WEEV-McMillan is 100% lethal regardless
of inoculation route but the Imperial 181 strain causes no mortality
despite both viruses successfully invading the CNS (Logue et al., 2009).
Comparison of a panel of WEEV isolates indicated variable virulence as
determined by mortality, and interestingly, more virulent lethal strains were
isolated from human or horse infections whereas less virulent strains were
originally derived from Culex tarsalis mosquitoes, suggesting a significant
host adaptation barrier for the virus to surmount before pathology occurs
(Logue et al., 2009). These data are consistent with studies in other outbred
mice (e.g., Swiss-Webster, Swiss-NIH) that indicate these epizootic WEEV
strains are unique in neuroinvasiveness (Bianchi et al., 1993). In contrast,
adult BALB/c mice infected with epizootic and enzootic WEEV isolates
uniformly succumb to infection, suggesting outbred mice may be more
useful for comparing viral determinants of virulence between epizootic
and enzootic WEEV strains (Nagata et al., 2006).
5.1.7 Sindbis and Semliki Forest viruses

Sindbis virus (SINV) infection of humans is characterized by fever, rash,
arthralgia and myalgia, and Semliki Forest virus (SFV) infection occasionally
causes similar disease signs (Adouchief et al., 2016; Mathiot et al., 1990; Zaid
et al., 2021). In contrast, SINV and SFV induce lethal CNS inflammation in
mice, making them useful models for investigating mechanisms of viral
CNS disease (Atkins et al., 1985; Fazakerley, 2004; Griffin, 1989). SINV
infection of mice by intracranial or intraperitoneal inoculation causes
encephalitis with age-dependent mortality. In addition, disease severity is
variable among mouse strains as C57BL/6 mice develop fatal encephalitis
up to 10 weeks of age, but BALB/c mice become resistant to fatal disease
by 4–5 weeks of age (Thach et al., 2000). Two canonical SINV strains used
to investigate determinants of encephalitic disease and virulence are the
age-restricted AR339 and a neuronal adapted strain (NSV), which is highly
virulent in older mice due to a glutamine (E) to histidine (H) mutation at E2
position 55 (Lustig et al., 1988; Tucker et al., 1993; Tucker and Griffin,
1991). Substitution of the NSV E2-E1 glycoprotein region in the avirulent
SINV strain, Toto1101, was sufficient to confer virulence in weanling mice,
although the 17-fold higher LD50 dose of this recombinant, called TE12,
compared to NSV indicates that additional residues outside the envelope
proteins are also important for neurovirulence (Lustig et al., 1988). NSV
is uniformly fatal in adult C57BL/6 mice following intracerebral inocula-
tion, with disease characterized by induction of proinflammatory cytokines
and cellular infiltrates in the CNS (Levine and Griffin, 1992; Thach et al.,
2000). In contrast, adult BALB/c mice recover from infection due to a
higher percentage of IL-10 producing CD4+ T cells in the CNS, however,
SINV RNA persists in the brain of BALB/c mice for at least 6 months post
infection with a virus-specific antibody response critical to preventing viral
reactivation (Griffin et al., 1997; Kulcsar et al., 2015; Levine and Griffin,
1992). SINV AR339 is lethal by intracranial inoculation in young mice,
and comparative studies in 4-week-old mice suggest interferon-stimulated
genes (ISGs), especially ISG12, are critical for protection against CNS
pathology (Labrada et al., 2002).
Similarly, SFV infection of C57BL/6, BALB/c, outbred ICR, and CBA
mice by intracranial or peripheral inoculation causes lethal encephalitis
characterized by hindlimb paralysis, virus-induced demyelination, and pro-
ductive viral replication in neurons and oligodendrocytes following crossing
of the blood-brain barrier (Coppenhaver et al., 1995; Fazakerley, 2004;
Taylor et al., 2015). SFV persists in the brains of adult C57BL/6 mice with
little to no pathology despite causing highly destructive fatal encepha-
lomyelitis in young mice; these differences may be related to the role of anti-
bodies in clearance of virus from the periphery and control of CNS
infection (Atkins et al., 1985; Fazakerley, 2004; Griffin et al., 1997). As with
SINV, several attenuated and virulent SFV strains are commonly used to
assess mechanisms of viral encephalitis, including attenuated SFV A7 or
SFV4 strains, and pathogenic SFV6 or SFV L10 strains (Michlmayr et al.,
2014). Attenuated SFV strains do cause acute encephalitis and demyelina-
tion in 4-week-old mice, but the disease resolves between 3 and 5 weeks
post-infection (Fazakerley, 2004; Mokhtarian et al., 1994; Taylor et al.,
2015). These murine models of SINV or SFV-mediated viral encephalitis
provide valuable insight into both mechanisms of virus-induced CNS
disease and pathogenic and protective immune responses in the CNS
(Griffin, 2010).
5.2 Other rodents

5.2.1 Hamsters
In addition to traditional laboratory mice, models of alphavirus infection
have been developed in other rodents (Fig. 5). Infection of 6–8 week old
golden hamsters with EEEV strain 79 by subcutaneous injection in the
quadriceps muscle causes high mortality with severe encephalitic disease
signs, including vasculitis and microhemorrhages, which are characteristics
of fatal human cases that have been difficult to replicate in mice (Dremov
et al., 1978; Paessler et al., 2004). Hamsters also have been used to model
WEEV infection, with intraperitoneal injection of WEEV into female ham-
sters resulting in high mortality and encephalitic lesions, although WEEV is
generally not fatal in humans and the short mean day-to-death limits use of
this model ( Julander et al., 2007). VEEV infection of hamsters has been
studied in greater depth. Hamsters infected with virulent VEEV (subtypes I,

II, III) develop severe histopathologic lesions in both lymphoid and
CNS tissues with rapid death thought to be associated with various
extra-neural pathologies, including extensive ileal lesions in Peyer’s patches
correlating with positive blood bacterial cultures of enteric origin and
massive bone marrow necrosis (Gorelkin and Jahrling, 1975; Jahrling and
Scherer, 1973; Walker et al., 1976). Hamsters also have been infected
with arthritogenic alphaviruses: 4–6-week-old male hamsters infected sub-
cutaneously with two different strains of CHIKV, of the East Central South
African lineage (ECSA), developed viremia and histopathological joint
lesions, including myositis and tenosynovitis, consistent with disease signs
in human patients (Bosco-Lauth et al., 2015; Cunha et al., 2020). These
hamsters did not display any overt clinical signs, however, and cleared the
virus rapidly—by 4 dpi—whereas CHIKV often establishes chronic symp-
toms in human patients possibly due to persistent infection (Bosco-Lauth
et al., 2015; McCarthy et al., 2019). It is possible that altering the inoculation
site from the abdomen to the ventral side of the footpad, such as was done to
improve CHIKV models in traditional inbred laboratory mice, or use of
CHIKV strains that were not passaged several times in suckling mice or
Vero cell culture, could improve this model (Alcorn and Klimstra, 2022;
Gardner et al., 2010; Prescott et al., 2017). Currently, hamsters are of
limited use for defining mechanisms of immunopathology and viral patho-
genesis due to their limited genetic tractability and the limited availability of
specific reagents, but are useful models for evaluating antiviral therapeutics
and vaccines (Miao et al., 2019).
5.2.2 Guinea pigs

As EEEV is capable of aerosol transmission, Roy et al. compared aerosol
infection of 8–9-week-old BALB/c mice and 8–9-week-old guinea pigs
with North American EEEV strain NJ1956 and MADV strain ArgM.
This work revealed that guinea pigs were modestly more resistant to infec-
tion but better recapitulated human disease signs of encephalitis, including
neuronal necrosis, meningeal inflammation, and vasculitis, making this a via-
ble model for studies of EEEV aerosol transmission and infection (Roy et al.,
2009). VEEV infection also has been assessed in guinea pigs as these animals
best predict the ability of different VEEV strains to induce equine viremia
(Greene et al., 2005a). However, early investigations highlighted that the
specific guinea pig strain used is critical for evaluating the potential virulence
of VEEV subtypes in equine hosts, and importantly, guinea pigs tend to
succumb to infection prior to the onset of clinical CNS disease, limiting their
use as models of human neurological disease (Scherer et al., 1979; Steele and
Twenhafel, 2010).
5.2.3 Rats
Laboratory rats are often used for studies of the CNS, as their larger size and
higher intelligence than traditional laboratory mice make them more rele-
vant for behavioral and psychiatric studies (Ellenbroek and Youn, 2016).
Adaptation of the SINV encephalitis mouse model to rats shows some
stark differences, as both murine neuroinvasive and attenuated SINV strains
fail to invade the CNS following intraperitoneal injection of adult rats.
Moreover, only one murine neurovirulent strain caused fatal disease follow-
ing intracranial virus inoculation of rats (Darman et al., 2004; Kobiler et al.,
1999). A key component of neurovirulent SINV pathogenesis is excitotoxic
motor neuron injury, which has also been observed in West Nile virus
(WNV) animal and human infection, and differences in susceptibility to
spinal motor neuron death between BALB/c mice, C57BL/6 mice, and
Sprague-Dawley rats may be attributed to the level of GLT-1-mediated
glutamate transport (Darman et al., 2004). SFV rodent encephalitis can
also be translated to the rat model, which shows viral strain-dependent
lethality following intranasal virus inoculation. Overt disease is associated
with necrosis of productively infected neurons and apoptosis of infiltrating
leukocytes, while immune-mediated demyelination occurs following
infection with both high and low virulence SFV strains (Atkins et al.,
1990; Sammin et al., 1999). The demyelination observed during infection
with both lethal and avirulent SFV strains suggests this rat model could
be useful for understanding the mechanistic basis of demyelinating diseases
in humans such as multiple sclerosis (Sammin et al., 1999). Rats also are
highly susceptible to epizootic VEEV infection, with VEEV strain
V-198 inducing lethal encephalomyelitis in 100% of animals regardless
of inoculation dose. Since metabolic functions in rats have been well
characterized in the context of other infectious diseases, this model could
be useful for defining the relationship between VEEV infection, metabo-
lism, and the course of clinical disease ( Jahrling et al., 1978). Finally,
VEEV infection of pregnant rats has been used to demonstrate structural
damage in placental and fetal tissues associated with VEEV infection
(Garcı́a-Tamayo et al., 1981).
5.3 Non-human primates

5.3.1 Arthritogenic alphaviruses in non-human primates
To date, studies of arthritogenic alphavirus infection in NHPs have focused
on CHIKV. Cynomolgus macaques (Macaca fascicularis) infected with
CHIKV by subcutaneous inoculation develop mild illness characterized
by a low, transient fever, mild to moderate malaise, and high level viremia
that peaks at 2 dpi (Labadie et al., 2010; Langsjoen et al., 2018). Multiple
additional studies found that in response to CHIKV infection at varying
doses, these macaques develop acute fever, rash, viremia, lymphopenia,
and display an increase in serum markers of liver damage. These results cor-
relate well with natural human infection. However, only high dose
(>107 PFU) inoculation results in the joint swelling indicative of arthritic
disease (Broeckel et al., 2015; Cirimotich et al., 2017; Roy et al., 2014).
These signs are consistent across CHIKV lineages as infection with either
the ECSA isolate Ross, Asian/American isolate YO123223, or IOL lineage
CHIKV-LR2006 isolate results in fever, malaise, anorexia, and viremia,
although interestingly, one study noted that the severity of this clinical
disease was greater in CHIKV-LR2006 infected macaques compared with
YO123223-infected macaques (Cirimotich et al., 2017; Higgs and Ziegler,
2010; Roy et al., 2014). CHIKV infection of rhesus macaques (Macaca
mulatta) also results in development of acute fever, rash, and viremia similar
to that observed in cynomolgus macaques, and erythema and swelling of
arm joints between 1 and 5 dpi (Broeckel et al., 2015; Broeckel et al.,
2017; Chen et al., 2010; Messaoudi et al., 2013). These disease signs are con-
sistent across CHIKV strains, including infection with CHIKV-LR2006
(Indian Ocean Lineage/ECSA), CHIKV 37997 (West African), and
ECSA strain DHS-4263 (Broeckel et al., 2017, Chen et al., 2010,
Messaoudi et al., 2013). Importantly, from the existing studies, it does
not appear that the change in virus dose between 103 PFU and 107 PFU
for subcutaneous inoculation or administration of high doses >107 PFU
by intravenous inoculation, alters disease severity in this rhesus macaque
model. Studies in this model have identified reduced B and T cell responses,
higher viral loads for a longer duration, and altered expansion of myeloid cell
populations in aged animals infected with CHIKV-LR (Messaoudi et al.,
2013). These findings reflect diminished immune responsiveness in the
elderly, providing a strong model for studying age-dependent clinical disease
severity (Salminen, 2022). Despite the presence of viral RNA in lymphoid
and joint-associated tissues in CHIKV 37997-infected pregnant macaques at
21 dpi (after viremia and clinical disease signs subside), a lack of detectable
viral RNA or germinal center development in fetal tissue indicated no trans-
placental transmission, suggesting CHIKV is not vertically transmitted
(Chen et al., 2010). Both the rhesus macaque and cynomolgus macaque
models of CHIKV infection have been used to investigate antiviral pre-
and post-prophylactic treatments including vaccination and intravenous
administration of human monoclonal antibodies against CHIKV during
acute disease, emphasizing how the usefulness of this model at mimicking
aspects of human disease supports the development of novel therapeutic
strategies (Broeckel et al., 2017; Roy et al., 2014). The cynomolgus
macaque model also provides strong evidence for persistent CHIKV infec-
tion. CHIKV RNA was detected in lymphoid organs, liver, synovial and
muscle tissues for months after inoculation. Moreover, infectious virus
was recovered from spleen, liver, and muscle tissue at 44 dpi and viral
antigen and RNA were detected in CD68+ macrophages, implicating
macrophages as a cellular site of viral persistence (Labadie et al., 2010).
5.3.2 Encephalitic alphaviruses in non-human primates

While EEEV, VEEV, and WEEV are naturally transmitted by mosquito
bite, which is usually mimicked in the laboratory using needle-based subcu-
taneous inoculation, studies in NHPs have primarily used aerosol-based
inoculation as these viruses are highly infectious biohazard threats when
in aerosolized form (Croddy et al., 2002). For more detailed description
of aerosol exposure, we direct the reader to the Section 6.1.1. Infection
of cynomolgus macaques with EEEV, VEEV, or WEEV by aerosol expo-
sure results in fever, viremia, lymphopenia, and encephalitis, and mortality is
dose-dependent for both EEEV and WEEV, although in one study compar-
ing high and low dose EEEV infection, survival was inversely correlated
with the development of apparent disease signs (Albe et al., 2021; Reed
et al., 2004; Reed et al., 2005). WEEV has been less studied in NHPs than
EEEV and VEEV, however, a small proportion of cynomolgus macaques
infected with the virulent WEEV CBA-87 strain by aerosol exposure suc-
cumb to disease with evidence of viral antigen and lymphocytic infiltrate
present in CNS tissue and a correlation between serum glucose levels and
severity of clinical encephalitis (Reed et al., 2005). Studies with EEEV
and VEEV using minimally invasive implantation of telemetry devices dem-
onstrate significant disruption of macaque behavior, including disrupted
circadian rhythm, cardiac abnormalities, disinterest in food and water, and
increased intracranial pressure following aerosol exposure; these parameters
and their measurement with telemetry are excellent quantitative ways of

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

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

infection
6.1 Transmission studies
Viral transmission is critical for the alphavirus life cycle and for the establish-
ment of disease (Fig. 6). In order for mosquito-transmitted alphaviruses
to spread, the virus must establish a viremia of sufficient magnitude and
duration in the vertebrate host to infect blood-feeding mosquitoes. Thus,
understanding the viral and host determinants of viremia is a key research
area of interest (Ander et al., 2022). To do this, researchers have utilized
a reductionist system to study alphavirus viremia in which a defined number
of viral particles or infectious units are inoculated intravenously into mice,
and the clearance rate of viral particles from the blood circulation is deter-
mined (Ander et al., 2022; Carpentier et al., 2019). This simplified system
bypasses the effects of de novo viral replication and allows for the determi-
nation of the fate of alphaviral particles as well as the identification of host
and viral factors that influence the rate of viral clearance, such as the scav-
enger receptor MARCO (Carpentier et al., 2019) and specific amino acid
residues in virion surface glycoproteins (Bernard et al., 2000; Carpentier
et al., 2019), respectively.
6.1.1 Aerosol transmission

Studies in mice and NHPs have been used to evaluate infection and disease
upon aerosol exposure, which is generally performed using head-only aero-
sol exposure in a class 3 biological safety cabinet with aerosol droplets gen-
erated using a nebulizer. For these studies, the respiratory capacity of the
Fig. 6 Utility of alphavirus transmission models. Alphavirus transmission has been stud-
ied using specific models of experimental viremia in which the fate of viral particles in
the blood circulation can be assessed following intravenous inoculation, following aero-
sol inoculation, and using mosquito and mosquito factor-based models to more closely
mimic natural infection. This figure was made using Biorender.com.
animals is determined prior to exposure, and the actual inhaled dose is deter-
mined by plaque assay on samples collected by impaction on gelatin-filters
(Reed et al., 2004; Taylor, 2007). These steps are essential as aerosol expo-
sure can be a more highly variable method of inoculation than needle-based
inoculation. In mice, aerosol exposure of 7–9-week-old female BALB/c
mice to EEEV Pe-6 causes dose-dependent mortality and recapitulates
the severe encephalitis observed in human patients infected following a
mosquito bite (Phelps et al., 2019). Histological evaluation of these mice
suggested that the brain supports productive EEEV infection, however,
further study of EEEV replication in cells of the murine CNS in the context
of aerosol infection is needed. In NHPs, aerosol exposure of adult
cynomolgus macaques and common marmosets to EEEV (FL91-4679
and FL93-939) also results in dose-dependent lethality and neurological dis-

ease similar to that observed in humans (Porter et al., 2017; Reed et al.,
2007). These studies highlight the infectiousness of aerosol-transmitted
EEEV, although as of yet, there have been no reported cases of aerosolized
EEEV infection in humans for comparison. Enhanced infectivity and disease
upon aerosol exposure of cynomolgus macaques to VEEV can be more
directly correlated with human infection due to early reports of laboratory
VEEV aerosol exposure and subsequent disease (Reed et al., 2004). In
addition, while no natural CHIKV infections by aerosol exposure have
been reported, CHIKV is capable of aerosol transmission in cynomolgus
macaques, causing viremia and increased markers of liver damage although
no overt clinical disease was observed (Cirimotich et al., 2017).
6.1.2 Mosquito and mosquito component-based infection models

One important consideration for alphavirus animal models is that
generally virus stocks are diluted in cell culture media or saline solution
prior to inoculation into the animal, while in natural infection, virus is trans-
mitted as part of mosquito saliva (Dudley et al., 2017; Thangamani et al.,
2010). There is a large interest in understanding the impact of mosquito
saliva and other arthropod feeding factors on the dynamics of alphavirus
infection as mosquito and tick salivary factors have been shown to influence
host susceptibility, tissue viral burden, viral dissemination, and immune
responses for a variety of arboviruses (Cox et al., 2012; Edwards et al.,
1998; Le Coupanec et al., 2013; Limesand et al., 2000; McCracken et al.,
2014; Moser et al., 2016; Osorio et al., 1996; Schneider et al., 2006;
Styer et al., 2011; Wanasen et al., 2004). One approach used is the
co-inoculation of mice with both a defined amount of virus and sonicated
salivary glands collected from virus-naı̈ve mosquitoes to mimic natural
infection (Schneider et al., 2004). When this approach was used with
SINV strain H55K70, which has similar mouse virulence phenotypes to
the commonly used strain AR339, the presence of salivary gland extract
(SGE) was associated with reduced type I and II IFN responses and an overall
shift from protective TH1 to TH2 cytokine responses (Schneider et al.,
2004). The most direct method to investigate natural mosquito transmission
of alphaviruses is to allow mosquitoes to blood feed on laboratory animals.
For example, infection of mosquitoes by blood feeding on EEEV-infected
chicks followed by blood feeding on naı̈ve laboratory mice was used as an
early method for estimating the amount of virus inoculated by mosquito
bite (Chamberlain et al., 1954). Since then, cell culture systems and infec-
tious virus derived from recombinant cDNA clones have allowed direct
infection of mosquitoes instead of blood feeding on chicks, for example, in

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

Alphaviruses circulate in the same regions of the world as other impor-
tant human pathogens, such as DENV, Zika (ZIKV), Plasmodium spp.,
Leishmania spp., and HIV, and in regions where people are more burdened
with helminth infections (Brooker, 2010; Mala et al., 2021; Pasaribu et al.,
2019). DENV and ZIKV are both transmitted by A. aegypti mosquitoes,
which also transmit CHIKV, making it important to characterize how
potential co-infection of both mosquito or mammalian hosts alters trans-
missibility, clinical disease, and development of protective immunity
(Fig. 7); a neglected area of public health concern (Vogels et al., 2019).
6.2.1 Plasmodium spp.

While Plasmodium species that cause malaria are primarily transmitted by
Anopheles gambiae mosquitoes, co-infection of humans remains a strong pos-
sibility due to the significant geographic overlap in malaria and CHIKV
endemic regions and the isolation of CHIKV from other Anopheles mosquito
species known to transmit malaria (Mala et al., 2021). In addition, ONNV is
transmitted by A. gambiae, making it vital to understand how ONNV and
Plasmodium co-infections of both mosquitoes and mammals impact transmis-
sion and disease (Pezzi et al., 2020). In one study, researchers found that
prior infection of WT C57BL/6 mice with rodent Plasmodium spp. (either
the lethal PbA strain or the non-lethal Py17x strain) prior to inoculation
Fig. 7 Utility of alphavirus co-infection models. Co-infection of hosts with alphaviruses

and other infectious agents, such as Plasmodium parasites or dengue virus, is an under-
studied area of research. However, some studies have assessed the impact of
co-infection on alphavirus pathogenesis and immunity in mammalian systems and
fecundity, transmission, and vector competence in mosquito models. This figure was
made using Biorender.com.
with CHIKV strain SGP11 reduced viremia and suppressed tissue viral
load and joint inflammation, whereas concomitant co-infection had no
effect on viremia but reduced peak joint inflammation (Teo et al., 2018).
Similar findings were observed following inoculation of ONNV into
Plasmodium-infected mice (Torres-Ruesta et al., 2022). Although these
studies investigated both an experimental cerebral malaria (ECM) model
and an uncomplicated malaria infection model that recapitulates liver stage
disease, metabolic acidosis, parasitemia, and severe anemia, inoculation of
CHIKV or ONNV occurred during the acute stage of Plasmodium infection
when the innate immune response is activated. Thus, it could be of interest
to modify these models and assess the outcome of CHIKV or ONNV infec-
tion following inoculation during the chronic stage of malaria infection
(De Niz and Heussler, 2018; Teo et al., 2018). In addition, testing the effect
of other Plasmodium species on CHIKV and ONNV disease, such as
P. chaubaudi, which establishes chronic malaria in mice (Stephens et al.,
2012), could enhance the utility and impact of these systems.
6.2.2 Dengue virus

A few studies have experimentally co-infected mosquitoes with CHIKV
and DENV to evaluate replication kinetics and transmission potential. Le
Coupanec et al. infected 7-day old female mosquitoes with a blood meal
containing the CHIKV 06.21 isolate from La Reunion with the A226V
mutation and a 1974 human serum DENV-2 isolate. Although CHIKV
RNA levels in the salivary glands (SGs) were lower in co-infected mosqui-
toes at 2 dpi, CHIKV RNA levels were significantly higher in SGs of
co-infected mosquitoes at 13 dpi; increased DENV-2 RNA was present
in SGs of co-infected mosquitoes consistently throughout infection. These

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

As arthropod-borne viruses that cycle between vertebrate and inver-
tebrate hosts, alphaviruses are constrained in the degree to which they can
adapt to one host over the other (Coffey et al., 2008; Vasilakis et al., 2009).
As viral genomic sequencing and development of robust recombinant
cDNA clone systems have become widespread, the complications of passag-
ing viral isolates in mammalian cell culture or laboratory animals prior to
experimental use has become more appreciated, since adaptive mutations
can confound studies of viral infection, pathogenesis, and immunity in
animal models (Alcorn and Klimstra, 2022; Ciota and Kramer, 2010;
DuPai et al., 2019; Gardner et al., 2012; Greene et al., 2005c). For example,
comparison of the cell-culture adapted CHIKV strain 181/25 with WT
CHIKV isolates in mice lacking the type I and II IFN receptors (AG129)
or the STAT1 transcription factor involved in type I and type II IFN
responses (STAT129) revealed that WT CHIKV infection was 100% lethal
in both AG129 and STAT129 mice, whereas CHIKV 181/25 was 100%
lethal in AG129 mice but only 50% lethal in STAT129 mice, emphasizing
the dramatic effect cell culture adaptations can have on infection outcome
(Gardner et al., 2012). This study, and many others, emphasizes the need
to utilize low passage viral isolates and/or cDNA clone-derived virus based
on direct viral genome sequencing of primary samples or low passage iso-
lates. We provide a table summarizing the origin and passage history of
alphaviruses commonly used in animal model systems (Table 1).
Table 1 Alphavirus strains commonly used in animal models
GenBank
Virus Lineage Strain Source Year Passage History Accession No. Key References
CHIKV IOL LR2006-OPY1 Human 2006 Vero-3, SM-1, Vero-1 DQ443544.2 Tsetsarkin et al. (2006),
Tsetsarkin et al. (2007)
IOL SL15649 Human 2006 Vero-3 GU189061.1 Hawman et al. (2013),
Morrison et al. (2011)
ECSA Ross Human 1953 SM-16, Vero-2, C6/36–1 AF490259.3 Langsjoen et al. (2018),
Powers et al. (2001)
WA 37,997 Aedes furcifer 1983 AP61 cells-1, Vero-1 AY726732.1 Tsetsarkin et al. (2007),
Vanlandingham et al. (2005)
Asian AF15561 Human 1962 Unk, Vero-2 EF452493.1 Gorchakov et al. (2012),
Hawman et al. (2016)
Asian 181/25 AF15561 1986 MRC-5 18 MW473668.1 Gorchakov et al. (2012),
derivative Levitt et al. (1986)
RRV I T48 Aedes vigilax 1959 SM-10, Vero-2 GQ433359.1 Morrison et al. (2006)
III SN11 Human 2009 C6/36-1, BHK-1 JF699395.1 Liu et al. (2011)
II DC5692 Aedes 1995 C6/36-1, Vero-1, BHK-1 HM234643.1 Jupille et al. (2011)
camptorhyncus
ONNV SG650 Human 1996 Vero-1 AF079456.1 Vanlandingham et al. (2006)
IMTSSA/5163 Human 2004 Vero-2 DQ399055.1 Chan et al. (2020)
BFV G2 BH2193 Culex 1974 SMB-2, Vero-2 U73745.1 Herrero et al. (2014)
annulirostris
MAYV genotype D CH Human 2001 Unk, Vero-1 DQ487410.1 Earnest et al. (2019), Weise
et al. (2014)
genotype D TRVL4675 Human 1954 SM-13, BHK-1 AF398378.1 Figueiredo et al. (2019),
Powers et al. (2001)
genotype D TRVL15537 Coquellitidia 1957 SM-5, Vero-2 MK573240.1 Chuong et al. (2019)
venezuelensis
genotype L BeH407 Human 1955 Unk-2 MK573238.1 Earnest et al. (2019), Fox
et al. (2015)
SINV I AR339 Culex pipiens 1952 SMB-9 JX682537.1 Labrada et al. (2002), Tucker
et al. (1993)
I TR339 AR339 consensus Pierro et al. (2008)
sequence derivative
I NSV AR339 1977 alternating SMB and Lustig et al. (1988), Thach
derivative WM-6 et al. (2000)
TE chimeric Toto1101, NSV, Lustig et al. (1988), Pierro
and SV1A (SV1A is et al. (2008)
SMB-7, BHK-5, CEF-2
version of AR339)
TE12 chimeric Toto1101 and Lustig et al. (1988)
NSV
I S.A.AR86 Culex spp. 1954 Unk U38305.1 Suthar et al. (2005)
SFV SFV4 Aedes 1942 Mouse-4 KP699763.1 Pingen et al. (2016)
abnormalis
SFV6 recombinant N/A Pingen et al. (2016)
derivative of
SFV4
A7 (74) AR2066 1959 SMB-7 X74425.1 Michlmayr et al. (2014)
derivative
Continued
Table 1 Alphavirus strains commonly used in animal models—cont’d
GenBank
Virus Lineage Strain Source Year Passage History Accession No. Key References
L10 Aedes 1942 Mouse-8, SMB-2 AY112987.1 Michlmayr et al. (2014)
abnormalis
EEEV NA FL91-4679 Aedes 1992 Vero-3 AY722102.1 Gardner et al. (2011), Vogel
albopictus et al. (2005)
NA FL93-939 Culiseta 1993 Vero-1, SMB-1 EF151502.1 Arrigo et al. (2010a),
melanura Honnold et al. (2015a)
NA V105-00210 Human 2005 Vero-3 KP282670.1 Yu et al. (2015)
VEEV IAB TrD (Trinidad) donkey brain 1943 GP-1, Vero-6, BHK-1 L01442.2 Kinney et al. (1989)
IAB TC-83 TrD 1967 FGHC-83 L01443.1 Kinney et al. (1989)
derivative
IC INH-9813 Human 1995 Vero-3 KP282671.1 Yu et al. (2015)
ID ZPC738 sentinel 1997 BHK-1 AF100566.1 Anishchenko et al. (2004)
hamster
WEEV Group A McMillan Human 1941 Mouse-2, SMB-1, Vero-2 GQ287640.1 Logue et al. (2009)
brain
Group B Imperial 181 Culex tarsalis 2005 Vero-2 Logue et al. (2009)
Group B Montana-64 Horse brain 1967 DE-2 GQ287643.1 Logue et al. (2009)
Group A Fleming Human 1938 SM-5, Vero-3 MN477208.1 Burke et al. (2020)
SM ¼ suckling mouse, SMB ¼ suckling mouse brain, WM ¼ weanling mouse, DE ¼ duck embryo cells, MRC-5 ¼ human fibroblast cell line, C3/36 ¼ A. albopictus cell line,
AP61 ¼ A. pseudocutellaris cell line, GP ¼ guinea pig, BHK ¼ baby hamster kidney cell line, FGHC ¼ fetal guinea pig heart cell cultures, Unk ¼ unknown, N/A ¼ no passage
history, NA ¼ North American, ECSA ¼ East/Central/South African, IOL ¼ Indian Ocean lineage, WA ¼ West African
* Bold text denotes strain with existing infectious clone system.
As mentioned above, alphavirus reverse genetics systems were

established more than 30 years age (Boyer and Haenni, 1994; Levis et al.,
1986; Rice et al., 1987). In addition to permitting the role of specific amino
acid or nucleotide mutations in replication and infection to be tested,
researchers have combined this technology with animal models in many
creative ways to advance our understanding of alphavirus infection, patho-
genesis and immunity. For example, to investigate the antiviral activity of
specific cytokines against SINV infection in the CNS, Binder and Griffin
engineered the cDNA for TNF-α or IFN-γ into the SINV genome down-
stream of a second subgenomic promoter, revealing a role for both cytokines
in promoting or hampering the clearance of SINV infection (Binder and
Griffin, 2001). Other examples include the generation of a recombinant
SINV library encoding a whole genome-targeting siRNA library to identify
host factors that impact SINV replication in vivo (Varble et al., 2013), a
recombinant CHIKV strain engineered to express the Cre recombinase
to identify cellular targets of infection following inoculation of tdTomato
flox-stop reporter mice (Young et al., 2019), numerous recombinant
alphaviruses engineered to encode luciferase or fluorescent proteins to track
viral infection and dissemination, including in live animals (Sun et al., 2014),
and the use of recombinant CHIKV and RRV strains encoding well-
characterized CD4+ and CD8+ T cell receptor epitopes to investigate T cell
responses during infection (Burrack et al., 2015; McCarthy et al., 2018).
8. Conclusions
As described above, there currently exist a variety of useful animal
models of alphavirus infection in both mammals and mosquitoes, however,
gaps remain in our ability to understand alphavirus infection using animal
models. For example, a robust small animal model of chronic arthralgia
induced by the arthritogenic alphaviruses remains elusive. Enhanced under-
standing of human clinical disease facilitated by patient cohort studies would
aid the future development of such a model. Also, commonly used strains of
laboratory mice often rapidly succumb to encephalitic alphavirus infection,
hindering study of chronic neurological sequelae observed in a large propor-
tion of symptomatic human patients. Use of chimeric SINV and EEE/
VEE/WEE viruses or generation of attenuated viruses and intracranial
inoculation could potentially facilitate development of a murine model of
chronic neurological disease following encephalitic alphavirus infection.
Moreover, reflection of the vast genetic and microbiome diversity of the
human population is significantly limited in inbred mice, and a link between

alphavirus pathogenesis and microbiome-regulated responses has been
reported (Winkler et al., 2020). To overcome these issues, future work
could apply alphavirus infection to more population-representative collab-
orative cross (CC) mice and non-laboratory “dirty” mice (Masopust et al.,
2017; Noll et al., 2020; Threadgill et al., 2011). As noted, while several stud-
ies have attempted to definitively characterize natural reservoir hosts of
alphaviruses in the laboratory, more extensive mechanistic studies to assess
why some animals develop disease or support viremia over others are needed
to better understand the ecology and host tropism that may dictate future
outbreaks and spillovers. Finally, other areas of under-investigation include
coinfection and comorbidity models. Alphaviruses share vector and regional
similarities with other arboviruses of public health concern, and with
helminth and parasitic diseases such as malaria. Moreover, patients with
comorbidities such as type I and II diabetes, other autoimmune disorders,
obesity, malnutrition, and cancer comprise an increasing proportion of
the human population. As mentioned, some studies have been done with
Plasmodium coinfection in mice and DENV-CHIKV coinfection in mosqui-
toes, and recently, a study comparing CHIKV, RRV, and MAYV infection
in lean, obese, and malnourished mice suggested significant differences in
morbidity (Teo et al., 2018; Weger-Lucarelli et al., 2019). Future studies
and models should incorporate coinfections and comorbidities to enhance
our understanding of their effect on alphavirus infection, pathogenesis,
and immunity.
Acknowledgments
We thank members of the Morrison laboratory for critical evaluation of this manuscript. This
work was supported by Public Health Service grants R01 AI141436 and R01 AI148144 from
the National Institute of Allergy and Infectious Diseases to T.E.M. C.J.L. was supported by an
RNA Biosciences Initiative Research Scholar Award.
References
Centers for Disease Control and Prevention (CDC), 1995. Venezuelan equine encephalitis-
-Colombia, 1995. MMWR Morb. Mortal. Wkly Rep. 44, 721–724.
Abeyratne, E., Reshamwala, R., Shelper, T., Liu, X., Zaid, A., Mahalingam, S., Taylor, A.,
2021. Altered spatial and temporal gait parameters in mice infected with ross river Virus.
mSphere 6, e0065921.
Adouchief, S., Smura, T., Sane, J., Vapalahti, O., Kurkela, S., 2016. Sindbis virus as a human
pathogen-epidemiology, clinical picture and pathogenesis. Rev. Med. Virol. 26,
221–241.
Ahearn, Y.P., Saredy, J.J., Bowers, D.F., 2020. The alphavirus sindbis infects
Enteroendocrine cells in the midgut of Aedes aegypti. Viruses 12, 848.
Albe, J.R., Ma, H., Gilliland, T.H., McMillen, C.M., Gardner, C.L., Boyles, D.A.,
Cottle, E.L., Dunn, M.D., Lundy, J.D., O’malley, K.J., Salama, N., Walters, A.W.,
Pandrea, I., Teichert, T., Klimstra, W.B., Reed, D.S., Hartman, A.L., 2021.
Physiological and immunological changes in the brain associated with lethal eastern
equine encephalitis virus in macaques. PLoS Pathog. 17, e1009308.
Alcorn, M.D.H., Klimstra, W.B., 2022. Glycosaminoglycan binding by arboviruses: a cau-
tionary tale. J. Gen. Virol. 103.
Althouse, B.M., Guerbois, M., Cummings, D.A.T., Diop, O.M., Faye, O., Faye, A.,
Diallo, D., Sadio, B.D., Sow, A., Faye, O., Sall, A.A., Diallo, M., Benefit, B.,
Simons, E., Watts, D.M., Weaver, S.C., Hanley, K.A., 2018. Role of monkeys in
the sylvatic cycle of chikungunya virus in Senegal. Nat. Commun. 9, 1046.
Alto, B.W., Civana, A., Wiggins, K., Eastmond, B., Shin, D., 2020. Effect of oral infection of
mayaro virus on fitness correlates and expression of immune related genes in Aedes
aegypti. Viruses 12, 719.
Ander, S.E., Li, F.S., Carpentier, K.S., Morrison, T.E., 2022. Innate immune surveillance of
the circulation: A review on the removal of circulating virions from the bloodstream.
PLoS Pathog. 18, e1010474.
Anishchenko, M., Bowen, R.A., Paessler, S., Austgen, L., Greene, I.P., Weaver, S.C., 2006.
Venezuelan encephalitis emergence mediated by a phylogenetically predicted viral
mutation. Proc. Natl. Acad. Sci. U. S. A. 103, 4994–4999.
Anishchenko, M., Paessler, S., Greene, I.P., Aguilar, P.V., Carrara, A.-S., Weaver, S.C.,
2004. Generation and characterization of closely related epizootic and enzootic infec-
tious cDNA clones for studying interferon sensitivity and emergence mechanisms of
Venezuelan equine encephalitis virus. J. Virol. 78, 1–8.
Appassakij, H., Khuntikij, P., Kemapunmanus, M., Wutthanarungsan, R., Silpapojakul, K.,
2013. Viremic profiles in asymptomatic and symptomatic chikungunya fever: a blood
transfusion threat? Transfusion 53, 2567–2574.
Armstrong, P.M., Andreadis, T.G., 2022. Ecology and epidemiology of eastern equine
encephalitis virus in the northeastern United States: an historical perspective. J. Med.
Entomol. 59, 1–13.
Aronson, J.F., Grieder, F.B., Davis, N.L., Charles, P.C., Knott, T., Brown, K.,
Johnston, R.E., 2000. A single-site mutant and revertants arising in vivo define early
steps in the pathogenesis of venezuelan equine Encephalitis virus. Virology 270,
111–123.
Arrigo, N.C., Adams, A.P., Watts, D.M., Newman, P.C., Weaver, S.C., 2010a. Cotton rats
and house sparrows as hosts for North and South American strains of eastern equine
encephalitis virus. Emerg. Infect. Dis. 16, 1373–1380.
Arrigo, N.C., Adams, A.P., Weaver, S.C., 2010b. Evolutionary patterns of eastern equine
encephalitis virus in North versus South America suggest ecological differences and tax-
onomic revision. J. Virol. 84, 1014–1025.
Atkins, G.J., Sheahan, B.J., 2016. Molecular determinants of alphavirus neuropathogenesis in
mice. J. Gen. Virol. 97, 1283–1296.
Atkins, G.J., Sheahan, B.J., Dimmock, N.J., 1985. Semliki forest virus infection of mice: a
model for genetic and molecular analysis of viral pathogenicity. J. Gen. Virol. 66,
395–408.
Atkins, G.J., Sheahan, B.J., Mooney, D.A., 1990. Pathogenicity of Semliki Forest virus
for the rat central nervous system and primary rat neural cell cultures: possible implica-
tions for the pathogenesis of multiple sclerosis. Neuropathol. Appl. Neurobiol. 16,
57–68.
Azar, S.R., Campos, R.K., Bergren, N.A., Camargos, V.N., Rossi, S.L., 2020. Epidemic
alphaviruses: ecology, emergence and outbreaks. Microorganisms 8, 1167.
Bantle, C.M., Phillips, A.T., Smeyne, R.J., Rocha, S.M., Olson, K.E., Tjalkens, R.B., 2019.
Infection with mosquito-borne alphavirus induces selective loss of dopaminergic
neurons, neuroinflammation and widespread protein aggregation. NPJ Parkinson’s
Dis. 5, 20.
Bastian, F.O., Wende, R.D., Singer, D.B., Zeller, R.S., 1975. Eastern equine encephalomy-
elitis. Histopathologic and ultrastructural changes with isolation of the virus in a human
case. Am. J. Clin. Pathol. 64, 10–13.
Bernard, K.A., Klimstra, W.B., Johnston, R.E., 2000. Mutations in the E2 glycoprotein
of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbid-
ity, and rapid clearance from blood of mice. Virology 276, 93–103.
Bianchi, T.I., Aviles, G., Monath, T.P., Sabattini, M.S., 1993. Western equine encephalo-
myelitis: virulence markers and their epidemiologic significance. Am. J. Trop. Med.
Hyg. 49, 322–328.
Binder, G.K., Griffin, D.E., 2001. Interferon-gamma-mediated site-specific clearance
of alphavirus from CNS neurons. Science 293, 303–306.
Blair, C.D., Olson, K.E., 2014. Mosquito immune responses to arbovirus infections. Curr.
Opin. Insect. Sci. 3, 22–29.
Bonifay, T., Prince, C., Neyra, C., Demar, M., Rousset, D., Kallel, H., Nacher, M.,
Djossou, F., Epelboin, L., 2018. Atypical and severe manifestations of chikungunya virus
infection in French Guiana: A hospital-based study. PLoS One 13, e0207406.
Bosco-Lauth, A.M., Han, S., Hartwig, A., Bowen, R.A., 2015. Development of a hamster
model for Chikungunya virus infection and pathogenesis. PLoS One 10, e0130150.
Bowen, G.S., Calisher, C.H., 1976. Virological and serological studies of Venezuelan equine
encephalomyelitis in humans. J. Clin. Microbiol. 4, 22–27.
Bowen, G.S., Rayfield, E.J., Monath, T.P., Kemp, G.E., 1980. Studies of glucose metabo-
lism in rhesus monkeys after Venezuelan equine encephalitis virus infection. J. Med.
Virol. 6, 227–234.
Boyer, J.C., Haenni, A.L., 1994. Infectious transcripts and cDNA clones of RNA viruses.
Virology 198, 415–426.
Broeckel, R., Fox, J.M., Haese, N., Kreklywich, C.N., Sukulpovi-Petty, S., Legasse, A.,
Smith, P.P., Denton, M., Corvey, C., Krishnan, S., Colgin, L.M.A., Ducore, R.M.,
Lewis, A.D., Axthelm, M.K., Mandron, M., Cortez, P., Rothblatt, J., Rao, E.,
Focken, I., Carter, K., Sapparapau, G., Crowe Jr., J.E., Diamond, M.S.,
Streblow, D.N., 2017. Therapeutic administration of a recombinant human monoclonal
antibody reduces the severity of chikungunya virus disease in rhesus macaques. PLoS
Negl. Trop. Dis. 11, e0005637.
Broeckel, R., Haese, N., Messaoudi, I., Streblow, D.N., 2015. Nonhuman primate models
of Chikungunya virus infection and disease (CHIKV NHP model). Pathogens (Basel,
Switzerland) 4, 662–681.
Brooke, C.B., Deming, D.J., Whitmore, A.C., White, L.J., Johnston, R.E., 2010. T cells
facilitate recovery from Venezuelan equine encephalitis virus-induced encephalomyelitis
in the absence of antibody. J. Virol. 84, 4556–4568.
Brooke, C.B., Sch€afer, A., Matsushima, G.K., White, L.J., Johnston, R.E., 2012. Early acti-
vation of the host complement system is required to restrict central nervous system inva-
sion and limit neuropathology during Venezuelan equine encephalitis virus infection.
J. Gen. Virol. 93, 797–806.
Brooker, S., 2010. Estimating the global distribution and disease burden of intestinal nem-
atode infections: Adding up the numbers – A review. Int. J. Parasitol. 40, 1137–1144.
Burke, C.W., Froude, J.W., Rossi, F., White, C.E., Moyer, C.L., Ennis, J., Pitt, M.L.,
Streatfield, S., Jones, R.M., Musiychuk, K., Kervinen, J., Zeitlin, L., Yusibov, V.,
Glass, P.J., 2019. Therapeutic monoclonal antibody treatment protects nonhuman pri-
mates from severe Venezuelan equine encephalitis virus disease after aerosol exposure.
Burke, C.W., Wiley, M.R., Beitzel, B.F., Gardner, C.L., Huang, Y.-J., Piper, A.E.,
Vanlandingham, D.L., Higgs, S., Palacios, G., Glass, P.J., Stedman, K.M., 2020.
Complete coding sequence of western equine Encephalitis virus strain fleming, isolated
from a human case. Microbiol. Resour. Announc. 9. e01223–19.
Burkett-Cadena, N.D., Day, J.F., Unnasch, T.R., 2022. Ecology of eastern equine enceph-
alitis virus in the southeastern United States: incriminating vector and host species
responsible for virus amplification, persistence, and dispersal. J. Med. Entomol. 59,
41–48.
Burrack, K.S., Montgomery, S.A., Homann, D., Morrison, T.E., 2015. CD8 + T cells con-
trol Ross River virus infection in musculoskeletal tissues of infected mice. J. Immunol.
194, 678–689.
Carpentier, K.S., Davenport, B.J., Haist, K.C., McCarthy, M.K., May, N.A., Robison, A.,
Ruckert, C., Ebel, G.D., Morrison, T.E., 2019. Discrete viral E2 lysine residues and
scavenger receptor MARCO are required for clearance of circulating alphaviruses.
Elife 8.
Carrara, A.-S., Coffey, L.L., Aguilar, P.V., Moncayo, A.C., Da Rosa, A.P.A.T.,
Nunes, M.R.T., Tesh, R.B., Weaver, S.C., 2007. Venezuelan equine encephalitis virus
infection of cotton rats. Emerg. Infect. Dis. 13, 1158–1165.
Carrara, A.-S., Gonzales, G., Ferro, C., Tamayo, M., Aronson, J., Paessler, S.,
Anishchenko, M., Boshell, J., Weaver, S.C., 2005. Venezuelan equine encephalitis virus
infection of spiny rats. Emerg. Infect. Dis. 11, 663–669.
Carrera, J.P., Forrester, N., Wang, E., Vittor, A.Y., Haddow, A.D., López-Vergès, S.,
Abadı́a, I., Castaño, E., Sosa, N., Báez, C., Estripeaut, D., Dı́az, Y., Beltrán, D.,
Cisneros, J., Cedeño, H.G., Travassos Da Rosa, A.P., Hernandez, H., Martı́nez-
Torres, A.O., Tesh, R.B., Weaver, S.C., 2013. Eastern equine encephalitis in Latin
America. N. Engl. J. Med. 369, 732–744.
Celone, M., Okech, B., Han, B.A., Forshey, B.M., Anyamba, A., Dunford, J.,
Rutherford, G., Mita-Mendoza, N.K., Estallo, E.L., Khouri, R., De Siqueira, I.C.,
Pollett, S., 2021. A systematic review and meta-analysis of the potential non-human ani-
mal reservoirs and arthropod vectors of the Mayaro virus. PLoS Negl. Trop. Dis. 15,
e0010016.
Chamberlain, R.W., Kissling, R.E., Sikes, R.K., 1954. Studies on the north american
arthropod-borne encephalitides: VII. Estimation of amount of eastern equine encepha-
litis virus inoculated by infected aedes Aegypti1. Am. J. Epidemiol. 60, 286–291.
Chan, Y.-H., Lum, F.-M., Ng, L.F.P., 2015. Limitations of current in vivo mouse models for
the study of Chikungunya virus pathogenesis. Med. Sci. 3, 64–77.
Chan, Y.H., Teo, T.H., Torres-Ruesta, A., Hartimath, S.V., Chee, R.S., Khanapur, S.,
Yong, F.F., Ramasamy, B., Cheng, P., Rajarethinam, R., Robins, E.G., Goggi, J.L.,
Lum, F.M., Carissimo, G., Renia, L., Ng, L.F.P., 2020. Longitudinal [18F]FB-IL-2
PET Imaging to Assess the Immunopathogenicity of O’nyong-nyong Virus Infection.
Front. Immunol. 11, 894.
Charles, P.C., Trgovcich, J., Davis, N.L., Johnston, R.E., 2001. Immunopathogenesis and
immune modulation of Venezuelan equine encephalitis virus-induced disease in the
mouse. Virology 284, 190–202.
Charles, P.C., Walters, E., Margolis, F., Johnston, R.E., 1995. Mechanism of neuroinvasion
of Venezuelan equine encephalitis virus in the mouse. Virology 208, 662–671.
Chen, C.-I., Clark, D.C., Pesavento, P., Lerche, N.W., Luciw, P.A., Reisen, W.K.,
Brault, A.C., 2010. Comparative pathogenesis of epidemic and enzootic Chikungunya
viruses in a pregnant Rhesus macaque model. Am. J. Trop. Med. Hyg. 83, 1249–1258.
Chen, H., Min, N., Ma, L., Mok, C.-K., Chu, J.J.H., 2020. Adenovirus vectored IFN-α
protects mice from lethal challenge of Chikungunya virus infection. PLoS Negl.
Trop. Dis. 14, e0008910.
Chen, R., Mukhopadhyay, S., Merits, A., Bolling, B., Nasar, F., Coffey, L.L., Powers, A.,
Weaver, S.C., ICTV Report, C, 2018. ICTV virus taxonomy profile: togaviridae.
J. Gen. Virol. 99, 761–762.
Chen, W., Foo, S.-S., Rulli, N.E., Taylor, A., Sheng, K.-C., Herrero, L.J., Herring, B.L.,
Lidbury, B.A., Li, R.W., Walsh, N.C., Sims, N.A., Smith, P.N., Mahalingam, S., 2014.
Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic
bone loss. Proc. Natl. Acad. Sci. 111, 6040–6045.
Chen, W., Foo, S.-S., Taylor, A., Lulla, A., Merits, A., Hueston, L., Forwood, M.R.,
Walsh, N.C., Sims, N.A., Herrero, L.J., Mahalingam, S., Diamond, M.S., 2015.
Bindarit, an inhibitor of monocyte chemotactic protein synthesis, protects against bone
loss induced by chikungunya virus infection. J. Virol. 89, 581–593.
Cheng, G., Liu, Y., Wang, P., Xiao, X., 2016. Mosquito defense strategies against viral infec-
tion. Trends Parasitol. 32, 177–186.
Chompoosri, J., Thavara, U., Tawatsin, A., Boonserm, R., Phumee, A., Sangkitporn, S.,
Siriyasatien, P., 2016. Vertical transmission of Indian Ocean Lineage of chikungunya
virus in Aedes aegypti and Aedes albopictus mosquitoes. Parasit. Vectors 9, 227.
Chuong, C., Bates, T.A., Weger-Lucarelli, J., 2019. Infectious cDNA clones of two
strains of Mayaro virus for studies on viral pathogenesis and vaccine development.
Virology 535, 227–231.
Chusri, S., Siripaitoon, P., Silpapojakul, K., Hortiwakul, T., Charernmak, B.,
Chinnawirotpisan, P., Nisalak, A., Thaisomboonsuk, B., Klungthong, C.,
Gibbons, R.V., Jarman, R.G., 2014. Kinetics of chikungunya infections during an out-
break in Southern Thailand, 2008–2009. Am. J. Trop. Med. Hyg. 90, 410–417.
Ciota, A.T., Kramer, L.D., 2010. Insights into arbovirus evolution and adaptation from
experimental studies. Viruses 2, 2594–2617.
Cirimotich, C.M., Vela, E.M., Garver, J., Barnewall, R.E., Miller, B.D., Meister, G.T.,
Rogers, J.V., 2017. Chikungunya virus infection in Cynomolgus macaques following
Intradermal and aerosol exposure. Virol. J. 14, 135.
Claflin, S.B., Webb, C.E., 2015. Ross river virus: many vectors and unusual hosts make for an
unpredictable pathogen. PLoS Pathog. 11, e1005070.
Clarke, D.H., 1961. Two nonfatal human infections with the virus of eastern encephalitis.
Am. J. Trop. Med. Hyg. 10, 67–70.
Coffey, L.L., Carrara, A.-S., Paessler, S., Haynie, M.L., Bradley, R.D., Tesh, R.B.,
Weaver, S.C., 2004. Experimental Everglades virus infection of cotton rats
(Sigmodon hispidus). Emerg. Infect. Dis. 10, 2182–2188.
Coffey, L.L., Vasilakis, N., Brault, A.C., Powers, A.M., Tripet, F., Weaver, S.C., 2008.
Arbovirus evolution in vivo is constrained by host alternation. Proc. Natl. Acad. Sci.
U. S. A. 105, 6970–6975.
Coppenhaver, D.H., Singh, I.P., Sarzotti, M., Levy, H.B., Baron, S., 1995. Treatment of
intracranial alphavirus infections in mice by a combination of specific antibodies and
an interferon inducer. Am. J. Trop. Med. Hyg. 52, 34–40.
Cotella, J.I., Sauce, A.L., Saldarriaga, C.I., Perez, G.E., Farina, J.M., Wyss, F., Sosa
Liprandi, A., Mendoza, I., Múnera, A.G., Alexander, B., Baranchuk, A., 2021.
Chikungunya and the Heart. Cardiology 146, 324–334.
Couderc, T., Chretien, F., Schilte, C., Disson, O., Brigitte, M., Guivel-Benhassine, F.,
Touret, Y., Barau, G., Cayet, N., Schuffenecker, I., Desprès, P., Arenzana-Seisdedos,
F., Michault, A., Albert, M.L., Lecuit, M., 2008. A mouse model for Chikungunya:
young age and inefficient type-I interferon signaling are risk factors for severe disease.
Couderc, T., Khandoudi, N., Grandadam, M., Visse, C., Gangneux, N., Bagot, S.,
Prost, J.F., Lecuit, M., 2009. Prophylaxis and therapy for Chikungunya virus
infection. J Infect Dis 200, 516–523.
Cox, J., Mota, J., Sukupolvi-Petty, S., Diamond, M.S., Rico-Hesse, R., 2012. Mosquito bite
delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice.
J. Virol. 86, 7637–7649.
Croddy, E., Perez-Armendariz, C., Hart, J., 2002. Who Has These Weapons? In:
Croddy, E., Perez-Armendariz, C., Hart, J. (Eds.), Chemical and Biological
Warfare: A Comprehensive Survey for the Concerned Citizen. Springer New York,
New York, NY.
Cunha, M.S., Costa, P.A.G., Correa, I.A., De Souza, M.R.M., Calil, P.T., Da Silva, G.P.D.,
Costa, S.M., Fonseca, V.W.P., Da Costa, L.J., 2020. Chikungunya virus: an emergent
arbovirus to the South American continent and a continuous threat to the world.
Frontiers in Microbiology, 11.
Curren, E.J., Lehman, J., Kolsin, J., Walker, W.L., Martin, S.W., Staples, J.E., Hills, S.L.,
Gould, C.V., Rabe, I.B., Fischer, M., Lindsey, N.P., 2018. West nile virus and other
nationally notifiable arboviral diseases–United States, 2017. MMWR Morb. Mortal.
Wkly Rep. 67, 1137–1142.
Darman, J., Backovic, S., Dike, S., Maragakis, N.J., Krishnan, C., Rothstein, J.D.,
Irani, D.N., Kerr, D.A., 2004. Viral-induced spinal motor neuron death is non-cell-
autonomous and involves glutamate excitotoxicity. J. Neurosci. 24, 7566–7575.
Davis, M., 1980. Neurochemical modulation of sensory-motor reactivity: acoustic and tactile
startle reflexes. Neurosci. Biobehav. Rev. 4, 241–263.
De La Monte, S., Castro, F., Bonilla, N.J., Gaskin De Urdaneta, A., Hutchins, G.M., 1985.
The systemic pathology of Venezuelan equine encephalitis virus infection in humans.
Am. J. Trop. Med. Hyg. 34, 194–202.
De Lima, S.T.S., De Souza, W.M., Cavalcante, J.W., Da Silva Candido, D., Fumagalli, M.J.,
Carrera, J.P., Simões Mello, L.M., De Carvalho Araújo, F.M., Cavalcante
Ramalho, I.L., De Almeida Barreto, F.K., De Melo Braga, D.N., Simião, A.R.,
Miranda Da Silva, M.J., Alves Barbosa Oliveira, R.M., Lima, C.P.S., De Sousa
Lins, C., Barata, R.R., Pereira Melo, M.N., Caldas De Souza, M.P., Franco, L.M.,
Fernandes Távora, F.R., Queiroz Lemos, D.R., De Alencar, C.H.M., De Jesus, R.,
De Souza Fonseca, V., Dutra, L.H., De Abreu, A.L., Lima Araújo, E.L., Ribas
Freitas, A.R., Vianez Júnior, J., Pybus, O.G., Figueiredo, L.T.M., Faria, N.R.,
Nunes, M.R.T., Cavalcanti, L.P.G., Miyajima, F., 2021. Fatal outcome of
Chikungunya virus infection in Brazil. Clin. Infect. Dis. 73, e2436–e2443.
De Niz, M., Heussler, V.T., 2018. Rodent malaria models: insights into human disease and
parasite biology. Curr. Opin. Microbiol. 46, 93–101.
Deardorff, E.R., Forrester, N.L., Travassos-Da-Rosa, A.P., Estrada-Franco, J.G.,
Navarro-Lopez, R., Tesh, R.B., Weaver, S.C., 2009. Experimental infection of poten-
tial reservoir hosts with Venezuelan equine encephalitis virus, Mexico. Emerg. Infect.
Dis. 15, 519–525.
Deresiewicz, R.L., Thaler, S.J., Hsu, L., Zamani, A.A., 1997. Clinical and
neuroradiographic manifestations of eastern equine encephalitis. N. Engl. J. Med.
336, 1867–1874.
Dremov, D.P., Solyanik, R.G., Miryutova, T.L., Laptakova, L.M., 1978. Attenuated
variants of eastern equine encephalomyelitis virus: pathomorphological, immunofluores-
cence and virological studies of infection in Syrian hamsters. Acta Virol. 22, 139–145.
Dudley, D.M., Newman, C.M., Lalli, J., Stewart, L.M., Koenig, M.R., Weiler, A.M.,
Semler, M.R., Barry, G.L., Zarbock, K.R., Mohns, M.S., Breitbach, M.E.,
Schultz-Darken, N., Peterson, E., Newton, W., Mohr, E.L., Capuano Iii, S.,
Osorio, J.E., O’connor, S.L., O’connor, D.H., Friedrich, T.C., Aliota, M.T., 2017.
Infection via mosquito bite alters Zika virus tissue tropism and replication kinetics in
rhesus macaques. Nat. Commun. 8, 2096.
DuPai, C.D., Mcwhite, C.D., Smith, C.B., Garten, R., Maurer-Stroh, S., Wilke, C.O.,
2019. Influenza passaging annotations: what they tell us and why we should listen.
Virus Evol. 5. vez016.
Earnest, J.T., Basore, K., Roy, V., Bailey, A.L., Wang, D., Alter, G., Fremont, D.H.,
Diamond, M.S., 2019. Neutralizing antibodies against Mayaro virus require Fc effector
functions for protective activity. J. Exp. Med. 216, 2282–2301.
Earnest, J.T., Holmes, A.C., Basore, K., Mack, M., Fremont, D.H., Diamond, M.S., 2021.
The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies. Cell
Rep. 35, 108962.
Edwards, J.F., Higgs, S., Beaty, B.J., 1998. Mosquito feeding-induced enhancement of
Cache Valley Virus (Bunyaviridae) infection in mice. J. Med. Entomol. 35, 261–265.
Ellenbroek, B., Youn, J., 2016. Rodent models in neuroscience research: is it a rat race? Dis.
Model. Mech. 9, 1079–1087.
Fazakerley, J.K., 2004. Semliki forest virus infection of laboratory mice: a model to study the
pathogenesis of viral encephalitis. Arch. Virol. Suppl., 179–190.
Figueiredo, C.M., Neris, R.L.D.S., Gavino-Leopoldino, D., Da Silva, M.O.L.,
Almeida, J.S., Dos-Santos, J.S., Figueiredo, C.P., Bellio, M., Bozza, M.T.,
Assunção-Miranda, I., 2019. Mayaro virus replication restriction and induction of
muscular inflammation in mice are dependent on age, type-I interferon response, and
adaptive immunity. Front. Microbiol. 10.
Fletcher, N.F., Meredith, L.W., Tidswell, E.L., Bryden, S.R., Gonçalves-Carneiro, D.,
Chaudhry, Y., Shannon-Lowe, C., Folan, M.A., Lefteri, D.A., Pingen, M.,
Bailey, D., Mckimmie, C.S., Baird, A.W., 2020. A novel antiviral formulation inhibits
a range of enveloped viruses. J. Gen. Virol. 101, 1090–1102.
Forrester, N.L., Palacios, G., Tesh, R.B., Savji, N., Guzman, H., Sherman, M.,
Weaver, S.C., Lipkin, W.I., 2012. Genome-scale phylogeny of the alphavirus genus sug-
gests a marine origin. J. Virol. 86, 2729–2738.
Fox, J.M., Long, F., Edeling, M.A., Lin, H., Van Duijl-Richter, M.K.S., Fong, R.H.,
Kahle, K.M., Smit, J.M., Jin, J., Simmons, G., Doranz, B.J., Crowe Jr., J.E.,
Fremont, D.H., Rossmann, M.G., Diamond, M.S., 2015. Broadly neutralizing
alphavirus antibodies bind an epitope on E2 and inhibit entry and egress. Cell 163,
1095–1107.
Franck, P.T., Johnson, K.M., 1970. An outbreak of Venezuelan encephalitis in man in the
Panamá Canal Zone. Am. J. Trop. Med. Hyg. 19, 860–865.
Franz, A.W.E., Kantor, A.M., Passarelli, A.L., Clem, R.J., 2015. Tissue barriers to arbovirus
infection in mosquitoes. Viruses 7, 3741–3767.
Fraser, J.R., Cunningham, A.L., Clarris, B.J., Aaskov, J.G., Leach, R., 1981. Cytology of
synovial effusions in epidemic polyarthritis. Aust. N. Z. J. Med. 11, 168–173.
Frolov, I., Hoffman, T.A., Prágai, B.M., Dryga, S.A., Huang, H.V., Schlesinger, S.,
Rice, C.M., 1996. Alphavirus-based expression vectors: strategies and applications.
Proc. Natl. Acad. Sci. U. S. A. 93, 11371–11377.
Furuya-Kanamori, L., Liang, S., Milinovich, G., Soares Magalhaes, R.J., Clements, A.C.A.,
Hu, W., Brasil, P., Frentiu, F.D., Dunning, R., Yakob, L., 2016. Co-distribution and
co-infection of chikungunya and dengue viruses. BMC Infect. Dis. 16, 84.
Gallichotte, E.N., Dobos, K.M., Ebel, G.D., Hagedorn, M., Rasgon, J.L., Richardson, J.H.,
Stedman, T.T., Barfield, J.P., 2021. Towards a method for cryopreservation of mosquito
vectors of human pathogens. Cryobiology 99, 1–10.
Garcı́a-Tamayo, J., Esparza, J., Martı́nez, A.J., 1981. Placental and fetal alterations due to
Venezuelan equine encephalitis virus in rats. Infect. Immun. 32, 813–821.
Gardner, C.L., Burke, C.W., Higgs, S.T., Klimstra, W.B., Ryman, K.D., 2012. Interferon-
alpha/beta deficiency greatly exacerbates arthritogenic disease in mice infected with
wild-type chikungunya virus but not with the cell culture-adapted live-attenuated
181/25 vaccine candidate. Virology 425, 103–112.
Gardner, C.L., Ebel, G.D., Ryman, K.D., Klimstra, W.B., 2011. Heparan sulfate binding by
natural eastern equine encephalitis viruses promotes neurovirulence. Proc. Natl. Acad.
Sci. U. S. A. 108, 16026–16031.
Gardner, J., Anraku, I., Le, T.T., Larcher, T., Major, L., Roques, P., Schroder, W.A.,
Higgs, S., Suhrbier, A., 2010. Chikungunya virus arthritis in adult wild-type mice.
J. Virol. 84, 8021–8032.
Garen, P.D., Tsai, T.F., Powers, J.M., 1999. Human eastern equine encephalitis: immuno-
histochemistry and ultrastructure. Mod. Pathol. 12, 646–652.
Gerardin, P., Couderc, T., Bintner, M., Tournebize, P., Renouil, M., Lemant, J.,
Boisson, V., Borgherini, G., Staikowsky, F., Schramm, F., Lecuit, M., Michault, A.,
2016. Chikungunya virus-associated encephalitis: A cohort study on La Reunion
Island, 2005-2009. Neurology 86, 94–102.
Gomes Ade, C., De Souza, J.M., Bergamaschi, D.P., Dos Santos, J.L., Andrade, V.R.,
Leite, O.F., Rangel, O., De Souza, S.S., Guimarães, N.S., De Lima, V.L., 2005.
Anthropophilic activity of Aedes aegypti and of Aedes albopictus in area under control
and surveillance. Rev. Saude Publica 39, 206–210.
Gonzalez-Salazar, D., Estrada-Franco, J.G., Carrara, A.S., Aronson, J.F., Weaver, S.C.,
2003. Equine amplification and virulence of subtype Ie Venezuelan equine encephalitis
viruses isolated during the 1993 and 1996 Mexican epizootics. Emerg. Infect. Dis. 9,
161–168.
Gorchakov, R., Wang, E., Leal, G., Forrester, N.L., Plante, K., Rossi, S.L., Partidos, C.D.,
Adams, A.P., Seymour, R.L., Weger, J., Borland, E.M., Sherman, M.B., Powers, A.M.,
Osorio, J.E., Weaver, S.C., 2012. Attenuation of Chikungunya virus vaccine strain 181/
clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein.
J. Virol. 86, 6084–6096.
Gorelkin, L., Jahrling, P.B., 1975. Virus-initiated septic shock. Acute death of Venezuelan
encephalitis virus-infected hamsters. Lab. Invest. 32, 78–85.
Goupil, B.A., McNulty, M.A., Martin, M.J., McCracken, M.K., Christofferson, R.C.,
Mores, C.N., 2016. Novel lesions of bones and joints associated with chikungunya virus
infection in two mouse models of disease: new insights into disease pathogenesis. PLoS
One 11, e0155243.
Greene, I.P., Paessler, S., Anishchenko, M., Smith, D.R., Brault, A.C., Frolov, I.,
Weaver, S.C., 2005a. Venezuelan equine encephalitis virus in the guinea pig model: evi-
dence for epizootic virulence determinants outside the E2 envelope glycoprotein gene.
Am. J. Trop. Med. Hyg. 72, 330–338.
Greene, I.P., Paessler, S., Austgen, L., Anishchenko, M., Brault, A.C., Bowen, R.A.,
Weaver, S.C., 2005b. Envelope glycoprotein mutations mediate equine amplification
and virulence of epizootic venezuelan equine encephalitis virus. J. Virol. 79, 9128–9133.
Greene, I.P., Wang, E., Deardorff, E.R., Milleron, R., Domingo, E., Weaver, S.C., 2005c.
Effect of alternating passage on adaptation of sindbis virus to vertebrate and invertebrate
cells. J. Virol. 79, 14253–14260.
Grieder, F.B., Davis, N.L., Aronson, J.F., Charles, P.C., Sellon, D.C., Suzuki, K.,
Johnston, R.E., 1995. Specific restrictions in the progression of Venezuelan equine
encephalitis virus-induced disease resulting from single amino acid changes in the glyco-
proteins. Virology 206, 994–1006.
Grieder, F.B., Vogel, S.N., 1999. Role of interferon and interferon regulatory factors in early
protection against Venezuelan equine encephalitis virus infection. Virology 257, 106–118.
Griffin, D., Levine, B., Tyor, W., Ubol, S., Desprès, P., 1997. The role of antibody in recov-
ery from alphavirus encephalitis. Immunol. Rev. 159, 155–161.
Griffin, D.E., 1989. Molecular pathogenesis of Sindbis virus encephalitis in experimental
animals. Adv. Virus Res. 36, 255–271.
Griffin, D.E., 2010. Recovery from viral encephalomyelitis: immune-mediated noncytolytic
virus clearance from neurons. Immunol. Res. 47, 123–133.
Gunn, B.M., Jones, J.E., Shabman, R.S., Whitmore, A.C., Sarkar, S., Blevins, L.K.,
Morrison, T.E., Heise, M.T., 2018. Ross River virus envelope glycans contribute to
disease through activation of the host complement system. Virology 515, 250–260.
Gunn, B.M., Morrison, T.E., Whitmore, A.C., Blevins, L.K., Hueston, L., Fraser, R.J.,
Herrero, L.J., Ramirez, R., Smith, P.N., Mahalingam, S., Heise, M.T., 2012.
Mannose binding lectin is required for alphavirus-induced arthritis/myositis. PLoS
Pathog. 8, e1002586.
Gupta, P., Sharma, A., Han, J., Yang, A., Bhomia, M., Knollmann-Ritschel, B., Puri, R.K.,
Maheshwari, R.K., 2017. Differential host gene responses from infection with neuro-
virulent and partially-neurovirulent strains of Venezuelan equine encephalitis virus.
BMC Infect. Dis. 17, 309.
Haese, N.N., Broeckel, R.M., Hawman, D.W., Heise, M.T., Morrison, T.E.,
Streblow, D.N., 2016. Animal models of chikungunya virus infection and disease. J
Infect Dis 214, S482–s487.
Hahn, C.S., Hahn, Y.S., Braciale, T.J., Rice, C.M., 1992. Infectious Sindbis virus transient
expression vectors for studying antigen processing and presentation. Proc. Natl. Acad.
Sci. U. S. A. 89, 2679–2683.
Hardy, J.L., Houk, E.J., Kramer, L.D., Reeves, W.C., 1983. Intrinsic factors affecting vector
competence of mosquitoes for arboviruses. Annu. Rev. Entomol. 28, 229–262.
Harley, D., Sleigh, A., Ritchie, S., 2001. Ross River virus transmission, infection, and dis-
ease: a cross-disciplinary review. Clin. Microbiol. Rev. 14, 909–932. table of contents.
Hawman, D.W., Fox, J.M., Ashbrook, A.W., May, N.A., Schroeder, K.M.S., Torres, R.M.,
Crowe Jr., J.E., Dermody, T.S., Diamond, M.S., Morrison, T.E., 2016. Pathogenic
Chikungunya Virus Evades B Cell Responses to Establish Persistence. Cell Rep. 16,
1326–1338.
Hawman, D.W., Stoermer, K.A., Montgomery, S.A., Pal, P., Oko, L., Diamond, M.S.,
Morrison, T.E., 2013. Chronic joint disease caused by persistent Chikungunya virus
infection is controlled by the adaptive immune response. J. Virol. 87, 13878–13888.
Hazelton, R.A., Hughes, C., Aaskov, J.G., 1985. The inflammatory response in the syn-
ovium of a patient with Ross River arbovirus infection. Aust. N. Z. J. Med. 15, 336–339.
Herrero, L.J., Lidbury, B.A., Bettadapura, J., Jian, P., Herring, B.L., Hey-Cunningham,
W.J., Sheng, K.C., Zakhary, A., Mahalingam, S., 2014. Characterization of Barmah
Forest virus pathogenesis in a mouse model. J. Gen. Virol. 95, 2146–2154.
Hibl, B.M., Dailey Garnes, N.J.M., Kneubehl, A.R., Vogt, M.B., Spencer Clinton, J.L.,
Rico-Hesse, R.R., 2021. Mosquito-bite infection of humanized mice with chi-
kungunya virus produces systemic disease with long-term effects. PLoS Negl. Trop.
Dis. 15, e0009427.
Higgs, S., Powers, A.M., Olson, K.E., 1993. Alphavirus expression systems: applications to
mosquito vector studies. Parasitol. Today 9, 444–452.
Higgs, S., Ziegler, S.A., 2010. A nonhuman primate model of chikungunya disease. J. Clin.
Invest. 120, 657–660.
Hoarau, J.J., Jaffar Bandjee, M.C., Krejbich Trotot, P., Das, T., Li-Pat-Yuen, G., Dassa, B.,
Denizot, M., Guichard, E., Ribera, A., Henni, T., Tallet, F., Moiton, M.P.,
Gauzere, B.A., Bruniquet, S., Jaffar Bandjee, Z., Morbidelli, P., Martigny, G.,
Jolivet, M., Gay, F., Grandadam, M., Tolou, H., Vieillard, V., Debre, P., Autran, B.,
Gasque, P., 2010. Persistent chronic inflammation and infection by Chikungunya
arthritogenic alphavirus in spite of a robust host immune response. J. Immunol. 184,
5914–5927.
Hollidge, B.S., Cohen, C.A., Akuoku Frimpong, J., Badger, C.V., Dye, J.M.,
Schmaljohn, C.S., 2021. Toll-like receptor 4 mediates blood-brain barrier permeability
and disease in C3H mice during Venezuelan equine encephalitis virus infection.
Virulence 12, 430–443.
Honnold, S.P., Mossel, E.C., Bakken, R.R., Fisher, D., Lind, C.M., Cohen, J.W.,
Eccleston, L.T., Spurgers, K.B., Erwin-Cohen, R., Bradfute, S.B.,
Maheshwari, R.K., Glass, P.J., 2015a. Eastern equine encephalitis virus in mice I: clinical
course and outcome are dependent on route of exposure. Virol. J. 12, 152.
Honnold, S.P., Mossel, E.C., Bakken, R.R., Lind, C.M., Cohen, J.W., Eccleston, L.T.,
Spurgers, K.B., Erwin-Cohen, R., Glass, P.J., Maheshwari, R.K., 2015b. Eastern equine
encephalitis virus in mice Ii: pathogenesis is dependent on route of exposure. Virol.
J. 12, 154.
Howard, A.T., 1974. Experimental infection and intracage transmission of Venezuelan
equine encephalitis virus (subtype IB) among cotton rats, Sigmodon hispidus (Say and
Ord). Am. J. Trop. Med. Hyg. 23, 1178–1184.
Huang, Y.-J.S., Higgs, S., Vanlandingham, D.L., 2019. Arbovirus-mosquito vector-host
interactions and the impact on transmission and disease pathogenesis of arboviruses.
Front. Microbiol. 10.
Hughes, H.R., Velez, J.O., Davis, E.H., Laven, J., Gould, C.V., Panella, A.J., Lambert, A.J.,
Staples, J.E., Brault, A.C., 2021. Fatal human infection with evidence of intrahost var-
iation of eastern equine encephalitis virus, Alabama, USA, 2019. Emerg. Infect. Dis. 27,
1886–1892.
Hwang, S.Y., Hertzog, P.J., Holland, K.A., Sumarsono, S.H., Tymms, M.J., Hamilton, J.A.,
Whitty, G., Bertoncello, I., Kola, I., 1995. A null mutation in the gene encoding a type I
interferon receptor component eliminates antiproliferative and antiviral responses to
interferons alpha and beta and alters macrophage responses. Proc. Natl. Acad. Sci.
U. S. A. 92, 11284–11288.
Jackson, A.C., Sengupta, S.K., Smith, J.F., 1991. Pathogenesis of Venezuelan equine
encephalitis virus infection in mice and hamsters. Vet. Pathol. 28, 410–418.
Jahrling, P.B., Depaoli, A., Powanda, M.C., 1978. Pathogenesis of a Venezuelan encephalitis
virus strain lethal for adult white rats. J. Med. Virol. 2, 109–116.
Jahrling, P.B., Scherer, F., 1973. Histopathology and distribution of viral antigens in hamsters
infected with virulent and benign Venezuelan encephalitis viruses. Am. J. Pathol. 72,
25–38.
Javelle, E., Ribera, A., Degasne, I., Ga€ uzère, B.-A., Marimoutou, C., Simon, F., 2015.
Specific management of post-chikungunya rheumatic disorders: A Retrospective
Study of 159 Cases in Reunion Island from 2006-2012. PLoS Negl. Trop. Dis. 9,
e0003603.
Johnson, K.M., Martin, D.H., 1974. Venezuelan equine encephalitis. Adv. Vet. Sci. Comp.
Med. 18, 79–116.
Julander, J.G., Siddharthan, V., Blatt, L.M., Schafer, K., Sidwell, R.W., Morrey, J.D., 2007.
Effect of exogenous interferon and an interferon inducer on western equine encephalitis
virus disease in a hamster model. Virology 360, 454–460.
Jupille, H.J., Oko, L., Stoermer, K.A., Heise, M.T., Mahalingam, S., Gunn, B.M.,
Morrison, T.E., 2011. Mutations in nsP1 and PE2 are critical determinants of Ross
River virus-induced musculoskeletal inflammatory disease in a mouse model.
Virology 410, 216–227.
Kafai, N.M., Diamond, M.S., Fox, J.M., 2022. Distinct cellular tropism and immune
responses to Alphavirus infection. Annu. Rev. Immunol. 40, 615–649.
Kain, M.P., Skinner, E.B., Van Den Hurk, A.F., McCallum, H., Mordecai, E.A., 2021.
Physiology and ecology combine to determine host and vector importance for Ross
River virus. Elife 10, e67018.
Kam, Y.W., Simarmata, D., Chow, A., Her, Z., Teng, T.S., Ong, E.K., Renia, L., Leo, Y.S.,
Ng, L.F., 2012. Early appearance of neutralizing immunoglobulin G3 antibodies is asso-
ciated with chikungunya virus clearance and long-term clinical protection. J Infect Dis
205, 1147–1154.
Khoo, C.C.H., Piper, J., Sanchez-Vargas, I., Olson, K.E., Franz, A.W.E., 2010. The RNA
interference pathway affects midgut infection- and escape barriers for Sindbis virus in
Aedes aegypti. BMC Microbiol. 10, 130.
Kinney, R.M., Johnson, B.J., Welch, J.B., Tsuchiya, K.R., Trent, D.W., 1989. The
full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan
equine encephalitis virus and its attenuated vaccine derivative, strain Tc-83. Virology
170, 19–30.
Kissling, R.E., Chamberlain, R.W., Nelson, D.B., Stamm, D.D., 1956. Venezuelan equine
encephalomyelitis in horses. Am. J. Hyg. 63, 274–287.
Kobiler, D., Rice, C.M., Brodie, C., Shahar, A., Dubuisson, J., Halevy, M., Lustig, S.,
1999. A single nucleotide change in the 5’ noncoding region of Sindbis virus confers
neurovirulence in rats. J. Virol. 73, 10440–10446.
Koterski, J., Twenhafel, N., Porter, A., Reed, D.S., Martino-Catt, S., Sobral, B., Crasta, O.,
Downey, T., Dasilva, L., 2007. Gene expression profiling of nonhuman primates
exposed to aerosolized Venezuelan equine encephalitis virus. FEMS Immunol. Med.
Microbiol. 51, 462–472.
Kulcsar, K.A., Baxter, V.K., Abraham, R., Nelson, A., Griffin, D.E., 2015. Distinct immune
responses in resistant and susceptible strains of mice during neurovirulent alphavirus
encephalomyelitis. J. Virol. 89, 8280–8291.
Kuno, G., Chang, G.-J.J., 2005. Biological transmission of arboviruses: reexamination of and
new insights into components, mechanisms, and unique traits as well as their evolution-
ary trends. Clin. Microbiol. Rev. 18, 608–637.
Kurkela, S., Manni, T., Vaheri, A., Vapalahti, O., 2004. Causative agent of Pogosta disease
isolated from blood and skin lesions. Emerg. Infect. Dis. 10, 889–894.
Labadie, K., Larcher, T., Joubert, C., Mannioui, A., Delache, B., Brochard, P., Guigand, L.,
Dubreil, L., Lebon, P., Verrier, B., De Lamballerie, X., Suhrbier, A., Cherel, Y., Le
Grand, R., Roques, P., 2010. Chikungunya disease in nonhuman primates involves
long-term viral persistence in macrophages. J. Clin. Invest. 120, 894–906.
Labrada, L., Liang, X.H., Zheng, W., Johnston, C., Levine, B., 2002. Age-dependent resis-
tance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central
nervous system and identification of a novel interferon-inducible protective gene, mouse
ISG12. J. Virol. 76, 11688–11703.
Langsjoen, R.M., Haller, S.L., Roy, C.J., Vinet-Oliphant, H., Bergren, N.A., Erasmus, J.H.,
Livengood, J.A., Powell, T.D., Weaver, S.C., Rossi, S.L., Denison, M.R.,
Morrison, T., Mahalingam, S., 2018. Chikungunya virus strains show lineage-specific
variations in virulence and cross-protective ability in murine and nonhuman primate
models. MBio 9. e02449–17.
Le Coupanec, A., Babin, D., Fiette, L., Jouvion, G., Ave, P., Misse, D., Bouloy, M.,
Choumet, V., 2013. Aedes mosquito saliva modulates Rift Valley fever virus pathoge-
nicity. PLoS Negl. Trop. Dis. 7, e2237.
Le Coupanec, A., Tchankouo-Nguetcheu, S., Roux, P., Khun, H., Huerre, M.,
Morales-Vargas, R., Enguehard, M., Lavillette, D., Misse, D., Choumet, V., 2017.
Co-infection of mosquitoes with chikungunya and dengue viruses reveals modulation
of the replication of both viruses in midguts and Salivary glands of Aedes aegypti
Mosquitoes. Int. J. Mol. Sci. 18, 1708.
Le Goff, G., Revollo, J., Guerra, M., Cruz, M., Barja Simon, Z., Roca, Y., Vargas Florès, J.,
Herve, J.P., 2011. Natural vertical transmission of dengue viruses by Aedes aegypti in
Bolivia. Parasite 18, 277–280.
Leung, C., Jia, Z., 2016. Mouse genetic models of human brain disorders. Front. Genet. 7.
Levine, B., Griffin, D.E., 1992. Persistence of viral RNA in mouse brains after recovery from
acute alphavirus encephalitis. J. Virol. 66, 6429–6435.
Levis, R., Weiss, B.G., Tsiang, M., Huang, H., Schlesinger, S., 1986. Deletion mapping of
Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replica-
tion and packaging. Cell 44, 137–145.
Levitt, N.H., Ramsburg, H.H., Hasty, S.E., Repik, P.M., Cole Jr., F.E., Lupton, H.W.,
1986. Development of an attenuated strain of chikungunya virus for use in vaccine
production. Vaccine 4, 157–162.
Lidbury, B.A., Simeonovic, C., Maxwell, G.E., Marshall, I.D., Hapel, A.J., 2000.
Macrophage-induced muscle pathology results in morbidity and mortality for Ross
River virus-infected mice. J Infect Dis 181, 27–34.
Limesand, K.H., Higgs, S., Pearson, L.D., Beaty, B.J., 2000. Potentiation of vesicular stoma-
titis New Jersey virus infection in mice by mosquito saliva. Parasite Immunol. 22,
461–467.
Liu, W.J., Rourke, M.F., Holmes, E.C., Aaskov, J.G., 2011. Persistence of multiple genetic
lineages within intrahost populations of Ross River virus. J. Virol. 85, 5674–5678.
Liu, Y., Cheng, G., 2016. Techniques for experimental infection of mosquitoes with West
Nile Virus. Methods Mol. Biol. 1435, 151–163.
Logue, C.H., Bosio, C.F., Welte, T., Keene, K.M., Ledermann, J.P., Phillips, A.,
Sheahan, B.J., Pierro, D.J., Marlenee, N., Brault, A.C., Bosio, C.M., Singh, A.J.,
Powers, A.M., Olson, K.E., 2009. Virulence variation among isolates of western equine
encephalitis virus in an outbred mouse model. J. Gen. Virol. 90, 1848–1858.
Lury, K.M., Castillo, M., 2004. Eastern equine encephalitis: CT and Mri findings in one case.
Emerg. Radiol. 11, 46–48.
Lustig, S., Jackson, A.C., Hahn, C.S., Griffin, D.E., Strauss, E.G., Strauss, J.H., 1988.
Molecular basis of Sindbis virus neurovirulence in mice. J. Virol. 62, 2329–2336.
Ma, H., Kim, A.S., Kafai, N.M., Earnest, J.T., Shah, A.P., Case, J.B., Basore, K.,
Gilliland, T.C., Sun, C., Nelson, C.A., Thackray, L.B., Klimstra, W.B.,
Fremont, D.H., Diamond, M.S., 2020a. LDLRAD3 is a receptor for Venezuelan equine
encephalitis virus. Nature 588, 308–314.
Ma, H., Lundy, J.D., Cottle, E.L., O’malley, K.J., Trichel, A.M., Klimstra, W.B.,
Hartman, A.L., Reed, D.S., Teichert, T., 2020b. Applications of minimally invasive
multimodal telemetry for continuous monitoring of brain function and intracranial
pressure in macaques with acute viral encephalitis. PLoS One 15, e0232381.
Ma, H., Lundy, J.D., O’malley, K.J., Klimstra, W.B., Hartman, A.L., Reed, D.S., 2019.
Electrocardiography Abnormalities in Macaques after Infection with Encephalitic
Alphaviruses. Pathogens (Basel, Switzerland) 8, 240.
Mala, W., Wilairatana, P., Kotepui, K.U., Kotepui, M., 2021. Prevalence of malaria and
chikungunya co-infection in febrile patients: a systematic review and meta-analysis.
Trop. Med. Infect Dis. 6.
Malherbe, H., Strickland-Cholmley, M., Jackson, A.L., 1963. Sindbis virus infection in man.
Report of a case with recovery of virus from skin lesions. S. Afr. Med. J. 37, 547–552.
Manimunda, S.P., Vijayachari, P., Uppoor, R., Sugunan, A.P., Singh, S.S., Rai, S.K.,
Sudeep, A.B., Muruganandam, N., Chaitanya, I.K., Guruprasad, D.R., 2010. Clinical
progression of chikungunya fever during acute and chronic arthritic stages and the
changes in joint morphology as revealed by imaging. Trans. R. Soc. Trop. Med.
Hyg. 104, 392–399.
Masopust, D., Sivula, C.P., Jameson, S.C., 2017. Of mice, dirty mice, and men: using mice to
understand human immunology. J. Immunol. 199, 383–388.
Mathiot, C.C., Grimaud, G., Garry, P., Bouquety, J.C., Mada, A., Daguisy, A.M.,
Georges, A.J., 1990. An outbreak of human Semliki Forest virus infections in Central
African Republic. Am. J. Trop. Med. Hyg. 42, 386–393.
McCarthy, M.K., Davenport, B.J., Reynoso, G.V., Lucas, E.D., May, N.A., Elmore, S.A.,
Tamburini, B.A., Hickman, H.D., Morrison, T.E., 2018. Chikungunya virus impairs
draining lymph node function by inhibiting HEV-mediated lymphocyte recruitment.
JCI Insight 3.
McCarthy, M.K., Davenport, B.J.J., Morrison, T.E., 2019. Chronic chikungunya virus
disease. Curr. Top. Microbiol. Immunol.
McCracken, M.K., Christofferson, R.C., Chisenhall, D.M., Mores, C.N., 2014. Analysis of
early dengue virus infection in mice as modulated by Aedes aegypti probing. J. Virol. 88,
1881–1889.
McFarlane, M., Arias-Goeta, C., Martin, E., O’hara, Z., Lulla, A., Mousson, L.,
Rainey, S.M., Misbah, S., Schnettler, E., Donald, C.L., Merits, A., Kohl, A.,
Failloux, A.-B., 2014. Characterization of aedes aegypti innate-immune pathways that
limit Chikungunya virus replication. PLoS Negl. Trop. Dis. 8, e2994.
Mclean, R.G., Crans, W.J., Caccamise, D.F., McNelly, J., Kirk, L.J., Mitchell, C.J.,
Calisher, C.H., 1995. Experimental infection of wading birds with eastern equine
Encephalitis virus. J. Wildl. Dis. 31, 502–508.
Messaoudi, I., Vomaske, J., Totonchy, T., Kreklywich, C.N., Haberthur, K., Springgay, L.,
Brien, J.D., Diamond, M.S., Defilippis, V.R., Streblow, D.N., 2013. Chikungunya virus
infection results in higher and persistent viral replication in aged rhesus macaques due to
defects in anti-viral immunity. PLoS Negl. Trop. Dis. 7, e2343.
Miao, J., Chard, L.S., Wang, Z., Wang, Y., 2019. Syrian hamster as an animal model for the
study on infectious diseases. Front. Immunol. 10.
Michie, A., Ernst, T., Chua, I.J., Lindsay, M.D.A., Neville, P.J., Nicholson, J., Jardine, A.,
Mackenzie, J.S., Smith, D.W., Imrie, A., 2020. Phylogenetic and timescale analysis
of barmah forest virus as inferred from genome sequence analysis. Viruses 12.
Michlmayr, D., Mckimmie, C.S., Pingen, M., Haxton, B., Mansfield, K., Johnson, N.,
Fooks, A.R., Graham, G.J., 2014. Defining the chemokine basis for leukocyte recruit-
ment during viral encephalitis. J. Virol. 88, 9553–9567.
Miller, M.R., Sorensen, M.R., Markle, E.D., Clarkson, T.C., Knight, A.L., Savran, M.J.,
Foy, B.D., 2021. Characterizing and quantifying arbovirus transmission by aedes aegypti
using forced salivation and analysis of bloodmeals. Insects 12, 304.
Mitchell, C.J., Mclean, R.G., Nasci, R.S., Crans, W.J., Smith, G.C., Caccamise, D.F., 1993.
Susceptibility parameters of aedes albopictus to per oral infection with eastern equine
Encephalitis virus. J. Med. Entomol. 30, 233–235.
Mogami, R., Vaz, J.L.P., Chagas, Y.D.F.B., Torezani, R.S., Vieira, A.D.A.,
Koifman, A.C.B., Barbosa, Y.B., De Abreu, M.M., 2017. Ultrasound of ankles in the
diagnosis of complications of chikungunya fever. Radiol. Bras. 50, 71–75.
Mokhtarian, F., Shi, Y., Zhu, P.F., Grob, D., 1994. Immune responses, and autoimmune
outcome, during virus infection of the central nervous system. Cell. Immunol. 157,
195–210.
Mombaerts, P., Iacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S., Papaioannou, V.E.,
1992. Rag-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877.
Monlux, W.S., Luedke, A.J., 1973. Brain and spinal cord lesions in horses inoculated with
Venezuelan equine encephalomyelitis virus (epidemic American and Trinidad strains).
Am. J. Vet. Res. 34, 465–473.
Morens, D.M., Folkers, G.K., Fauci, A.S., 2019. Eastern equine encephalitis virus — another
emergent arbovirus in the United States. N. Engl. J. Med. 381, 1989–1992.
Morrison, T.E., Fraser, R.J., Smith, P.N., Mahalingam, S., Heise, M.T., 2007. Complement
contributes to inflammatory tissue destruction in a mouse model of Ross River
virus-induced disease. J. Virol. 81, 5132–5143.
Morrison, T.E., Oko, L., Montgomery, S.A., Whitmore, A.C., Lotstein, A.R., Gunn, B.M.,
Elmore, S.A., Heise, M.T., 2011. A mouse model of chikungunya virus-induced
musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis,
and persistence. Am. J. Pathol. 178, 32–40.
Morrison, T.E., Simmons, J.D., Heise, M.T., 2008. Complement receptor 3 promotes
severe ross river virus-induced disease. J. Virol. 82, 11263–11272.
Morrison, T.E., Whitmore, A.C., Shabman, R.S., Lidbury, B.A., Mahalingam, S.,
Heise, M.T., 2006. Characterization of Ross River virus tropism and virus-induced
inflammation in a mouse model of viral arthritis and myositis. J. Virol. 80, 737–749.
Moser, L.A., Lim, P.Y., Styer, L.M., Kramer, L.D., Bernard, K.A., 2016. Parameters of
mosquito-enhanced west nile virus infection. J. Virol. 90, 292–299.
Myles, K.M., Wiley, M.R., Morazzani, E.M., Adelman, Z.N., 2008. Alphavirus-derived
small RNAs modulate pathogenesis in disease vector mosquitoes. Proc. Natl. Acad.
Sci. 105, 19938–19943.
Nagata, L.P., Hu, W.G., Parker, M., Chau, D., Rayner, G.A., Schmaltz, F.L., Wong, J.P.,
2006. Infectivity variation and genetic diversity among strains of Western equine
encephalitis virus. J. Gen. Virol. 87, 2353–2361.
Ng, L.F.P., 2017. Immunopathology of Chikungunya Virus Infection: Lessons Learned from
Patients and Animal Models. Annu. Rev. Virol. 4, 413–427.
Noll, K.E., Whitmore, A.C., West, A., McCarthy, M.K., Morrison, C.R., Plante, K.S.,
Hampton, B.K., Kollmus, H., Pilzner, C., Leist, S.R., Gralinski, L.E.,
Menachery, V.D., Sch€afer, A., Miller, D., Shaw, G., Mooney, M., Mcweeney, S.,
Pardo-Manuel De Villena, F., Schughart, K., Morrison, T.E., Baric, R.S.,
Ferris, M.T., Heise, M.T., 2020. Complex genetic architecture underlies regulation
of influenza-a-virus-specific antibody responses in the collaborative cross. Cell
Rep. 31, 107587.
Oberste, M.S., Fraire, M., Navarro, R., Zepeda, C., Zarate, M.L., Ludwig, G.V.,
Kondig, J.F., Weaver, S.C., Smith, J.F., Rico-Hesse, R., 1998. Association of
Venezuelan equine encephalitis virus subtype IE with two equine epizootics in
Mexico. Am. J. Trop. Med. Hyg. 59, 100–107.
Osorio, J.E., Godsey, M.S., Defoliart, G.R., Yuill, T.M., 1996. La Crosse viremias in
white-tailed deer and chipmunks exposed by injection or mosquito bite. Am.
J. Trop. Med. Hyg. 54, 338–342.
Ozden, S., Huerre, M., Riviere, J.P., Coffey, L.L., Afonso, P.V., Mouly, V., De
Monredon, J., Roger, J.C., El Amrani, M., Yvin, J.L., Jaffar, M.C., Frenkiel, M.P.,
Sourisseau, M., Schwartz, O., Butler-Browne, G., Desprès, P., Gessain, A.,
Ceccaldi, P.E., 2007. Human muscle satellite cells as targets of Chikungunya virus
infection. PLoS One 2, e527.
Paessler, S., Aguilar, P., Anishchenko, M., Wang, H.-Q., Aronson, J., Campbell, G.,
Cararra, A.-S., Weaver, S.C., 2004. The hamster as an animal model for eastern equine
encephalitis—and its use in studies of virus entrance into the Brain. J Infect Dis 189,
2072–2076.
Paessler, S., Yun, N.E., Judy, B.M., Dziuba, N., Zacks, M.A., Grund, A.H., Frolov, I.,
Campbell, G.A., Weaver, S.C., Estes, D.M., 2007. Alpha-beta T cells provide protection
against lethal encephalitis in the murine model of VEEV infection. Virology 367,
307–323.
Pasaribu, A.P., Alam, A., Sembiring, K., Pasaribu, S., Setiabudi, D., 2019. Prevalence and
risk factors of soil-transmitted helminthiasis among school children living in an agricul-
tural area of North Sumatera, Indonesia. BMC Public Health 19, 1066.
Pezzi, L., Diallo, M., Rosa-Freitas, M.G., Vega-Rua, A., Ng, L.F.P., Boyer, S., Drexler, J.F.,
Vasilakis, N., Lourenco-De-Oliveira, R., Weaver, S.C., Kohl, A., De Lamballerie, X.,
Failloux, A.B., Brasil, P., Busch, M., Diamond, M.S., Drebot, M.A., Gallian, P.,
Jaenisch, T., Labeaud, A.D., Lecuit, M., Neyts, J., Reusken, C.B., Ribeiro, G.S.,
Rios, M., Rodriguez-Morales, A.J., Sall, A., Simmons, G., Simon, F.,
Siqueira, A.M., 2020. GloPID-R report on chikungunya, o’nyong-nyong and
Mayaro virus, part 5: Entomological aspects. Antiviral Res. 174, 104670.
Pezzi, L., Rodriguez-Morales, A.J., Reusken, C.B., Ribeiro, G.S., Labeaud, A.D.,
Lourenço-De-Oliveira, R., Brasil, P., Lecuit, M., Failloux, A.B., Gallian, P.,
Jaenisch, T., Simon, F., Siqueira, A.M., Rosa-Freitas, M.G., Vega Rua, A.,
Weaver, S.C., Drexler, J.F., Vasilakis, N., De Lamballerie, X., 2019. GloPID-R report
on chikungunya, o’nyong-nyong and Mayaro virus, part 3: Epidemiological distribution
of Mayaro virus. Antiviral Res. 172, 104610.
Phelps, A.L., O’brien, L.M., Eastaugh, L.S., Davies, C., Lever, M.S., Ennis, J., Zeitlin, L.,
Nunez, A., Ulaeto, D.O., 2019. Aerosol infection of Balb/c mice with eastern equine
encephalitis virus; susceptibility and lethality. Virol. J. 16, 2.
Phillips, A.T., Rico, A.B., Stauft, C.B., Hammond, S.L., Aboellail, T.A., Tjalkens, R.B.,
Olson, K.E., 2016. Entry sites of venezuelan and western equine Encephalitis viruses
in the mouse central nervous system following peripheral infection. J. Virol. 90,
5785–5796.
Phillips, A.T., Stauft, C.B., Aboellail, T.A., Toth, A.M., Jarvis, D.L., Powers, A.M.,
Olson, K.E., 2013. Bioluminescent imaging and histopathologic characterization of
WEEV neuroinvasion in outbred CD-1 mice. PLoS One 8, e53462.
Pierro, D.J., Powers, E.L., Olson, K.E., 2008. Genetic determinants of Sindbis virus mos-
quito infection are associated with a highly conserved alphavirus and flavivirus envelope
sequence. J. Virol. 82, 2966–2974.
Pingen, M., Bryden, S.R., Pondeville, E., Schnettler, E., Kohl, A., Merits, A.,
Fazakerley, J.K., Graham, G.J., Mckimmie, C.S., 2016. Host inflammatory response
to mosquito bites enhances the severity of arbovirus infection. Immunity 44, 1455–1469.
Porter, A.I., Erwin-Cohen, R.A., Twenhafel, N., Chance, T., Yee, S.B., Kern, S.J.,
Norwood, D., Hartman, L.J., Parker, M.D., Glass, P.J., Dasilva, L., 2017.
Characterization and pathogenesis of aerosolized eastern equine encephalitis in the com-
mon marmoset (Callithrix jacchus). Virol. J. 14, 25.
Powers, A.M., Brault, A.C., Shirako, Y., Strauss, E.G., Kang, W., Strauss, J.H.,
Weaver, S.C., 2001. Evolutionary relationships and systematics of the alphaviruses.
J. Virol. 75, 10118–10131.
Prescott, J., Feldmann, H., Safronetz, D., 2017. Amending Koch’s postulates for viral disease:
When "growth in pure culture" leads to a loss of virulence. Antiviral Res. 137, 1–5.
Ratsitorahina, M., Harisoa, J., Ratovonjato, J., Biacabe, S., Reynes, J.-M., Zeller, H.,
Raoelina, Y., Talarmin, A., Richard, V., Soares, J.L., 2008. Outbreak of dengue and
chikungunya fevers, Toamasina, Madagascar, 2006. Emerg. Infect. Dis. 14, 1135.
Reddy, A.J., Woods, C.W., Welty-Wolf, K.E., 2008. Eastern equine encephalitis leading to
multi-organ failure and sepsis. J. Clin. Virol. 42, 418–421.
Reed, D.S., Lackemeyer, M.G., Garza, N.L., Norris, S., Gamble, S., Sullivan, L.J.,
Lind, C.M., Raymond, J.L., 2007. Severe Encephalitis in cynomolgus macaques exposed
to aerosolized eastern equine Encephalitis virus. J Infect Dis 196, 441–450.
Reed, D.S., Larsen, T., Sullivan, L.J., Lind, C.M., Lackemeyer, M.G., Pratt, W.D.,
Parker, M.D., 2005. Aerosol exposure to western equine Encephalitis virus causes fever
and Encephalitis in cynomolgus macaques. J Infect Dis 192, 1173–1182.
Reed, D.S., Lind, C.M., Sullivan, L.J., Pratt, W.D., Parker, M.D., 2004. Aerosol Infection of
Cynomolgus Macaques with Enzootic Strains of Venezuelan Equine Encephalitis
Viruses. J Infect Dis 189, 1013–1017.
Renault, P., Solet, J.L., Sissoko, D., Balleydier, E., Larrieu, S., Filleul, L., Lassalle, C.,
Thiria, J., Rachou, E., De Valk, H., Ilef, D., Ledrans, M., Quatresous, I., Quenel, P.,
Pierre, V., 2007. A major epidemic of chikungunya virus infection on Reunion
Island, France, 2005-2006. Am. J. Trop. Med. Hyg. 77, 727–731.
Rice, C.M., Levis, R., Strauss, J.H., Huang, H.V., 1987. Production of infectious RNA
transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a
temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.
J. Virol. 61, 3809–3819.
Riswari, S.F., Ma’roef, C.N., Djauhari, H., Kosasih, H., Perkasa, A., Yudhaputri, F.A.,
Artika, I.M., Williams, M., Van Der Ven, A., Myint, K.S., Alisjahbana, B.,
Ledermann, J.P., Powers, A.M., Jaya, U.A., 2016. Study of viremic profile in febrile
specimens of chikungunya in Bandung, Indonesia. J. Clin. Virol. 74, 61–65.
Rohani, A., Zamree, I., Joseph, R.T., Lee, H.L., 2008. Persistency of transovarial
dengue virus in Aedes aegypti (Linn.). Southeast Asian J. Trop. Med. Public Health
39, 813–816.
Ronca, S.E., Dineley, K.T., Paessler, S., 2016. Neurological sequelae resulting from
Encephalitic alphavirus infection. Front. Microbiol. 7, 959.
Ronca, S.E., Smith, J., Koma, T., Miller, M.M., Yun, N., Dineley, K.T., Paessler, S., 2017.
Mouse model of neurological complications resulting from Encephalitic alphavirus
infection. Front. Microbiol. 8, 188.
Rosen, L., Shroyer, D.A., Tesh, R.B., Freier, J.E., Lien, J.C., 1983. Transovarial transmis-
sion of dengue viruses by mosquitoes: Aedes albopictus and Aedes aegypti. Am. J. Trop.
Med. Hyg. 32, 1108–1119.
Roy, C.J., Adams, A.P., Wang, E., Plante, K., Gorchakov, R., Seymour, R.L.,
Vinet-Oliphant, H., Weaver, S.C., 2014. Chikungunya vaccine candidate is highly
attenuated and protects nonhuman primates against telemetrically monitored disease
following a single dose. J Infect Dis 209, 1891–1899.
Roy, C.J., Reed, D.S., Wilhelmsen, C.L., Hartings, J., Norris, S., Steele, K.E., 2009.
Pathogenesis of aerosolized Eastern Equine Encephalitis virus infection in guinea pigs.
Virol. J. 6, 170.
Rudd, P.A., Wilson, J., Gardner, J., Larcher, T., Babarit, C., Le, T.T., Anraku, I.,
Kumagai, Y., Loo, Y.M., Gale Jr., M., Akira, S., Khromykh, A.A., Suhrbier, A.,
2012. Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic
fever and shock. J. Virol. 86, 9888–9898.
Ryman, K.D., Gardner, C.L., Meier, K.C., Biron, C.A., Johnston, R.E., Klimstra, W.B.,
2007. Early restriction of alphavirus replication and dissemination contributes to
age-dependent attenuation of systemic hyperinflammatory disease. J. Gen. Virol. 88,
518–529.
Ryzhikov, A.B., Ryabchikova, E.I., Sergeev, A.N., Tkacheva, N.V., 1995. Spread of
Venezuelan equine encephalitis virus in mice olfactory tract. Arch. Virol. 140,
2243–2254.
Sahu, S.P., Pedersen, D.D., Jenny, A.L., Schmitt, B.J., Alstad, A.D., 2003. Pathogenicity of a
Venezuelan equine encephalomyelitis serotype IE virus isolate for ponies. Am. J. Trop.
Med. Hyg. 68, 485–494.
Salminen, A., 2022. Clinical perspectives on the age-related increase of immunosuppressive
activity. J. Mol. Med. (Berl) 100, 697–712.
Sammin, D.J., Butler, D., Atkins, G.J., Sheahan, B.J., 1999. Cell death mechanisms in the
olfactory bulb of rats infected intranasally with Semliki forest virus. Neuropathol.
Appl. Neurobiol. 25, 236–243.
Santiago, F.W., Halsey, E.S., Siles, C., Vilcarromero, S., Guevara, C., Silvas, J.A., Ramal, C.,
Ampuero, J.S., Aguilar, P.V., 2015. Long-term arthralgia after mayaro virus infection
correlates with sustained pro-inflammatory cytokine response. PLoS Negl. Trop. Dis.
9, e0004104.
Saxton-Shaw, K.D., Ledermann, J.P., Borland, E.M., Stovall, J.L., Mossel, E.C., Singh, A.J.,
Wilusz, J., Powers, A.M., 2013. O’nyong nyong virus molecular determinants of unique
vector specificity reside in non-structural protein 3. PLoS Negl. Trop. Dis. 7, e1931.
Scherer, W.F., Chin, J., Ordonez, J.V., 1979. further observations on infections of guinea
pigs with venezuelan Encephalitis virus strains. Am. J. Trop. Med. Hyg. 28, 725–728.
Scherer, W.F., Ordonez, J.V., Jahrling, P.B., Pancake, B.A., Dickerman, R.W., 1972.
Observations of equines, humans and domestic and wild vertebrates during the 1969
equine epizootic and epidemic of Venezuelan encephalitis in Guatemala. Am.
J. Epidemiol. 95, 255–266.
Schilte, C., Buckwalter, M.R., Laird, M.E., Diamond, M.S., Schwartz, O., Albert, M.L.,
2012. Cutting edge: independent roles for IRF-3 and IRF-7 in hematopoietic and
nonhematopoietic cells during host response to Chikungunya infection. J. Immunol.
188, 2967–2971.
Schneider, B.S., Soong, L., Girard, Y.A., Campbell, G., Mason, P., Higgs, S., 2006.
Potentiation of West Nile encephalitis by mosquito feeding. Viral Immunol. 19, 74–82.
Schneider, B.S., Soong, L., Zeidner, N.S., Higgs, S., 2004. Aedes aegypti Salivary Gland
Extracts Modulate anti-viral and TH1/TH2 Cytokine responses to Sindbis virus infec-
tion. Viral Immunol. 17, 565–573.
Seabaugh, R.C., Olson, K.E., Higgs, S., Carlson, J.O., Beaty, B.J., 1998. Development of a
chimeric sindbis virus with Enhancedper OsInfection ofAedes aegypti. Virology 243,
99–112.
Seymour, R.L., Rossi, S.L., Bergren, N.A., Plante, K.S., Weaver, S.C., 2013. The role of
innate versus adaptive immune responses in a mouse model of O’nyong-nyong virus
infection. Am. J. Trop. Med. Hyg. 88, 1170–1179.
Sharp, T.M., Keating, M.K., Shieh, W.J., Bhatnagar, J., Bollweg, B.C., Levine, R.,
Blau, D.M., Torres, J.V., Rivera, A., Perez-Padilla, J., Munoz-Jordan, J.,
Sanabria, D., Fischer, M., Rivera Garcia, B., Tomashek, K.M., Zaki, S.R., 2021.
Clinical characteristics, histopathology, and tissue immunolocalization of chikungunya
virus antigen in fatal cases. Clin. Infect. Dis. 73, e345–e354.
Simon, F., Parola, P., Grandadam, M., Fourcade, S., Oliver, M., Brouqui, P., Hance, P.,
Kraemer, P., Mohamed, A.A., De Lamballerie, X., Charrel, R., Tolou, H., 2007.
Chikungunya infection: an emerging rheumatism among travelers returned from
Indian Ocean islands. Report of 47 cases. Medicine (Baltimore) 86, 123–137.
Sinclair, J.B., Asgari, S., 2020. Ross river virus provokes differentially expressed MicroRNA
and RNA interference responses in Aedes aegypti mosquitoes. Viruses 12, 695.
Smith, D.R., Schmaljohn, C.S., Badger, C., Ostrowski, K., Zeng, X., Grimes, S.D.,
Rayner, J.O., 2020. Comparative pathology study of Venezuelan, eastern, and western
equine encephalitis viruses in non-human primates. Antiviral Res. 182, 104875.
Soden, M., Vasudevan, H., Roberts, B., Coelen, R., Hamlin, G., Vasudevan, S., La
Brooy, J., 2000. Detection of viral ribonucleic acid and histologic analysis of inflamed
synovium in Ross River virus infection. Arthritis Rheum. 43, 365–369.
Soto, R.A., Hughes, M.L., Staples, J.E., Lindsey, N.P., 2022. West Nile virus and other
domestic nationally notifiable arboviral diseases - United States, 2020. MMWR
Morb. Mortal. Wkly Rep. 71, 628–632.
Steele, K.E., Davis, K.J., Stephan, K., Kell, W., Vogel, P., Hart, M.K., 1998. Comparative
neurovirulence and tissue tropism of wild-type and attenuated strains of Venezuelan
equine encephalitis virus administered by aerosol in C3H/HeN and BALB/c mice.
Vet. Pathol. 35, 386–397.
Steele, K.E., Twenhafel, N.A., 2010. REVIEW PAPER: pathology of animal models of
alphavirus encephalitis. Vet. Pathol. 47, 790–805.
Stephens, R., Culleton, R.L., Lamb, T.J., 2012. The contribution of Plasmodium chabaudi
to our understanding of malaria. Trends Parasitol. 28, 73–82.
Stephenson, E.B., Peel, A.J., Reid, S.A., Jansen, C.C., McCallum, H., 2018. The
non-human reservoirs of Ross River virus: a systematic review of the evidence.
Parasit. Vectors 11, 188.
Styer, L.M., Kent, K.A., Albright, R.G., Bennett, C.J., Kramer, L.D., Bernard, K.A., 2007.
Mosquitoes inoculate high doses of west nile virus as they probe and feed on live hosts.
Styer, L.M., Lim, P.Y., Louie, K.L., Albright, R.G., Kramer, L.D., Bernard, K.A., 2011.
Mosquito saliva causes enhancement of West Nile virus infection in mice. J. Virol.
85, 1517–1527.
Sucupira, P.H.F., Ferreira, Á.G.A., Leite, T.H.J.F., De Mendonça, S.F., Ferreira, F.V.,
Rezende, F.O., Marques, J.T., Moreira, L.A., 2020. The RNAi pathway is important
to control mayaro virus infection in Aedes aegypti but not for wolbachia-mediated
protection. Viruses 12, 871.
Sun, C., Gardner, C.L., Watson, A.M., Ryman, K.D., Klimstra, W.B., 2014. Stable,
high-level expression of reporter proteins from improved alphavirus expression vectors
to track replication and dissemination during encephalitic and arthritogenic disease.
J. Virol. 88, 2035–2046.
Suthar, M.S., Shabman, R., Madric, K., Lambeth, C., Heise, M.T., 2005. Identification of
adult mouse neurovirulence determinants of the Sindbis virus strain AR86. J. Virol. 79,
4219–4228.
Taylor, A., Herrero, L.J., Rudd, P.A., Mahalingam, S., 2015. Mouse models of
alphavirus-induced inflammatory disease. J. Gen. Virol. 96, 221–238.
Taylor, K., 2007. Biodefense: research methodology and animal models. Emerg. Infect. Dis.
13, 523.
Taylor, K., Kolokoltsova, O., Patterson, M., Poussard, A., Smith, J., Estes, D.M., Paessler, S.,
2012. Natural killer cell mediated pathogenesis determines outcome of central nervous
system infection with Venezuelan equine encephalitis virus in C3H/HeN mice. Vaccine
30, 4095–4105.
Teo, T.-H., Lum, F.-M., Ghaffar, K., Chan, Y.-H., Amrun, S.N., Tan, J.J.L., Lee, C.Y.P.,
Chua, T.-K., Carissimo, G., Lee, W.W.L., Claser, C., Rajarethinam, R., Renia, L.,
Ng, L.F.P., 2018. Plasmodium co-infection protects against chikungunya virus-induced
pathologies. Nat. Commun. 9, 3905.
Thach, D.C., Kimura, T., Griffin, D.E., 2000. Differences between C57bl/6 and BALB/cBy
mice in mortality and virus replication after intranasal infection with neuroadapted
Sindbis virus. J. Virol. 74, 6156–6161.
Thangamani, S., Higgs, S., Ziegler, S., Vanlandingham, D., Tesh, R., Wikel, S., 2010. Host
immune response to mosquito-transmitted chikungunya virus differs from that elicited
by needle inoculated virus. PLoS One 5, e12137.
Thavara, U., Tawatsin, A., Pengsakul, T., Bhakdeenuan, P., Chanama, S.,
Anantapreecha, S., Molito, C., Chompoosri, J., Thammapalo, S., Sawanpanyalert, P.,
Siriyasatien, P., 2009. Outbreak of chikungunya fever in Thailand and virus detection
in field population of vector mosquitoes, Aedes aegypti (L.) and Aedes albopictus
Skuse (Diptera: Culicidae). Southeast Asian J. Trop. Med. Public Health 40, 951–962.
Threadgill, D.W., Miller, D.R., Churchill, G.A., De Villena, F.P., 2011. The collaborative
cross: a recombinant inbred mouse population for the systems genetic era. ILAR J. 52,
24–31.
Torres-Ruesta, A., Teo, T.H., Chan, Y.H., Amrun, S.N., Yeo, N.K., Lee, C.Y.,
Nguee, S.Y., Tay, M.Z., Nosten, F., Fong, S.W., Lum, F.M., Carissimo, G.,
Renia, L., Ng, L.F., 2022. Malaria abrogates O’nyong-nyong virus pathologies by
restricting virus infection in nonimmune cells. Life Sci. Alliance 5.
Trefry, J.C., Rossi, F.D., Accardi, M.V., Dorsey, B.L., Sprague, T.R., Wollen-Roberts,
S.E., Shamblin, J.D., Kimmel, A.E., Glass, P.J., Miller, L.J., Burke, C.W.,
Cardile, A.P., Smith, D.R., Bavari, S., Authier, S., Pratt, W.D., Pitt, M.L., Nasar, F.,
2021. The utilization of advance telemetry to investigate critical physiological parameters
including electroencephalography in cynomolgus macaques following aerosol challenge
with eastern equine encephalitis virus. PLoS Negl. Trop. Dis. 15, e0009424.
Tsetsarkin, K., Higgs, S., McGee, C.E., De Lamballerie, X., Charrel, R.N.,
Vanlandingham, D.L., 2006. Infectious clones of Chikungunya virus (La Reunion
isolate) for vector competence studies. Vector Borne Zoonotic Dis. 6, 325–337.
Tsetsarkin, K.A., Vanlandingham, D.L., McGee, C.E., Higgs, S., 2007. A single mutation
in Chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog.
3, e201.
Tucker, P.C., Griffin, D.E., 1991. Mechanism of altered Sindbis virus neurovirulence asso-
ciated with a single-amino-acid change in the E2 Glycoprotein. J. Virol. 65, 1551–1557.
Tucker, P.C., Strauss, E.G., Kuhn, R.J., Strauss, J.H., Griffin, D.E., 1993. Viral determinants
of age-dependent virulence of Sindbis virus for mice. J. Virol. 67, 4605–4610.
Turell, M.J., Beaman, J.R., Neely, G.W., 1994. Experimental transmission of eastern equine
encephalitis Virus by strains of Aedes albopictus and A. taeniorhynchus (Diptera:
Culicidae). J. Med. Entomol. 31, 287–290.
Uhrlaub, J.L., Pulko, V., Defilippis, V.R., Broeckel, R., Streblow, D.N., Coleman, G.D.,
Park, B.S., Lindo, J.F., Vickers, I., Anzinger, J.J., Nikolich-Žugich, J., 2016.
Dysregulated TGF-β Production Underlies the Age-Related Vulnerability to
Chikungunya Virus. PLoS Pathog. 12, e1005891.
Vanlandingham, D.L., Hong, C., Klingler, K., Tsetsarkin, K., McElroy, K.L., Powers, A.M.,
Lehane, M.J., Higgs, S., 2005. Differential infectivities of o’nyong-nyong and chi-
kungunya virus isolates in Anopheles gambiae and Aedes aegypti mosquitoes. Am.
J. Trop. Med. Hyg. 72, 616–621.
Vanlandingham, D.L., Tsetsarkin, K., Klingler, K.A., Hong, C., McElroy, K.L.,
Lehane, M.J., Higgs, S., 2006. Determinants of vector specificity of o’nyong nyong
and chikungunya viruses in Anopheles and Aedes mosquitoes. Am. J. Trop. Med.
Hyg. 74, 663–669.
Varble, A., Benitez, A.A., Schmid, S., Sachs, D., Shim, J.V., Rodriguez-Barrueco, R.,
Panis, M., Crumiller, M., Silva, J.M., Sachidanandam, R., Tenoever, B.R., 2013. An
in vivo RNAi screening approach to identify host determinants of virus replication.
Cell Host Microbe 14, 346–356.
Vasilakis, N., Deardorff, E.R., Kenney, J.L., Rossi, S.L., Hanley, K.A., Weaver, S.C., 2009.
Mosquitoes put the brake on arbovirus evolution: experimental evolution reveals
slower mutation accumulation in mosquito than vertebrate cells. PLoS Pathog. 5,
e1000467.
Vazeille, M., Mousson, L., Failloux, A.B., 2009. Failure to demonstrate experimental vertical
transmission of the epidemic strain of Chikungunya virus in Aedes albopictus from La
Reunion Island, Indian Ocean. Mem. Inst. Oswaldo Cruz 104, 632–635.
Vazeille, M., Mousson, L., Martin, E., Failloux, A.-B., 2010. orally co-infected aedes
albopictus from la reunion island, indian ocean, can deliver both dengue and
chikungunya infectious viral particles in Their Saliva. PLoS Negl. Trop. Dis. 4, e706.
Vazeille, M., Moutailler, S., Coudrier, D., Rousseaux, C., Khun, H., Huerre, M., Thiria, J.,
Dehecq, J.S., Fontenille, D., Schuffenecker, I., Despres, P., Failloux, A.B., 2007. Two
Chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different
patterns of infection in the mosquito, Aedes albopictus. PLoS One 2, e1168.
Vogel, P., Kell, W.M., Fritz, D.L., Parker, M.D., Schoepp, R.J., 2005. Early events in
the pathogenesis of eastern equine encephalitis virus in mice. Am. J. Pathol. 166,
159–171.
Vogels, C.B.F., R€ uckert, C., Cavany, S.M., Perkins, T.A., Ebel, G.D., Grubaugh, N.D.,
2019. Arbovirus coinfection and co-transmission: A neglected public health concern?
PLoS Biol. 17, e3000130.
Walker, D.H., Harrison, A., Murphy, K., Flemister, M., Murphy, F.A., 1976.
Lymphoreticular and myeloid pathogenesis of Venezuelan equine encephalitis in
hamsters. Am. J. Pathol. 84, 351–370.
Wanasen, N., Nussenzveig, R.H., Champagne, D.E., Soong, L., Higgs, S., 2004.
Differential modulation of murine host immune response by salivary gland extracts from
the mosquitoes Aedes aegypti and Culex quinquefasciatus. Med. Vet. Entomol. 18,
191–199.
Wang, E., Bowen, R.A., Medina, G., Powers, A.M., Kang, W., Chandler, L.M.,
Shope, R.E., Weaver, S.C., 2001. Virulence and viremia characteristics of 1992
epizootic subtype IC Venezuelan equine encephalitis viruses and closely related enzootic
subtype ID strains. Am. J. Trop. Med. Hyg. 65, 64–69.
Weaver, S.C., Barrett, A.D., 2004. Transmission cycles, host range, evolution and emer-
gence of arboviral disease. Nat. Rev. Microbiol. 2, 789–801.
Weaver, S.C., Chen, R., Diallo, M., 2020. Chikungunya virus: role of vectors in emergence
from enzootic cycles. Annu. Rev. Entomol. 65, 313–332.
Weaver, S.C., Ferro, C., Barrera, R., Boshell, J., Navarro, J.C., 2004. Venezuelan equine
encephalitis. Annu. Rev. Entomol. 49, 141–174.
Weaver, S.C., Salas, R., Rico-Hesse, R., Ludwig, G.V., Oberste, M.S., Boshell, J.,
Tesh, R.B., 1996. Re-emergence of epidemic Venezuelan equine encephalomyelitis
in South America. VEE Study Group. Lancet 348, 436–440.
Weger-Lucarelli, J., Carrau, L., Levi, L.I., Rezelj, V., Vallet, T., Blanc, H., Boussier, J.,
Megrian, D., Coutermarsh-Ott, S., Leroith, T., Vignuzzi, M., 2019. Host nutritional
status affects alphavirus virulence, transmission, and evolution. PLoS Pathog. 15,
e1008089.
Weger-Lucarelli, J., Chu, H., Aliota, M.T., Partidos, C.D., Osorio, J.E., 2014. A novel
MVA vectored chikungunya virus vaccine elicits protective immunity in mice. PLoS
Negl. Trop. Dis. 8, e2970.
Weise, W.J., Hermance, M.E., Forrester, N., Adams, A.P., Langsjoen, R., Gorchakov, R.,
Wang, E., Alcorn, M.D., Tsetsarkin, K., Weaver, S.C., 2014. A novel live-attenuated
vaccine candidate for mayaro Fever. PLoS Negl. Trop. Dis. 8, e2969.
Williams, J.A., Long, S.Y., Zeng, X., Kuehl, K., Babka, A.M., Davis, N.M., Liu, J.,
Trefry, J.C., Daye, S., Facemire, P.R., Iversen, P.L., Bavari, S., Pitt, M.L., Nasar, F.,
2022. Eastern equine encephalitis virus rapidly infects and disseminates in the brain
and spinal cord of cynomolgus macaques following aerosol challenge. PLoS Negl.
Trop. Dis. 16, e0010081.
Winkler, E.S., Shrihari, S., Hykes Jr., B.L., Handley, S.A., Andhey, P.S., Huang, Y.S.,
Swain, A., Droit, L., Chebrolu, K.K., Mack, M., Vanlandingham, D.L.,
Thackray, L.B., Cella, M., Colonna, M., Artyomov, M.N., Stappenbeck, T.S.,
Diamond, M.S., 2020. The Intestinal Microbiome Restricts Alphavirus Infection and
Dissemination through a Bile Acid-Type I IFN Signaling Axis. Cell 182, 901–918.e18.
Winter Jr., W.D., 1956. Eastern equine encephalomyelitis in Massachusetts in 1955; report
of two cases in infants. N. Engl. J. Med. 255, 262–267.
Young, A.R., Locke, M.C., Cook, L.E., Hiller, B.E., Zhang, R., Hedberg, M.L.,
Monte, K.J., Veis, D.J., Diamond, M.S., Lenschow, D.J., 2019. Dermal and muscle
fibroblasts and skeletal myofibers survive chikungunya virus infection and harbor
persistent RNA. PLoS Pathog. 15, e1007993.
Yu, G.-Y., Wiley, M.R., Kugelman, J.R., Ladner, J.T., Beitzel, B.F., Eccleston, L.T.,
Morazzani, E.M., Glass, P.J., Palacios, G.F., 2015. Complete coding sequences of eastern
equine encephalitis virus and venezuelan equine encephalitis virus strains isolated from
human cases. Genome Announc. 3. e00243–15.
Zacks, M.A., Paessler, S., 2010. Encephalitic alphaviruses. Vet. Microbiol. 140, 281–286.
Zaid, A., Burt, F.J., Liu, X., Poo, Y.S., Zandi, K., Suhrbier, A., Weaver, S.C.,
Texeira, M.M., Mahalingam, S., 2021. Arthritogenic alphaviruses: epidemiological
and clinical perspective on emerging arboviruses. Lancet Infect. Dis. 21, e123–e133.
Ziegler, S.A., Lu, L., Da Rosa, A.P., Xiao, S.Y., Tesh, R.B., 2008. An animal model for
studying the pathogenesis of chikungunya virus infection. Am. J. Trop. Med. Hyg.
79, 133–139.

1 s2 - 0 S0065352722000197 Main

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

1 s2 - 0 S0065352722000197 Main

Uploaded by

Copyright:

Available Formats

CHAPTER TWO

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.,

For example, two studies reported contradictory findings regarding the

5. Animal models of human disease

neonatal mice causes lethal encephalitis and is generally used as a challenge

5.1.1 Ross river virus

dysfunction characterized by the loss of gripping ability, altered gait, and

5.1.2 Barmah forest virus

objective video-based assessment of movement and behavior in small

5.1.3 Mayaro and o’nyong nyong viruses

5.1.4 Eastern equine encephalitis virus

of longer-term sequelae. Nevertheless, these models have been invaluable

5.1.5 Venezuelan equine encephalitis virus

have found a critical role for αβ T cells in controlling infection of the

encephalitis in mice similar to epizootic VEEV strains and can be studied at

5.1.6 Western equine encephalitis virus

5.1.7 Sindbis and Semliki Forest viruses

5.2 Other rodents

studied in greater depth. Hamsters infected with virulent VEEV (subtypes I,

5.2.2 Guinea pigs

5.3 Non-human primates

5.3.2 Encephalitic alphaviruses in non-human primates

and their measurement with telemetry are excellent quantitative ways of

meninges, hemorrhage in the cerebrum, and perivascular cuffing in multiple

6. Animal models of unique aspects of alphavirus

6.1.1 Aerosol transmission

and FL93-939) also results in dose-dependent lethality and neurological dis-

6.1.2 Mosquito and mosquito component-based infection models

infection of mosquitoes instead of blood feeding on chicks, for example, in

6.2 Co-infection studies

6.2.1 Plasmodium spp.

Fig. 7 Utility of alphavirus co-infection models. Co-infection of hosts with alphaviruses

6.2.2 Dengue virus

in SGs of co-infected mosquitoes consistently throughout infection. These

7. Virus strains used in animal models

As mentioned above, alphavirus reverse genetics systems were

human population is significantly limited in inbred mice, and a link between

You might also like