Eilat virus, a unique alphavirus with host range
restricted to insects by RNA replication
Farooq Nasara,1, Gustavo Palaciosb,1,2, Rodion V. Gorchakova, Hilda Guzmana, Amelia P. Travassos Da Rosaa,
Nazir Savjib,3, Vsevolod L. Popova, Michael B. Shermanc, W. Ian Lipkinb, Robert B. Tesha, and Scott C. Weavera,c,4
aInstitute for Human Infections and Immunity and Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555;bCenter for Infection
and Immunity, Mailman School of Public Health, Columbia University, New York, NY 10032; andcSealy Center for Structural Biology and Molecular Biophysics
and Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555
Edited* by Barry J. Beaty, Colorado State University, Fort Collins, CO, and approved July 18, 2012 (received for review March 23, 2012)
Mostalphavirusesand manyotherarbovirusesare mosquito-borne
and exhibit a broad host range, infecting many different verte-
brates including birds, rodents, equids, humans, and nonhuman
primates. Consequently, they can be propagated in most verte-
brate and insect cell cultures. This ability of arboviruses to infect
arthropods and vertebrates is usually essential for their mainte-
nance in nature. However, several flaviviruses have recently been
described that infect mosquitoes but not vertebrates, although the
mechanism of their host restriction has not been determined. Here
we describe a unique alphavirus, Eilat virus (EILV), isolated from
a pool of Anopheles coustani mosquitoes from the Negev desert
of Israel. Phylogenetic analyses placed EILV as a sister to the West-
ern equine encephalitis antigenic complex within the main clade of
mosquito-borne alphaviruses. Electron microscopy revealed that,
and budded from the plasma membrane of mosquito cells in culture.
EILV readily infected a variety of insect cells with little overt cyto-
pathic effect. However, in contrast to typical mosquito-borne alpha-
viruses, EILV could not infect mammalian or avian cell lines, and viral
as well as RNA replication could not be detected at 37 °C or 28 °C.
vertebrate cells. Thus, EILV seems to be mosquito-specific and repre-
sents a previously undescribed complex within the genus Alphavirus.
Reverse genetic studies of EILV may facilitate the discovery of deter-
minants of alphavirus host range that mediate disease emergence.
tive-sense, 11- to 12-kb RNA genomes that contains two ORFs
(1): the 5′ two thirds of the genome encodes four nonstructural
proteins (nsPs; nsP1–nsP4); the 3′ third encodes five structural
proteins (sPs; Capsid, E3, E2, 6K, and E1). Alphaviruses enter
the host cell via receptor-mediated endocytosis. Following in-
ternalization, low endocytic pH induces a conformational change
that exposes an E1 fusion peptide resulting in the cytoplasmic
release of the nucleocapsid. The genomes of alphaviruses are
capped and polyadenylated and serve as mRNA for translation
of the nsPs. The resulting polyprotein is sequentially cleaved into
four nsPs responsible for RNA replication, modification, and
proteolytic cleavage. The nsPs facilitate the synthesis of negative
and positive strands as well as the transcription of subgenomic
mRNA encoding the sPs. Following translation, glycosylated E1/
E2 heterodimers are inserted into the plasma membrane. Capsid
proteins interact with one genomic RNA copy to form nucleo-
capsids, which interact with the cytoplasmic tail of E2 to initiate
virion budding from host cell membranes (1).
The genus Alphavirus currently includes 29 species grouped into
10 complexes based on antigenic and/or genetic similarities (2, 3).
The Barmah Forest, Ndumu, Middelburg, and Semliki Forest
complexes occur almost exclusively in the Old World, whereas the
Venezuelan equine encephalitis (VEE), eastern equine encepha-
litis (EEE), and Trocara complexes comprise New World viruses
he genus Alphavirus in the family Togaviridae comprises
small, spherical, enveloped viruses with single strand, posi-
(2, 3). The western equine encephalitis (WEE) complex contains
and New World [Aura virus (AURAV)] viruses as well as recom-
binant viruses [WEE virus (WEEV), Highlands J, Fort Morgan,
and Buggy Creek] (2–5). The latter are decedents of an ancient
recombinant virus that obtained nonstructural and capsid genes
from an EEE-like virus and the remaining genes from a Sindbis-
like ancestor (4, 5). Last, the aquatic alphaviruses comprise two
groups, Southern elephant seal virus and salmon pancreas disease
virus (SPDV) (6, 7). SPDV and its subtype sleeping disease virus
are distantly related to all other alphaviruses (7).
Most alphaviruses infect terrestrial vertebrates via mosquito-
borne transmission and thereby exhibit a broad host range (8).
Occasionally, these cycles spill over into humans and domesti-
cated animals to cause disease. Human infections with Old World
viruses such as Ross River virus, chikungunya virus, and SINV are
typically characterized by fever, rash, and polyarthritis, whereas
infections with the New World viruses VEE virus (VEEV), EEE
virus (EEEV), and WEEV can cause fatal encephalitis (8).
Alphaviruses infect a wide range of vertebrate and insect hosts,
including mosquito species encompassing at least six genera as
well as ticks and lice (6, 8–10). Vertebrate hosts include fish,
equids, birds, amphibians, reptiles, rodents, pigs, humans, and
nonhuman primates (9). Consequently, alphaviruses can be cul-
tured in many vertebrate and insect cell lines (11–13). In contrast,
the distantly related fish alphaviruses, which are not known to
have arthropod vectors, exhibit a narrow host range (fish cells
only) that is at least partially a result of temperature sensitivity
(14–16). However, the viral factor(s) that underlie the varying
host range of alphaviruses are poorly understood. Host-restricted
alphaviruses that group within the mosquito-borne clade may
provide insights into these factor(s); however, to date, none has
been identified. Here we describe a host-restricted alphavirus
of mosquitoes and demonstrate that its inability to infect verte-
brates is caused at least in part by restricted RNA replication.
Virus Isolation. Eilat virus (EILV) was one of 91 virus isolates
obtained during an arbovirus survey the Negev desert, including
in the city of Eilat, in Israel, during 1982 to 1984 (17). EILV was
Author contributions: F.N., R.V.G., R.B.T., and S.C.W. designed research; F.N., G.P., R.V.G.,
A.P.T.D.R., N.S., V.L.P., and M.B.S. performed research; F.N., R.V.G., H.G., R.B.T., and S.C.W.
contributed new reagents/analytic tools; F.N., R.V.G., A.P.T.D.R., V.L.P., M.B.S., W.I.L.,
R.B.T., and S.C.W. analyzed data; and F.N. and S.C.W. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1F.N. and G.P. contributed equally to this work.
2Present address: US Army Medical Research Institute for Infectious Diseases, Fort Detrick,
Frederick, MD 21702.
3Present address: School of Medicine, New York University, New York, NY 10016.
4To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 4, 2012
| vol. 109
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originally isolated in mosquito cells by Joseph Peleg (Hebrew
University, Jerusalem) from a pool of Anopheles coustani mos-
quitoes, and was subsequently sent to one of the authors (R.B.T.)
for further study. Preliminary characterization showed that EILV
was unable to infect mammalian cells or to kill infant mice in-
oculated intracerebrally, but could replicate to high titers in a
variety of insect cells.
Genomic Analysis. The complete genomic EILV sequence, de-
termined by 454 pyrosequencing, was translated and compared
with that of SINV to determine the length of each gene product;
a schematic illustration is shown in Fig. 1A. The lengths of the
UTRs and intergenic regions, as well as of each gene, were similar
to those of other alphaviruses. Nucleotide and amino acid se-
quence identity ofEILV withotheralphavirusesrangedfrom 57%
to 43% and 58% to 28%, respectively (Dataset S1). In both anal-
yses, EILV had greater similarity to WHATV, AURAV, SINV,
and Trocara virus (TROV), and had the lowest sequence identity
to SPDV. The EILV nsPs displayed higher amino acid identity to
those of other alphavirusthan did the sPs, with nsP4exhibiting the
highest amino acid identity and nsP3 the least (Dataset S2).
Analyses ofputative EILVconserved sequence elements (CSEs)
based on mFold estimates indicated that the EILV 5′ UTR formed
hairpin structures similar to those of SINV, and the nsP1 CSE had
>70% nt sequence identity with AURAV, WHATV, and SINV
(Fig. S1 A and B). Like the 5′ CSE, the EILV nsP1 CSE formed
hairpin structures similar to those of SINV. The EILV subgenomic
promoter shared 88% ntsequence identity with WEEV and EEEV
(Fig.S1C),andthe3′CSEwas almost identicalto that ofAURAV,
EEEV, VEEV, and SFV (Fig. S1D).
Last, the putative EILV nonstructural and structural poly-
protein cleavage sites had greater sequence identity with TROV,
AURAV, WHATV, and SINV (Fig. S2A), whereas the E1 fusion
peptide was identical to that of WHATV and shared significant
sequence identity with SINV, WEEV, EEEV, VEEV, and chi-
kungunya virus (Fig. S2B). The ribosomal binding site showed
greater sequence divergence (Fig. S2B), but was most similar to
that of AURAV and SINV.
In Vitro Characterization. An EILV genomic cDNA clone was con-
structed and rescued by electroporation of transcribed RNA. EILV
infection did not cause any overt cytopathic effects on C7/10 cells,
although they grew at a slower rate than uninfected cells. EILV
formed 3- to 4-mm plaques 3 d after infection of C7/10 cells (Fig.
1B). RNA analysis of EILV-infected C7/10 cells revealed the syn-
thesis of genomic as well as subgenomic RNA, characteristic of all
alphaviruses (Fig. 1C).
EM. Transmission EM and cryoEM imaging of EILV virions
showed that they were spherical, 70 nm in diameter, and budded
from the plasma membrane of mosquito cells (Fig. 2 A and B). A
20-Å-resolution cryoEM reconstruction (Fig. 2A) revealed an
unusual protrusion on the glycoprotein spikes that is absent in
SINV. The observed volume of this protrusion was consistent
with the expected volume of the E3 protein.
Phylogenetic and Serological Analysis. Neighbor-joining, maximum-
likelihood, and Bayesian methods were used to determine the
relationship of EILV within the genus Alphavirus. Trees were
generated using full-length as well as nonstructural and structural
polyprotein gene nucleotide alignments. All three methods placed
EILV within the clade of mosquito-borne alphaviruses (Fig. 3 and
Figs. S3 and S4). The genomic and structural nucleotide analyses
placed EILV as a sister to the WEE complex (Fig. 3 and Fig. S3)
with high posterior probability support. Analyses of the non-
structural alignment showed some inconsistency. Neighbor joining
placed EILV as a sister to WEE complex, whereas Bayesian and
maximum-likelihood analyses placed it within the WEE complex
basal to WHATV (Fig. S4).
assays werealsoperformed to determine theantigenic relationship
of EILV within the Alphavirus genus. By CF, EILV did not cross
react with sera against most alphaviruses and had only minimal
cross-reactivity with TROV, AURAV, SINV, EEEV, and VEEV
antisera(Fig.4A).ByHI, EILVantiserum cross-reactedminimally
with TROV, SINV, WEEV, and EEEV (Fig. 4B). Purified EILV
did not hemagglutinate, and EILV antiserum reacted non-
specifically with mosquito cell antigens, confounding HI results.
Amino acid size of each protein is denoted below.
The intergenic region, 5′ and 3′ UTR nucleotide
lengths are above in gray. EILV plaques 3 d after
infection on C7/10 cells (B). Synthesis of virus-specific
RNAs in C7/10 cells infected with EILV or SINV 7 hpi,
analyzed by agarose gel electrophoresis (C). G, ge-
nomic RNA; SG, subgenomic RNA.
Schematic diagram of the EILV genome (A).
Nasar et al.PNAS
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In Vitro Host Range.Representative vertebrate and insect cell lines
[Vero (African green monkey), BHK-21 (baby hamster kidney),
HEK-293 (human embryonic kidney), NIH 3T3 (mouse fibro-
blast), duck embryo fibroblast (DEF), A6 (Xenopus laevis), Aedes
albopictus (C6/36 and C7/10), Culex tarsalis, and Phlebotomus
papatasi (PP-9)] were used to determine the in vitro host range of
EILV. SINV, which has a broad in vitro host range, was used as
a positive control (12–14). EILV and SINV infected C. tarsalis,
P. papatasi, C6/36, and C7/10 cells (Fig. 5A and Fig. S5A), and
replicated to high titers (>107pfu/mL) 12 h postinfection (hpi)
with peak titers of 5 × 107to 5 × 108pfu/mL at 48 hpi; however,
the infections did not produce overt cytopathic effects (Fig. S6).
All vertebrate cell lines were readily infected by SINV and showed
extensive cytopathic effects at 12 hpi (Fig. S5B), whereas EILV
was unable to infect any of the vertebrate cell lines and no cy-
topathic effects were observed (Fig. 5B and Fig. S6). The EILV
inocula decayed significantly by 72 hpi and were barely greater
than the limit of detection at 96 hpi.
The inability of EILV to infect vertebrate cells was confirmed
by infection with the EILV-expressing red fluorescent protein
(eRFP) from a second subgenomic promoter. The red fluores-
cent protein was readily observed in mosquito but not vertebrate
cells (Fig. S6). In contrast, the SINV-eGFP control expressed
efficiently in mosquito and vertebrate cells.
Analysis of EILV Genomic RNA Replication in Vertebrate Cells. To
ascertain whether the EILV host range was limited at the level of
RNA replication, the EILV-eRFP cDNA clone was transcribed
in vitro, and ∼10-μg RNA aliquots were electroporated into
vertebrate and insect cells. EILV-eRFP produced no detectable
RFP expression in vertebrate cells incubated at 37 °C or 28 °C as
long as 4 d after electroporation, whereas it readily replicated in
insect cells 24 hpi (Fig. 6). This lack of replication and resultant
absence of eRFP expression was not a result of inefficient elec-
troporation of EILV RNA into the vertebrate cells, as our
electroporation efficiency was ∼35% to 95% with the equivalent
SINV-eGFP replicon (Fig. S7).
Here we describe a host-restricted alphavirus that groups phylo-
genetically within the mosquito-borne clade. Viruses with similar
host restriction have been described for the family Flaviviridae.
The mosquito-specific flaviviruses can be divided into two distinct
groups. The first group includes cell fusing agent, Kamiti River
virus, and Culex flaviviruses, which are distantly related phylo-
genetically to the main branch of mosquito- and tick-borne path-
ogenic vertebrate viruses (19–21). These viruses likely represent
an ancestral lineage that could only infect invertebrates and
A 20-Å cryoEM reconstruction of EILV glycoprotein spikes on the virion
surface (A). The protrusion possibly representing the E3 protein is high-
lighted in purple. SINV glycoprotein spikes are shown as a comparison (45).
EILV virions are shown budding from the surface of C7/10 cells (B).
Eilat virion morphology determined by cryoEM and transmission EM.
probabilities <1 shown on major branches. Alphavirus complexes are denoted in bold.
Bayesian phylogenetic tree based on nucleotide sequences of the alphavirus structural ORF. A midpoint rooted tree is shown with all posterior
| www.pnas.org/cgi/doi/10.1073/pnas.1204787109Nasar et al.
subsequently gained the ability to infect vertebrates. It is probable
that genus Alphavirus also contains additional yet-undiscovered
host-restrictedalphavirusescomprising a similarly outlying lineage.
The second flavivirus group includes the newly identified Nounané
virus (NOUV) and Lammi virus (LAMV), which are closely re-
lated to the mosquito-borne pathogens such as dengue fever, yel-
low fever, and West Nile virus (22, 23). NOUV and LAMV, like
EILV, replicate in insect cells but not in mammalian or avian cells
(22,23).The phylogeneticplacementoftheseflavivirusesaswell as
EILV within the mosquito-borne clades of their respective genera
suggests that they have lost the ability to infect vertebrate cells or
that the mosquito-borne viruses independently and convergently
most parsimonious explanation, which requires the fewest host
range changes, is that EILV and the ancestral parent of NOUV
and LAMV lost their ability to infect vertebrate cells.
are poorly understood. Available data suggest that mutations in
the CSEs or glycoproteins can alter host range (24–33). However,
these mutations result in change in fitness in vertebrate or insect
host but do not completely abolish replication. Alphavirus host
range can be restricted, at least in part, by temperature. SINV can
be cultured to high titers from 15 °C to 40 °C, suggesting a wide
permissive temperature range (24, 34), whereas the distantly re-
lated aquatic SPDV appears to have a very narrow temperature
range of 10 °C to 15 °C (16). Our genetic analysis of EILV CSEs
and other key elements could not explain its observed host range
restriction, as they showed no major differences compared with
mosquito-borne alphaviruses. There are several possible steps at
which the EILV host restriction could occur: (i) attachment and
entry, (ii) incompatibility with host cell factors, or (iii) tempera-
ture sensitivity. We generated strong evidence for the second
hypothesis, as we were unable to detect eRFP expression in ver-
tebrate cell lines incubated at 37 °C or 28 °C, indicating that the
EILV replication was unable to express subgenomic mRNA and
is likely not temperature-sensitive. Our results suggest that EILV
RNA replication is restricted by improper interactions between
its RNA or gene products with vertebrate cell cofactors. Addi-
tionally, the first hypothesis could also represent redundant
blocks to vertebrate cell infection; further studies are under way
to assess the ability of EILV to enter cells. The EILV viral genes
or RNA elements potentially responsible for the host restriction
are also currently under investigation.
Our in vitro characterization EILV showed no overt cytopathic
effects in insect cells. However, a reduction in the growth of in-
fected cells was observed, which facilitated the development of
a plaque assay. EILV virions, similar to other alphaviruses, were
spherical in shape, 70 nm in diameter, and budded from the
plasma membrane. A protrusion was observed in the glycoprotein
spikes of EILV that appeared to correspond to the E3 protein.
Semliki Forest virus and VEEV are also reported to incorporate
E3 into virions (35, 36). We are attempting to produce higher-
resolution EILV cryoEM maps to confirm this interpretation.
tests with EILV and other alphavirus antigens and hyperimmune mouse
ascitic fluids (MIAF). Asterisk indicates the reciprocal of heterologous titer
(Ht)/reciprocal of homologous titer (Ho).
Complement fixation (A) and Hemagglutination inhibition (B)
of 10 (measured in mosquito cells). Supernatants were collected at indicated intervals postinfection and titrated on C7/10 cell monolayers. Each data point
represents the mean titer of samples taken from triplicate infections ± SD. A6 cells were incubated at 28 °C.
Replication kinetics of EILV on representative insect (gray, 28 °C) (A) and vertebrate (black, 37 °C) (B) cell lines. Monolayers were infected at an MOI
Nasar et al. PNAS
| September 4, 2012
| vol. 109
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EILV expressed genomic and subgenomic RNA species sim-
ilar in size to those of SINV. However, the EILV subgenomic
RNA expression level in mosquito cells was lower than that of
SINV. One possible explanation is that the EILV labeling was
not performed at the appropriate time to visualize greater sub-
genomic RNA levels. Another possible explanation is that EILV
packages subgenomic RNA like another alphavirus, AURAV
(37). Finally, EILV may possess a more efficient mechanism of
virion assembly. The latter possibility has been suggested for
other alphaviruses (38).
The phylogenetic analyses placed EILV within the clade of
mosquito-borne alphaviruses. Analyses based on concatenated
nsP/sP ORFs, as well as the sP ORF, consistently placed EILV
at the base of WEE complex with strong support. However, the
nsP ORF alone placed EILV within the WEE complex basal to
WHATV. We believe the concatenated, full-length genome anal-
ysis provides the most accurate placement of EILV within the
genus, as it is based on both ORFs containing more informative
characters. Our analysis also suggests that EILV is not the de-
scendent of a major recombination event like WEEV and others,
as its placement did not change significantly with nsP or sP ORF
analyses. Additionally, the placement of EILV within the genus
did not alter previously determined relationships within the genus
(3). Our serological analysis showed minimal cross-reactions with
other alphaviruses (mainly TROV, AURAV, and SINV). These
data suggest a distant relationship between EILV and these
viruses, consistent with the phylogenetic placements. The latter
was also supported by the genetic analysis of EILV that revealed
considerable divergence relative toother alphaviruses, both at the
nucleotide (43%–57%) and amino acid (32%–78%) levels. The
results of these analyses indicate that EILV represents a pre-
viously undescribed complex within the genus Alphavirus.
The discovery of EILV was fortuitous, as it does not produce
overt cytopathic effects in vertebrate or insect cells and does not
kill infant mice. In the initial isolation, extensive cytopathic
effects were observed in insect cells. However, deep sequencing
revealed the presence of two unique viruses. The second virus,
designated Negev virus, will be described in another publication.
Negev virus was responsible for the observed cytopathic effects
and replicated to higher titers in mosquito cells than EILV; only
after generation of a cDNA clone could EILV be isolated. This
serendipitous discovery of EILV thus highlights the value of
large-scale molecular screening techniques to identify new viru-
ses. It also underscores our limited knowledge of the mosquito
virome and the likelihood that other viruses like EILV are
present in other families or genera of arthropod-borne viruses.
Finally, EILV provides a unique opportunity to study the evo-
lution and molecular determinants of alphavirus host range, and,
more importantly, the fundamental factors that underlie their
pathogenesis in animals and humans. Additionally, EILV may also
be useful to genetically engineer alphavirus chimeras as a vaccine
platform or to express foreign genes in mosquitoes with the po-
tential to render them refractory to pathogen transmission.
Materials and Methods
Viruses and Cells. EILV and SINV (Eg 339) as well as C. tarsalis and P. papatasi
cells were obtained from the World Reference Center for Emerging Viruses
and Arboviruses at the University of Texas Medical Branch. Both viruses were
amplified on C7/10 cells and stored at −80 °C. BHK-21, HEK-293, duck em-
bryo fibroblast, NIH 3T3, A6, and C6/36 cell lines were obtained from the
American Type Culture Collection. Cell lines were propagated at 37 °C or 28 °C
with 5% CO2in DMEM containing 10% (vol/vol) FBS, sodium pyruvate (1 mM),
and penicillin (100 U/mL)–streptomycin (100 μg/mL). C6/36, C7/10, and C. tar-
salis media were additionally supplemented with 1% (vol/vol) tryptose phos-
phate broth (Sigma). P. papatasi cells were maintained in Schneider media
(Sigma) supplemented with 10% (vol/vol) FBS and penicillin (100 U/mL)–
streptomycin (100 μg/mL).
Genomic Sequencing, Cloning, and Rescue of Full-Length Infectious EILV Clone.
EILV genome was sequenced by 454 sequencing as described previously
(3). The EILV cDNA clone was constructed by using standard molecular
of RNA were electroporated into vertebrate and insect cells. Phase-contrast and fluorescent field photographs were taken at day 4 after electroporation.
Replication of EILV genomic RNA in vertebrate (37 °C) and insect (28 °C) cell lines. RNA was transcribed in vitro from the cDNA clone and ∼10-μg aliquots
| www.pnas.org/cgi/doi/10.1073/pnas.1204787109Nasar et al.
Phylogenetic Analysis. Phylogenetic analyses were performed as previously Download full-text
described (3). Alphavirus sequences were downloaded from GenBank
(Dataset S3 lists accession numbers). The two ORFs were concatenated; the
C terminus of nsP3 and the N terminus of the capsid genes, which cannot
be reliably aligned, were removed; and the complete alignment was split
into nsP and sP ORFs. E2-6k-E1 sequence was used for structural ORF
analysis. Three analyses were performed: neighbor-joining, maximum-
likelihood, and Bayesian. The robustness of the neighbor-joining phylog-
eny was evaluated by bootstrap resampling with 1,000 replicates. Mod-
eltest in PAUP was used to identify the best-fit nucleotide substitution
model, GTR+I+G (39). The robustness of maximum-likelihood and Bayesian
phylogenies was evaluated by bootstrap resampling of 100 and 5 million
Serologic Tests. CF and HI tests were performed as described previously (40).
Transmission EM. Thin-section and cryoEM were performed as described
RNA Analysis. C7/10 monolayers were infected with SINV or EILV at a multi-
plicity of infection (MOI) of 10; 4 h postinfection cells were labeled with [3H]
uridine (20 μCi/mL) in the presence of dactinomycin (1 μg/mL) for 3 h. RNA
was analyzed by agarose gel electrophoresis as described previously (44).
Plaque Assay. Virus titration was performed on freshly confluent C7/10 cell
monolayers in six-well plates. Duplicate wells were infected with 0.1-mL
aliquots from serial 10-fold dilutions in growth medium, 0.4 mL of growth
media was added to each well to prevent cell desiccation, and virus was
adsorbed for 2 h. Following incubation, the inoculum was removed, and
monolayers were overlaid with 3 mL containing a 1:1 mixture of 2% trag-
acanth and 2× MEM with 10% (vol/vol) FBS, 2% tryptose phosphate broth
solution, and 2% (vol/vol) penicillin/streptomycin. Cells were incubated at
28 °C in 5% CO2for 3 d for plaque development, the overlay was removed,
and monolayers were fixed with 10% formaldehyde. Cells were stained with
0.2% (wt/vol) crystal violet in 30% methanol and plaques were counted.
One-Step Replication Curves. Replication curves were performed on repre-
sentative cell lines in triplicate with an MOI of 10 (EILV titered on mosquito
cells only). Virus was adsorbed to 50% confluent cells for 2 h at 37 °C (ver-
tebrate) or 28 °C (insect and A6). After the inoculum was removed, mono-
layers were rinsed five times with PBS solution to remove unbound virus,
and 5 mL of growth medium was added to each flask. Aliquots of 0.5 mL
were taken immediately afterward as a “time 0” sample and replaced with
0.5 mL of fresh medium. Flasks were incubated at 37 °C or 28 °C, and further
samples were taken at 12, 24, 48, 72, and 96 hpi.
ACKNOWLEDGMENTS. The authors thank Dr. Frederick A. Murphy for help
in interpreting the electromicrographs, Amy Schuh and Dr. Naomi Forrester
for their helpful discussions on phylogenetics, and Drs. Andrew Haddow and
Konstantin Tsetsarkin for their helpful discussions in manuscript prepara-
tion. This work was supported by National Institutes of Health Contract
HHSN272201000040I/HHSN27200004/D04 (to R.B.T.).
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| vol. 109
| no. 36