JOURNAL OF VIROLOGY, Apr. 2005, p. 4219–4228
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 7
Identification of Adult Mouse Neurovirulence Determinants of
the Sindbis Virus Strain AR86
Mehul S. Suthar,1Reed Shabman,1Kenya Madric,1Cassandra Lambeth,1
and Mark T. Heise1,2*
Department of Genetics2and Department of Microbiology and Immunology,1The University of
North Carolina at Chapel Hill, Chapel Hill, North Carolina
Received 11 August 2004/Accepted 12 November 2004
Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to
virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to
adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related
(22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3
[positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that
genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence.
Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to
Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys;
avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent
to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with
those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in
the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations
did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain
and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence deter-
minants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing
the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type
The infection of mice with Sindbis-group viruses provides an
excellent model for studying virus-induced neurologic disease.
The outcome of Sindbis virus infection in the mouse model has
been found to correlate with the age and strain of the animal,
virus dose, route of inoculation, and virus strain (5, 6, 13, 31).
The infection of neonatal mice with Sindbis virus results in a
lethal disease characterized by elevated levels of proinflamma-
tory cytokines and high viral titers in the muscles, brain, and
serum in the absence of overt encephalitis (11, 26, 27). Increas-
ing the age of the animal or infecting with an attenuated
mutant virus results in a shift from systemic disease toward a
neurologic disease; however, most Sindbis-group viruses are
avirulent in mice greater than 14 days of age (28).
Previous neurovirulence studies with Sindbis-group viruses
have identified virulence determinants in both the E2 glyco-
protein gene (29, 30) and the 5? noncoding region (3, 12).
Studies with the neuroadapted Sindbis virus (NSV) identified a
His at amino acid position 55 in the E2 glycoprotein that plays
a major role in adult mouse neurovirulence (8, 30). The mech-
anism underlying this change is not yet completely understood;
however, E2 His 55 was shown to correlate with improved
binding and entry into neuronal cells (30), increased levels of
viral replication (2), and the ability to overcome the protective
effect of bcl-2 overexpression in neurons (15, 16). Additionally,
a single substitution of a G at position 8 in the 5? noncoding
region of NSV was responsible for conferring neurovirulence
in adult rats (12).
In addition to demonstrating the role of the structural genes
in adult mouse neurovirulence, studies with the Sindbis virus
S.A.AR86 (AR86) and Semliki Forest virus (SFV) indicate
that the viral nonstructural genes contribute to adult mouse
neurovirulence (9, 32, 33). Studies with AR86 have shown that
the presence of a Thr at position 538 in nsP1 plays an impor-
tant role in contributing to adult mouse neurovirulence. Re-
placing this Thr of AR86 with an Ile, found in most Sindbis-
group viruses, attenuates the virus, while introduction of the
Thr into a nonneurovirulent laboratory strain of Sindbis virus
led to an increase in neurovirulence (9). The introduction of
the attenuating Ile at position 538 in nsP1 did not affect viral
growth, as viruses that contained this change replicated as well
as wild-type AR86 both in cell culture and in the brains of
infected animals. Furthermore, the presence of an Ile at posi-
tion 538 accelerated processing of the nonstructural protein
precursor (P123) into the mature nonstructural proteins, lead-
ing to earlier induction of viral 26S RNA synthesis during
infection (10). In the case of SFV, mapping studies performed
using virulent and avirulent strains demonstrated the impor-
tance of the nsP3 gene, including replacement of the opal
termination codon (located within nsP3) with a sense codon, in
reconstituting adult mouse neurovirulence (32, 33).
In this study, we utilized a newly generated clone of the
Sindbis virus Girdwood to map determinants of adult mouse
neurovirulence within AR86. While AR86 causes a lethal dis-
* Corresponding author. Mailing address: The Carolina Vaccine
Institute, The University of North Carolina at Chapel Hill, 827 Mary
Ellen Jones Building, CB #7292, Chapel Hill, NC 27599. Phone: (919)
966-1492. Fax: (919) 843-6924. E-mail: email@example.com.
ease in adult mice, Girdwood is avirulent, even when admin-
istered intracranially (i.c.). However, these viruses differ by
only 22 single amino acid coding changes, as well as an 18-
amino-acid sequence in nsP3 that is present in Girdwood but
deleted from AR86. Detailed mapping studies localized deter-
minants to both the nonstructural and structural genes. Fur-
thermore, the virus containing the attenuating mutations did
not adversely affect in vitro viral replication, and the attenu-
ated viruses established infection in the brain and spinal cord
as efficiently as the virulent viruses. However, at late times
postinfection, the virus containing the virulence determinants
grew to higher levels in the spinal cord, suggesting that the
viruses containing the attenuating determinants either fail to
spread or are cleared more efficiently than the virulent virus.
MATERIALS AND METHODS
Virus stocks and cell culture. Plasmids containing the viral cDNA are desig-
nated by the prefix “p,” while the infectious virus derived from the cDNA clone
does not contain the “p” designation; i.e., S300 represents virus derived from the
plasmid pS300. Clone pS300 was derived from AR86 RNA, and S300 is compa-
rable to AR86 virus both in its growth and in its virulence properties (23). Clone
pS300 is identical to clone pS55 (23) except for a PmeI restriction site engineered
at the 3? end of the viral 3? untranslated region (UTR) to facilitate generating
chimeras with the Girdwood clone. Virus derived from the Girdwood cDNA
clone also exhibits in vitro and in vivo phenotypes similar to the those of the
Girdwood natural isolate (M. Suthar, unpublished data).
Virus stocks were made as described previously (9). Briefly, viral cDNA plas-
mids were linearized with PmeI and used as templates for the synthesis of
full-length transcripts by using SP6-specific mMessage Machine in vitro tran-
scription kits (Ambion). Transcripts were electroporated into BHK-21 cells
grown in ?-minimal essential medium (10% fetal calf serum [Gibco], 10% tryp-
tose phosphate broth ?, and 0.29 mg of L-glutamine [Gibco] per ml). Superna-
tants were harvested 24 to 27 h after electroporation, subjected to centrifugation
at 3,000 rpm (Sorvall rotor RTH-250) for 20 min at 4°C, and frozen in 1-ml
aliquots. Virus stocks were titrated on BHK-21 cells as previously described (23).
BHK-21 cells were maintained at 37°C in ?-minimal essential medium for a
maximum of 10 passages. Single-step in vitro growth curves were performed as
previously described (9). Briefly, BHK-21 cells were plated at 105cells/well in
24-well plates (Sarstedt) for 18 h at 37°C. Medium was removed, and wells were
infected with virus in triplicate at a multiplicity of infection of 5.0. Cells were
incubated at 37°C for 1 h. Wells were then washed three times with 1 ml of
room-temperature phosphate-buffered saline (PBS) supplemented with 1% do-
nor calf serum and Ca2?-Mg2?. One milliliter of growth medium was then added
to each well, and cells were incubated at 37°C. Samples of supernatant were
removed at various time points, with an equal volume of fresh medium added to
maintain a constant volume within each well. Samples were frozen at ?80°C until
analysis by plaque assay.
Construction of full-length cDNAs and chimeric viruses. Standard recombi-
nant DNA techniques were used to construct cDNA clones as previously de-
scribed (21). The chimeric cDNA clone pS350, carrying the nsP1, nsP2, and nsP3
genes of Girdwood in the AR86 genome, was constructed by replacing the MfeI
(nucleotide [nt] 43)-to-BstBI (nt 6411) fragment of pS300 with that of pG100
(Fig. 1). The chimeric cDNA clone pG106, carrying the structural genes of
Girdwood in the AR86 genome, was constructed by exchanging the MfeI (nt
43)-to-BstBI (nt 6411) fragment of pG100 with that of pS300 (Fig. 1).
Clone pS341 (with changes of Ile to Val at position 648 and Leu to Glu at
position 651 in nsP2), clone pS342 (with a Gly-to-Glu change at nsP3 position
344), and clone pS344 (with a Cys-to-opal termination codon change at nsP3
position 537) were created by PCR-mediated site-directed mutagenesis on the
wild-type pS300 background (Fig. 2). Clone pS343, containing the Girdwood
18-amino-acid region in nsP3 between positions 386 and 403, was constructed by
replacing the SfiI (nt 5122)-to-AatII (nt 5285) fragment of pS300 with that of
FIG. 1. Both the nonstructural and structural genes of AR86 contain neurovirulence determinants. A diagram of the Alphavirus genome
organization is located at the top. On the left are the names of the cDNAs encoding full-length virus, beginning with the parental strains pS300
(wild-type AR86) and pG100 (wild-type Girdwood) and followed by the chimeric viruses. The cDNA constructs are diagrammed to show the se-
quences derived from pS300 (shaded box) and pG100 (open box). The chimeric clone pS350 contains AR86 nucleotide sequences between 1 and
6411, and the chimeric pG106 clone contains AR86 nucleotide sequences from 6411 to 11343. Groups (n, number of mice per group) of either 4-
week-old (A) or 6-week-old (B) female CD-1 mice were inoculated i.c. with 103PFU of each virus and observed daily for clinical signs. Virulence was
assessed by morbidity (average clinical score [CS] on day 5), mortality, and average survival time (AST; number of days ? standard deviation [SD]).
4220 SUTHAR ET AL.J. VIROL.
pG100. However, the introduction of this region also included a single amino
acid change of a Glu at nsP3 position 344, which was subsequently corrected to
a Gly by primer-directed mutagenesis.
For the following panel of nonstructural chimeric cDNA clones, AR86
genomic fragments or point mutations were introduced into the chimeric virus
pS350 (Fig. 3). Clone pS351 was derived by replacing the thymidine with cytidine
at nucleotide position 1672 using PCR megaprimer mutagenesis (22), which
replaced an Ile with a Thr codon at nsP1 position 538. Clone pS352 was made by
replacing the adenine with a thymidine at nucleotide position 5793 to replace the
opal termination codon at nsP3 position 537 with a Cys codon. Clone pS353 was
constructed by deleting the sequences between nucleotide positions 5257 and
5311, corresponding to amino acid positions 386 to 403 of nsP3 in Girdwood, by
primer-directed mutagenesis. Clones pS354, pS355, pS356, and pS364, which
contain different combinations of the changes at nsP1 position 538, nsP3 posi-
tions 386 to 403, and nsP3 position 537, were constructed by exchanging restric-
tion fragments from clones pS351, pS352, and pS353.
For the panel of structural gene chimeric viruses listed in Fig. 4, AR86 geno-
mic fragments or point mutations were introduced into the chimeric virus pG106.
The StuI-BssHII fragment (nt 8110 to 9766), including most of the E2 coding
region from clone pS300, was introduced into clone pG106 to create the chimeric
clone pG107, and the BssHII-PmeI fragment (nt 9766 to 11343), encompassing
most of the E1 coding region of clone pS300, was introduced into clone pG106
to create chimeric clone pG108. Clone pG117 was constructed by replacing the thy-
midine at position 9319 of pG106 with cytidine by PCR megaprimer mutagenesis,
resulting in a coding change from a Ser to a Leu at E2 position 243. Chimeric clone
pG163 was constructed by primer-directed mutagenesis of pG100 as follows: sub-
stitution of thymidine for cytidine at nucleotide 1672 (Ile to Thr at nsP1 538), de-
letion of nucleotides between 5257 and 5311 (18-amino-acid deletion from nsP3 386
to 403), substitution of adenine for thymidine at nucleotide 5765 (opal to Cys at
nsP3 537), and substitution of thymidine for cytidine at nucleotide 9319 (Leu to
Ser at E2 243). The reciprocal set of changes was made in pS300 to create clone
pS363. Introduction of the various mutations was confirmed by sequencing at the
FIG. 2. Mutational analysis of neurovirulence determinants within the nonstructural genes of AR86. The cDNA constructs are diagrammed to
show sequences derived from AR86 (pS300) along with amino acids located in nsP2 at positions 648 and 651 and nsP3 positions 344, 386 to 403,
and 537. Groups (n) of 6-week-old female CD-1 mice were inoculated i.c. with 103PFU of either wild-type AR86 (S300) or each mutant virus and
observed daily for clinical signs. Virulence was assessed by morbidity (average CS on day 5), mortality, and AST (number of days ? SD). The
mortalities for the mutant viruses S343 and S344 were statistically significant when compared to S300 (P ? 0.05).
FIG. 3. AR86 neurovirulence determinants within the nonstructural genes. The cDNA constructs are diagrammed to show the sequences
derived from pS300 (shaded box) and pG100 (open box) along with amino acids located in nsP1 at position 538 (Thr in pS300 and Ile in pG100),
AR86 nucleotide sequences between 6411 and 11343. Groups (n) of 6-week-old female CD-1 mice were inoculated i.c. with 103PFU of each virus and
observed daily for clinical signs. Virulence was measured by morbidity (average CS on day 5), mortality, and AST (average number of days ? SD). The
mortalities for the chimeric viruses S354, S355, and S356 were statistically significant when compared to the parental chimeric virus S350 (P ? 0.05).
VOL. 79, 2005NEUROVIRULENCE DETERMINANTS OF SINDBIS VIRUS AR86 4221
University of North Carolina at Chapel Hill (UNC-CH) Genome Analysis Facil-
ity with a model 373A DNA sequencing apparatus (Applied Biosystems). Chi-
meric virus stocks derived from full-length cDNAs were made as described above.
Animal studies. Specific-pathogen-free female CD-1 mice were obtained from
Charles River Breeding Laboratories (Raleigh, N.C.). Animal housing and care
were in accordance with all UNC-CH Institutional Animal Care and Use Com-
mittee guidelines. Six-week-old mice were anesthetized with either isoflurane
(Halocarbon Laboratories) or ketamine supplemented with xylazine (Barber
Med.) prior to i.c. inoculation with a standard dose of 103PFU of virus in diluent
(PBS, pH 7.4) supplemented with 1% donor calf serum (Gibco). Mock-infected
mice received diluent alone. Mice were monitored daily for body weight and scored
for clinical signs as follows: (i), ruffled fur and/or hunched body posture; (ii), mild
hind-limb dysfunction; (iii), prominent hind-limb dysfunction; (iv), severe hind-limb
dysfunction; (v), paralysis of hind limbs; and (vi), terminal morbidity. The average
clinical score was calculated on day 5. This day was chosen because by day 6
infected mice reproducibly showed severe disease progression and occasionally
were euthanized, with death being recorded for the following day. As required by
the UNC-CH animal protocol, infected mice were euthanized during the exper-
iment either when mice dropped below 30% of initial body weight or when mice
exhibited severe disease signs. Statistical analysis for mortality was performed
using Fisher’s exact chi-square test, and differences were considered significant
when P was ?0.05 (Instat 3.0). For the in vivo growth studies, mice were
inoculated as described above and sacrificed by exsanguination followed by
perfusion with 10 ml of PBS at 6, 12, 24, 48, 72, 96, and 120 h postinfection. The
whole brain was removed and weighed, and viral load was assessed by plaque
assay on BHK-21 cells (9, 23). The spinal cord was removed, divided into thoracic
and lumbar regions, and weighed, and viral loads were evaluated by plaque assay
on BHK-21 cells.
Neurovirulence determinants are localized within both non-
structural and structural genes of AR86. Sindbis virus adult
mouse neurovirulence determinants have previously been found
in the viral 5? noncoding region UTR and the E2 glycoprotein
(12, 29). In addition, previous studies with AR86 identified a
single coding change at nsP1 position 538 that was essential for
adult mouse neurovirulence (9). In an effort to identify addi-
tional virulence determinants within AR86, comparisons were
made between AR86 and Girdwood virus, a Sindbis virus that
is closely related to AR86 but nonneurovirulent in adult mice.
To confirm neurovirulence phenotypes, mice were inoculated
i.c. with 103PFU of either S300 (AR86) or G100 (Girdwood)
virus and monitored for virus-induced morbidity and mortality.
Mice were observed daily and scored for clinical signs, such as
weight loss, ruffled fur, hunched body posture, hind-limb dys-
function (mild, moderate, and severe), paralysis of hind limbs,
and terminal morbidity. The average clinical score (CS) for each
group was calculated from observations made on day 5. Con-
sistent with previous reports, S300 caused 100% morbidity and
100% mortality in 4-week-old mice, while G100 caused no
detectable morbidity or mortality in the infected mice (Fig.
FIG. 4. AR86 neurovirulence determinants within the structural genes. The cDNA constructs are diagrammed to show the sequences derived
from pS300 (shaded box) and pG100 (open box) along with the amino acid located in E2 at position 243 (Ser in pS300 and Leu in pG100). All
of the chimeric clones contain AR86 nucleotide sequences between 1 and 6411. Groups (n) of 6-week-old female CD-1 mice were inoculated i.c.
with 103PFU of virus and observed daily for clinical signs. Virulence was assessed by morbidity (average CS on day 5), mortality, and AST (average
number of days ? SD). The mortalities for the chimeric viruses G107 and G117 were statistically significant when compared to the parental
chimeric virus G106 (P ? 0.05).
TABLE 1. Amino acid sequence comparison between
AR86 and Girdwood
Amino acid in:
aAmino acid numbering is from AR86.
bnsP3 18-amino-acid deletion found in AR86.
cFirst amino acid after 18-amino-acid deletion.
4222SUTHAR ET AL.J. VIROL.
1A). G100 and S300 differ at 22 single amino acid positions and
by the presence (G100) or absence (S300) of an 18-amino-acid
sequence in nsP3 (Table 1). Given that the coding differences
between S300 and G100 were distributed throughout the ge-
nome, our initial strategy involved defining whether the viral
nonstructural or structural genes contained the major deter-
minants of AR86 virulence. Therefore, two chimeras contain-
ing either the AR86 nonstructural genes with the G100 struc-
tural genes (G106) or the G100 nonstructural genes with the
AR86 structural genes (S350) were constructed and evaluated
for virulence following i.c. infection of 4-week-old CD-1 mice.
G106 caused 91.7% mortality and 100% morbidity, while S350
caused neither morbidity nor mortality (Fig. 1A), though this
virus grew as well as either wild-type parent (S300 or G100)
both in vitro and in vivo (data not shown). Furthermore, in
vitro transcripts of pS300, pG100, pS350, and pG106 exhibited
similar specific infectivities (data not shown). These results
strongly suggested that the major determinants of AR86 neu-
rovirulence were located in the nonstructural coding region.
However, when older mice (6 weeks old) were inoculated i.c.
with G106, only 26.1% mortality was observed, in comparison
to 95.5% mortality with S300 (Fig. 1B). Therefore, it was likely
that determinants in both the nonstructural and structural
genes were required for the full neurovirulence of AR86.
Since AR86 virulence determinants appeared to be located
in both the structural and nonstructural genes, mapping of
these virulence determinants was done separately, with the
initial focus on the nonstructural region. While it was possible
that noncoding differences were at least partly responsible for
the virulence differences between AR86 and Girdwood, the
initial analysis focused on the coding differences, which were
located in nsP1, nsP2, and nsP3 but not nsP4 (Table 1). Pre-
vious analysis indicated that eight of the coding differences, as
well as the nsP3 deletion, were unique to AR86 when it was
compared to the known coding sequences of five nonvirulent
Sindbis viruses (23). Furthermore, in previous studies, the
unique Thr at nsP1 538 of AR86 was shown to be essential for
adult mouse neurovirulence, as viruses with Ile at this position
no longer caused lethal disease in adult mice (9). This result
suggested that other coding changes that were unique to AR86
might be virulence determinants. Therefore, each of the
AR86-specific coding changes, with the exception of two
changes at codons 442 and 446 in nsP3, were changed in the
S300 background to the codon found at the corresponding
position in Girdwood (clones pS341 and pS342). The role of
the 18-amino-acid deletion was also assessed by reintroducing
the 18-amino-acid sequence found at nsP3 positions 386 to 403
of Girdwood and the other nonneurovirulent Sindbis viruses
back into the S300 clone (clone pS343), and an opal codon was
introduced at nsP3 position 537 (pS344). The specific changes
introduced into each clone were confirmed by sequencing.
Viruses derived from these clones were evaluated for a loss of
virulence following i.c. inoculation of 103PFU into 6-week-old
CD-1 mice. In the process of performing this analysis, it be-
came apparent that the reported Arg codon at position 256 of
nsP2 in the AR86 infectious clone (23) was actually an Ala
codon, which is present in all of the other Sindbis virus ge-
nomes. Therefore, this position was dropped from our analysis.
Three changes at positions 648 and 651 of nsP2 (clone pS341)
and at position 344 of nsP3 (clone pS342) did not appear to be
major contributors to the adult mouse neurovirulence pheno-
type of AR86, since changing these codons to the correspond-
ing codon found in Girdwood did not affect virulence in S300
(Fig. 2). However, reintroduction of the 18-amino-acid dele-
tion in nsP3 (clone pS343), or changing the unique Cys codon
at nsP3 position 537 back to the opal termination codon (clone
pS344) found in most nonneurovirulent Sindbis viruses altered
S300 virulence (Fig. 2). In addition, both of these viruses ex-
hibited growth kinetics equivalent to the wild-type S300 virus
in vitro, suggesting that the decreased virulence was not simply
due to a decrease in virus growth (data not shown). These
results suggested that in addition to nsP1 position 538 (9), ad-
ditional AR86 virulence determinants included the 18-amino-
acid deletion in nsP3 and the Cys codon at nsP3 537, while the
unique codons at nsP2 648 and 651 or nsP3 344 did not appear
to contribute to adult mouse virulence. The role of nsP3 442
and 446 was not addressed in this analysis, but the contribution
of these determinants to virulence was evaluated in later stud-
ies (see below).
wood background results in a gain of virulence. Though the
mutation of nsP3 537 (Cys to opal) or the reintroduction of the
18-amino-acid deletion into nsP3 on the S300 background re-
sulted in a loss of virulence, it was possible that rather than
being true virulence determinants, these changes simply de-
creased virus fitness and thereby caused a subtle decrease in
virus replication which affected virulence. Therefore, a more
rigorous test of whether these or other determinants within the
nonstructural genes were virulence determinants was per-
formed by introducing changes into the nonneurovirulent Gird-
wood background (clone pG100) and assaying for a gain of
virulence. Since the AR86 nonstructural genes did not confer
wild-type levels of virulence in the absence of the AR86 struc-
tural genes, all nonstructural mapping studies were performed
using the clone pS350, which contained the Girdwood non-
structural region and the AR86 structural genes. Therefore, if
all of the nonstructural determinants of virulence were intro-
duced into the pS350 clone, the resulting virus should exhibit a
level of neurovirulence comparable to that of AR86. As an
initial step in this process, the three loci that were implicated
in the S300 analysis (Fig. 2) (9) were analyzed in the S350
background by introducing the codon found in the virulent
AR86 virus back into the corresponding position in the Gird-
wood nonstructural region and looking for a gain of virulence
The introduction of a Thr codon in place of the Ile at nsP1
538 in the nonneurovirulent Sindbis virus TR339 was previ-
ously shown to increase virulence in 18- to 21-day-old mice (9),
which strongly suggests that this determinant plays a major role
in adult mouse neurovirulence. When a Thr codon was placed
at nsP1 538 of the pS350 clone, the resulting virus (S351)
caused 100% morbidity, with a day 5 average clinical score of
2.2, and 16.7% mortality (Fig. 3), compared to 27.3% morbid-
ity (with a day 5 average clinical score of 0.4) and 0% mortality
with the S350 virus. A virus (S352) where the consensus opal
termination codon at nsP3 position 537 was replaced with the
AR86-derived Cys codon caused 66.7% morbidity (day 5 av-
erage clinical score of 1.3) but no mortality (Fig. 3). Likewise,
deleting the 18 amino acids from positions 386 to 403 in nsP3
of clone S350 (virus designated S353) gave 78.6% morbidity
VOL. 79, 2005NEUROVIRULENCE DETERMINANTS OF SINDBIS VIRUS AR86 4223
(day 5 average clinical score of 1.2) but no mortality (Fig. 3).
Therefore, all three changes independently increased S350 vir-
ulence as determined by an increase in virus-induced morbid-
ity; however, none of the individual determinants were able to
raise S350 virulence to the level observed with AR86 (clone
S300). Therefore, the individual coding changes were evalu-
ated in combination. The introduction of both the 18-amino-
acid deletion and the Cys codon at nsP3 position 537 of clone
ps350 resulted in a virus (S364) that caused 91.7% morbidity
(day 5 average clinical score of 1.5) and 6.7% mortality (Fig. 3).
However, when the Thr at nsP1 position 538 was introduced
into clone pS350 in combination with either the 18-amino-acid
deletion (clone ps355) or the Cys codon at nsP3 position 537
(clone ps354), the resulting viruses were significantly more
virulent than the parental S350 virus (27.3% morbidity and 0%
mortality) (Fig. 3). The S355 virus (Thr at nsP1 538 and 18-
amino-acid deletion at nsP3 386 to 403) caused 100% morbid-
ity and 65.4% mortality in 6-week-old mice, while the S354
virus (Thr at nsP1 538 and Cys at nsP3 537) caused 100%
morbidity and 76.9% mortality (Fig. 3). Finally, when all three
changes were introduced into the same virus (clone pS356), the
resulting virus caused 100% morbidity and 100% mortality in
6-week-old mice following i.c. inoculation. Therefore, the Thr
at nsP1 position 538, the 18-amino-acid deletion from nsP3 386
to 403, and the Cys at nsP3 537 are the major determinants of
AR86 neurovirulence within the AR86 nonstructural genes.
Minor roles for other coding changes, such as nsP3 positions
442 and 446, or noncoding changes cannot be ruled out; how-
ever, they were not necessary for the full adult mouse neuro-
virulence phenotype in the Girdwood background.
Serine at position 243 in the E2 glycoprotein is a major
determinant of AR86 adult mouse neurovirulence. Both the
AR86 nonstructural genes determinants and the AR86 struc-
tural genes were required for complete neurovirulence (Fig. 1).
Therefore, studies were initiated to identify the structural gene
determinants that contributed to adult mouse virulence. Since
complete virulence required the AR86 nonstructural genes, all
structural gene mapping studies were conducted using the
G106 clone, which contains the AR86 nonstructural genes and
the Girdwood structural genes and causes 87.0% morbidity
(day 5 average clinical score of 2.0) and 26.1% mortality. Gene
segments or individual coding changes from the structural
genes of wild-type AR86 (pS300) were introduced back into
pG106, and the resulting viruses were evaluated for increased
virulence following i.c. inoculation of adult mice. The AR86
and Girdwood structural genes contained five coding differ-
ences: one difference in the E2 glycoprotein, one in the 6k
protein, and three in the E1 glycoprotein. The relative contri-
bution of the change in E2 versus the changes in the 6k and E1
protein coding regions were assessed by using two chimeras
where the E2 coding region or the 6k and E1 coding region of
AR86 was introduced into the G106 background. When these
viruses were evaluated for virulence, the virus with the AR86
E2 gene (G107) caused 100% morbidity and 82.4% mortality
(Fig. 4), while the virus with the AR86 6k and E1 coding region
caused 100% morbidity (day 5 average clinical score of 2.2)
and 33.3% mortality (Fig. 4). While these results suggest that
minor determinants of AR86 neurovirulence reside within the
6k and E1 coding region, it is clear that a major determinant of
adult mouse neurovirulence was located in the E2 glycopro-
tein. Since AR86 and Girdwood differ only at position 243 of
E2, it was likely that this was the major determinant of adult
mouse neurovirulence within the AR86 structural proteins.
This possibility was directly tested by introducing the Ser co-
don at E2 position 243 into the G106 clone to create the virus
G117. While G106 caused 87.0% morbidity (day 5 average
clinical score of 2.0) and 26.1% mortality, clone G117 caused
100% morbidity (day 5 average clinical score of 2.0) and 92.6%
mortality, suggesting that the Ser at E2 position 243 was a
major determinant of adult mouse neurovirulence (Fig. 4).
Although the average peak clinical scores for G106 and G117
are the same on day 5, G117-infected mice progress to show
more severe disease and increased mortality.
Determinants at nsP1 538, nsP3 386 to 430, nsP3 537, and
E2 243 are able to confer an adult mouse neurovirulence
phenotype in the Girdwood genetic background. In order to
determine whether the three nonstructural determinants and
the Ser codon at E2 position 243 were the major determinants
of adult mouse neurovirulence in the AR86 virus, the four
determinants were introduced into the G100 background.
While G100 caused no morbidity or mortality in 6-week-old
mice, the virus G163, which was identical to G100 except for
the presence of an Ile-to-Thr change at nsP1 538, an 18-amino-
acid deletion from nsP3 positions 386 to 403, an opal-to-Cys
change at nsP3 537, and a Leu-to-Ser change at E2 243, caused
100% morbidity (day 5 average clinical score of 3.0) and 84.6%
FIG. 5. Identification of the major determinants of neurovirulence within AR86. The cDNA constructs are diagrammed to show the sequences
derived from pS300 (shaded box) and pG100 (open box) along with amino acids located in nsP1 at position 538 (Thr in pS300 and Ile in pG100),
nsP3 between positions 386 and 403 (del 386-403 in pS300), nsP3 at position 537 (Cys in pS300 and Opal in pG100), and E2 at position 243 (Ser
in pS300 and Leu in pG100). Groups (n) of 6-week-old female CD-1 mice were inoculated i.c. with 103PFU of virus and observed daily for clinical
signs. Virulence was measured by morbidity (average CS on day 5), mortality, and AST (average number of days ? SD).
4224 SUTHAR ET AL.J. VIROL.
mortality (Fig. 5). For comparison, the same four changes were
introduced into the S300 background; however, in this case the
changes were Thr to Ile at nsP1 538, reconstitution of nsP3
codons 386 to 403, Cys to opal at nsP3 537, and Ser to Leu at
E2 243. This virus, which was designated S363, caused 71.4%
morbidity (day 5 average clinical score of 0.9) and 0% mortal-
ity, in contrast to the parental S300 virus, which caused 100%
morbidity (day 5 average clinical score of 3.1) and 95.5% mor-
tality (Fig. 5). These results show that the introduction of the
four AR86 determinants back into the nonvirulent Girdwood
background resulted in a virus that exhibited neurovirulence
that was comparable to AR86, demonstrating that these four
changes play a major role in the adult mouse neurovirulence
phenotype of AR86. However, minor contributions from other
coding or noncoding differences between Girdwood and AR86
cannot be ruled out.
In vitro and in vivo growth analysis of the attenuated and
virulent viruses. The differences in virulence between the at-
tenuated viruses (G100 or S363 chimera) and the virulent
viruses (S300 or G163 chimera) could simply be explained by
replication efficiency or the ability to successfully establish in-
fection within neural tissues. Therefore, a single-step in vitro
growth assay was performed for each virus in BHK-21 cells. As
shown in Fig. 6A, the attenuated viruses grew at least as well as
the virulent viruses at all time points tested. Viral growth in the
brains and spinal cords of infected mice was evaluated next.
Though G100 and S363 are significantly attenuated with regard
to causing disease, both of these viruses grew at least as well as
the virulent S300 and G163 viruses in the brains of infected
mice at all times postinfection (Fig. 6B). Furthermore, when
viral growth in the spinal cord was evaluated for S363 (atten-
uated) and S300 (virulent), viral titers were equivalent at 48 h
postinfection, suggesting that both viruses were able to estab-
lish infection within the spinal cord. However, at 96 h postin-
fection, which is a time point when mice are starting to develop
virus-induced disease signs, the virulent S300 virus exhibited
significantly higher titers (P ? 0.05) in the spinal cord than the
attenuated S363 virus (Fig. 6C and D). This result suggests that
the virus containing the four attenuating determinants is either
defective in its ability to spread within the spinal cord or
cleared more efficiently than the virus containing the four
virulence determinants. Additional studies to evaluate the ex-
FIG. 6. In vitro and in vivo growth analysis of virulent and attenuated viruses. (A) A single-step in vitro growth curve was performed on BHK-21
cells infected with S300 (solid line, filled circle), S363 (broken line, open circle), G100 (solid line, open square), or G163 (broken line, filled square)
at an MOI of 5.0. Shown are data from a representative experiment where each point represents the average of results from three independent
samples ? SD. (B) Six-week-old female CD-1 mice were infected i.c. with 103PFU of S300 (solid line, filled circle), S363 (broken line, open circle),
G100 (solid line, open square), and G163 (broken line, filled square). Mice were sacrificed by exsanguination at 6, 12, 24, 48, 72, 96, and 120 h
postinfection and perfused with PBS (pH 7.4). The brain was harvested and evaluated for viral load by plaque assay on BHK-21 cells. The data
shown represent results from one of three experiments for the brain. (C and D) Six-week-old CD-1 mice were infected with S300 (solid line, filled
circle) or S363 (broken line, open circle) as in panel B. Mice were sacrificed at 48, 96, or 144 h postinfection and perfused with PBS, and viral titers
in the thoracic or lumbar spinal cord were determined by plaque assay. (C) Viral titers in the thoracic spinal cord (n ? 3 mice per time point; data
shown represent results from one of two identical experiments). (D) Viral loads in the lumbar spinal cord. Data were pooled from two experiments
and six mice per time point. Differences in viral loads in the thoracic and lumbar spinal cord at 96 h postinfection are statistically significant (P
? 0.05) as measured by two-tailed Student’s t test.
VOL. 79, 2005 NEUROVIRULENCE DETERMINANTS OF SINDBIS VIRUS AR864225
tent of viral replication within these tissues will be required to
address this question.
The identification of the molecular determinants of alpha-
virus virulence represents an important step in understanding
the pathogenesis of alphavirus-induced neurologic disease.
The majority of studies looking at alphavirus neurovirulence
determinants have identified the structural gene E2 and the 5?
noncoding region as major determinants of virulence in both
neonatal and adult mice (3, 19, 29). However, recent work with
the Sindbis virus AR86 and Semliki Forest virus has demon-
strated a role for the nonstructural genes in mediating alpha-
virus neurovirulence (9, 32, 33). In this report, we describe the
identification of four coding changes as major determinants of
AR86 neurovirulence: at nsP1 position 538, at nsP3 position
537, an 18-amino-acid deletion from nsP3 386 to 403, and at E2
position 243. The fact that these four changes conferred adult
mouse neurovirulence on a normally nonneurovirulent Sindbis
virus argues that these changes represent the major determi-
nants of AR86 neurovirulence, although contributions from
additional AR86 determinants cannot be ruled out. It is also
important to note that the neurovirulence determinants did
not appear to simply affect viral replication in vitro or in neural
tissue at early times postinfection (Fig. 6). However, studies in
the spinal cord suggest that the attenuated viruses are either
defective in spreading at late times in the infection or may be
cleared more efficiently than the virulent viruses (Fig. 6). Ad-
ditional work is required to assess these possibilities and also
determine whether the differences between the virulent and
attenuated viruses are spinal cord specific or whether there was
a lack of sensitivity in detecting differences in the brain by
It is striking that all four of the identified AR86 neuroviru-
lence determinants are, to our knowledge, unique to AR86 and
not present in the published sequences of nonadult mouse-
neurovirulent Sindbis viruses (23). Furthermore, these deter-
minants do not appear to be shared with other adult mouse-
neurovirulent Sindbis viruses, such as the NSV strain (3, 19,
31). This result suggests that AR86 and NSV have evolved
different strategies for causing neurologic disease in adult mice.
For instance, while the nonstructural gene determinants of
AR86 are essential for neurovirulence in adult mice, the E2
243 change is only essential in mice greater than 4 weeks of age
(Fig. 1). In contrast, the structural genes of the NSV strain of
Sindbis are the major determinants of adult mouse neuroviru-
lence, with a His at E2 position 55 being particularly important
(31). There are several potential explanations for the differ-
ences in virulence determinants between AR86 and NSV, in-
cluding passage histories and strain variation in the original
viral isolates from which these neurovirulent viruses were de-
rived. NSV was originally derived from the AR339 strain of
Sindbis (7), while AR86 and Girdwood, which are closely re-
lated to each other, were isolated in South Africa. In fact,
Girdwood is one of the few Sindbis viruses isolated from an
infected human (20). Therefore, it is possible that genetic
differences between the AR339 strain and the South African
Sindbis virus strains, such as AR86 and Girdwood, biased these
viruses toward different types of virulence determinants. NSV
and AR86 were also placed under different types of selective
pressure during their early passages. NSV was derived from a
virus that was passaged an unknown number of times in cell
culture and then selected through six rounds of passage in
neonatal and weanling mice (7). In contrast, AR86 was isolated
from a mosquito pool and subjected to 45 to 60 alternating
rounds of intracranial passage in neonatal and weanling mice
(34), with very limited exposure to cell culture prior to the
generation of the infectious clone (23). It is likely that these
vastly different passage histories are in part responsible for the
differences in virulence determinants between AR86 and NSV.
While all four changes are essential for the complete mouse
neurovirulence phenotype of AR86, it appears that the Thr at
nsP1 538 plays a particularly important role. Previous studies
have shown that changing the Thr at nsP1 538 in AR86 to the
consensus Ile found in nonneurovirulent viruses results in a
complete loss of virus-induced mortality, though this virus was
still capable of causing disease (9). Likewise, the introduction
of a Thr codon at nsP1 position 538 into S350 (which contains
the AR86 structural genes in the Girdwood virus background),
creating S351, did not result in a complete restoration of neu-
rovirulence, but a partial gain of virulence was observed (100%
morbidity and 16.7% mortality [Fig. 3]). As for the mechanism
underlying the role for nsP1 538 in virulence, we have previ-
ously reported that the substitution of the wild-type Thr with
an Ile residue accelerates processing of the P123 polyprotein
precursor into the mature nsP1, nsP2, and nsP3 proteins (10).
This coincided with a more rapid induction of 26S RNA syn-
thesis, contributing to earlier expression from the 26S pro-
moter in infected cells, but did not measurably affect the levels
of viral minus- or plus-strand RNA synthesis (10). The role of
altered nonstructural polyprotein processing and/or accelerat-
ed induction of 26S RNA synthesis in adult mouse neuroviru-
lence has yet to be determined. They may be acting through
one or more mechanisms, including induction of immune me-
diators by the earlier 26S RNA expression, enhanced cyto-
pathic effect due to high-level expression of the viral structural
genes, or differential effects on host macromolecular synthesis
due to differences in the ratios of the nonstructural polyprotein
precursor to mature nonstructural proteins.
The 18-amino-acid deletion between residues 386 and 403 in
nsP3 is located within the C-terminal region, which is highly
variable between alphaviruses (as reviewed in reference 25).
The function for the nsP3 protein has yet to be defined. How-
ever, it has been shown to be a phosphoprotein that is required
for the synthesis of both viral minus-strand and subgenomic
RNA (14, 17). It is interesting that the 18-amino-acid deletion
results in the removal of 7 Ser residues, which may affect the
overall phosphorylation of nsP3. Mutational analysis within the
nonconserved C-terminal region of Sindbis virus nsP3 altered
levels of viral minus-strand RNA synthesis, along with levels of
nsP3 phosphorylation (14). Therefore, it will be important to
assess the effect of the 18-amino-acid deletion on viral minus-
strand synthesis. Recently, it was reported that mutations
within the nsP3 gene of SFV, including a 7-amino-acid deletion
within the C-terminal region, fully restored neurovirulence in
adult mice (32), which suggests that nsP3 may contribute to the
virulence of multiple alphaviruses. However, more analysis is
required to determine the exact role for the AR86 nsP3 in
adult mouse neurovirulence.
4226 SUTHAR ET AL.J. VIROL.
While most alphaviruses carry an opal termination codon
proximal to the 3? end of the nsP3 gene, the Sindbis virus strain
AR86 (23) and the SFV strain SFV4 carry a sense codon,
which has been shown to be an important contributor to neu-
rovirulence in adult mice (23, 32, 33). Tuittila and Hinkkanen
performed a detailed mapping study within the replicase genes
using virulent and avirulent SFV strains and found that an Arg
(virulent SFV4 strain) at nsP3 position 469, in place of the opal
termination codon, was an important contributor to the adult
mouse neurovirulence phenotype (32). The mechanism of this
change in pathogenesis is not well understood. Translational
readthrough of the opal termination codon occurs at a fre-
quency of about 5 to 10% (as reviewed in reference 4), leading
to limiting quantities of the nsP4 protein relative to the other
nonstructural proteins. Interestingly, the nsP4 protein was
found to be tightly regulated within infected cells, in that ex-
cess nsP4 was shown to be rapidly degraded by the N-end rule
pathway (1). It is also worth noting that a different nsP3 C
terminus is produced in viruses carrying either an opal termi-
nation codon or a sense codon. In the presence of an opal
termination codon, the predominate nsP3 C terminus is pro-
duced by the translational stop codon, while in the presence of
a sense codon, an extra 7 amino acids are added to the C
terminus of nsP3. It is well known that the opal termination
codon regulates both nonstructural polyprotein processing and
viral RNA synthesis. Li and Rice replaced the opal termination
codon of Sindbis virus with different sense codons and found
increased levels of the nsP3/4 polyprotein precursor and re-
duced levels of mature nsP3 early during infection (18). This
study also demonstrated that replacing the opal termination
codon with a sense codon led to reduced levels of both 49S
genomic and 26S subgenomic viral RNA synthesis early during
infection (18). Based on these studies, it will be important to
determine whether the Cys codon in AR86 affects neuroviru-
lence through alterations of nonstructural polyprotein process-
ing and/or viral RNA synthesis, which may exert subtle effects
on viral replication or affect a yet to-be-defined interaction
with the host.
The neurovirulence determinant within the E2 glycoprotein
at position 243, where AR86 encodes a unique serine residue,
most likely affects early viral interactions with neurons or other
cell types in the infected animal. This change is near a region
of E2 that is associated with receptor attachment, which raises
the strong possibility that the Ser residue might affect virus-
receptor interactions either through direct receptor interac-
tions or by changing the conformation of the E2 glycoprotein
(24). This is supported by findings within the E2 glycoprotein
of Sindbis virus, in that a Gly residue at position 172 enhanced
viral binding to neuronal cells (29). Therefore, additional stud-
ies to evaluate the effect of Ser versus Leu at E2 position 243
on virus binding or infection of neurons may provide useful
information on the role of this determinant in regulating viral
infection of neurons or other cell types. However, the lack of a
clearly defined neuronal receptor for alphaviruses currently
prevents a direct analysis of this determinant’s role in virus-
In conclusion, we have identified major determinants of
Sindbis virus adult mouse neurovirulence by using two closely
related neurovirulent and nonneurovirulent Sindbis-group vi-
ruses. These determinants are nsP1 position 538, a deletion in
nsP3 between 386 and 403, nsP3 position 537, and E2 position
243. Further studies to determine whether these virulence de-
terminants act by affecting viral RNA synthesis or cell tropism
or through some as yet undefined interaction with the infected
host are under way.
This research was supported by NIH research grant R01 AR47190.
We thank the members of the Carolina Vaccine Institute and the
Johnston Laboratory for helpful scientific discussions. We also thank
Dwayne Muhammad for providing excellent technical support with cell
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