Journal of Medical Virology 82:304310 (2010)

Immunomodulatory Cytokines Determine the

Outcome of Japanese Encephalitis Virus Infection
in Mice
S.M. Biswas,1 S. Kar,1 R. Singh,1 D. Chakraborty,2 V. Vipat,1 C.G. Raut,1 A.C. Mishra,1
M.M. Gore,1 and D. Ghosh1*
1

National Institute of Virology, Sus Road Campus, Pashan, Pune, Maharashtra, India
DSS Imagetech Pvt Ltd, South Extension Part II, New Delhi, India

Japanese encephalitis virus (JEV) induces an

acute infection of the central nervous system,
the pathogenic mechanism of which is not fully
understood. To investigate host response to JEV
infection, 14-day-old mice were infected via the
extraneural route, which resulted in encephalitis
and death. Mice that received JEV immune
splenocyte transfer were protected from extraneural JEV infection. Pathology and gene expression profiles were then compared in brains of
mice that either succumbed to JEV infection or
were protected from infection by JEV immune
cell transfer. Mice undergoing progressive JEV
infection had increased expression of proinflammatory cytokines, chemokines, and signal transducers associated with the interferon (IFN)
pathway. In contrast, mice receiving immune cell
transfer had increased production of the Th2
cytokine IL-4, and of IL-10, with subdued expression of IFN-g. We observed IL-10 to be an
important factor in determining clinical outcome
in JEV infection. Data obtained by microarray
analysis were further confirmed by quantitative
RT-PCR. Together, these data suggest that JEV
infection causes an unregulated inflammatory
response that can be countered by the expression
of immunomodulatory cytokines in mice that
survive lethal infection. J. Med. Virol. 82:304
310, 2010. 2009 Wiley-Liss, Inc.
KEY WORDS: Japanese encephalitis; microarray;
adoptive
transfer;
splenocytes

INTRODUCTION
Japanese encephalitis (JE) is a flaviviral disease of
great medical importance. Japanese encephalitis virus
(JEV) infection occurs throughout most of Asia and is
also spreading to new geographical areas like Australia.
Children are at greatest risk of infection in endemic
areas [Ruben and Gajanana, 1997].
2009 WILEY-LISS, INC.

Factors governing the establishment of JEV infection

in the central nervous system (CNS) are poorly understood. The course of disease in humans can effectively
be replicated in laboratory rodents. Young mice are
susceptible to extraneural inoculation with JEV and
succumb to infection soon after the establishment of
infection in the CNS [Johnson et al., 1985; Hase et al.,
1990]. The adoptive transfer of JEV immune splenocytes
into these young mice has been shown to confer
protection against subsequent lethal challenge with
JEV, thus demonstrating a role for cellular immunity in
JEV infection [Biswas et al., 2009]. Differential expression profiling of genes using high throughput system
enables the identification of critical genes responsible
for modulation of infection and a subsequent understanding of the cellular and molecular pathways
associated with disease progression.
Most gene profiling studies in flavivirus infection have
been carried out using permissive cell lines. Microarray
analysis of human glioblastoma cells infected with West
Nile Virus revealed the differential expression of
173 cellular genes, of which 23 were identified that
might play a role in cellular neuro-degeneration [Koh
and Ng, 2005]. Apoptosis was identified as the mechanism of cell death induced by JEV replication in several
neuronal and non-neuronal cell lines [Liao et al., 1997].
In addition, activation of tumor necrosis factor receptor
1 (TNFR1) and signaling through the TNFR-associated
death domain (TRADD) following JEV infection was
assumed to trigger downstream apoptotic cascades in
mouse and human neuroblastoma cell lines [Swarup
et al., 2007a].

Additional Supporting Information may be found in the online

version of this article.
*Correspondence to: D. Ghosh, National Institute of Virology,
Sus Road Campus, Pashan, Pune 411021, India.
E-mail: dhrubaa2010@gmail.com
Accepted 11 September 2009
DOI 10.1002/jmv.21688
Published online in Wiley InterScience
(www.interscience.wiley.com)

Gene Profiling in JEV Infection

Studies on flavivirus infection in the mouse brain

provide an opportunity to study the complex interplay of
factors involved in vivo. Subtraction hybridization
revealed 24 genes in the brains of JEV infected mice,
most of which were identified to be IFN inducible genes
[Saha et al., 2006]. Comparison of gene expression
profiles in mice infected with West Nile virus strains of
different neurovirulence identified genes involved in
IFN signaling pathways, protein degradation, T-cell
recruitment, MHC class I and II antigen presentation
and apoptosis [Venter et al., 2005].
In the present study, gene expression profiling of nave
mice at an early time point following extraneural
infection with JEV was carried out. This time point was
selected in order to detect early host gene responses to JE
virus infection, as changes in host gene expression to
virus infection at this stage might prove critical to the
outcome of infection. Further, profiling was done in
brains from mice that were protected from JEV challenge
through the adoptive transfer of JEV immune splenocytes. Thus, the role played by immune cells in preventing virus entry and spread into the brains of JEV infected
mice could be studied. A major observation was the
marked increase in expression of the immunomodulatory
cytokine IL-10 in the brains of mice that were protected
from lethal JEV infection. In addition, expression of the
proinflammatory cytokine IFN-g and signal transducers
STAT-1 and STAT-2 were down-modulated in these mice.
Conversely, mice that succumbed to JEV infection had
significantly down-regulated expression of IL-10, which
probably helped in the establishment of an unregulated
proinflammatory environment in these animals. The
results obtained upon microarray analysis were further
confirmed by quantitative RT-PCR of selected genes.
MATERIALS AND METHODS
Virus and Animals
JEV strain 733913 was obtained from the Institutes
virus repository. Mice were procured from the small
animal facility of the Institute and were maintained
according to the Committee for Protection, Supervision
and Control of Experiments on Animals (CPSCEA)
guidelines. A 20% mouse brain derived stock of the virus
was titrated in 14-day-old mice and used for all virus
experiments. Fourteen-day-old nave BALB/c mice were
infected through the intra peritoneal (IP) route with
50 LD50 (50 times the lethal dose50) titer of JEV.
Experimental controls were mock injected with 0.75%
bovine serum albumin (BSA) in phosphate-buffered saline
(PBS). In addition, mice were adoptively transferred with
2
107 splenocytes from JEV immune syngenic animals
by the intravenous route and challenged 24 hr postinfection (PI) with 50 LD50 of JEV [Biswas et al., 2009].
Quantitation of Viral RNA From JEV
Infected Mouse Brains
Infected and control mice were euthanized by CO2
asphyxiation at various time points PI. Blood was

305

drained by cardiac puncture and brains were harvested

and weighed. A 10% mouse brain homogenate was made
in PBS 0.75% BSA, and RNA was isolated by the
QIAMP viral RNA kit (Qiagen, Hilden, Germany). Viral
RNA was quantitated using the Taqman based Geno
Sens JEV quantitative RT-PCR (Professional Biotech,
New Delhi, India) kit on an ABI 7300 real-time PCR
system [Biswas et al., 2009].
Histology
Mice that were either lethally infected with JEV or
protected from infection by immune cell transfer were
sacrificed at various time points PI. Blood was drained
by cardiac puncture and brains were excised and fixed in
10% buffered formalin. Organs were processed by
treating with three changes of acetone and a 1:1 mix of
acetone benzene, followed by two changes of benzene.
Organs were then impregnated with paraffin wax
and sectioned. Sections were stained with hematoxylin
and eosin and observed.
Preparation of Total Cellular RNA and
Hybridization to Gene Chip
Infected mice and age-matched controls were sacrificed as described on day 3 PI. Brains were also
harvested at the same time point from mice that were
adoptively transferred with splenocytes from JEV
immune animals prior to virus challenge. A 10% mice
brain homogenate was prepared in TRI reagent (MRC,
Cincinnati, OH) and total RNA was purified using the
RNeasy kit (Qiagen). Ten micrograms of RNA was
reverse transcribed with amino allyl dNTPs using the
ChipShot Indirect Labeling System (Promega, Madison,
WI). The purified amino allyl cDNA was labeled with
Cy3 and Cy5 (Amersham, Uppsala, Sweden) for control
and experimental samples respectively. Dye swap labeling was carried out to prevent bias in Cy Dye
incorporation. Purified samples were lyophilized, resuspended in hybridization buffer (Pronto Universal
Hybridization kit, Corning) and hybridized on the
Discovery mouse chip provided by Arrayit (Telechem
International, Sunnyvale, CA). Hybridization was carried out in a Hybstation (Genomic Solutions, Ann Arbor,
MI) and the profile used was 558c for 6 hr, 508c for 6 hr,
and 428c for 6 hr. Scanning was performed at 10-mm
resolutions with the Scan array express (PerkinElmer,
Waltham, MI). Grid alignment was done using gene
annotation files and raw data were extracted into MS
EXCEL.
Data Analysis
Data were analyzed using Genowiz Microarray
and Pathway analysis tool (Ocimum Biosolutions,
Hyperabad, India). Replicated genes were merged and
median values of the expression ratios of these replicated genes were further considered for the dataset.
Empty spots were removed by filtering. The final dataset
consisted of 381 genes/probes out of 785. Dye bias was
dealt with by applying loess normalization. Log transJ. Med. Virol. DOI 10.1002/jmv

306

Biswas et al.

formation (log2) was carried out to stabilize the variation

in dataset and median centering was performed to bring
down data distribution of dataset close to zero. In order
to detect highly expressed genes, fold change analysis
was done. Genes with twofold up/down-regulation were
considered as differentially expressed. Functional classification of the genes was performed using Gene
Ontology and pathway analysis.
Quantitative RT-PCR of Host Genes
Using SYBR Green I
The differential expression data was further validated
by quantitative RT-PCR. Primers were selected from
Primer Bank (http://pga.mgh.harvard.edu/primerbank).
Two hundred fifty nanograms of total RNA from
brains of virus infected, control and mice receiving
JEV immune splenocytes were used for quantitative
RT-PCR analysis. Reaction was performed using the
QuantiTect SYBR Green RT-PCR kit (Qiagen) according to the manufacturers instructions. Reaction efficiency was calculated by using serial 10-fold dilutions
of the housekeeping gene b-actin and sample genes
(Supplementary Fig. 1). Reactions were carried out on
an ABI 7300 real-time PCR system and the thermal
profile used was Stage 1: 508C for 30 min; Stage 2: 958C
for 15 min; Stage 3: 948C for 15 sec, 608C for 30 sec; and
728C for 30 sec, repeated for 40 cycles. Melting curve
analysis was performed to verify product specificity.
RESULTS
Quantitative RT-PCR of JEV Infected
Mouse Brains
Real-time kinetics of JEV replication in 14-day-old
mouse brain following peripheral infection revealed
an initial phase of virus growth starting from day 2 PI,
which increased steadily and reached a peak by
day 8 (Fig. 1). Mice started showing symptoms of
sickness by day 6 and death was observed on days 7
and 8 PI. Based on viral titers, the early time point
selected for microarray analysis of infected mice brain
was day 3 PI.

Viral RNA copies / gm brain (log10)

10

0
day2

day4

day6

day8

Days post infection

Fig. 1. Fourteen-day-old mice were infected by the IP route with JEV

and brains harvested every alternate day PI. Viral titers were
calculated by quantitative RT-PCR for JEV.

J. Med. Virol. DOI 10.1002/jmv

Histological Analysis of JEV Infected

Mouse Brains
Brains from mice infected with JEV were observed
for histological analysis. Mice that succumbed to JEV
infection had evidence of inflammation in the brain at
all time-points PI. Major changes recorded were perivascular cuffing (Fig. 2a), cellular infiltrates (Fig. 2b)
and mild vascular damage. The focal infiltration of
polymorphonuclear (PMN) cells and mononuclear cells
(MNC) in the meninges and cerebral tissues was
observed at later time points PI (Fig. 2c). Areas of
necrosis with neuronal shrinkage were observed
(Fig. 2d). Inflammatory infiltrates were associated with
neuronal damage. Infiltrating inflammatory cells in
JEV infected mouse brain have mostly been identified as
lymphocytes and macrophages [German et al., 2006].
We observed the infiltration of mainly PMN cells into
the brains of infected animals. Brains of mice that were
protected from JEV infection by immune cell transfer
mostly showed no abnormalities other than slight
congestion (Fig. 3a,b). Necrosis and glial nodule formation was observed on day 8 PI (Fig. 3c), while
neuronophagia around necrotic foci was also observed
(Fig. 3d), suggesting viral presence in the CNS of
protected mice.
Analysis of Differentially Expressed Genes and
Validation of Their Expression by SYBR Green
Quantitative RT-PCR Analysis
Differentially expressed genes were acquired from
a final dataset of 381 probes by microarray analysis.
This was further validated by quantitative RT-PCR
which determined fold differences in gene expression
by the 2DDCt method. Maximally up-regulated genes
were STAT1, STAT2, IFN-g and b2M, which were upregulated 20-, 9-, 18-, and 10-fold respectively (Table I).
These genes were also found to show highest fold
increase upon quantitative RT-PCR analysis (13.5-,
3.6-, and 2.9-fold up-regulation respectively). Most of
the up-regulated genes were recognized to be Interferon
Stimulated Genes (ISGs) and downstream mediators of
TNF signaling. Other important up-regulated genes
were those involved in cell cycle progression-cyclins and
cyclin-dependent kinases (CDKs), receptor tyrosine
kinases, and the co-stimulatory molecules CD40 and
CD70.
Down-regulated genes were mainly identified as cell
cycle inhibitors and cell death associated genesFAS
and BCL2 (Table II). Type I receptor cytokines and
their receptors were also mostly down-regulated. The
immunoregulatory cytokine IL-10 showed the highest
level of down-regulation (0.004-fold). IL-10 levels were
also seen to be maximally down-regulated upon quantitative RT-PCR analysis (0.3-fold down-regulation).
It was earlier shown that 14-day-old mice receiving splenocytes from JEV immunized animals
were protected from subsequent lethal infection with
JEV [Biswas et al., 2009]. Brains of mice that were
protected from infection had diminished expression of

Gene Profiling in JEV Infection

307

Fig. 2. Histological analysis from brains of mice lethally infected with JEV, harvested at various time
points post-JEV infection. a: Perivascular cuffing on day 2 PI (original magnification (OM) 20
).
b: Inflammatory foci on day 4 PI (OM 20
). c: PMN cell infiltration on day 8 PI (OM 40
). d: PMN cell
infiltration and neuronal shrinkage on day 8 PI (OM 20
).

the proinflammatory cytokine IFN-g and associated

transcription factors STAT1 and STAT2 as observed by
both differential gene expression profiling and quantitative RT-PCR analysis. An enhanced expression of the
chemokines CXCL10, RANTES, and MCP-1 was also
observed in protected animals.
Genes that were down-regulated in JEV infected mice
brain strikingly had elevated expressions in mice that
were adoptively transferred with JEV immune splenocytes. Notable among these was IL-10 (4.5-fold upregulation). The immunomodulatory cytokine TGF-b
was also seen to be up-regulated, as was RIPK, the
TNFR associated adaptor which is crucial for NFkB
mediated antiapoptotic signaling. IL-10 also showed
high levels of up-regulation (42.4-fold) in protected
mice brains when assayed by quantitative RT-PCR.
This increased expression of IL-10 in mice that
were protected from JEV infection was particularly
interesting, considering that mice that succumbed to

JEV infection had significantly decreased levels of

IL-10.
DISCUSSION
The pattern of host gene expression in virus infected
brain may contribute to our understanding of encephalitis. Differential gene expression profiling provides a
tool to explore the molecular interaction of JEV with
host cells.
Here, we investigated the differential expression of
genes in the brain during early infection of 14-day-old
mice through the IP route, which mimics the natural
course of disease progression into the brain. We also
studied host gene expression in age-matched mice that
were subjected to JEV immune cell transfer. Factors
involved in cell cycle progression, growth factor receptors, transcriptional activators, cytokines and immunomodulators, and genes involved in signal transduction
were seen to be modulated in response to JEV infection.
J. Med. Virol. DOI 10.1002/jmv

308

Biswas et al.

Fig. 3. Histological analysis from brains of mice protected from lethal JEV infection by the adoptive
transfer of JEV immune splenocytes. a: Slight congestion on day 2 PI (original magnification (OM) 20
).
b: Slight congestion on day 4 PI (OM 20
). c: Necrosis and glial nodule formation on day 8 PI (OM 40
).
d: Neuronophagia on day 10 PI (OM 40
).

TABLE I. 14 Genes of Our Interest Which Were Up-Regulated Upon JEV Infection Are Represented
Fold change in mice infected with JEV
GenBank Acc. No.
NM_009283
AF187231
K00083
NM_011609
NM_011610
NM_009735
NM_008689
NM_011611
NM_011617
NM_016756
X64713
NM_021274
NM_013653
AF065929

Gene name
STAT1
STAT2
IFN-g
TNFR1
TNFR2
b2M
NFkB1
CD40
CD70
CDK2
CCNB1 (cyclin B1)
CXCL10
RANTES
MCP-1

Microarray
20.436
9.650
18.354
2.291
1.339
10.189
4.31
8.515
3.311
1.164
1.407
1.356
Not detected
Not detected

Real-time PCR
16.13
3.618
1.488
1.209

(4.39)
(0.78)
(0.32)
(0.16)

2.982 (1.01)
1.529 (0.22)

Fold change in mice receiving JEV

immune splenocytes before infection
Microarray
6.5
Not detected
3.67
10.090
4.789
11.202
5.455
5.093
2.687
3.241
7.416
15.068
10.024
6.3

Real-time PCR
0.886
1.089
0.820
1.385

(0.22)
(0.12)
(0.19)
(0.13)

1.161 (0.25)
1.113 (0.37)

Comparisons in gene expression levels between mice infected with JEV and mice protected from JEV infection by immune cell transfer are shown.
Fold change values obtained by quantitative RT-PCR for a subset of these genes are also indicated along with standard deviations.

J. Med. Virol. DOI 10.1002/jmv

Gene Profiling in JEV Infection

309

TABLE II. In the Microarray Analysis, 15 Genes That Were Down-Regulated Upon JEV Infection Are Represented
Fold change in mice infected with JEV
GenBank Acc. No.
NM_007987
NM_009741
NM_009068
NM_009367
NM_011640
U41504
NM_008369
NM_021283
NM_010548
NM_010549
NM_008350
NM_008354
S80963
NM_008358
NM_010927

Fold change in mice receiving JEV

immune splenocytes before infection

Gene name

Microarray

Real-time PCR

Microarray

Real-time PCR

FAS
BCL2
RIPK1
TGFb
TRP53
IL-2
IL-3Ra
IL-4
IL-10
IL-11RA1
IL-11
IL-12RB2
IL-13RA1
IL-15Ra
NOS2

0.189
0.315
0.114
0.239
0.683
0.318
0.832
0.326
0.004
0.236
0.540
0.063
0.528
0.542
0.808

0.881 (0.10)
0.873 (0.33)

6.417
2.206
9.767
3.725
2.75
4.442
9.694
2.809
4.5
2.969
5.667
4.286
9.791
7.155
Not detected

2.151 (0.16)
0.933 (0.39)

0.433 (0.23)
0.816 (0.08)

0.322 (0.01)

0.903 (0.03)

0.942 (0.28)
1.041 (0.30)

42.419 (3.98)

1.381 (0.12)

Comparisons in gene expression levels between mice infected with JEV and mice protected from JEV infection by immune cell transfer are shown.
Fold change values obtained by quantitative RT-PCR for a subset of these genes are also indicated along with standard deviations.

In mice that succumbed to JEV infection, signal transducers STAT1, STAT2 and the proinflammatory cytokine IFN-g were highly up-regulated, as were many
ISGs. Interestingly, in case of protected mice, the levels
of STAT1, STAT2, and IFN-g gene expression were
down-modulated as compared to levels in susceptible
mice. These results were similar to cytokine serum
protein levels obtained in our earlier study, where mice
that were susceptible to lethal JEV infection had high
serum levels of the proinflammatory cytokines TNF-a
and IFN-g compared with mice that were protected from
infection by immune cell transfer [Biswas et al., 2009].
IFN-g also primes recruited macrophages and resident microglial cells in the brain to induce expression of
NOS2 and the proinflammatory cytokine TNF-a [Ghoshal et al., 2007]. In our study, NOS2 mRNA was
unaffected under both diseased and protected conditions, suggesting that NO has a limited role in the
immunopathogenesis of JEV.
Among other up-regulated genes, both infected and
protected mice expressed increased transcription of the
2 M gene (MHC-I). The increased expression of MHC-I
is induced via NFkB in response to JEV replication,
though IFN-g is also known to up-regulate this molecule
[Lobigs et al., 2003]. The up-regulation of MHC and
chemokines like MCP-1, RANTES and the IFN-g early
response gene CXCL10 produced by resident CNS cells
results in the infiltration of macrophages and effector
T cells from the periphery [Chen et al., 2004; Klein
et al., 2005; Bhowmick et al., 2007]. The infiltration of
inflammatory cells into the CNS was evident on
histological analysis of JEV infected mouse brains
(Figs. 2 and 3).
The proinflammatory cytokine TNF-a can mediate its
effects through either of its receptors, TNFR1 (p55) and
TNFR2 (p75), both of which were up-regulated in mice
that succumbed to JEV infection [Chen and Goeddel,
2002]. TNFR1 signaling via TRADD has been observed
to promote apoptosis in JEV infected neuroblastoma cell

lines [Swarup et al., 2007a]. In lethally infected mice,

the TRADD interacting adaptor kinase, receptor interacting protein kinase I (RIPK1) was down-regulated,
while up-regulation of this molecule was observed in
protected mice, possibly signifying that this molecule is
a crucial intermediate in the response to infection
[Festjens et al., 2007].
Various cell cycle progressors like CDK2 and cyclin B1
were also up-regulated upon JEV infection. Cyclins and
CDKs are used for viral transcription during infection
[Schang et al., 1999]. It has also been shown in a West
Nile model of infection that cycling cells produce
significantly more virus than cells in the G0 phase
[Kesson et al., 2002].
An interesting observation was the down-regulation
of the type I receptor cytokines IL-2, 3, 4, 10, 11, 12, 13,
15 or their receptors in mice that succumbed to JEV
infection. Expression of TGF-b was also downregulated. IL-4 and IL-13 are Th2 cytokines with
immunomodulatory activities. In our earlier study,
expression of serum protein levels of IL-2 and the Th2
cytokines IL-4 and IL-5 was seen to be down-regulated
in JEV susceptible mice as infection progressed compared with mice that were protected from infection,
which had sustained levels of IL-4 and IL-5 at all time
points PI [Biswas et al., 2009]. These results further
validate our initial findings and also establish the role of
antiinflammatory cytokines like IL-10 as a negative
regulator of inflammation and antagonist of IFN-g
expression [Liu et al., 2003]. Mice that were protected
from JEV infection had highly elevated levels of IL-10.
The role of IL-10 in JEV infection has been reported
recently [Swarup et al., 2007b; Saxena et al., 2008].
These studies however, were performed using intracerebral infection or subclinical infection with JEV
respectively. The current study not only confirms the
earlier results, but uses a route of infection which closely
mimics the route of entry of JEV into the CNS during
natural infection, thus reflecting natural disease proJ. Med. Virol. DOI 10.1002/jmv

310

Biswas et al.

gression. In addition, the observation of increased IL-10

levels in mice that are protected from infection further
substantiates the function of IL-10 as an immunomodulatory molecule that mediates protection from acute
encephalitis. IL-10 may therefore, play a central role in
determining the clinical outcome in JEV infection.
The installment of a primarily proinflammatory
environment in the CNS upon JEV infection, mediated
mainly by cytokines and chemokines which are either
recruited from the periphery or produced by resident
microglia and macrophages has been demonstrated by
many studies on JEV pathogenesis [Winter et al., 2004;
Bhowmick et al., 2007]. In many cases, the virus is not
directly involved in the destruction of brain tissue, but
instead causes indirect damage through cell-mediated
immune responses. Activated inflammatory cells
secrete cytokines such as IL-1b, IL-8, IL-18, and TNFa, which can cause apoptosis of neuronal cells [Singh
et al., 2000; Ghoshal et al., 2007; Das et al., 2008]. The
production of the antiinflammatory cytokine IL-10 in
such an environment may therefore, provide the balance
between activation and modulation of antiviral effectors. A recent study has documented the release of IL-10
upon JEV infection of dendritic cells (DCs) which led to
reduced priming of CD8 T cells, thus demonstrating
that IL-10 produced by JEV-infected DCs function as a
mediator of the suppression of T cell activation, at least
in CD8 T cells [Aleyas et al., 2009].
In conclusion, the stimulation of an antiviral response
through the up-regulation of IFN and MHC-1 and the
large-scale recruitment of effector lymphocytes into the
brain, in addition to the up-regulation of TNF-a may
result in the perpetuation of a primarily proinflammatory environment in the brain that could result in death
of the host through immunopathogenesis. In contrast,
increased production of the Th2 cytokine IL-4 and of IL10 and TGF-b, coupled with subdued expression of IFNg, was seen to directly control unrestrained neuronal
death caused by an unregulated immune response.
ACKNOWLEDGMENTS
S. M. Biswas is in receipt of Senior Research fellowship and Dr. R. Singh is a Research Associate from the
Indian Council of Medical Research, New Delhi.
REFERENCES
Aleyas AG, George JA, Han YW, Rahman MM, Kim SJ, Han SB, Kim
BS, Kim K, Eo SK. 2009. Functional modulation of dendritic cells
and macrophages by Japanese encephalitis virus through MyD88
adaptor molecule-dependent and independent pathways. J Immunol 183:24622474.
Bhowmick S, Duseja R, Das S, Appaiahgiri MB, Vrati S, Basu A. 2007.
Induction of IP-10 (CXCL10) in astrocytes following Japanese
encephalitis. Neurosci Lett 414:4550.
Biswas SM, Ayachit VM, Sapkal GN, Mahamuni SA, Gore MM. 2009.
Japanese encephalitis virus produces a CD4-Th2 response and
associated immunoprotection in an adoptive transfer murine
model. J Gen Virol 90:818826.
Chen C-J, Chen J-H, Chen S-Y, Liao S-L, Raung S-L. 2004. Upregulation of RANTES gene expression in neuroglia by Japanese
encephalitis virus infection. J Virol 78:1210712119.

J. Med. Virol. DOI 10.1002/jmv

Chen G, Goeddel DV. 2002. TNF-R1 signaling: A beautiful pathway.

Science 296:16341635.
Das S, Mishra MK, Ghosh J, Basu A. 2008. Japanese encephalitis virus
infection induces IL-18 and IL-1b in microglia and astrocytes:
Correlation with in vitro cytokine responsiveness of glial cells and
subsequent neuronal death. J Neuroimmunol 195:6072.
Festjens N, Vanden Berghe T, Cornelis S, Vandenabeele P. 2007. RIP1,
a kinase on the crossroads of a cells decision to live or die. Cell Death
Differ 14:400410.
German AC, Myint KSA, Mai NTH, Pomeroye I, Phu NH, Tzartos J,
Winter P, Collett J, Farrar J, Barrett A, Kipar A, Esiri MM, Solomon
T. 2006. A preliminary neuropathological study of Japanese
encephalitis in humans and a mouse model. Trans R Soc Trop
Med Hyg 100:11351145.
Ghoshal A, Das S, Ghosh S, Mishra MK, Sharma V, Koli P, Sen E, Basu
A. 2007. Proinflammatory mediators released by activated microglia induces neuronal death in Japanese encephalitis. Glia 55:483
496.
Hase T, Dubois DR, Summers PL. 1990. Comparative study of mouse
brains infected with Japanese encephalitis virus by intracerebral or
intraperitoneal inoculation. Int J Exp Pathol 71:857869.
Johnson RT, Burke DS, Elwell M, Leake CJ, Nisalak A, Hoke CH,
Lorsomrudee W. 1985. Japanese encephalitis: Immunocytochemical studies of viral antigen and inflammatory cells in fatal cases.
Ann Neurol 18:567573.
Kesson AM, Cheng Y, King NJC. 2002. Regulation of immune
recognition molecules by flavivirus, West Nile. Viral Immunol 15:
273283.
Klein RS, Lin E, Zhang B, Luster AD, Tollet J, Samuel MA, Engle M,
Diamond MS. 2005. Neuronal CXCL10 directs CD8 T-cell
recruitment and control of West Nile virus encephalitis. J Virol
79:1145711466.
Koh W-L, Ng M-L. 2005. Molecular mechanisms of West Nile virus
pathogenesis in brain cells. Emerg Infect Dis 11:629632.
Liao C-L, Lin Y-L, Wang J-J, Huang Y-L, Yeh C-T, Ma S-H, Chen L-K.
1997. Effect of enforced expression of human bcl-2 on Japanese
encephalitis virus-induced apoptosis in cultured cells. J Virol 71:
59635971.
Liu XS, Xu Y, Hardy L, Khammanivong V, Zhao W, Fernando GJ,
Leggatt GR, Frazer IH. 2003. IL-10 mediates suppression of the
CD8 T cell IFN-gamma response to a novel viral epitope in a primed
host. J Immunol 171:47654772.
Lobigs M, Mu llbacher A, Regner M. 2003. MHC-class I up-regulation by
flaviviruses: Immune interaction with unknown advantage to host
or pathogen. Immunol Cell Biol 81:217223.
Ruben R, Gajanana A. 1997. Japanese encephalitis in India. Indian
J Pediatr 64:243251.
Saha S, Sugumar P, Bhandari P, Rangarajan PN. 2006. Identification
of Japanese encephalitis virus-inducible genes in mouse brain and
characterization of GARG39/IFIT2 as a microtubule-associated
protein. J Gen Virol 87:32853289.
Saxena V, Mathur A, Krishnani N, Dhole TN. 2008. Kinetics of cytokine
profile during intraperitoneal inoculation of Japanese encephalitis
virus in BALB/c mice model. Microbes Infect 10:12101217.
Schang LM, Rosenberg A, Schaffer PA. 1999. Transcription of Herpes
Simplex virus immediate-early and early genes is inhibited by
roscovitine, an inhibitor specific for cellular cyclin-dependent
kinases. J Virol 73:21612172.
Singh A, Kulshreshtha R, Mathur A. 2000. Secretion of the chemokine
interleukin-8 during Japanese encephalitis virus infection. J Med
Microbiol 49:607612.
Swarup V, Das S, Ghosh S, Basu A. 2007a. Tumor necrosis factor
receptor-1 induced neuronal death by TRADD contributes to the
pathogenesis of Japanese encephalitis. J Neurochem 103:771783.
Swarup V, Ghosh J, Tuseja R, Ghosh S, Basu A. 2007b. Japanese
encephalitis virus infection decrease endogenous IL-10 production:
Correlation with microglial activation and neuronal death. Neurosci Lett 420:144149.
Venter M, Myers TG, Wilson MA, Kindt TJ, Paweska JT, Burt FJ,
Leman PA, Swanepoel R. 2005. Gene expression in mice infected
with West Nile virus strains of different neurovirulence. Virology
342:119140.
Winter PM, Dung NM, Loan HT, Kneen R, Wills B, Thu LT, House D,
White NJ, Farrar JJ, Hart CA, Solomon T. 2004. Proinflammatory
cytokines and chemokines in humans with Japanese encephalitis.
J Infect Dis 190:16181626.