Sarkar et al.

Virology Journal 2012, 9:271

http://www.virologyj.com/content/9/1/271

SHORT REPORT Open Access

Molecular evidence for the occurrence of

Japanese encephalitis virus genotype I and III
infection associated with acute Encephalitis in
Patients of West Bengal, India, 2010
Arindam Sarkar1, Debjani Taraphdar1, Subhra Kanti Mukhopadhyay2, Sekhar Chakrabarti1
and Shyamalendu Chatterjee1*

Abstract
Background: Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is the sole etiologic agent of
Japanese Encephalitis (JE); a neurotropic killer disease which is one of the major causes of viral encephalitis
worldwide with prime public health concern. JE was first reported in the state of West Bengal, India in 1973. Since
then it is being reported every year from different districts of the state, though the vaccination has already been
done. Therefore, it indicates that there might be either partial coverage of the vaccine or the emergence of
mutated/new strain of JEV. Considering this fact, to understand the JEV genotype distribution, we conducted a
molecular epidemiological study on a total of 135 serum/cerebrospinal fluid (CSF) samples referred and/or collected
from the clinically suspected patients with Acute encephalitis syndrome (AES), admitted in different district hospitals
of West Bengal, India, 2010.
Findings: JEV etiology was confirmed in 36/135 (26.6%) and 13/61 (21.3%) 2–15 days’ febrile illness samples from
AES cases by analyzing Mac-ELISA followed by RT-PCR test respectively. Phylogenetic analysis based on complete
envelope gene sequences of 13 isolates showed the emergence of JEV genotype I (GI), co-circulating with
genotype III (GIII).
Conclusion: This study represents the first report of JEV GI with GIII, co-circulating in West Bengal. The efficacy of
the vaccine (derived from JEV GIII strain SA-14-14-2) to protect against emerging JEV GI needs careful evaluation.
In future, JE outbreak is quite likely in the state, if this vaccine fails to protect sufficiently against GI of JEV.
Keywords: Acute encephalitis syndrome, Japanese encephalitis virus, Genotype I, Genotype III, West Bengal

Background problem. Worldwide case-fatality rate of JE was recorded

The mosquito-borne Japanese encephalitis virus (JEV) is to be 30% approximately with 30-50% of survivors devel-
an enveloped, positive-sense single-stranded RNA virus, oping permanent neurologic deficit/sequelae [3].
member of the genus Flavivirus under the family Flavi- Recent studies have shown that the envelope (E) gene
viridae [1]. JEV is the sole etiologic agent of Japanese En- is an established phylogenetic marker for JEV, since this
cephalitis (JE); a neurotropic killer disease being one of region is free from selective pressure that supports ob-
the major causes of viral encephalitis in human. Since scure long-term evolutionary relationship [4]. Altogether
the isolation of this virus in Japan in 1935 [2], it has 5 distinct genotypes have been identified among the JEV
spread worldwide becoming a major public health strains [5] of which genotype III (GIII) is mostly circu-
lated in the Southeast Asian countries, including Japan,
South Korea, China, Taiwan, Vietnam, Philippines, and
* Correspondence: shyamalenduchatterjee@gmail.com
1
ICMR virus unit, ID & BG Hospital, 57, Dr. S. C. Banerjee Road, Beliaghata, India [6]. However, it was recently documented that GIII
Kolkata-700010, West Bengal, India is replaced by genotype I (GI) in South Korea, Thailand
Full list of author information is available at the end of the article

© 2012 Sarkar et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Sarkar et al. Virology Journal 2012, 9:271 Page 2 of 6
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and China [7]. Though GIII is predominant in India, GI syndrome (AES), showing high grade fever (≥39°c) for
has been introduced in the country recently [7]. In 1973 JE 2–15 days including any two of the following symptoms,
outbreak was first recorded in the districts of Burdwan and viz. headache, vomiting, stupor, delirium, abnormal
Bankura in West Bengal where 700 cases and 300 deaths movements, presence of kernig’s sign, convulsions, neck
were reported [8,9]. Thereafter, several JE outbreaks took rigidity, altered sensorium, unconsciousness admitted in
place in the state [10-12]. Every year sporadic JE cases are 8 different district hospitals, West Bengal during the
being reported indicating its endemicity in this state des- period from July to December in 2010 (Figure 1).
pite the vaccination programme undertaken by the State All the samples were tested for IgM antibody against
Health Department, Government of West Bengal [13]. In JEV by using IgM antibody-capture (Mac) ELISA kit
addition, the geographic features, environmental factors (National institute of virology, Pune, India), according to
and socio-economic status of this state also favor JEV the manufacturer’s protocol.
transmission [14]. Moreover, the reports of JE incidences Only 61 JEV IgM negative samples with a history of ≤
in the state are the indications of either partial coverage of 3 days’ illness were screened and 200 μl of each of them
the vaccine or the emergence of mutated/new strain of were used for virus isolation on C6/36 cell line accord-
JEV. Genetic variation of JEV circulating in West Bengal ing to the standard protocol [13]. The tissue culture
has not yet been investigated and hence to ascertain the fluids were collected from the samples producing prom-
same a molecular epidemiological study was undertaken. inent cytopathic effect (CPE) and subjected for RNA ex-
traction by QIAamp RNA viral kit (Qiagen, GmbH,
Materials and Methods Hilden, Germany), following the manufacturer’s protocol.
A total of 92 serum and 43 cerebrospinal fluid (CSF) To identify the isolates as JEV, reverse transcription-
samples were referred and/or collected from 135 clinic- PCR (RT-PCR) was carried out with the extracted RNA
ally suspected pediatric-adolescent (0–20 years old) and (50 pg to 1μg) by Qiagen one step RT-PCR kit (Qiagen,
adult (≥ 21 years old) individuals with Acute encephalitis GmbH, Hilden, Germany), in accordance with the

Figure 1 Map of West Bengal showing the location of sample collection areas.
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Table 1 Background information of selected strains/isolates of JEV referenced in this study

Strains/isolates Country and Year Source Genotype GenBank
of isolation Accession no.
Nakayama Japan, 1935 Human brain III U70413
JaGAr01 Japan, 1959 Mosquito III AF069076
JaoArS982 Japan, 1982 Mosquito III M18370
Ishikawa Japan, 1998 Mosquito I AB051292
JaNAr0102 Japan, 2002 Mosquito I AY377577
Fu Australia,1995 Human II AF217620
WTP-70-22 Malaysia ,1970 Mosquito II U70421
PhAn1242 Philipines,1984 Pig serum III U70417
Muar Malaysia, 1952 Human V HM596272
691004 Srilanka, 1969 Human III Z34097
H49778 Srilanka, 1987 Human III U70395
DH20 Nepal, 1985 Human III U03690
VN118 Vietnam, 1979 Mosquito III U70420
K94P05 Korea, 1994 Mosquito I AF045551
KV1899 Korea, 1999 Pig I AY316157
K91P55 Korea, 1991 Mosquito I U34928
GP78 India, 1978 Human III AF075723
733913 India, 1973 Human brain III Z34095
P20778 India, 1958 Human III Z34096
R53567 India, unavailable unavailable III U70418
782219 India, 1982 Human III U70402
826309 India, 1982 Human brain III U70403
R53567 India, unavailable unavailable III U70418
014178 India, 2001 Human blood III EF623987
04940-4 India, 2002 Mosquito III EF623989
057434 India, 2005 Human blood III EF623988
78124 India, 1978 Human III U70387
JEV-GKP-0945054 India, 2009 Human CSF I HM156572
P3 China, 1949 Mosquito III U47032
SA14 China, 1958 Mosquito III U14163
SA14-14-2 China, unavailable Vaccine III D90195
GZ04-36 China, 2004 Mosquito III DQ404112
XZ0934 China, 2009 Mosquito V JF915894
SH-53 China, 2001 Mosquito I AY555757
JKT1749 Indonesia, 1979 Mosquito II U70405
JKT9092 Indonesia, 1981 Mosquito IV U70409
JKT7003 Indonesia, 1981 Mosquito IV U70408
JKT5441 Indonesia, 1981 Mosquito II U70406
2372 Thailand, 1979 Human I U70401
Chiang Mai Thailand, 1964 Human III U70393
HK8256 Taiwan, 1972 Mosquito III U03691
IND/10/WB India, 2010 Human CSF III JN189785
IND/10/WB/JEV28 Midnapore, India, 2010 Human CSF I JN703381
IND/10/WB/JEV21 Midnapore, India, 2010 Human CSF I JN703382
IND/10/WB/JEV38 South 24 Pgs, West Bengal, India, 2010 Human CSF III JN968470
IND/10/WB/JEV37 Hooghly, West Bengal, India, 2010 Human serum III JN968471
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Table 1 Background information of selected strains/isolates of JEV referenced in this study (Continued)
IND/10/WB/JEV44 Malda, West Bengal, India, 2010 Human CSF III JN968468
IND/10/WB/JEV41 Malda, West Bengal, India, 2010 Human CSF III JN968469
IND/10/WB/JEV40 Howrah, West Bengal, India, 2010 Human CSF III JN968472
IND/10/WB/JEV39 Birbhum, West Bengal, India, 2010 Human CSF III JN968473
IND/10/WB/JEV42 Murshidabad, West Bengal, India, 2010 Human serum III JN968474
IND/10/WB/JEV43 Murshidabad, West Bengal, India, 2010 Human serum III JN968475
IND/10/WB/JEV35 Nadia, West Bengal, India, 2010 Human serum III JN968476
IND/10/WB/JEV36 Nadia, West Bengal, India, 2010 Human serum III JN968477
MVEV-1-51 Australia, 1951 Human AF161266
All strains/isolates are JEV, except Murray valley encephalitis virus strain (MVE-1-51) used as out group for phylogenetic analysis in this study.

manufacture’s specifications, using 0.6 μM of primer previously published JEV strains, including 12 from India
pairs [13] that specific for structural E gene sequence of and 29 from worldwide (Table 1). Dendrogram showed 2 E
JEV. The PCR products were separated by electrophor- gene sequences of the isolates (GenBank: JN703381,
esis on 1% agarose gel, stained with ethidium bromide. JN703382) belonging to GI and comprising 89%-91%
RT-PCR amplicons were purified using the Qiagen gel nucleotide (nt) identity with 11 E gene sequences of other
extraction kit (Qiagen, GmbH, Hilden, Germany), accord- isolates (GenBank: JN968468- JN968477, JN189785)
ing to the manufacturer’s protocol, followed by direct se- belonging to GIII (Figure 2). Moreover, these 2 GI E gene
quencing using the BigDye Terminator Cycle Sequencing sequences showed 99% nt similarity with each other
Ready Reaction Kit (Applied Biosystems, Foster City, CA, and were most similar (96%) with Japanese GI strain Ishi-
USA), as per the manufacturer’s instructions and the pro- kawa (GenBank: AB051292), followed by 94%-95% nt simi-
ducts were analyzed using an automated DNA sequencer, larity with Indian isolate JEV-GKP-0945054 (GenBank:
3130XL Genetic Analyzer (PE Applied Biosystems, Foster HM156572). Eleven GIII E gene sequences showed 97%-
city, CA, USA). The 1,500 nucleotides generated complete 99% nt similarity with each other and 93%-98% nt similarity
sequences of the JEV E gene that were edited and cor- with other Indian GIII strains, having the highest similarity
rected using the Finch TV software (http://www.geospiza. (97%-98%) with Indian P20778 strain (GenBank: Z34096).
com). Multiple sequence alignment and phylogenetic ana-
lysis were performed by using CLUSTALW (www.ebi.ac. Discussion
uk/Tools/clustalw2/index.html) and MEGA version 5.0 JEV infection is considered as a prime issue on public
software (www.megasofteware. net). The phylogenetic tree health concern in West Bengal. The present study
was constructed by the neighbor-joining method, tested reveals that 36/135 (26.6%) and 13/61 (21.3%) samples
with Kimura 2-parameter model. were positive to JE by Mac-ELISA and RT-PCR method
respectively. This observation is the proof of JEV infec-
Results tion in recent time and to detect the total number of JE
Out of 135 samples, only 36 (26.6%) samples were reactive cases, ELISA negative acute samples (from ≤ 3 days’
to JEV specific IgM antibody, of which 23 (63.8%) and 13 febrile illness) should be confirmed by RT-PCR test.
(36.1%) samples were CSF and serum respectively. Only 61 JE incidences (61.2% vs. 38.7%) were higher in pediatric-
of the remaining 99 JEV IgM negative samples having the adolescent age group than adult because pediatrics were
history of ≤ 3 days of febrile illness were selected and subse- infected possibly due to lack of immunity and adolescents
quently subjected to tissue culture resulting in 19 samples were directly exposed to the mosquito vector (Culex sp.) bite,
producing prominent CPE of which only 13 (21.3%) sam- as they usually took active part in cultivation in crop-fields
ples were identified as JE positive by RT-PCR method, con- where vectors usually breed. In the present study, JE was
sisting of 8 (61.5%) from CSF and 5 (38.4%) from serum. found to occur in the monsoon period with the maximum
We have a total of 49 (36 IgM + 13 RT-PCR positive) JE number of cases in September when the Culex mosquitoes
cases (36.2%) of which 30 (19 IgM + 11 RT-PCR positive) breed in the paddy fields covered with stagnant rain water.
were pediatric-adolescent (61.2%) and remaining 19 (17 IgM However, we found that 86 [(99–61)+(61–13)] samples
+ 2 RT-PCR positive) were found to be adult cases (38.7%). with a history of 2–15 days’ illness were true JE negative
Moreover, the occurrence of JEV infection was recorded dur- possibly due to either mishandling of samples which
ing the month of July to December with the maximum num- damaged the IgM antibody/the viral titre or the presence
ber of cases (46.9%) observed in the month of September. of another etiology responsible for AES.
The Figure 2 shows the phylogenetic tree derived from In our previous reports we have achieved 36 JEV isolates
13 E gene sequences of JEV isolates along with 41 [13] belonging to GIII whose E gene sequences were
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U70401/2372/Thailand/1979
U34928/K91P55/Korea/1991
60
88 AY316157/KV1899/Korea/1999
AY377577/JaNAr0102/Jap an/2002
64
AY555757/SH-53/China/2001 GI
91
AF045551/K94P05/Korea/1994
97
HM 156572/JEV-GKP-0945054/India/2009
100 AB051292/Ishikawa/Jap an/1998

97 JN703382/IND/10/WB/JEV21/M idnap ore/India/2010

JN703381/IND/10/WB/JEV28/M idnap ore/India/2010
U70405/JKT1749/Indonesia/1979

73 U70421/WTP-70-22/M alay sia/1970

100
GII
U70406/JKT5441/Indonesia/1981
AF217620/Fu/Australia/1995

93 U70413/Nakay ama/Jap an/1935

70 Z34097/691004/Srilanka/1969
U70420/VN118/Vietnam/1979
Z34096/P20778/India/1958
JN968471/IND/10/WB/JEV37/Hooghly /India/2010
75
100 JN968470/IND/10/WB/JEV38/South 24 Parganas/India/2010
89
67
84 JN968472/IND/10/WB/JEV40/Howrah/India/2010
85
JN189785/IND/10/WB/M idnap ore/India/2010
JN968469/IND/10/WB/JEV41/M alda/India/2010
98 JN968468/IND/10/WB/JEV44/M alda/India/2010
51
JN968474/IND/10/WB/JEV42/M urshidabad/India/2010
JN968473/IND/10/WB/JEV39/Birbhum/India/2010

93 JN968475/IND/10/WB/JEV43/M urshidabad/India/2010

100 JN968476/IND/10/WB/JEV35/Nadia/India/2010
JN968477/IND/10/WB/JEV36/Nadia/India/2010
U70402/782219/India/1982 GIII
99 U70387/78124/India/1978
U03690/DH20/Nep al/1985

100 EF623989/04940-4/India/2002
EF623988/057434/India/2005

100 51 U70395/H49778/Srilanka/1987

98 U70403/826309/India/1982
96
U70418/R53567/India
AF075723/GP78/India/1978

60
100 Z34095/733913/India/1973
EF623987/014178/India/2001
71
U70393/Chiang M ai/Thailand/1964

96 M 18370/JaoArS982/Jap an 1982
56
U70417/PhAn1242/Philip p ines/1984

98 U14163/SA14/China/1954
62
D90195/SA14-14-2/China

99 AF069076/JaGAr01/Jap an/1959
U70396/HK8256/Taiwan/1972

93 U47032/P3/China/1949
DQ404112/GZ 04-36/China/2004

100 U70409/JKT 9092/Indonesia/1981 GIV

U70408/JKT7003/Indonesia/1981

100 HM 596272/M uar/M alay sia/1952

GV
JF915894/XZ 0934/China/2009
AF161266/M VEV-1-51/Australia/1951

0.05

Figure 2 Phylogenetic analysis of 13 JEV isolates in serum/CSF samples from AES patients, West Bengal. The Neighbor-Joining (NJ)
Phylogenetic tree based on complete envelope (E) gene nucleotide sequences of Japanese encephalitis virus (JEV) isolates/strains. The Murray
valley encephalitis virus strain (MVEV-1-51) was used as an out group for generating the rooted tree. The robustness of dendrogram was
evaluated by 1000 bootstrap pseudo replicates. Bootstrap values (≥50% of replicates) were shown in corresponding nodes. Horizontal branch
lengths are proportional to genetic distance and vertical branch lengths have no significance. Each taxon is named systematically by mentioning
the accession number, strain name, country of origin and year of isolation. The isolates’ sequences used in this study were marked with filled
circle and triangle symbols. Genotypes are indicated on the right. Scale bar indicates nucleotide substitutions per site.
Sarkar et al. Virology Journal 2012, 9:271 Page 6 of 6
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submitted to NCBI GenBank database like GenBank: Received: 3 February 2012 Accepted: 23 October 2012
JN189782, JN189783, JN189784 and HQ891146 etc. (un- Published: 15 November 2012

published data). The present study, therefore, constitutes References

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Competing interests Serological and molecular diagnosis of Japanese encephalitis reveals an
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Acknowledgements Japanese encephalitis into Australia. Emerg Infect Dis 2001,
This work was funded by Department of Science and Technology, Govt. of 7:900–903.
West Bengal, India [Grant No. 396(Sanc.)/ST/P/S&T/9G-27/2007]. The authors
express their heartfelt and sincere thanks to the members of DBT centre for
doi:10.1186/1743-422X-9-271
bioinformatics, Presidency University, Kolkata for their support during
Cite this article as: Sarkar et al.: Molecular evidence for the occurrence
bioinformatics (phylogenetic) analysis. The enthusiastic help obtained from of Japanese encephalitis virus genotype I and III infection associated
the physicians of District Hospitals in West Bengal, for providing us with the with acute Encephalitis in Patients of West Bengal, India, 2010. Virology
clinically suspected samples for this study, is gratefully acknowledged. Journal 2012 9:271.

Author details
1
ICMR virus unit, ID & BG Hospital, 57, Dr. S. C. Banerjee Road, Beliaghata,
Kolkata-700010, West Bengal, India. 2Department of Microbiology, The
University of Burdwan Golapbag, Burdwan, West Bengal, India.