Accepted Manuscript Title: West Nile Encephalitis Outbreak in Kerala, India, 2011 Author: B. Anukumar Gajanan N. Sapkal Babasheb V. Tandale R. Balasubramanian Daya Gangale PII: DOI: Reference:
S1386-6532(14)00217-0 http://dx.doi.org/doi:10.1016/j.jcv.2014.06.003 JCV 3048
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
11-4-2014 29-5-2014 2-6-2014
Please cite this article as: Anukumar B, Sapkal GN, Tandale BV, Balasubramanian R, Gangale D, West Nile Encephalitis Outbreak in Kerala, India, 2011, Journal of Clinical Virology (2014), http://dx.doi.org/10.1016/j.jcv.2014.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
West Nile Encephalitis Outbreak in Kerala, India, 2011
2
Anukumar. B *, Gajanan N.Sapkal , Babasheb V.Tandale . , and Balasubramanian. R and
3
Daya Gangale
4 5
Author affiliations: a National Institute of Virology, Kerala, Govt. T. D. Medical College Hospital
6
Alappuzha, Kerala, India, PIN code:688005, India.
7
AB: [email protected]. [email protected]
8
BR: [email protected]
a
b
b
a
us
cr
ip t
b
b
National Institute of Virology, 20-A, Dr.Ambedkar Road, Post box No.11,Pune , Maharashtra India. PIN code: 411001, India GNS: [email protected] BVT: [email protected] DG: [email protected]
16
*Dr. Anukumar. B
17
Scientist,
18
National Institute of Virology (Kerala Unit)
19
(Indian Council of Medical Research)
20
Govt. T. D. Medical College Hospital
21
Alappuzha, Kerala, India, PIN code:688005, India
22
Tel: + 91 – 477 - 2970004
23
Fax: + 91 – 20 – 2280100
24
Email: [email protected]. [email protected]
25
Abstract: 250 words
26
Text: 1250 words
M
an
9 10 11 12 13 14 15
Ac ce p
te
d
Address for correspondence:
1 Page 1 of 14
Abstract
28
Background: An outbreak of acute encephalitis syndrome (AES) was reported in Kerala in India
29
in May 2011. The outbreak features were unusual in terms of seasonality, geographical
30
distribution, age group, and clinical manifestations in comparison to the epidemiological features
31
of Japanese Encephalitis.
32
Objective: To detect the etiology of the acute encephalitis syndrome outbreak.
33
Study design: Investigation of outbreak was undertaken by collection of brief clinical history
34
and epidemiological details along with the specimens for viral diagnosis. The serum/CSF
35
samples (patients=208) received from the sentinel hospitals were subjected to IgM capture
36
ELISA and RT-PCR specific for Japanese encephalitis (JE) virus and West Nile virus (WNV).
37
The JE/WN IgM positive samples were further tested by serum neutralization assay for the
38
presence of JE and WNV specific neutralizing antibody.
39
Result: Most of the affected patients were aged above 15 years. No spatial clustering of the
40
disease was noticed. Cases were observed in premonsoon and early monsoon season and in JE
41
non-endemic area of Kerala. A total of 47 patient samples were positive for in-house JE IgM
42
capture ELISA and WNV IgM capture ELISA. Serum neutralization assay result revealed that 32
43
of 42 (76.19%) sera were positive for WNV neutralization antibodies. WNV was isolated from a
44
clinical specimen. Phylogenetic analysis of WNV envelope gene revealed 99% homology with
45
Russian Lineage 1 WNV.
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2 Page 2 of 14
Conclusion: West Nile virus (WNV) etiology was confirmed by virus isolation and detection of
47
virus specific antibody from clinical specimen. Phylogenetic analysis grouped the current strain
48
in lineage I West Nile virus.
49
Keywords:
50
outbreak, Kerala, India
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cr
Flavivirus, Acute encephalitis syndrome, Lineage I West Nile virus, Disease
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3 Page 3 of 14
51
Background
52
An outbreak of acute encephalitis syndrome (AES) was reported during May through July 2011 in Kerala, India. As per the records of Kerala state health services, over 300 cases with
54
4 deaths were recorded. This region of India is surrounded by the coast of Arabian Sea on the
55
West and is bound by the Western Ghats in the East and has over 3% population of the country.
56
Also, the geographic region is conducive for mosquito breeding. In India, many encephalitis
57
outbreaks have been associated with Japanese encephalitis (JE) [1]. Such outbreaks due to JE
58
were also documented in Kerala during 1996 and 1997 [2]. However, there were no reports of
59
increased incidence in the area in the recent past. JE was suspected initially based on the history,
60
but the affected age group and epidemiologic features indicated a closely related Flavivirus.
61
Within the Flaviviridae family, the genus flavivirus comprises tick-borne, mosquito-
M
an
us
cr
ip t
53
borne, and no-known-vector (NKV) viruses, and are further placed into groups with shared
63
antigenic cross-reactivity. Serological cross-reactivity amongst flaviviruses has been well
64
documented [3]. A number of flaviviruses constitute a significant threat to global health.
65
Amongst the mosquito-borne diseases, JE and West Nile virus (WNV) are endemic in many
66
parts of Southeast Asia including India. WNV is endemic throughout Africa, the Middle East,
67
West and Central Asia, and the Mediterranean; however, most early epidemics occurred mainly
68
in rural populations with few cases of severe neurological disease [4]. In India, antibodies against
69
WNV were first detected in human sera from Bombay in 1952 [5] and subsequently at South
70
Arcot district of Tamil Nadu [6]. Fatal cases were seen in children unlike in older age groups in
71
other countries [7].
Ac ce p
te
d
62
72
We report the detection of WNV etiology in AES outbreak in Kerala, India. 4 Page 4 of 14
Objective
74
To detect the incriminated agent causing the AES outbreak in Kerala, India
75
Study design and setting
ip t
73
76
cr
Field investigation of AES outbreak was undertaken for identifying etiology of the
outbreak. AES case was defined as a person of any age, at any time of year with the acute onset
78
of fever and a change in mental status and/or new onset of seizures. All the cases reported from
79
southern part of Kerala during May to July, 2011 were included. A total of 208 patients who had
80
a diagnosis of AES were sampled with sera and/or cerebrospinal fluid (CSF).
81
Serological assays
M
an
us
77
82
Serum and CSF samples were subjected to JEV IgM capture ELISA using an in-house kit [8]. Detection of WNV IgM antibody was carried out on sera and CSF using WNV IgM capture
84
ELISA kit (InBios, Germany) according to manufacturer’s instructions. Results were confirmed
85
by detection of virus neutralizing antibody in serum against JE and WN viruses in porcine stable
86
kidney (PS) cells using 733913 (JEV) and G22886 (WNV) viruses by cytopathic effect (CPE)
87
method in 96 well tissue culture plates [8]. Mouse polyclonal anti JEV and WNV along with
88
non-immune serum was always included in the assay.
89
PCR detection and sequencing analysis
Ac ce p
te
d
83
90
The PCR was performed for the samples (n=77) with less than five post onset days of
91
illness (POD).Viral nucleic acids were extracted using QIAamp viral RNA mini kits (Qiagen,
92
Germany). RT-PCR was performed for JEV and WNV using primers as described earlier [8, 9].
93
PCR products were purified using QIAquick gel extraction kit (Qiagen, Germany). Both strands 5 Page 5 of 14
were sequenced using Big Dye Terminator cycle sequencing Ready Reaction Kit (Applied
95
Biosystems, USA) in ABI PRISM 3130 XL Genetic Analyser (Applied Biosystems, CA, USA).
96
The phylogenetic tree was constructed with Jukes–Cantor parameter with bootstrap analysis of
97
1,000 replicates with the MEGA version 5.0 program [11].
98
Virus isolation
99
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standard protocol [10].
101
Results
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Virus isolation was attempted in Vero cells from RT-PCR positive samples according to
Among 208 AES cases, 69 (33%) belonged to pediatric age group (15 years). Clinically, the cases had high fever with headache and
104
one or more of altered sensorium, seizures or neurological deficits. The trend of the epidemic
105
showed that most cases were reported in the month of May and June. No spatial clustering of the
106
cases was noted in the affected area.
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Anti JE IgM antibodies were detected in either sera or CSF of 47 cases (CSF & Serum=33, Serum=14). All these samples (47) were also anti WNV IgM antibody positive.
109
Thirty-eight of 47 (80%) IgM positive cases were above 45 years while only 5 (10%) were in
110
pediatric age group. Therefore, to confirm the etiology, sera available from 42 IgM positive
111
cases were further tested for neutralization assay against JEV and WNV. Thirty-two of 42
112
(76.19%) sera were positive for WNV in neutralization assay. However, only 2 (5%) sera tested
113
positive for antibodies against JEV and 2 (5%) showed equivocal results. Sera available from
114
remaining 161 AES cases which were negative for IgM were further tested for neutralization
115
assay against JEV and WNV. Of these 161 cases, 26 (14.52%) and 54 (54. 74%) were positive
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6 Page 6 of 14
116
for the presence of JEV and WNV neutralizing antibodies respectively. Eighteen (10.05%)
117
samples showed equivocal results.
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A total of 3/77 samples were positive for WNV (104 bp amplicon) specific RNA. Sequences of all three amplicons showed 99% homology with Lineage I WNV strains
120
(Accession No AY278441).
cr
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Cytopathic effect was observed in Vero cells inoculated with only one CSF (sample no.
122
Ind-KLU181) and showed amplicon of WNV specific RNA (104 bp). Further analysis of 1500
123
nucleotide sequence in envelop (E) gene of the isolate showed 99% homology with WNV on
124
BLAST search. Phylogenetic analysis of the isolate revealed 99% homology with Russian
125
Lineage 1 WNV (Accession No AY278441 - Ast99-901). The isolate showed a nucleotide
126
sequence identity of 95 % and 80 % with Indian WNV 68856 (Lineage I) and WNV 804994
127
(Lineage V) respectively (Fig 1).
128
Discussion
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129
We report the lineage I WNV activity first time in Kerala. Lineage V WNV prevalence in
130
newer areas of India has been reported [12]. However, the present investigation showed the
131
emergence of potentially epidemic strain of lineage I WNV after 43 years in the country. We
132
recorded 2 deaths in WNV confirmed cases while one death was from the case with equivocal
133
results. In majority of the cases, fever with central nervous system involvement was observed.
134
However, the signs and symptoms seem to differ considerably in the reported outbreaks [13, 14].
135 136
We observed the highest degree of cross reactivity in IgM antibodies between WNV and JEV. Neutralizing antibody profile on IgM positive sera confirmed recent WNV infection in 76% 7 Page 7 of 14
of the cases while 5% of the cases were due to JEV. On the contrary to earlier reports, we
138
observed unusual features of the current outbreak. JEV and WNV cases were reported during the
139
premonsoon and early monsoon season in JE non-endemic area of Kerala. Also, the clustering of
140
cases as reported in earlier outbreak in 1997 was not noticed in the current outbreak [2]. Not
141
even a single case was reported from the known JE endemic areas. In order to avoid WNV
142
related morbidity, mortality and complications in non-endemic areas of India, estimation of the
143
actual disease burden and development of appropriate control measures need to be undertaken.
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an
In conclusion, WNV might be the cause for substantial number of acute encephalitis cases in the current outbreak. To our knowledge, no study has reported the WNV activity in this
146
region. It is thus necessary to launch studies on vector incrimination and epidemiologic
147
surveillance to monitor the spread of these viruses in this region, which will help to control
148
infection and device effective management strategies.
149
Author’s contributions
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AB planned and organized the study and wrote the manuscript, GNS did viral
151
neutralization assay and reviewed the manuscript, BVT helped in the epidemiological aspect of
152
the study and reviewed the manuscript, BR helped for field investigation, and DG carried out
153
sequencing and phylogenetic analysis.
154
Funding
155
This study was supported through Viral Diagnostic Laboratory network program by Indian
156
Council of Medical Research, Ministry of Health and Family Welfare, Government of India
157
(VIR/9/2011‐ECD‐1).
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Competing Interests 8 Page 8 of 14
The authors declare that they have no competing interest.
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Ethical Approval
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The project was approved by the Institute Human Ethical Committee, National Institute of
162
Virology, Pune-1. The reference number is No. 110(01)/EC-I/1141.
163
Acknowledgements
The authors gratefully acknowledge Mr.Shankar Vidhate , D.K.Butte and Jijo for their excellent technical assistance.
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References
[1] Rodrigues FM, New E. Epidemiology of Japanese encephalitis in India: A brief overview. In
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Proceedings of the National Conference on Japanese Indian Council of Medical Research.1984
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SRC - GoogleScholar:1-9.
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[2] Kumar S. Japanese encephalitis outbreak in Kerala has unusual features. Lancet.
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1996;347:678.
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[3] Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, et al.
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Antigenic relationships between flaviviruses as determined by cross-neutralization tests with
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polyclonal antisera. The Journal of general virology. 1989;70 ( Pt 1):37-43.
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[4] Gyure KA. West Nile virus infections. Journal of neuropathology and experimental
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neurology. 2009;68:1053-60.
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[5] Banker DD, J. Preliminary observations on antibody patterns against certain viruses among
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inhabitants of Bombay city. Indian Sci. 1952;6 SRC - GoogleScholar:733-66.
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[6] Risbud AR, Sharma V, Rao CV, Rodrigues FM, Shaikh BH, Pinto BD, et al. Post-epidemic
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serological survey for JE virus antibodies in south Arcot district (Tamil Nadu). The Indian
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journal of medical research. 1991;93:1-5.
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[7] Gajanana A, Thenmozhi V, Samuel PP, Reuben R. A community-based study of subclinical
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flavivirus infections in children in an area of Tamil Nadu, India, where Japanese encephalitis is
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endemic. Bulletin of the World Health Organization. 1995;73:237-44.
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[8] Sapkal GN, Wairagkar NS, Ayachit VM, Bondre VP, Gore MM. Detection and isolation of
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Japanese encephalitis virus from blood clots collected during the acute phase of infection. The
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American journal of tropical medicine and hygiene. 2007;77:1139-45.
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[9] Grinev A, Daniel S, Stramer S, Rossmann S, Caglioti S, Rios M. Genetic variability of West
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Nile virus in US blood donors, 2002-2005. Emerging infectious diseases. 2008;14:436-44.
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[10] Rao BL, Basu A, Wairagkar NS, Gore MM, Arankalle VA, Thakare JP, et al. A large
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outbreak of acute encephalitis with high fatality rate in children in Andhra Pradesh, India, in
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2003, associated with Chandipura virus. Lancet. 2004;364:869-74.
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[11] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular
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evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum
195
parsimony methods. Molecular biology and evolution. 2011;28:2731-9.
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[12] Khan SA, Dutta P, Khan AM, Chowdhury P, Borah J, Doloi P, et al. West Nile virus
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infection, Assam, India. Emerging infectious diseases. 2011;17:947-8.
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[13] Nash D, Mostashari F, Fine A, Miller J, O'Leary D, Murray K, et al. The outbreak of West
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Nile virus infection in the New York City area in 1999. The New England journal of medicine.
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2001;344:1807-14.
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[14] Brien JD, Uhrlaub JL, Hirsch A, Wiley CA, Nikolich-Zugich J. Key role of T cell defects in
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age-related vulnerability to West Nile virus. The Journal of experimental medicine.
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2009;206:2735-45.
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[15] Reisen WK, Fang Y, Martinez VM. Effects of temperature on the transmission of west nile
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virus by Culex tarsalis (Diptera: Culicidae). Journal of medical entomology. 2006;43:309-17.
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[16] Ilkal MA, Mavale MS, Prasanna Y, Jacob PG, Geevarghese G, Banerjee K. Experimental
207
studies on the vector potential of certain Culex species to West Nile virus. The Indian journal of
208
medical research. 1997;106:225-8.
209
Legend
210
Figure 1. Phylogenetic tree based on 1500 nucleotide sequence corresponding to E region of
211
Indian WNV. JEV was used as an out group. The tree was constructed with the program Mega
212
5.0 by neighbor-joining with Jukes–Cantor parameter distances (bar). Bootstrap confidence level
213
(1000 replicates) and a confidence probability value based on the standard error test were
214
calculated using mega. GenBank accession numbers for the complete genomic sequences
215
included in the phylogenetic analysis are: WNFCG Wengler (human, Uganda, 1937; M12294),
216
B956 (human, Uganda, 1937; AY532665), Eg-101 (human, Egypt, 1951; AF260968), RO97-50
217
(Culex pipiens, Romania, 1996; AF260969), Rabensburg 97-103 (Culex pipiens, Czech
218
Republic, 1997; AY765264), IS-98-STD (stork, Israel, 1998; AF481864), Italy-1998-Eq (equine,
219
Italy, 1998; AF404757), LEIV-Krnd88-190 (Dermacentor marginatus, Russia, 1998;
220
AY277251), Ast99-901 (human, Russia, 1999; AY278441), LEIV-Vlg00-27924 (human, Russia,
221
2000; AY278442), Sarafend (Israel; AY688948), Mexico TM171-03 (Mexico;
222
AY660002),Chin-01 (China; AY490240), Kunjin (strain MRM61C, Culex annulirostris, MRM
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11 Page 11 of 14
Australia, 1980; D00246), NY99 (USA, 2005; DQ211652), PaH001 (France, 2003; AY268133),
224
Ast02-2-692 (Anopheles messeae ,Russia, 2006; DQ411035), WN96-111 (Horse, 1996, France;
225
AY701412), Ind68856 (Rousettus leschenaultia, 1968, India; EU249803), Ind804994 (Human,
226
1980, India; DQ256376), SA381/00(Human, 2000, South Africa; EF429199), JEV-GP78 (India;
227
AF075723).
cr
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12 Page 12 of 14
72 100 80
NY99
Mexico TM171‐03 IS-98-STD PaH001
73
100
Ast99‐901
99
Ast02-2-692
71
WN96-111
Italy‐1998‐Eq 100 100
RO97-50
us
100
cr
98
ip t
Ind-KLU181
LEIV‐Vlg00‐27924 Chin-01
an
100 100 99
100
Eg101
Ind 68856 Kunjin
M
50
Ind 804994
Sarafend
SA381/00
100
d
85
WNFCG-Wengler
100
B956 Rabensburg 97-103 LEIV-Krnd88-190
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JEV‐GP78
0.02
228 229
13 Page 13 of 14
Highlights
230
Outbreak of acute encephalitis syndrome with unusal features
231
The outbreak was detected by JE and WN Mac ELISA and Neutralization test
232
West Nile virus was isolated from the clinical samples
233
The virus belongs to lineage I and closely related to Russian strain
234
First report of isolation of WNV from Kerala state
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14 Page 14 of 14
S1386-6532(14)00217-0 http://dx.doi.org/doi:10.1016/j.jcv.2014.06.003 JCV 3048
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
11-4-2014 29-5-2014 2-6-2014
Please cite this article as: Anukumar B, Sapkal GN, Tandale BV, Balasubramanian R, Gangale D, West Nile Encephalitis Outbreak in Kerala, India, 2011, Journal of Clinical Virology (2014), http://dx.doi.org/10.1016/j.jcv.2014.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
West Nile Encephalitis Outbreak in Kerala, India, 2011
2
Anukumar. B *, Gajanan N.Sapkal , Babasheb V.Tandale . , and Balasubramanian. R and
3
Daya Gangale
4 5
Author affiliations: a National Institute of Virology, Kerala, Govt. T. D. Medical College Hospital
6
Alappuzha, Kerala, India, PIN code:688005, India.
7
AB: [email protected]. [email protected]
8
BR: [email protected]
a
b
b
a
us
cr
ip t
b
b
National Institute of Virology, 20-A, Dr.Ambedkar Road, Post box No.11,Pune , Maharashtra India. PIN code: 411001, India GNS: [email protected] BVT: [email protected] DG: [email protected]
16
*Dr. Anukumar. B
17
Scientist,
18
National Institute of Virology (Kerala Unit)
19
(Indian Council of Medical Research)
20
Govt. T. D. Medical College Hospital
21
Alappuzha, Kerala, India, PIN code:688005, India
22
Tel: + 91 – 477 - 2970004
23
Fax: + 91 – 20 – 2280100
24
Email: [email protected]. [email protected]
25
Abstract: 250 words
26
Text: 1250 words
M
an
9 10 11 12 13 14 15
Ac ce p
te
d
Address for correspondence:
1 Page 1 of 14
Abstract
28
Background: An outbreak of acute encephalitis syndrome (AES) was reported in Kerala in India
29
in May 2011. The outbreak features were unusual in terms of seasonality, geographical
30
distribution, age group, and clinical manifestations in comparison to the epidemiological features
31
of Japanese Encephalitis.
32
Objective: To detect the etiology of the acute encephalitis syndrome outbreak.
33
Study design: Investigation of outbreak was undertaken by collection of brief clinical history
34
and epidemiological details along with the specimens for viral diagnosis. The serum/CSF
35
samples (patients=208) received from the sentinel hospitals were subjected to IgM capture
36
ELISA and RT-PCR specific for Japanese encephalitis (JE) virus and West Nile virus (WNV).
37
The JE/WN IgM positive samples were further tested by serum neutralization assay for the
38
presence of JE and WNV specific neutralizing antibody.
39
Result: Most of the affected patients were aged above 15 years. No spatial clustering of the
40
disease was noticed. Cases were observed in premonsoon and early monsoon season and in JE
41
non-endemic area of Kerala. A total of 47 patient samples were positive for in-house JE IgM
42
capture ELISA and WNV IgM capture ELISA. Serum neutralization assay result revealed that 32
43
of 42 (76.19%) sera were positive for WNV neutralization antibodies. WNV was isolated from a
44
clinical specimen. Phylogenetic analysis of WNV envelope gene revealed 99% homology with
45
Russian Lineage 1 WNV.
Ac ce p
te
d
M
an
us
cr
ip t
27
2 Page 2 of 14
Conclusion: West Nile virus (WNV) etiology was confirmed by virus isolation and detection of
47
virus specific antibody from clinical specimen. Phylogenetic analysis grouped the current strain
48
in lineage I West Nile virus.
49
Keywords:
50
outbreak, Kerala, India
ip t
46
cr
Flavivirus, Acute encephalitis syndrome, Lineage I West Nile virus, Disease
Ac ce p
te
d
M
an
us
51
3 Page 3 of 14
51
Background
52
An outbreak of acute encephalitis syndrome (AES) was reported during May through July 2011 in Kerala, India. As per the records of Kerala state health services, over 300 cases with
54
4 deaths were recorded. This region of India is surrounded by the coast of Arabian Sea on the
55
West and is bound by the Western Ghats in the East and has over 3% population of the country.
56
Also, the geographic region is conducive for mosquito breeding. In India, many encephalitis
57
outbreaks have been associated with Japanese encephalitis (JE) [1]. Such outbreaks due to JE
58
were also documented in Kerala during 1996 and 1997 [2]. However, there were no reports of
59
increased incidence in the area in the recent past. JE was suspected initially based on the history,
60
but the affected age group and epidemiologic features indicated a closely related Flavivirus.
61
Within the Flaviviridae family, the genus flavivirus comprises tick-borne, mosquito-
M
an
us
cr
ip t
53
borne, and no-known-vector (NKV) viruses, and are further placed into groups with shared
63
antigenic cross-reactivity. Serological cross-reactivity amongst flaviviruses has been well
64
documented [3]. A number of flaviviruses constitute a significant threat to global health.
65
Amongst the mosquito-borne diseases, JE and West Nile virus (WNV) are endemic in many
66
parts of Southeast Asia including India. WNV is endemic throughout Africa, the Middle East,
67
West and Central Asia, and the Mediterranean; however, most early epidemics occurred mainly
68
in rural populations with few cases of severe neurological disease [4]. In India, antibodies against
69
WNV were first detected in human sera from Bombay in 1952 [5] and subsequently at South
70
Arcot district of Tamil Nadu [6]. Fatal cases were seen in children unlike in older age groups in
71
other countries [7].
Ac ce p
te
d
62
72
We report the detection of WNV etiology in AES outbreak in Kerala, India. 4 Page 4 of 14
Objective
74
To detect the incriminated agent causing the AES outbreak in Kerala, India
75
Study design and setting
ip t
73
76
cr
Field investigation of AES outbreak was undertaken for identifying etiology of the
outbreak. AES case was defined as a person of any age, at any time of year with the acute onset
78
of fever and a change in mental status and/or new onset of seizures. All the cases reported from
79
southern part of Kerala during May to July, 2011 were included. A total of 208 patients who had
80
a diagnosis of AES were sampled with sera and/or cerebrospinal fluid (CSF).
81
Serological assays
M
an
us
77
82
Serum and CSF samples were subjected to JEV IgM capture ELISA using an in-house kit [8]. Detection of WNV IgM antibody was carried out on sera and CSF using WNV IgM capture
84
ELISA kit (InBios, Germany) according to manufacturer’s instructions. Results were confirmed
85
by detection of virus neutralizing antibody in serum against JE and WN viruses in porcine stable
86
kidney (PS) cells using 733913 (JEV) and G22886 (WNV) viruses by cytopathic effect (CPE)
87
method in 96 well tissue culture plates [8]. Mouse polyclonal anti JEV and WNV along with
88
non-immune serum was always included in the assay.
89
PCR detection and sequencing analysis
Ac ce p
te
d
83
90
The PCR was performed for the samples (n=77) with less than five post onset days of
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illness (POD).Viral nucleic acids were extracted using QIAamp viral RNA mini kits (Qiagen,
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Germany). RT-PCR was performed for JEV and WNV using primers as described earlier [8, 9].
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PCR products were purified using QIAquick gel extraction kit (Qiagen, Germany). Both strands 5 Page 5 of 14
were sequenced using Big Dye Terminator cycle sequencing Ready Reaction Kit (Applied
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Biosystems, USA) in ABI PRISM 3130 XL Genetic Analyser (Applied Biosystems, CA, USA).
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The phylogenetic tree was constructed with Jukes–Cantor parameter with bootstrap analysis of
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1,000 replicates with the MEGA version 5.0 program [11].
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Virus isolation
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standard protocol [10].
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Results
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Virus isolation was attempted in Vero cells from RT-PCR positive samples according to
Among 208 AES cases, 69 (33%) belonged to pediatric age group (15 years). Clinically, the cases had high fever with headache and
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one or more of altered sensorium, seizures or neurological deficits. The trend of the epidemic
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showed that most cases were reported in the month of May and June. No spatial clustering of the
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cases was noted in the affected area.
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Anti JE IgM antibodies were detected in either sera or CSF of 47 cases (CSF & Serum=33, Serum=14). All these samples (47) were also anti WNV IgM antibody positive.
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Thirty-eight of 47 (80%) IgM positive cases were above 45 years while only 5 (10%) were in
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pediatric age group. Therefore, to confirm the etiology, sera available from 42 IgM positive
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cases were further tested for neutralization assay against JEV and WNV. Thirty-two of 42
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(76.19%) sera were positive for WNV in neutralization assay. However, only 2 (5%) sera tested
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positive for antibodies against JEV and 2 (5%) showed equivocal results. Sera available from
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remaining 161 AES cases which were negative for IgM were further tested for neutralization
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assay against JEV and WNV. Of these 161 cases, 26 (14.52%) and 54 (54. 74%) were positive
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for the presence of JEV and WNV neutralizing antibodies respectively. Eighteen (10.05%)
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samples showed equivocal results.
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A total of 3/77 samples were positive for WNV (104 bp amplicon) specific RNA. Sequences of all three amplicons showed 99% homology with Lineage I WNV strains
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(Accession No AY278441).
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Cytopathic effect was observed in Vero cells inoculated with only one CSF (sample no.
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Ind-KLU181) and showed amplicon of WNV specific RNA (104 bp). Further analysis of 1500
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nucleotide sequence in envelop (E) gene of the isolate showed 99% homology with WNV on
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BLAST search. Phylogenetic analysis of the isolate revealed 99% homology with Russian
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Lineage 1 WNV (Accession No AY278441 - Ast99-901). The isolate showed a nucleotide
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sequence identity of 95 % and 80 % with Indian WNV 68856 (Lineage I) and WNV 804994
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(Lineage V) respectively (Fig 1).
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Discussion
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We report the lineage I WNV activity first time in Kerala. Lineage V WNV prevalence in
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newer areas of India has been reported [12]. However, the present investigation showed the
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emergence of potentially epidemic strain of lineage I WNV after 43 years in the country. We
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recorded 2 deaths in WNV confirmed cases while one death was from the case with equivocal
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results. In majority of the cases, fever with central nervous system involvement was observed.
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However, the signs and symptoms seem to differ considerably in the reported outbreaks [13, 14].
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We observed the highest degree of cross reactivity in IgM antibodies between WNV and JEV. Neutralizing antibody profile on IgM positive sera confirmed recent WNV infection in 76% 7 Page 7 of 14
of the cases while 5% of the cases were due to JEV. On the contrary to earlier reports, we
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observed unusual features of the current outbreak. JEV and WNV cases were reported during the
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premonsoon and early monsoon season in JE non-endemic area of Kerala. Also, the clustering of
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cases as reported in earlier outbreak in 1997 was not noticed in the current outbreak [2]. Not
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even a single case was reported from the known JE endemic areas. In order to avoid WNV
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related morbidity, mortality and complications in non-endemic areas of India, estimation of the
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actual disease burden and development of appropriate control measures need to be undertaken.
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In conclusion, WNV might be the cause for substantial number of acute encephalitis cases in the current outbreak. To our knowledge, no study has reported the WNV activity in this
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region. It is thus necessary to launch studies on vector incrimination and epidemiologic
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surveillance to monitor the spread of these viruses in this region, which will help to control
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infection and device effective management strategies.
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Author’s contributions
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AB planned and organized the study and wrote the manuscript, GNS did viral
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neutralization assay and reviewed the manuscript, BVT helped in the epidemiological aspect of
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the study and reviewed the manuscript, BR helped for field investigation, and DG carried out
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sequencing and phylogenetic analysis.
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Funding
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This study was supported through Viral Diagnostic Laboratory network program by Indian
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Council of Medical Research, Ministry of Health and Family Welfare, Government of India
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(VIR/9/2011‐ECD‐1).
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Competing Interests 8 Page 8 of 14
The authors declare that they have no competing interest.
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Ethical Approval
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The project was approved by the Institute Human Ethical Committee, National Institute of
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Virology, Pune-1. The reference number is No. 110(01)/EC-I/1141.
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Acknowledgements
The authors gratefully acknowledge Mr.Shankar Vidhate , D.K.Butte and Jijo for their excellent technical assistance.
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[10] Rao BL, Basu A, Wairagkar NS, Gore MM, Arankalle VA, Thakare JP, et al. A large
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outbreak of acute encephalitis with high fatality rate in children in Andhra Pradesh, India, in
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[12] Khan SA, Dutta P, Khan AM, Chowdhury P, Borah J, Doloi P, et al. West Nile virus
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infection, Assam, India. Emerging infectious diseases. 2011;17:947-8.
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[13] Nash D, Mostashari F, Fine A, Miller J, O'Leary D, Murray K, et al. The outbreak of West
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[16] Ilkal MA, Mavale MS, Prasanna Y, Jacob PG, Geevarghese G, Banerjee K. Experimental
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medical research. 1997;106:225-8.
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Legend
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Figure 1. Phylogenetic tree based on 1500 nucleotide sequence corresponding to E region of
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Indian WNV. JEV was used as an out group. The tree was constructed with the program Mega
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5.0 by neighbor-joining with Jukes–Cantor parameter distances (bar). Bootstrap confidence level
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(1000 replicates) and a confidence probability value based on the standard error test were
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calculated using mega. GenBank accession numbers for the complete genomic sequences
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included in the phylogenetic analysis are: WNFCG Wengler (human, Uganda, 1937; M12294),
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B956 (human, Uganda, 1937; AY532665), Eg-101 (human, Egypt, 1951; AF260968), RO97-50
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(Culex pipiens, Romania, 1996; AF260969), Rabensburg 97-103 (Culex pipiens, Czech
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Republic, 1997; AY765264), IS-98-STD (stork, Israel, 1998; AF481864), Italy-1998-Eq (equine,
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Italy, 1998; AF404757), LEIV-Krnd88-190 (Dermacentor marginatus, Russia, 1998;
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AY277251), Ast99-901 (human, Russia, 1999; AY278441), LEIV-Vlg00-27924 (human, Russia,
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2000; AY278442), Sarafend (Israel; AY688948), Mexico TM171-03 (Mexico;
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AY660002),Chin-01 (China; AY490240), Kunjin (strain MRM61C, Culex annulirostris, MRM
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Australia, 1980; D00246), NY99 (USA, 2005; DQ211652), PaH001 (France, 2003; AY268133),
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Ast02-2-692 (Anopheles messeae ,Russia, 2006; DQ411035), WN96-111 (Horse, 1996, France;
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AY701412), Ind68856 (Rousettus leschenaultia, 1968, India; EU249803), Ind804994 (Human,
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1980, India; DQ256376), SA381/00(Human, 2000, South Africa; EF429199), JEV-GP78 (India;
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AF075723).
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72 100 80
NY99
Mexico TM171‐03 IS-98-STD PaH001
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100
Ast99‐901
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Ast02-2-692
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WN96-111
Italy‐1998‐Eq 100 100
RO97-50
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Ind-KLU181
LEIV‐Vlg00‐27924 Chin-01
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100 100 99
100
Eg101
Ind 68856 Kunjin
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50
Ind 804994
Sarafend
SA381/00
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WNFCG-Wengler
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B956 Rabensburg 97-103 LEIV-Krnd88-190
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JEV‐GP78
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Highlights
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Outbreak of acute encephalitis syndrome with unusal features
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The outbreak was detected by JE and WN Mac ELISA and Neutralization test
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West Nile virus was isolated from the clinical samples
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The virus belongs to lineage I and closely related to Russian strain
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First report of isolation of WNV from Kerala state
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14 Page 14 of 14
