Journal of Medical Virology 87:1258–1267 (2015)

Japanese Encephalitis Associated Acute Encephalitis Syndrome Cases in West Bengal, India: A Sero-Molecular Evaluation in Relation to Clinico-Pathological Spectrum Arindam Sarkar,1* Somenath Datta,2 Bani K. Pathak,3 Subhra K. Mukhopadhyay,4 and Shyamalendu Chatterjee1 1

ICMR Virus, ID & BG Hospital, Kolkata, West Bengal, India IPGME&R, Kolkata, West Bengal, India 3 Department of Biotechnology, St. Xavier’s College, Kolkata, West Bengal, India 4 Department of Microbiology, The University of Burdwan, Golapbag, Burdwan, West Bengal, India 2

Japanese encephalitis (JE) is a major public health problem in Asia and worldwide and it is responsible mainly for viral acute encephalitis syndrome (AES). The sole etiologic agent of JE is Japanese encephalitis virus (JEV). Although JE/AES cases have been regarded traditionally as a disease of children, a growing number of patients with JE/AES cases are also seen in the adult age group every year in the state of West Bengal, India in spite of vaccination. Therefore, a systematic study was performed to differentiate and characterize the clinico-pathological parameters and viral diversity among the patients of different age groups. Viral diversity was also evaluated from the JE/AES cases, depending on their disease severity. A total of 441 JE/AES cases were included in this study. By MAC–ELISA, 111 samples were found JEV IgM positive and among the IgM negative cases, 26 samples were found RT–PCR positive against JEV infection. Neck rigidity, abnormal behavior, convulsion, protein in CSF, WBC in CSF, and aspartate transaminase in blood differed significantly among the patients of pediatric-adolescent and adult group in both IgM positive and RT–PCR positive cases. Viral diversity was increased significantly in the pediatric-adolescent group compared to adult patients. Interestingly, with the rise in disease severity the viral diversity was found to be increased among the patients, irrespective of their age distribution. Based on clinico-pathological parameters and analysis of viral diversity, it can be concluded that viral diversity which occurs naturally is likely to affect disease severity, especially in the patients of pediatricadolescent group. J. Med. Virol. 87:1258– 1267, 2015. # 2015 Wiley Periodicals, Inc. C 2015 WILEY PERIODICALS, INC. 

KEY WORDS:

Japanese encephalitis virus; Dengue virus; pediatric-adolescents; IgM-capture ELISA; RT-PCR; molecular phylogeny

INTRODUCTION Japanese encephalitis (JE), a mosquito-borne neurotropic viral disease, is a major public health problem in Asia and worldwide. Japanese encephalitis virus (JEV), an enveloped, positive-sense singlestranded RNA virus belonging to the genus Flavivirus within the family Flaviviridae [Lindenbach and Rice, 2001], is the major causative agent of JE being associated with viral acute encephalitis syndrome [Solomon et al., 2008] in human with prime public health concern. Worldwide the incidental rate of JE is 30,000–50,000 cases per year and the estimated mortality is 10,000 per year, whereas about 30–50% of survivors develop serious permanent neuropsychiatric problem [Sarkar et al., 2012a]. Despite vaccination, the increasing trend of JE associated acute encephalitis syndrome patients and their deaths in India indicate a serious threat to public health in Conflict of Interest: none. Grant sponsor: Indian Council of Medical Research, New Delhi, India.  Correspondence to: Arindam Sarkar, ICMR Virus Unit, GB-4, 1st Floor, ID & BG Hospital, 57, Dr. S. C. Banerjee Road, Beliaghata, Kolkata 700010, India. E-mail: [email protected] Accepted 20 January 2015 DOI 10.1002/jmv.24165 Published online 4 May 2015 in Wiley Online Library (wileyonlinelibrary.com).

Persistence of JE Associated AES Cases in West Bengal

near future [Communicable Diseases_ Report of the Working Group on Disease Burden for the 12th Five Year Plan, WG3 (1), 2011]. The natural transmission cycle of JEV involves an enzoonotic (sylvatic) mosquito– bird–mosquito cycle, involving primarily Culex spp. mosquitoes as primary vectors [Geevarghese et al., 2004], wading ardeid birds as reservoir host [Japanese Encephalitis_The Center for Food Security and Public Health, 2007], pigs as amplifying host [Reuben and Gajanana, 1997], and humans as accidental “dead end” hosts [Diagana et al., 2007]. Although most of the JE patients were asymptomatic with sub-clinical infections [Halstead and Grosz, 1962; Grossman et al., 1973; Chakraborty et al., 1981], JEV infection typically causes a selflimiting but debilitating classical illness characterized by acute encephalitis syndrome [Solomon et al., 2008]. The origin of JEV is thought to be in the Indonesia–Malaysia region and from there it evolved into five distinct genotypes [based on envelope (E) gene] of which genotype III (GIII) is prevalent mostly in South-East Asian countries including India [Solomon et al., 2003] and recently genotype I (GI) was also found sharing a close genetic relationship with GI strains from Japan and Korea [Fulmali et al., 2011; Sarkar et al., 2012b]. The state of West Bengal is situated at the eastern part of India and the first major outbreak of JE took place in the year 1973 in the two districts of West Bengal, namely Burdwan and Bankura [Sarkar et al., 2012c]. The endemicity of JEV in West Bengal has been proved by earlier publications [Sarkar et al., 2012b,c, 2013]. Although vaccination programs have been conducted against JE in different districts of West Bengal by the State Health Department, Govt. of West Bengal, yet every year sporadic JEassociated acute encephalitis syndrome patients and their deaths are continuously being reported from the state [Disease control programme_Scheme/program under national rural health mission: Programme Implementation Plan 2011-12/National Rural Health Mission/Health and Family Welfare Department, West Bengal, 2010; Sarkar et al., 2012c] where the people are dependent mainly on cultivation and the stagnant water in the paddy fields serve as congenial home for mosquito breeding [Sarkar et al., 2012a]. Besides, to raise their economic status they take up piggery and mini-poultry usually in their own hut bringing animals in their close association. In addition, environmental factors also favor JEV transmission in this state [Sarkar et al., 2012a]. On the other hand, the reports of JE incidences or endemicity of JEV in the state might be the indications of partial vaccination although the emergence of mutated strain of JEV could not be excluded. Molecular evolutionary analysis based on envelope sequences of many viruses such as HIV, HCV, small ruminant lentivirus, Epstein-Barr viruses etc. have established clearly that during infection the virus experience host immune pressure and undergo selection

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and independent evolution [Stumpf and Pybus, 2002; Sitki-Green et al., 2003; Richman et al., 2004; Philpott et al., 2005; Roque-Afonso et al., 2005; Pisoni et al., 2007; Lim et al., 2007; Ismail et al., 2014]. By this way the selection pressure of the host immune system can increase the viral genetic diversity [Ismail et al., 2014]. During JEV infection, the virus also experiences immune selection pressure. Therefore, the molecular characterization of JEV strains is important for disease monitoring and clinical outcomes. Till date, there is no such systematic study indicating the correlation of viral diversity with patient’s age distribution and also with disease severity in the state. Hence, in the present study, it was aimed at detecting JE-associated hospitalized acute encephalitis syndrome patients with clinico-pathological, sero-molecular investigations for its prevention, and clinical management in the population. It also tried to find out the genetic diversity of JEV among patients with two different age categories having different clinical manifestations. MATERIALS AND METHODS Patients and Samples Collection A total of 441 [283 serum and 158 cerebrospinal fluid (CSF)] samples were referred to and/or collected from clinically diagnosed 441 acute encephalitis syndrome patients of pediatric-adolescent (0–20 years old) and adult (21 years old) individuals showing high grade fever (39˚C) for 15 days including the following symptoms, namely headache, abdominal pain, myalgia, nausea, vomiting, neck rigidity, altered sensorium, abnormal behavior, convulsion, presence of Kernig’s sign admitted to government hospitals in the state of West Bengal during the study period 2005–2008. The study was approved duly by the joint ethical committee of the Indian Council of Medical Research Virus Unit and the National Institute of Cholera and Enteric Diseases, Kolkata, India. Informed consents were obtained in prescribed proforma from the patients or legal guardians or relatives of the patients before the collection of the samples. All the samples were transported to the Indian Council of Medical Research Virus Unit, Kolkata, maintaining the cold chain, for the detection of JEV infection. The details of clinical findings provided by the concerned physicians were also maintained in a register. Cerebral malaria and bacteriological as well as other viral etiologies were ruled out by the physicians of the hospitals concerned. Sera were separated from the clotted blood samples and both sera and CSF samples were stored in aliquots at 80˚C until use. JE Diagnostic Tests Serology: IgM-capture ELISA. Firstly, JE virus exposure was tested serologically by MAC–ELISA (IgM antibody capture-Enzyme Linked Immunosorbent Assay) in all the referred and/or collected J. Med. Virol. DOI 10.1002/jmv

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samples. The ELISA test kits (all ready to use reagents including 96-well plates) were supplied by the National Institute of Virology (NIV), Pune, India. ELISA measurements were undertaken following the protocol supplied by manufacturer. The test was standardized and reported by the NIV in 1984 [Gadkari and Shaikh, 1984]. Diluted patient sera (1:100) or CSF (1:10) were added to the plates. Absorbance of experimental, positive and negative control samples were measured at 450 nm in duplicate in 96-well plates using a microtitre plate reader (Titertek Multiskan Plus, Lab systems Finland, Type-314). In addition, serological cross-reactions are also common within the flaviviruses i.e., JEV and Dengue virus (DENV) [Gadkari and Shaikh, 1984; Ghosh and Basu, 2009] and both are prevalent in the state [Hati, 2006; Sarkar et al., 2010, 2012c,d]. Therefore, the JEV IgM positive samples were tested further for the presence of IgM antibody against the another flavivirus namely DENV by using DENV IgM capture ELISA kit, also obtained from the NIV, Pune, India and the test was performed according to manufacturer’s instructions. Isolation of virus from samples. Only JEV IgM negative samples were subjected to tissue culture using C6/36 cell line for the purpose of virus isolation as described previously [Sarkar et al., 2012c]. The tissue culture fluids were collected from the samples producing prominent cytopathic effect (CPE), and centrifuged at 1000g for 5 min and the supernatants of tissue culture fluids (STF) were kept in aliquots at 80˚C till the isolation/extraction of viral RNA, followed by E gene amplification through reverse transcriptase (RT)-polymerase chain reaction (PCR) test. Molecular assays: RNA extraction, E protein gene amplification and nucleotide sequencing. RNA extraction was done using QIAamp RNA viral kit (Qiagen, GmbH, Hilden, Germany), as per manufacturer’s protocol followed by RT–PCR that was carried out by Qiagen one step RT–PCR kit (Qiagen, GmbH) for the molecular detection of JEV etiology, in accordance with the manufacturer’s specifications where the steps are stated briefly as follows: in a single tube, the target RNA was converted to cDNA, followed by amplification in 25 ml mixture (per reactions) volumes containing the following components: 10 ml DNase, RNase-free water, 5 ml of 5 Qiagen onestep RT–PCR buffer, 1 ml of 10 mM dNTPs, 1.5 mL of 10 mM/mL primer pairs [both forward and reverse primers i.e., JEnvF (W) and JEnvR (W), Supplemental Table SI], specific for structural E protein gene sequence of JEV [Sarkar et al., 2012c], 1 ml of Qiagen one-step RT–PCR enzyme mix (containing Omniscript and Sensiscript reverse transcriptase along with HotStar Taq DNA polymerase) and 5 ml of template RNA (50 pg to 1 mg). The PCR tube was placed in ABI 9700 thermal cycler. The RT–PCR conditions were as follows: in a reverse transcription step, the reaction J. Med. Virol. DOI 10.1002/jmv

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mixture was allowed initially to synthesize cDNA at 50˚C for 20 min. The denatured cDNA was used subsequently for PCR amplification at 95˚C for 15 min under initial PCR activation step facilitating HotStar Taq DNA polymerase activation and Omniscript and Sensiscript reverse transcriptase inactivation, followed by 35 cycles of denaturation (at 95˚C for 30 sec), annealing of primers (at 67˚C for 32 sec) and extension of primers (at 72˚C for 1 min 45 sec). Then, a final extension step was carried out at 72˚C for 2 min, followed by rapid thermal ramp to 4 ˚C. Finally, the RT–PCR products were kept at 20˚C until use or the same were separated electrophoretically on 1% agarose gel, stained with ethidium bromide followed by gel documentation using gel-doc apparatus (Bio-Rad, Hercules, CA) or UV Transilluminator (Allied scientific products, Kolkata, India). RT–PCR amplicons were purified with the help of the Qiagen gel extraction kit (Qiagen, GmbH), according to manufacturer’s protocol and then subjected to direct sequencing or sequence PCR using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA), in accordance with manufacturer’s specifications. Here, three individual/different sequence PCRs were performed with three different primers (Supplemental Table SI) to amplify desired PCR product (1.5 kb DNA for JEV E gene). Briefly, the dye termination sequence PCR was performed in a total of 10 mL mixture volume where 4 mL DNase, RNase-free water, 1 mL of 5 reaction buffer, 2 mL BigDye Terminator Cycle Sequencing Ready Reaction mix (containing dNTP mix; dye terminators ddUTP, ddGTP, ddCTP, ddATP, and DNA polymerase enzyme), 2.5 mL of purified PCR product/DNA (30–60 ng), and 0.5 mL of 10 mM/mL primer (Supplemental Table SI) were added subsequently, mixed gently, and the PCR tube was placed finally in ABI 9700 thermal cycler where reaction conditions were as follows: initial denaturation (at 96˚C for 5 min), followed by 25 cycles of denaturation (at 96˚C for 10 sec), annealing (at 50˚C for 10 sec), and elongation (at 60˚C for 4 min), followed by rapid thermal ramp to 4 ˚C. Finally, the sequence PCR product was kept at 20˚C until ready to purify. The products were purified by ethanol precipitation and were analyzed by an automated DNA sequencer, 3130XL Genetic Analyzer (PE Applied Biosystems). The freely available Finch TV software (http://www. geospiza.com) was used to edit and/or correct combined the 1,500 nucleotides which generated complete E protein gene sequences of JEV isolates. Those sequences were subjected to BLAST search. Multiple sequence alignment (MSA) and phylogenetic relationship. The E protein gene sequences of JEV strains/isolates used in MSA and phylogenetic analysis in this study were enlisted in Supplemental Table SII. MSA and phylogenetic analysis were performed by CLUSTALW (http://www.ebi. ac.uk/Tools/clustalw2/index.html) and MEGA version 6.0 software (http://www.megasofteware. net). The

Persistence of JE Associated AES Cases in West Bengal

phylogenetic tree was constructed by the neighborjoining method, tested with Kimura 2-parameter model and evaluated by 1,000 bootstrap pseudo replicates. The strain MVEV-1-51 was used as an outgroup for generating the rooted tree. In addition, the viral diversity that included the mean genetic distance (d), the number of synonymous substitutions per synonymous site (dS), and the number of nonsynonymous substitutions per non-synonymous site (dN), was also calculated for each patient. The genetic distances at the nucleotide level were calculated under the Tamura 3-parameter model whereas genetic distances at the amino acid level were calculated under the JTT model using MEGA version 6.0 software (http://www.megasofteware. net). The dS represented ‘silent’ nucleotide changes that did not alter the amino acid encoded, whereas the dN represented ‘replacement’ nucleotide changes that resulted in a different amino acid. The dS and dN were calculated under the modified Nei-Gojobori model with Jukes-Cantor correction by MEGA version 6.0 software (http://www.megasofteware. net). Statistical Analysis To evaluate or correlate the impact of clinico-pathological profile of pediatric-adolescent and adult acute encephalitis syndrome patients with JE virus infection, statistical analysis was performed by x2 test with Yates’ correction as well as differences in means were assessed by Fisher’s t-test using SPSS version 16.0. P-values of 40 U/L; P < 0.0001 and P < 0.05 in IgM positive and RT–PCR positive, respectively, Table II). Interestingly, the level (mean  SD) of these parameters was changed

TABLE I. Clinical Characteristics of the Pediatric-adolescent and the Adult Patients With JE Virus Infection IgM positive (n ¼ 111) Clinical characteristics Fever Headache Abdominal pain Myalgia Nausea Vomiting Neck rigidity Altered sensorium Abnormal behavior Convulsion Presence of Kernig’s sign

Pediatric-adolescent n ¼ 76 76 (100) 59 (77.6) 12 (15.8) 18 (23.7) 35 (46) 41 (53.9) 63 (82.8) 46 (60.5) 38 (50) 44 (57.8) 42 (55.2)

RT-PCR positive (n ¼ 26)

Adult n ¼ 35 35 30 3 7 13 15 20 19 9 12 18

(100) (85.7) (8.6) (20) (37.1) (42.8) (57.1) (54.2) (25.7) (34.2) (51.4)

P-value

Pediatric-adolescent n ¼ 12

Adult n ¼ 14

P-value

— 0.462 0.462 0.851 0.5 0.378 0.008* 0.68 0.028* 0.035* 0.864

12 (100) 11 (91.6) 2 (16.6) 3 (25) 7 (58.3) 8 (66.6) 10 (83.3) 7 (58.3) 8 (66.6) 9 (75) 7 (58.3)

14 (100) 13 (92.8) 2 (14.2) 3 (21.4) 6 (42.8) 7 (50) 5 (35.7) 3 (21.4) 2 (14.2) 3 (21.4) 7 (50)

— 0.532 0.706 0.802 0.694 0.646 0.04* 0.128 0.02* 0.019* 0.976

Data are no. (%) of patients; *Significant (P < 0.05).

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Sarkar et al. TABLE II. Pathological Findings of the Pediatric-adolescent and the Adult Patients With JE Virus Infection Mean  SD

No (%)

Pathological findings IgM positive (n ¼ 111) Protein in CSF, 50-70 mg/100 mL Sugar in CSF, 45-65 mg/100mL WBC in CSF,  6/mm3 Blood urea, >40 mg/100 mL Alanine transaminase, >40 U/L Aspartate transaminase, >40 U/L

Pediatric-adolescent n ¼ 76 35 37 29 7 50 58

(46) (48.6) (38.1) (9.2) (65.7) (76.3)

Adult n ¼ 35 15 15 11 5 21 8

(42.8) (42.8) (31.4) (14.2) (60) (22.8)

P-value

Pediatric-adolescent n ¼ 76

0.913 0.714 0.636 0.638 0.706 40 mg/100 mL Alanine transaminase, >40 U/L Aspartate transaminase, >40 U/L

Pediatric-adolescent n ¼ 12 7 6 4 1 8 9

(58.3) (50) (33.3) (8.3) (66.6) (75)

67.6  2.8 50.7  7.5 37.7  5.0 80.1  20.0 77.8  24.4 104.7  52.3

10.4  103 substitution/site, respectively, P ¼ 0.09, Fig. 3c) and non-synonymous substitutions (dN, 4.2  103 > 7.1  103 > 7.5  103 substitution/site, respectively, P ¼ 0.26, Fig. 3d). DISCUSSION Acute encephalitis syndrome patients with JEV infections have become a major public health problem in the state of West Bengal due to its complexity and lack of any specific treatment. The present study aimed to identify the association of different clinico-pathological, sero-molecular investigations in JE-associated

Fig. 2. The dynamic changes of viral genetic distance (d), dS and dN within complete E gene nucleotide sequences of JEV among patients of the pediatric-adolescent (n ¼ 12) and the adult (n ¼ 14) group. In the pediatric-adolescent groups the genetic distance in both nucleotide (a) and amino acid (b) level were found to be higher significantly compared to the adult groups. The dS (the number of synonymous substitutions per synonymous site) and dN (the number of nonsynonymous substitutions per non-synonymous site) were also found to be higher significantly in the pediatric-adolescent group compare to the adult groups (c and d, respectively).

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Fig. 3. The increasing trend of viral genetic distance (d), dS and dN within complete E gene nucleotide sequences of JEV among the patients with mild (n ¼ 8), moderate (n ¼ 8) and severe (n ¼ 10) disease categories. Although the viral diversity were not found significant (

Japanese encephalitis associated acute encephalitis syndrome cases in West Bengal, India: A sero-molecular evaluation in relation to clinico-pathological spectrum.

Japanese encephalitis (JE) is a major public health problem in Asia and worldwide and it is responsible mainly for viral acute encephalitis syndrome (...
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