Molecular epidemiology of Japanese Encephalitis Virus in pig population of Odisha, Assam and Manipur states of India

Japanese encephalitis virus (JEV) comes under the family Flaviviridae and genus flavivirus. It predominantly infects the children under the age of 10 years and the case fatality rate can stretch out as high as 30%. Pigs act as reservoir and amplifying intermediate host for JEV. Recent report suggested longer persistence of JEV in tonsil than in circulation of experimentally infected pigs. The current investigation was conducted to understand the prevalence and molecular epidemiology of JEV infection in pigs in three different geographical sites in India (Odisha, Assam and Manipur). Serum samples were tested by ELISA and RT-PCR for detection of JEV, while only RT-PCR was done in case of tonsils tissues collected from pigs slaughtered in abattoir. Prevalence of JEV was highest in Manipur (25.45% in serum and 10.08% in tonsil) but lower in Assam (3.75% in serum and 0% in tonsils) and Odisha (1.49% in serum and 3.7% in tonsils). The percentage of sero-positivity was found to be 3.75% of IgM and 9.9% of IgG in Assam and Odisha respectively. Genotype III (GIII) of JEV was the dominant genotype and sporadic mutations of S83G, H76P, E78Q, C55S, and S64W along with two consistent mutations V46S and V51I were observed in all the GIII strains. Analysis of the E gene sequence revealed a single mutation, S118N in the GI strain. Older pigs (above 7 months) were found to be infected relatively more (8.6%) than younger pigs (age group 3-7 months). In conclusion, the high JE virus infection rate of pig in the current locations suggests the need for continuous surveillance of this virus in pigs which will ultimately help to adopt an effective control strategy to prevent the spread of JE infection to human. Author summary Japanese encephalitis is one of the contributing factors in acute encephalitis syndrome cases reported across India as well as Asia. Primarily young naive human population are affected with JEV. The death rate can be as high as 30% and in about 30%-50% surviving population paralysis, brain damage or other serious permanent sequelae may be observed. The viral load gets amplified in pigs and thus plays a crucial role in transmitting the infection in human communities living in close proximity to pig dwelling. The current study was conducted to demonstrate prevalence of JEV in pig population of three geographical regions of India viz. the States of Odisha, Assam and Manipur that have reported JE outbreaks in human population. The current study demonstrates that the rate of infection is 3.28% among pigs in Manipur followed by Assam and Odisha. GIII was found to be the most predominant JEV genotype, while only one GI genotype strain was detected from Odisha region. These findings suggested the need of continuous surveillance of this virus in pigs and proper implementation of human and animal vaccination programme to control the infection.

reported across India as well as Asia. Primarily young naive human population are affected 48 with JEV. The death rate can be as high as 30% and in about 30%-50% surviving population 49 paralysis, brain damage or other serious permanent sequelae may be observed. The viral load 50 gets amplified in pigs and thus plays a crucial role in transmitting the infection in human 51 communities living in close proximity to pig dwelling. The current study was conducted to 52 demonstrate prevalence of JEV in pig population of three geographical regions of India viz. 53 the States of Odisha, Assam and Manipur that have reported JE outbreaks in human 54 population. The current study demonstrates that the rate of infection is 3.28% among pigs in 55 Manipur followed by Assam and Odisha. GIII was found to be the most predominant JEV 56 genotype, while only one GI genotype strain was detected from Odisha region. These     in the abattoir as a part of the routine process after obtaining necessary consent for the same.

131
The serum was separated from the blood sample by centrifugation at 5000 rpm for 5 mins at  The viral RNA was extracted from serum samples as referred by Desingu et al, 2016 [19].

141
For this, 140μl of swine serum was used and extraction was done, using viral RNA isolation 142 kit (QIAmp Viral RNA Mini Kit Qiagen, Germany) according to the manufacturer's protocol.

143
The viral RNA was quantified and cDNA was prepared with random hexamers using 1μg of 144 viral RNA by Superscript III first strand cDNA synthesis kit (Invitrogen, USA). This was 145 followed by a PCR reaction using Taq DNA polymerase (Thermoscientific, USA) with initial   The IgM antibody detection against JEV in pig serum was also carried out by using Porcine 162 Japanese Encephalitis IgM antibody (JE-IgM AB) ELISA kit (Genexbio Health Science Pvt. 163 Ltd.) as per manufacturer's protocol.

164
Sequencing and phylogenetic analysis using E gene sequence 165 The positive serum and tissue samples were subjected to bi-directional sequencing with 166 overlapping primers using an automated DNA sequencer (Applied biosystem®3500 series 167 genetic analyser), based on the Sanger's dye terminating method. The sequence obtained was 168 analyzed for its correct identity using the BLAST tool of NCBI and confirmed.

169
The obtained sequences were aligned by the ClustalW tool [29] and the phylogenetic analysis

189
In the present study, the total number of serum samples screened from Odisha, Assam and  (Fig 2A and 2B). On the other hand, 15 out of 400 (3.75%) of IgM and 40 195 out of 402 (9.9%) of IgG positive cases were reported from Assam and Odisha respectively.  As per the district-wise distribution for all the three states, the overall prevalence of JEV for 202 serum samples was found to be 1.49% in Malkangiri, 25.45% in Imphal West, whereas 203 3.33% in Jorhat, 5.71% in Lakhimpur, 4.00% in Dhemaji and 1.67% in Kamrup (S1A Table).

204
Besides this, percentage of positivity for tonsils tissues by RT-PCR were found to be 3.7% in 205 Malkangiri, 8.23% in Imphal West, 35.89% in Imphal East, 3.4% in Kakching and 100% in 206 Bishnupur (S1B Table). However, district wise distribution was not available for Assam Assam (S1B Table).

210
The number of JEV positive cases detected in pig serum in the above mentioned states was 211 found to be 5.21%, 1.67% and 77.7% respectively for the age group 3-7 months, whereas 212 above 7 months, it was found to be 0%, 6.88% and 15.2%, respectively in Odisha, Assam and 213 Manipur (Fig 3A and 3B). However, for tonsil samples from Manipur 11.12% (3 out of 27) 214 was found to be positive under age group 3-7 months, whereas 9.95% (22 out of 221) was   Table S2.

232
The genotype I and III strains were found to be circulating among the pigs of the three states 233 under study, of which, genotype III was found to be the predominant one. The genotype III Taiwan and Japan (Fig 5). However, the rest of the strains clustered in genotype III including 242 the prototype Nakayama strain. The amino acid sequence of the translated protein coded by the E gene of the detected GI 252 strain was aligned with that of the respective prototype strain as well as to the strain isolated 253 from human in 2005 from Gorakhpur, India using the Clustal Omega tool. It was noticed that 254 the JEV GI strain isolated from Odisha was approximately 99% identical with a single 255 mutation (S118N) observed within the amino acid region of 1-156 of the E gene (S3A Table   256 and S1A Fig). Similarly, after the alignment of GIII E amino acid sequences with the 257 prototype strain, it was observed that they were approximately 96-98% identical. The

297
Here, the genotype III strain was found to be circulating predominantly among pig population 298 of the three states. This was reported earlier that genotype III is the most prevalent strain in   The current study limited to three geographical sites has indicated that JEV prevalence in pigs 318 can be very high in areas where human JEV outbreak has been reported. We suggest that JEV 319 outbreaks in human population might be controlled by vaccinating pigs in areas of high 320 prevalence of the viral load in the animal reservoir -a strategy that has not been widely 321 adopted. 322 We are grateful to Dr Anirban Basu (NBRC, Haryana, India) for kindly providing the JEV 324 strain, GP78. We are thankful to Dr Balachandran Ravindran for conceptualization, reviewing 325 and editing the manuscript. We would like to thank Ujjwal Mahata and BirSingh Mahata for 326 collecting the pig samples from Malkangiri region of Odisha. We are also grateful to 327 Konthoujam Abung Meitei for providing samples from Imphal West district of Manipur.