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Table of Contents
SHORT RESEARCH COMMUNICATION
Year : 2022  |  Volume : 59  |  Issue : 2  |  Page : 190-192

First report on the detection of Japanese encephalitis virus in fruit bats from India


1 Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India
2 Centre for Wildlife Conservation Management & Disease Surveillance, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India

Date of Submission06-Aug-2021
Date of Acceptance26-Nov-2021
Date of Web Publication08-Sep-2022

Correspondence Address:
Himani Dhanze
Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, UP-243122
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.335769

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  Abstract 

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

Keywords: Japanese encephalitis virus; TaqMan real-time RT-PCR; Fruit bat


How to cite this article:
Dhanze H, Karikalan M, Mehta D, Gupta M, Mote A, Kumar M S, Pawde A M. First report on the detection of Japanese encephalitis virus in fruit bats from India. J Vector Borne Dis 2022;59:190-2

How to cite this URL:
Dhanze H, Karikalan M, Mehta D, Gupta M, Mote A, Kumar M S, Pawde A M. First report on the detection of Japanese encephalitis virus in fruit bats from India. J Vector Borne Dis [serial online] 2022 [cited 2022 Sep 28];59:190-2. Available from: https://www.jvbd.org/text.asp?2022/59/2/190/335769

Japanese encephalitis (JE) is a re-emerging mosquito-borne zoonotic disease responsible for causing several children deaths every year in Asian countries. Japanese encephalitis virus (JEV) is a member of the arthropodborne genus Flavivirus of the family Flaviviridae[1]. JEV is the main cause of severe encephalitis in Asia with 70,000 cases reported every year[2]. The zoonotic cycle of JEV involves ardeid birds as reservoir, swine as amplifying host and mosquitoes of mainly Culex vishnui subgroup as vector[2]. In the recent past the role of bats has been elucidated in transmitting several zoonotic viruses like Hendra virus, Nipah virus, and SARS-like coronavirus[3],[4],[5]. The presence of JEV in bats has been detected by researchers in other countries but the role of bats in transmission of the virus has not been defined yet[6]. To the best of our knowledge this is the first report on the detection of JEV in fruits bats from India and suggests their possible role in maintenance of JEV in the region.

During the last decade, maximum number of human JE cases were reported from Assam and Uttar Pradesh (UP) states of India[7]. Since 1978, repeated occurrence of JE cases has been recorded in Deoria and Gorakhpur districts in eastern UP[8]. During 2011–2018, four districts of Gorakhpur Division and three districts of Basti Division contributed to 86 per cent of total AES (Acute encephalitis syndrome) cases of UP[9].

In Gorakhpur District of UP, 52 bats were found dead on 26 May 2020[10]. The representative bat carcass samples (n=8) were submitted by the State Wildlife Department to Indian Veterinary Research Institute, Bareilly. The bat species was identified as Pteropus spp. The post mortem examination revealed heat stroke as the cause of mortality. The carcass was dehydrated and subcutaneous contusions and hemorrhages were observed at the head while skeletal muscles were dried and sticky with the cooked appearance. The histopathological examination revealed engorged and multifocal areas of hemorrhages in brain and other vital organs.

Gorakhpur is a known endemic region for JE occurrence[8] and hence we decided to investigate the fruit bat (Pteropus spp.) brain samples for the presence of JEV using RT-PCR and Taq-Man real time RT-PCR. A total of 8 representative brain samples were chosen for the study.

The brain tissue samples were collected aseptically from bats and processed for RNA extraction. The brain tissue homogenate was prepared by triturating brain tissue in 1 ml Trizol reagent using sterile pestle and mortar, followed by centrifugation at 12,000 rpm for 15 min at 4°C. The supernatant was harvested for further RNA extraction using TRIzol-LS reagent as per manufacturer instructions (Life Technologies, Invitrogen, Carlsbad, CA).

The RNA was reverse transcribed into cDNA using 100 units of M-MuLV reverse transcriptase enzyme (Thermo Scientific) and 1 μg of oligo (dT) primer (Thermo Scientific) per reaction. The previously published primers targeting envelope gene[11] and NS1 gene[12] of JEV were used for cDNA amplification in two different PCR assays. In both the assays 25 μl of reaction mixture containing 2.5 μl of 10x DreamTaq buffer, 0.3 μl of DreamTaq DNA polymerase (5 U/μl, Thermo Scientific), 2 μl of 2 mM dNTPs (Thermo Scientific), 1.5 μl of forward primer (10 pmol), 1.5 μl of reverse primer (10 pmol), 5 μl of template cDNA and 12.2 μl of nuclease free water, was prepared. The reaction was carried out in a thermal cycler (Bio-Rad, T100 thermal cycler) and thermal conditions for envelope gene primers were initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 45 sec, followed by a final extension at 72°C for 7 min. The amplification cycle for NS1 gene primers was carried out with the same thermal conditions except annealing temperature of 49°C for 45 sec.

The TaqMan real-time PCR assay was performed with previously published probe-primer combination targeting the conserved region of envelope gene so that both genotype I and genotype III prevalent in India could be detected[13]. The reaction was set up with 5 μl of mastermix (TaqMan Universal Master Mix II, Thermo Fisher Scientific), 1 μl (100 ng) of cDNA, 0.5 μl of probe-primer mix (concentration of 5 and 18 mM, respectively) and 3.5 μl of nuclease free water (Thermo Scientific, Fermentas) with the following thermal conditions: 50°C for 2 min, 95°C for 10 min and 45 cycles of 95°C for 15 sec and 60°C for 1 min. Positive (quantified JEV RNA) control and ‘No Template Control’ in duplicates were included to rule out any false positive. A test result was considered positive if a sigmoid amplification curve crossed the threshold before 45 cycles. Results were analyzed by observing Ct values in amplification curves.

A total of eight brain samples were tested by conventional RT-PCR and TaqMan real-time RT-PCR. None of the samples were detected as positive in the conventional RT-PCR, while five out of the eight brain samples tested positive for JEV by TaqMan real-time RT-PCR. Quantification of JEV in fruit bat brain samples by TaqMan realtime RT-PCR was estimated based on the regression line equation of standard curve, using the formula: log10 copy numbers = Ct - y-intercept/slope.

The JEV copy numbers in positive bat brain samples ranged between 8 to 18 copies/reaction [Table 1]. The low viral load in the bat brain samples might be the reason for failure in JEV detection by conventional RT-PCR. It has already been proven that TaqMan real-time RT-PCR assay is a highly sensitive assay[13]. Nowadays, real-time RT- PCR assay has earned wider acceptance due to the improved sensitivity, rapidity and reproducibility, and has been used for diagnosis of various viruses including JEV[14],[15].
Table 1: JEV copy numbers in bat brain samples from Gorakhpur, India

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Bats are reservoir hosts for several emerging and reemerging viral pathogens causing morbidity and mortality in wildlife, livestock and humans[16]. Previously, JEV or antibodies against JEV have been detected in bats in Japan and China, although the role of bats in cycle of JEV is still not clear[17],[18]. In another study, researchers have inoculated bats with JEV and reported infection in mosquitoes that have fed on the inoculated bats. However, the studies did not reveal any detectable viremia and clinical signs in the bats[19]. The present study is the first report from India on the detection of JEV in bats. Researchers have suggested quantifying JEV in bats of different species, which would provide a better assessment of risks posed by JEV in bats[6]. In our study, we found low viral load in bat brain samples which might be the reason for no obvious clinical signs in bats and points towards their role in maintenance of virus in nature. Bats can harbor JEV for a long time when the temperature drops[20], which suggests that they might be possible hosts for the virus overwintering. It is evident from our study that JE virus is circulating in bat population of Gorakhpur District of Uttar Pradesh and thus there is a need to screen more number of bat samples to elucidate their role in the epidemiology of JEV.

Ethical statement: Not applicable

Conflict of interest: None



 
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Thiel H-J, Collett MS, Gould EA, Heinz FX, Meyers G, Purcell RH, et al. Flaviviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus taxonomy: VIIIth report of the International Committee on Taxonomy of Viruses. San Diego (CA). Elsevier Academic Press 2005; 981–98.  Back to cited text no. 1
    
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NVBDCP, 2020. Japanese encephalitis. www.nvbdcp.gov.in/ Doc/jes-aes-cd-. (Accessed on November 26, 2020).  Back to cited text no. 7
    
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Kumari R, Joshi PL. A review of Japanese encephalitis in Uttar Pradesh, India. WHO South-East Asia Journal of Public Health 2012; 1(4): 374–95.  Back to cited text no. 8
    
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Times of India. Panic after bats found dead in Gorakhpur.27.5.2020. https://timesofindia.indiatimes.com/city/lucknow/panic-after-bats-found-dead-in-gorakhpur/articleshow/76023274.cms (Assessed on October 13, 2021).  Back to cited text no. 10
    
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Santhosh SR, Parida MM, Dash PK, Pateriya A, Pattnaik B, Pradhan HK, et al. Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus. Journal of Virological Methods 2007; 143(1): 73–80.  Back to cited text no. 14
    
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