Detection of Nipah virus in Pteropus medius in 2019 outbreak from Ernakulam district, Kerala, India

Background In June 2019, Nipah virus (NiV) infection was detected in a 21-year-old male (index case) of Ernakulum, Kerala, India. This study was undertaken to determine if NiV was in circulation in Pteropus species (spp) in those areas where the index case had visit history in 1 month. Methods Specialized techniques were used to trap the Pteropus medius bats (random sampling) in the vicinity of the index case area. Throat and rectal swabs samples of 141 bats along with visceral organs of 92 bats were collected to detect the presence of NiV by real-time reverse transcriptase-polymerase chain reaction (qRTPCR). Serum samples of 52 bats were tested for anti-NiV Immunoglobulin (Ig) G antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). The complete genome of NiV was sequenced by next-generation sequencing (NGS) from the tissues and swab samples of bats. Results One rectal swab sample and three bats visceral organs were found positive for the NiV. Interestingly, 20.68% (12/58) of Pteropus were positive for anti-NiV IgG antibodies. NiV sequences of 18,172; 17,200 and 15,100 nucleotide bps could be retrieved from three Pteropus bats. Conclusion A distinct cluster of NiV sequences, with significant net-evolutionary nucleotide divergence, was obtained, suggesting the circulation of new genotype (I-India) in South India. NiV Positivity in Pteropus spp. of bats revealed that NiV is circulating in many districts of Kerala state, and active surveillance of NiV should be immediately set up to know the hotspot area for NiV infection. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-021-05865-7.

which shares a border with Bangladesh [5,6]. In 2018, the NiV outbreak was reported in Kozhikode, Kerala, with a CFR of approximately 89% [7,8]. It was found that Pteropus spp. of bats were the probable source of NiV infection in the 2018 outbreak in Kerala [8].
During the present outbreak, a 21-year-old male student was admitted to a private hospital of Ernakulam district, Kerala state, with persistent fever for 10 days, followed by acute encephalitic symptoms for 2 days. The clinical samples, including blood, serum, urine, and cerebrospinal fluid (CSF) were referred to the Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV), Pune to rule out NiV infection in the case. Nipah viral RNA was detected from CSF and urine samples by real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Anti-Immunoglobulin (Ig) M antibodies were detected in serum samples by Enzyme-Linked Immunosorbent Assay (ELISA) [7,8].
The present study was undertaken to ascertain the NiV virus circulation in Pteropus and Rousettus bats in those areas where index case visited in the near month to understand the probable exposure. Bats were trapped and screened for NiV RNA and anti-NiV IgG antibodies.

Ethics statement
The study was approved by the Institutional Animal Ethics Committee (IAEC) of ICMR-NIV, Pune (IAEC/2019/ MEZ/04). The permission from Principal Chief Conservator of Forest, Kerala state was obtained for trapping of bats and performing this study.

Study area
Bats were captured from the five-kilometer radius of the index case residence (Ernakulum district) and the college area (Idukki district) between 5th and 10th day after detection of index case. The five sites from where the bats were trapped were: Vavakkad, Aluva, Thuruthipuram in Ernakulam district and Thodupuzha, Muttam in Idukki district as per the proximity to the residence and college (Fig. 2).
The selected sites except for Thuruthipuram had bat roosts with numbers ranging from a hundred to several thousand. Thuruthipuram was chosen due to the presence of guava and almond tree plantations, frequently visited by bats, which were just 100-200 m away from the residence of the index case. Vavakkad site was within 5 km from the index case house and had a permanent roost with approximately 200 bats. Thodupuzha site was within a 5 km radius from the college, where the index case was studying and had a massive roost (nearly 10-20 thousand bats), spread over about half an acre of land. Muttam was towards the Idukki dam area and was one of the oldest roosts in the area spread over an acre of land with approximately 50,000 bats.

Trapping of bats and collection of samples
Bats [Pteropus medius (109) and Rousettus leschenaultii (32)] were trapped randomly using mist nests hoisted on aluminum inter-connectible poles at the height of 48 to 56 ft. The nets were set up to trap the bats between 6 and 7 PM before the foraging activities of the bats were initiated. The trapped bats were collected in the early morning (4 to 6 am) and were anesthetized with isoflurane inhalation. Throat and rectal swabs specimens were collected from 141 bats in virus transport medium (HiViral™ Transport Medium, HIMEDIA) and stored immediately in dry ice. The blood samples (around 2-3 ml) were collected from the wing (cephalic) vein of 78 bats [Pteropus spp (n = 58) and Rousettus spp (n = 32)] and serum was separated. After recovery from anesthesia, the bats were released back [7,8]. All the samples (serum, throat, and rectal swabs) were transported to ICMR-NIV, Pune in dry ice (Table 1). Ninety-two bats from five sites were euthanized with Isoflurane inhalation overdose. They were transported to ICMR-NIV, Pune, in liquid nitrogen to check for the presence of the NiV RNA in visceral organs. Necropsy was performed on 92 captured bats taking all biosafety precautions in the Biosafety Level (BSL)-4 laboratory at ICMR-NIV, Pune. The organs (brain, heart, lung, liver, spleen, intestine, kidney, and reproductive organs) were collected aseptically. These organs were then triturated in minimum essential medium (MEM) (Life Technologies Corporation, USA), and tissue homogenate was stored at − 80 0 C for further use.

Detection of NiV RNA by qRTPCR
RNA was extracted from the throat, rectal swabs and tissue homogenates using the Magmax RNA extraction kit (Applied Biosystems, USA) as per manufacturer's instructions. NiV-specificqRT-PCR was performed as described earlier [9].

Detection of anti-NiV bat IgG antibodies by ELISA
The bat serum samples were heat-inactivated (56 0 C for 30 min). ELISA plates were coated overnight at 4°C with NiV infected Vero CCL81 cell lysate antigen in 1:20 dilution, and uninfected Vero CCL81 cell lysate was used as a negative antigen. A volume of 100 (micro-liter) μl (1: 100 dilution) bat sera samples were added and incubated for 1 hour at 37°C. 100 μl of anti-bat IgG horseradish peroxidase (HRP) conjugate (Thermo fisher scientific) in 1:2000 dilution was added and incubated for 1 hour at 37°C. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Cat No29994, Clinical Science Product incorporation NeA blue, USA) was added and incubated at 37°C for 12-15 min. The reaction was stopped by adding 1 normal (N) Sulfuric acid (H 2 SO 4 ), and plates were read at 450 nm (nm). The plates were washed four times using 10 millimolar (mM) phosphate buffer saline (PBS) pH 7.4 with 0.1% Tween-20 (Sigma, USA) at the end of each step. Positive and negative controls available at ICMR-NIV, Pune were included in the test. For sensitivity and specificity, indigenously developed ELISA was compared with anti-Nipah bat IgG ELISA reagents provided by the Center for Disease Control and Prevention (CDC), Atlanta USA. The sensitivity and specificity of indigenous anti-NiV bat IgG ELISA were 100.0 and 83.3%, respectively, in comparison to ELISA developed by CDC reagents, which is considered as the gold standard [10].

Next-generation sequencing (NGS) and phylogenetic analysis of NiV positive bat specimens
Efforts were made for the amplification of bat specimens using the PCR multiplexing method. The multiplexing reaction was performed with the primer pools designed using the Primal Schema tool http://primal.zibraproject.org/ using Kerala NiV sequences MH523640 [11]. The primers designed for the complete NiV sequencing were of 400 base pairs (bps) with an overlap of 75 bps. The steps involved are as follows: 5 μl of the RNA (liver, spleen, kidney, lung, and rectal swab) was amplified using 35 cycles of PCR. The amplified PCR product was loaded on 1.5% agarose gel and electrophoresed. The PCR products were purified using the Qiagen gel extraction kit (Qiagen, Germany). The amplicon generated from this extracted product was used from the second strand synthesis step, as described in the TruSeq Stranded mRNA LT Library preparation kit (Illumina, USA). This purified amplicon was then used further for library preparation as described in the TruSeq Stranded mRNA LT Library preparation kit (Illumina, USA), beginning at the second strand synthesis step as described earlier [12]. The generated reads were analyzed using CLC Genomics workbench version 11.0. Reference-based mapping was used to retrieve the genomic sequence of the NiV. Reference NiV sequences were downloaded from the GenBank database and were aligned using MEGA software version 7 [13]. An evolutionary tree was generated using the best-predicted model for the N gene of the Nipah virus. A bootstrap replication of 1000 cycles were performed to assess statistical robustness.

Results
Detection of NiV RNA in the bat samples  Table 1).

Detection of anti-NiV IgG antibodies in bat serum samples
Anti NiV-IgG bat antibodies were detected in 12/58 (20.68% positivity) Pteropus bat sera from sites 1,3,4 and 5, whereas none of the Rousettus sera were found positive (Table 1).

NGS and phylogenetic analysis of NiV positive bat specimens
The complete genome of the NiV (18,172 nucleotide bps) could be retrieved from the kidney sample of one Pteropus bat, using the reference-based mapping approach [9]. NiV sequences could also be retrieved from the liver/spleen sample of the two Pteropus bats with a length of approximately 17,200 and 15,100 bps. The details of the NiV genomic regions retrieved from the bat samples are given in Supplementary Figure 1. The evolutionary tree demonstrated a distinct branch for the Indian NiV sequences (Fig. 3). NiV human sequences from Kozhikode, Kerala 2018 outbreak, and the NiV bat sequences from the current 2019 study area branched out from the Bangladesh sequences. The NiV strains circulating in the southern region of India are distinct from the B genotype and formed a separate cluster. The netevolutionary nucleotide divergence found between the NiV strains from South India (Kerala) to Bangladesh and Malaysia strains is 1.96 and 8.24%, respectively. Genewise nucleotide and amino acid net-evolutionary divergence for Malaysia and Bangladesh strains concerning Indian sequences are given in Table 2. Bangladesh and West Bengal sequences showed a divergence of 0.75%, while Kerala and Bangladesh sequences showed the divergence of 2.2%. Hence we hypothesize the existence of two types of genotypic sequences in India and propose a separate genotype for Kerala, Indian strains as 'I-India' genotype. The percent nucleotide divergence for the sequences used in the study and the amino acids changes for the representative sequences are given as Supplementary files 1, and 2 respectively. The pairwise comparison for each NiV gene with respect to its reference Bangladesh strain is given in supplementary Table 3.

Discussion
India has once again garnered the world's attention with the outbreak of NiV in Ernakulum district of Kerala, within a year of an outbreak in Kozhikode district [8,9]. Even though the mode of transmission of NiV to humans was well studied in Malaysia, Singapore, Philippines and Bangladesh outbreaks, the transmission dynamics in recent outbreaks in India in 2018 and 2019 needs to be further studied. Bat NiV positivity from within 5 km radius of the residence and college of the index case probably explains the role of Pteropus spps. in NiV transmission. The rectal swab, liver/spleen, lung, kidneys, intestine, and reproductive organ in Pteropus medius collected from Thodupuzha, possibly demonstrated bat as a potential source of NiV infection to the index case although, the route of transmission remains unclear. More intensive studies are needed to understand the dynamics of NiV transmission. The mode of transmission of NiV differed at each outbreak and has shown the presence of an intermediate host in Malaysia, Singapore and Philippines [2,14]. However, in India and Bangladesh, no intermediate host could be determined even though pigs in Bangladesh were experimentally shown to shed the virus in body fluids and excreta [15,16]. The Pteropus spps were also identified as the most likely source of NiV transmission during the 2018 outbreak in Kozhikode [8,9]. Since no effective drugs or prophylaxis are available for NiV infection, preparedness for early diagnosis is essential. The timely detection and confirmation of the etiological agent coupled with the lessons learned from the 2018 Nipah virus outbreak in the Kerala state have helped in early isolation of the patient, further preventing human to human transmission.
No NiV positivity could be detected in 32 Rousettus spps, sampled from the 2019 and 2018 Kerala outbreak area, indicating Rousettus spp. may not have a role in the NiV transmission cycle. The presence of anti-NiV IgG antibodies in 20.68% of the P. medius suggests NiV seropositivity in the current outbreak region. Four sampling sites showing the presence of anti-NiV IgG bat antibodies warrants active human surveillance for early detection of NiV cases.
Further, the presence of distinct clusters in the sequence analysis from the outbreaks in southern India (Kozhikode and Ernakulum districts of Kerala state in humans and bats) guides us to hypothesize the presence of a new 'Indian (I)' genotype circulating in India.

Conclusions
NiV positivity in Pteropus medius during the current outbreak (2019), suggest the probable role of bats in NiV transmission in Ernakulam, Kerala state. Authors would like to put forward the proposal for the circulation of new NiV strain "India (I)" in the Southern part of India, which is different from Bangladesh and Indian northeast NiV strains. Further studies are needed to understand the disease implication of this new NiV strain on humans, along with the development of new diagnostics and treatment modalities.