Background & objectives:Nipah virus (NiV) is a zoonotic paramyxovirus that causes fatal encephalitis in humans. Enzyme Linked Immunosorbent Assay (ELISA) is a safe, sensitive, specific, and affordable diagnostic tool that can be used during screening of large-scale epidemiological investigations. Development and evaluation of IgM and IgG ELISA for screening serum samples of NiV suspected cases would also help in planning public health interventions.Methods:An IgM capture (MAC) ELISA and an indirect IgG ELISA were developed using NiV antigen to detect IgM and IgG antibodies against NiV in human sera. The sensitivity, specificity, and cross-reactivity of the assays were evaluated using NiV IgM, IgG positive, negative human sera and measles, mumps, rubella, Crimean-Congo haemorrhagic fever, Kyasanur forest disease IgM, IgG positive sera, respectively.Results:The developed anti-NiV IgM and IgG ELISAs have shown specificity of 99.28 per cent and sensitivity of 100 per cent compared to reference test from Centers for Disease Control and Prevention, USA. Assays demonstrated negative predictive value of 100 per cent and positive predictive value as 90 and 93.94 per cent for anti-Nipah IgM ELISA and IgG ELISA respectively with test accuracy of 99.33 per cent.Interpretation & conclusions:Timely diagnosis of NiV is crucial for the management of cases, which could prevent further spread of infection in the community. IgM ELISA can be used as primary diagnostic tool followed by polymerase chain reaction. These assays have advantages of its applicability during outbreak investigations and surveillance activities at hospital or onsite laboratories with basic biosafety practices.

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.

© 2022 Indian Journal of Medical Research, published by Wolters Kluwer Medknow for Director-General, Indian Council of Medical Research Nipah virus (NiV) is one of the re-emerging virus, from the Paramyxoviridae family, mainly affecting the Southeast Asia region1. Pteropus species of bats are known natural reservoirs of NiV. Spillover of NiV can occur among the resident human population or visitors, in places where these bats roost. Humans can also get the infection from the pigs, the intermediate host for the transmission of NiV, as observed during the outbreak of 1998-99 in Malaysia and Singapore2-4. Human-tohuman transmission has also been reported during the outbreaks of Nipah in Bangladesh and India5-10. India has witnessed many outbreaks of Nipah in the States of West Bengal and Kerala with a high mortality of 68-100 per cent during the period of 2001-19. The sudden emergence of NiV from Kerala affected the public health system because of lack of preparedness to such a health emergency. However, the NiV spread was successfully contained during the 2018 outbreak and subsequently the preparedness helped in quickly containing the infection in 201911,with no secondary cases reported.

As per the World Health Organization, Research and Development Blueprint List of epidemic threats, Nipah virus (NiV) disease is one of the priority diseases that needs urgent action1. Since its detection in 1998, many outbreaks of Nipah have been reported from Malaysia, Singapore, Bangladesh and India234567. It is a serious public health threat for the countries in Southeast Asia. In the last two decades from (2001 to 2021), India has reported five Nipah outbreaks among human population in West Bengal and Kerala4567. In 2018, the sudden emergence of NiV was observed in Kozhikode and Malappuram districts of Kerala6. The outbreak had a case-fatality rate of 89 per cent with two cases survived the NiV infection. Subsequently, another NiV outbreak was reported from Ernakulum district, Kerala, during 20198. A single individual was affected with NiV who survived the infection and recovered completely8. Nipah outbreaks have been found to occur in sporadic form with a few cases. Consequently, NiV-specific antibody response has been studied only in Nipah symptomatic survivors. There is no information available on the persistence of the antibodies during follow up among asymptomatic contacts of Nipah-positive cases. Hence, the present study was carried out to evaluate the antibody response among symptomatic survivors of NiV infection and their asymptomatic contacts identified during 2018 and 2019 NiV outbreaks from Kerala, India. The study was approved by the Institutional Human Ethics Committee of the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, Maharashtra, India. The NiV infection survivors (n=3) and their asymptomatic contacts (n=3) of 2018 and 2019 outbreaks were identified, and blood samples were collected at different time intervals. The samples were transported to ICMR-NIV, Pune, under cold chain at 4°C. A total of 28 follow up blood samples were collected from the survivors of NiV outbreak during May 2018 (cases 1 and 2) till 438th day post-onset of disease (POD). Five follow up samples from 2019 NiV outbreak (case 3)8 were collected from 11th to 113th days POD. Seven follow up samples were collected from asymptomatic contacts (contacts 1, 2 and 3) identified in 2018 NiV outbreak on 49th to 476th days post-exposure. Serum was separated from these samples and screened for the presence of anti-NiV human immunoglobulin (Ig) M and IgG using an in-house–developed indirect ELISA. Each sample was tested in duplicate, and reproducibility of the assay was checked. Receiver operating characteristic (ROC) curve was used to determine the cut-off of the assay. Samples were considered as positive if average optical density (OD) of negative control was greater than 0.2 and P/N ratio was more than 1.5. Both the anti-NiV IgM and IgG assays demonstrated specificity of 99.28 per cent and sensitivity of 100 per cent compared to the reference test from the US Centers for Disease Control and Prevention (unpublished data). Three symptomatic and two asymptomatic contact cases were tested positive for anti-Nipah IgM and IgG antibodies. However, one asymptomatic contact showed positivity only for anti-NiV IgM antibodies. Anti-NiV IgM was detectable from 5th POD to 27th POD, while anti-NiV IgG was detected for more than one year among symptomatic NiV cases, respectively. Asymptomatic contacts had detectable levels of anti-NiV IgG from 49th POD to approximately 13 months post-exposure (Table). Contact 3 was positive for anti-NiV IgM at days post-exposure (DPE) 63 but did not show the presence of IgM and IgG antibodies in further collections till 300 till 450 days post-exposure. Considering this, Contact 3 was excluded from IgG analysis. The trends of IgM and IgG response in these cases was represented as a scatter plot (Figure 1A and B).Table: Anti-Nipah antibodies among symptomatic survivors of Nipah virus infection and their asymptomatic contacts from Kerala, IndiaFigure: ELISA for anti-Nipah virus immunoglobulin M (IgM) and G (IgG) optical density (OD) read at 450 nm for Nipah positive cases - cases 1, 2 and 3 and contacts 1, 2 and 3. (A) Anti-Nipah virus IgM and (B) anti-Nipah virus IgG antibody OD at different post-onset of disease (POD)/days post-exposure. The curves represent IgM and IgG response trends in the individual cases.The samples showing IgG positivity among survivors/contacts (cases 1-3; contacts 1 and 2) were tested with plaque reduction neutralization test (PRNT) to determine the neutralizing antibody titre at the Maximum Containment Facility of ICMR-NIV, Pune9. NiV-positive human serum sample was used as positive control. Briefly, ten-fold dilution of heat-inactivated (56°C for 1 h) serum samples to a dilution factor of 10−6 was mixed with an equal amount of virus suspension (10−4) and was incubated for one hour. Subsequently, 200 µl of serum–virus mixture was added to each well (in duplicates) of 24-well pre-seeded Vero cell plates and incubated in a CO2 incubator for one hour. Inoculum was removed, and 3 ml overlay medium (2% CMC+2X MEM+2% FBS) was added to each well followed by incubation at 37°C in a CO2 incubator for four days. Overlay medium was removed, and cells were washed with 1x PBS (phosphate buffered saline) and stained with amido black stain. The neutralization titre (PRNT50) of the test serum sample is defined as the reciprocal of the highest test serum dilution, for which the virus infectivity is reduced by 50 per cent when compared with the average plaque count of the challenge virus control9101112. Neutralizing antibody titres of cases 1, 2 and 3 at POD 483, 432 and 113 were 11482, 2291 and 661 whereas those of contacts 1 and 2 at DPE 380 and 385 were 457 and 1145, respectively. The findings suggest the persistence of neutralizing antibody response in symptomatic as well as asymptomatic cases after one-year POD/DPE. Among the Paramyxovirus family, NiV has demonstrated a high zoonotic potential along with high fatality rates8. Serology plays an important role in the diagnosis of NiV infection; however, antibody kinetics in Nipah infection is poorly studied. A study conducted by Nikolay et al10 reported the absence of anti-NiV antibodies in asymptomatic cases. Earlier studies by Ramasundram et al11 revealed anti-NiV IgM and IgG positivity in patients with symptomatic NiV infections till 3-7 months and eight months, respectively. Our study demonstrated the presence of IgM antibodies for more than two months and IgG for more than one year after infection in symptomatic and asymptomatic cases. Irrespective of the presence of clinical symptoms, we observed comparable IgM and IgG immune response against NiV infection. Both the survivors and asymptomatic contacts showed detectable levels of neutralizing antibody till 14 and 11 months, respectively. Further research is needed to determine long-term immune response or waning immunity. Financial support & sponsorship: Financial support was provided by the ICMR-NIV, Pune, India. Conflicts of Interest: None. Acknowledgment Authors aknowledge Prof Balram Bhargava, Secretary, Department of Health Research, Ministry of Health and Family Welfare, Government of India and Director-General, ICMR, New Delhi for encouragement and support and thank Servshree Prasad Sarkale, Deepak Mali, Shrimati Savita Patil and Triparna Majumdar from Maximum Containment Laboratory, ICMR-NIV, Pune, for the technical support extended during the study. Authors also thank Dr Anu Kumar, Servshree Jijo Koshy and T. Nikil, ICMR-NIV, Kerala Unit, Alappuzha, for co-ordination of sample transportation to ICMR-NIV, Pune.

The present manuscript deals with experimental infections of bonnet macaques (Macaca radiata) to study disease progression for better insights into the Kyasanur Forest Disease (KFD) pathogenesis and transmission. Experimentally, 10 monkeys were inoculated with KFD virus (KFDV) (high or low dose) and were regularly monitored and sampled for various body fluids and tissues at preset time points. We found that only 2 out of the 10 animals showed marked clinical signs becoming moribund, both in the low dose group, even though viremia, virus shedding in the secretions and excretions were evident in all inoculated monkeys. Anti-KFDV immunoglobulin (Ig)M antibody response was observed around a week after inoculation and anti-KFDV IgG antibody response after two weeks. Anaemia, leucopenia, thrombocytopenia, monocytosis, increase in average clotting time, and reduction in the serum protein levels were evident. The virus could be re-isolated from the skin during the viremic period. The persistence of viral RNA in the gastrointestinal tract and lymph nodes was seen up to 53 and 81 days respectively. Neuro-invasion was observed only in moribund macaques. Re-challenge with the virus after 21 days of initial inoculation in a monkey did not result in virus shedding or immune response boosting.

Background & objectives:Bats are considered to be the natural reservoir for many viruses, of which some are potential human pathogens. In India, an association of Pteropus medius bats with the Nipah virus was reported in the past. It is suspected that the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also has its association with bats. To assess the presence of CoVs in bats, we performed identification and characterization of bat CoV (BtCoV) in P. medius and Rousettus species from representative States in India, collected during 2018 and 2019.Methods:Representative rectal swab (RS) and throat swab specimens of Pteropus and Rousettus spp. bats were screened for CoVs using a pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. A single-step RT-PCR was performed on the RNA extracted from the bat specimens. Next-generation sequencing (NGS) was performed on a few representative bat specimens that were tested positive. Phylogenetic analysis was carried out on the partial sequences of RdRp gene sequences retrieved from both the bat species and complete viral genomes recovered from Rousettus spp.Results:Bat samples from the seven States were screened, and the RS specimens of eight Rousettus spp. and 21 Pteropus spp. were found positive for CoV RdRp gene. Among these, by Sanger sequencing, partial RdRp sequences could be retrieved from three Rousettus and eight Pteropus bat specimens. Phylogenetic analysis of the partial RdRp region demonstrated distinct subclustering of the BtCoV sequences retrieved from these Rousettus and Pteropus spp. bats. NGS led to the recovery of four sequences covering approximately 94.3 per cent of the whole genome of the BtCoVs from Rousettus bats. Three BtCoV sequences had 93.69 per cent identity to CoV BtRt-BetaCoV/GX2018. The fourth BtCoV sequence was 96.8 per cent identical to BtCoV HKU9-1.Interpretation & conclusions:This study was a step towards understanding the CoV circulation in Indian bats. Detection of potentially pathogenic CoVs in Indian bats stresses the need for enhanced screening for novel viruses in them. One Health approach with collaborative activities by the animal health and human health sectors in these surveillance activities shall be of use to public health. This would help in the development of diagnostic assays for novel viruses with outbreak potential and be useful in disease interventions. Proactive surveillance remains crucial for identifying the emerging novel viruses with epidemic potential and measures for risk mitigation.

Background & objectives: The presence of Cat Que virus (CQV) in Culex mosquitoes and pigs has been reported in China and Vietnam. Due to the spread of similar species of the Culex mosquitoes in India, there is a need to understand the replication kinetics of this virus in mosquito models. As a part of preparedness and to identify the presence of this CQV in humans and swine, this study was carried out to develop diagnostic tests. Methods: Serological and molecular diagnostic assays were developed for testing the mosquito population, human and swine serum samples. In this line, RNA-dependent RNA polymerase (L), glycoprotein (M) and nucleocapsid (S) genes-based reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for CQV. Real-time RT-PCR was used for screening of retrospectively collected human serum samples (n=1020) with acute febrile illness during 2014-2017. Simultaneously, an in-house anti-CQV swine and human IgG ELISAs were also developed to detect anti-CQV IgG antibody. Human serum samples (n=883) with post-onset of disease (POD) >4 days and swine serum samples (n=459) were tested for the presence of anti-CQV IgG antibodies. CQV NIV 612,045 isolate was used for susceptibility and replication kinetics experiment using three different species of mosquitoes to understand its behaviour in Indian mosquitoes. Results: All human serum samples (n=1020) screened for the presence of CQV using real-time RT-PCR were found to be negative. Anti-CQV IgG antibody positivity was recorded in two of 883 human serum samples tested. Virus susceptibility experiments indicated that three species of mosquito, namely Aedes aegypti, Culex quinquefasciatus and Cx. tritaeniorhynchus supported multiplication of CQV by intrathoracic as well as artificial membrane/oral feeding routes. Interpretation & conclusions: Anti-CQV IgG antibody positivity in human serum samples tested and the replication capability of CQV in mosquitoes indicated a possible disease causing potential of CQV in Indian scenario. Screening of more human and swine serum samples using these assays is required as a proactive measure for understanding the prevalence of this neglected tropical virus.

Background & objectives: Kyasanur forest disease (KFD) is an infectious disease discovered in Karnataka State of India in 1957; since then, the State has been known to be enzootic for KFD. In the last few years, its presence was observed in the adjoining five States of the Western Ghats of India. The present study was conducted to understand the kinetics of viral RNA, immunoglobulin M (IgM) and IgG antibody in KFD-infected humans for developing a diagnostic algorithm for KFD. Methods: A prospective follow up study was performed among KFD patients in Sindhudurg district of Maharashtra State, India. A total of 1046 suspected patients were tested, and 72 KFD patients were enrolled and followed for 17 months (January 2016 to May 2017). Serum samples of KFD patients were screened for viral RNA, and IgM and IgG antibodies. Results: KFD viral positivity was observed from 1st to 18th post-onset day (POD). Positivity of anti-KFD virus (KFDV) IgM antibodies was detected from 4th till 122nd POD and anti-KFDV IgG antibodies detected from 5th till 474th POD. A prediction probability was determined from statistical analysis using the generalized additive model in R-software to support the laboratory findings regarding viral kinetics. Interpretation & conclusions: This study demonstrated the presence of KFD viral RNA till 18th POD, IgM antibodies till 122nd POD and IgG till the last sample collected. Based on our study an algorithm was recommended for accurate laboratory diagnosis of KFDV infection. A sample collected between 1 and 3 POD can be tested using KFDV real-time reverse transcriptase polymerase chain reaction (RT-PCR); between 4 and 24 POD, the combination of real-time RT-PCR and anti-KFDV IgM enzyme-linked immunosorbent assay (ELISA) tests can be used; between POD 25 and 132, anti-KFDV IgM and IgG ELISA are recommended.

We retrieved Nipah virus (NiV) sequences from 4 human and 3 fruit bat (Pteropus medius) samples from a 2018 outbreak in Kerala, India. Phylogenetic analysis demonstrated that NiV from humans was 96.15% similar to a Bangladesh strain but 99.7%-100% similar to virus from Pteropus spp. bats, indicating bats were the source of the outbreak.

Crimean Congo Hemorrhagic Fever (CCHF) is a highly infectious zoonotic disease of humans transmitted by Hyalomma ticks. Earlier studies have shown CCHF seroprevalence in livestock throughout India, yet sporadic outbreaks have been recorded mostly from the Gujarat state of India since 2011. Occupational vulnerability to CCHF for animal handlers, veterinarians, abattoir workers, and healthcare workers has been documented. The current study was planned to determine the seroprevalence of CCHF with an intention to identify the high -risk population and high -risk areas from Gujarat state, India.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.