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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.

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.

Additional file 2: Supplementary Table 1. Percentage nucleotide and amino acid similarity for each gene of Nipah virus with respect to the Nipah virus retrieved from human sequence of Bangladesh, India, 2004 (Accession Number: AY988601.1) outbreak.

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.

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.

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.