Differential Cell Line Susceptibility to the SARS-CoV-2 Omicron BA.1.1 Variant of Concern.

The unique mutations of the SARS-CoV-2 Omicron variant are associated with increased transmissibility, immune escape, increased binding affinity to ACE-2, and increased viral load. Omicron exhibited a shift in tropism infecting the upper respiratory tract compared to other variants of concern which have tropism for the lower respiratory tract. The tropism of omicron variants in cell lines of different hosts and tissue origins still remains unclear. Considering this, we assessed the susceptibility of different cell lines to the SARS-CoV-2 omicron BA.1.1 variant and permissiveness among different cell lines for omicron replication. Susceptibility and permissiveness of a total of eleven cell lines, including six animal cell lines and five human cell lines for omicron BA.1.1 infection, were evaluated by infecting individual cell lines with omicron BA.1.1 isolate at a 0.1 multiplicity of infection. Virus replication was assessed by observation of cytopathic effects followed by viral load determination by real-time PCR assay and virus infectivity determination by TCID50 assay. The characteristic cytopathic effect, increased viral load, and productive omicron replication was detected in Vero CCL-81, Vero E6, Vero/hSLAM, MA-104, and Calu-3 cells. Although LLC MK-2 cells showed an increased TCID50 titer at the second infection, the viral load did not show much difference in both infections. Caco-2 cells did not show evident CPE, but they supported omicron replication at a low level. A549, RD, MRC-5, and BHK-21 cells supported omicron BA.1.1 replication without the CPE. This is the first study on the comparison of susceptibility of different cell lines to Omicron variant BA.1.1, which might be useful for future studies on emerging SARS-CoV-2 variants.

Neutralizing antibody responses to SARS-CoV-2 in COVID-19 patients.

BACKGROUND & OBJECTIVES: The global pandemic caused by SARS-CoV-2 virus has challenged public health system worldwide due to the unavailability of approved preventive and therapeutic options. Identification of neutralizing antibodies (NAb) and understanding their role is important. However, the data on kinetics of NAb response among COVID-19 patients are unclear. To understand the NAb response in COVID-19 patients, we compared the findings of microneutralization test (MNT) and plaque reduction neutralization test (PRNT) for the SARS-CoV-2. Further, the kinetics of NAb response among COVID-19 patients was assessed. METHODS: A total of 343 blood samples (89 positive, 58 negative for SARS-CoV-2 and 17 cross-reactive and 179 serum from healthy individuals) were collected and tested by MNT and PRNT. SARS-CoV-2 virus was prepared by propagating the virus in Vero CCL-81 cells. The intra-class correlation was calculated to assess the correlation between MNT and PRNT. The neutralizing endpoint as the reduction in the number of plaque count by 90 per cent (PRNT90) was also calculated. RESULTS: The analysis of MNT and PRNT quantitative results indicated that the intra-class correlation was 0.520. Of the 89 confirmed COVID-19 patients, 64 (71.9%) showed NAb response. INTERPRETATION & CONCLUSIONS: The results of MNT and PRNT were specific with no cross-reactivity. In the early stages of infection, the NAb response was observed with variable antibody kinetics. The neutralization assays can be used for titration of NAb in recovered/vaccinated or infected COVID-19 patients.

Study of Kyasanur forest disease viremia, antibody kinetics, and virus infection in target organs of Macaca radiata.

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.

Development of indigenous IgG ELISA for the detection of anti-SARS-CoV-2 IgG.

BACKGROUND & OBJECTIVES: Since the beginning of the year 2020, the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacted humankind adversely in almost all spheres of life. The virus belongs to the genus Betacoronavirus of the family Coronaviridae. SARS-CoV-2 causes the disease known as coronavirus disease 2019 (COVID-19) with mild-to-severe respiratory illness. The currently available diagnostic tools for the diagnosis of COVID-19 are mainly based on molecular assays. Real-time reverse transcription-polymerase chain reaction is the only diagnostic method currently recommended by the World Health Organization for COVID-19. With the rapid spread of SARS-CoV-2, it is necessary to utilize other tests, which would determine the burden of the disease as well as the spread of the outbreak. Considering the need for the development of such a screening test, an attempt was made to develop and evaluate an IgG-based ELISA for COVID-19. METHODS: A total of 513 blood samples (131 positive, 382 negative for SARS-CoV-2) were collected and tested by microneutralization test (MNT). Antigen stock of SARS-CoV-2 was prepared by propagating the virus in Vero CCL-81 cells. An IgG capture ELISA was developed for serological detection of anti-SARS-CoV-2 IgG in serum samples. The end point cut-off values were determined by using receiver operating characteristic (ROC) curve. Inter-assay variability was determined. RESULTS: The developed ELISA was found to be 92.37 per cent sensitive, 97.9 per cent specific, robust and reproducible. The positive and negative predictive values were 94.44 and 98.14 per cent, respectively. INTERPRETATION & CONCLUSIONS: This indigenously developed IgG ELISA was found to be sensitive and specific for the detection of anti-SARS-CoV-2 IgG in human serum samples. This assay may be used for determining seroprevalence of SARS-CoV-2 in a population exposed to the virus.

Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever.

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.

Nipah Virus Sequences from Humans and Bats during Nipah Outbreak, Kerala, India, 2018.

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.

Isolation of Tioman virus from Pteropus giganteus bat in North-East region of India.

Bat-borne viral diseases are a major public health concern among newly emerging infectious diseases which includes severe acute respiratory syndrome, Nipah, Marburg and Ebola virus disease. During the survey for Nipah virus among bats at North-East region of India; Tioman virus (TioV), a new member of the Paramyxoviridae family was isolated from tissues of Pteropus giganteus bats for the first time in India. This isolate was identified and confirmed by RT-PCR, sequence analysis and electron microscopy. A range of vertebrate cell lines were shown to be susceptible to Tioman virus. Negative electron microscopy study revealed the "herringbone" morphology of the nucleocapsid filaments and enveloped particles with distinct envelope projections a characteristic of the Paramyxoviridae family. Sequence analysis of Nucleocapsid gene of TioV demonstrated sequence identity of 99.87% and 99.99% nucleotide and amino acid respectively with of TioV strain isolated in Malaysia, 2001. This report demonstrates the first isolation of Tioman virus from a region where Nipah virus activity has been noticed in the past and recent years. Bat-borne viruses have become serious concern world-wide. A Survey of bats for novel viruses in this region would help in recognizing emerging viruses and combating diseases caused by them.

Establishment of cell line from embryonic tissue of Pipistrellus ceylonicus bat species from India & its susceptibility to different viruses.

BACKGROUND & OBJECTIVES: Pipistrellus ceylonicus bat species is widely distributed in South Asia, with additional populations recorded in China and Southeast Asia. Bats are the natural reservoir hosts for a number of emerging zoonotic diseases. Attempts to isolate bat-borne viruses in various terrestrial mammalian cell lines have sometimes been unsuccessful. The bat cell lines are useful in isolation and propagation of many of the viruses harboured by bats. New stable bat cell lines are needed to help such investigations and to assist in the study of bat immunology and virus-host interactions. In this study we made an attempt to develop a cell line from P. ceylonicus bats. METHODS: An effort was made to establish cell line from embryo of P. ceylonicus species of bat after seeding to Dulbecco's modified eagle medium (DMEM) supplemented with 10 per cent foetal bovine serum; a primary cell line was established and designated as NIV-BtEPC. Mitochondrial DNA profile analysis was done using cyt-b and ND-1 gene sequences from the cell line. Phylogenetic tree was constructed using neighbour-joining algorithm for cyt-b and ND-1 genes with 1000-bootstrap replicates. RESULTS: NIV-BtEPC cell line was susceptible to Chandipura (CHPV) and novel adenovirus (BtAdv-RLM) isolated from Rousettus leschenaulti from India but did not support multiplication of a number of Bunyaviruses, Alphaviruses and Flavivirus. This might be useful for isolation of a range of viruses and investigation of unknown aetiological agents. INTERPRETATION & CONCLUSIONS: In this study, a new bat cell line was developed from P. ceylonicus. This cell line was successfully tested for the susceptibility to Chandipura and BtAdv-RLM virus isolated from bats. The approach developed and optimised in this study may be applicable to the other species of bats and this established cell line can be used to facilitate virus isolation and basic research into virus-host interaction.