Surveillance of Nipah virus in Pteropus medius of Kerala state, India, 2023.

Introduction: Since 2018, the Indian state of Kerala has reported four Nipah virus (NiV) disease outbreaks, raising concerns about NiV spillover from bats to the human population. Considering this, a cross-sectional study was undertaken in the Pteropus medius bat population around the Nipah virus-affected regions of Kozhikode, Kerala, India, during February, July, and September 2023. Methods: Throat swabs, rectal swabs, and organ samples were collected from bats to test for NiV using the real-time reverse transcriptase polymerase chain reaction (RT-PCR), while serum samples were screened for anti-Nipah IgG antibodies through ELISA. Results: An overall seroprevalence of 20.9% was observed in 272 P. medius bats tested. The throat and rectal swab samples of 321 bats were negative for NiV RNA. However, 4 of 44 P. medius bats tested positive for NiV in their liver/spleen samples. The partial N gene retrieved showed more than 99% similarity with the earlier reported NiV genome from Kerala state, India. Discussion: The findings of the study caution that there is a spillover risk in the region and necessary precautions should be taken.

Needle-free injection system delivery of ZyCoV-D DNA vaccine demonstrated improved immunogenicity and protective efficacy in rhesus macaques against SARS-CoV-2.

The apprehension of needles related to injection site pain, risk of transmitting bloodborne pathogens, and effective mass immunization have led to the development of a needle-free injection system (NFIS). Here, we evaluated the efficacy of the NFIS and needle injection system (NIS) for the delivery and immunogenicity of DNA vaccine candidate ZyCoV-D in rhesus macaques against SARS-CoV-2 infection. Briefly, 20 rhesus macaques were divided into 5 groups (4 animals each), that is, I (1 mg dose by NIS), II (2 mg dose by NIS), III (1 mg dose by NFIS), IV (2 mg dose by NFIS) and V (phosphate-buffer saline [PBS]). The macaques were immunized with the vaccine candidates/PBS intradermally on Days 0, 28, and 56. Subsequently, the animals were challenged with live SARS-CoV-2 after 15 weeks of the first immunization. Blood, nasal swab, throat swab, and bronchoalveolar lavage fluid specimens were collected on 0, 1, 3, 5, and 7 days post infection from each animal to determine immune response and viral clearance. Among all the five groups, 2 mg dose by NFIS elicited significant titers of IgG and neutralizing antibody after immunization with enhancement in their titers postvirus challenge. Besides this, it also induced increased lymphocyte proliferation and cytokine response. The minimal viral load post-SARS-CoV-2 challenge and significant immune response in the immunized animals demonstrated the efficiency of NFIS in delivering 2 mg ZyCoV-D vaccine candidate.

In-silico screening and in-vitro assay show the antiviral effect of Indomethacin against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the worldwide spread of coronavirus disease 19 (COVID-19), and till now, it has caused death to more than 6.2 million people. Although various vaccines and drug candidates are being tested globally with limited to moderate success, a comprehensive therapeutic cure is yet to be achieved. In this study, we applied computational drug repurposing methods complemented with the analyses of the already existing gene expression data to find better therapeutics in treatment and recovery. Primarily, we identified the most crucial proteins of SARS-CoV-2 and host human cells responsible for viral infection and host response. An in-silico screening of the existing drugs was performed against the crucial proteins for SARS-CoV-2 infection, and a few existing drugs were shortlisted. Further, we analyzed the gene expression data of SARS-CoV-2 in human lung epithelial cells and investigated the molecules that can reverse the cellular mRNA expression profiles in the diseased state. LINCS L1000 and Comparative Toxicogenomics Database (CTD) were utilized to obtain two sets of compounds that can be used to counter SARS-CoV-2 infection from the gene expression perspective. Indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), and Vitamin-A were found in two sets of compounds, and in the in-silico screening of existing drugs to treat SARS-CoV-2. Our in-silico findings on Indomethacin were further successfully validated by in-vitro testing in Vero CCL-81 cells with an IC50 of 12 µM. Along with these findings, we briefly discuss the possible roles of Indomethacin and Vitamin-A to counter the SARS-CoV-2 infection in humans.

Serosurvey for Nipah virus in bat population of southern part of India.

Nipah virus (NiV) is one of the priority pathogens with pandemic potential. Though the spread is far slower than SARS-CoV-2, case fatality is the biggest concern. Fruit bats belonging to genus Pteropus are identified to be the main reservoir of the virus causing sporadic cases and outbreaks in Malaysia, Bangladesh and India. The sudden emergence of Nipah in Kerala, India during 2018-2019 has been astonishing with respect to its introduction in the unaffected areas. With this, active Nipah virus surveillance was conducted among bat populations in Southern part of India viz., Karnataka, Kerala, Tamil Nadu, Telangana, Puducherry and Odisha during January-November 2019. Throat swabs/rectal swabs (n = 573) collected from Pteropus medius and Rousettus leschenaultii bat species and sera of Pteropus medius bats (n = 255) were screened to detect the presence of Nipah viral RNA and anti-Nipah IgG antibodies respectively. Of 255 P. medius bats sera samples, 51 bats (20%) captured from Karnataka, Kerala, Tamil Nadu and Puducherry demonstrated presence of anti-Nipah IgG antibodies. However, the presence of virus couldn't be detected in any of the bat specimens. The recent emergence of Nipah virus in Kerala in September 2021 warrants further surveillance of Nipah virus among bat populations from the affected and remaining states of India.

Nipah Virus Outbreak in Kerala State, India Amidst of COVID-19 Pandemic.

We report here a Nipah virus (NiV) outbreak in Kozhikode district of Kerala state, India, which had caused fatal encephalitis in a 12-year-old boy and the outbreak response, which led to the successful containment of the disease and the related investigations. Quantitative real-time reverse transcription (RT)-PCR, ELISA-based antibody detection, and whole genome sequencing (WGS) were performed to confirm the NiV infection. Contacts of the index case were traced and isolated based on risk categorization. Bats from the areas near the epicenter of the outbreak were sampled for throat swabs, rectal swabs, and blood samples for NiV screening by real-time RT-PCR and anti-NiV bat immunoglobulin G (IgG) ELISA. A plaque reduction neutralization test was performed for the detection of neutralizing antibodies. Nipah viral RNA could be detected from blood, bronchial wash, endotracheal (ET) secretion, and cerebrospinal fluid (CSF) and anti-NiV immunoglobulin M (IgM) antibodies from the serum sample of the index case. Rapid establishment of an onsite NiV diagnostic facility and contact tracing helped in quick containment of the outbreak. NiV sequences retrieved from the clinical specimen of the index case formed a sub-cluster with the earlier reported Nipah I genotype sequences from India with more than 95% similarity. Anti-NiV IgG positivity could be detected in 21% of Pteropus medius (P. medius) and 37.73% of Rousettus leschenaultia (R. leschenaultia). Neutralizing antibodies against NiV could be detected in P. medius. Stringent surveillance and awareness campaigns need to be implemented in the area to reduce human-bat interactions and minimize spillover events, which can lead to sporadic outbreaks of NiV.

Immunogenicity and protective efficacy of inactivated SARS-CoV-2 vaccine candidate, BBV152 in rhesus macaques.

The COVID-19 pandemic is a global health crisis that poses a great challenge to the public health system of affected countries. Safe and effective vaccines are needed to overcome this crisis. Here, we develop and assess the protective efficacy and immunogenicity of an inactivated SARS-CoV-2 vaccine in rhesus macaques. Twenty macaques were divided into four groups of five animals each. One group was administered a placebo, while three groups were immunized with three different vaccine candidates of BBV152 at 0 and 14 days. All the macaques were challenged with SARS-CoV-2 fourteen days after the second dose. The protective response was observed with increasing SARS-CoV-2 specific IgG and neutralizing antibody titers from 3rd-week post-immunization. Viral clearance was observed from bronchoalveolar lavage fluid, nasal swab, throat swab and lung tissues at 7 days post-infection in the vaccinated groups. No evidence of pneumonia was observed by histopathological examination in vaccinated groups, unlike the placebo group which exhibited interstitial pneumonia and localization of viral antigen in the alveolar epithelium and macrophages by immunohistochemistry. This vaccine candidate BBV152 has completed Phase I/II (NCT04471519) clinical trials in India and is presently in phase III, data of this study substantiates the immunogenicity and protective efficacy of the vaccine candidates.

How artificial intelligence may help the Covid-19 pandemic: Pitfalls and lessons for the future.

The clinical severity, rapid transmission and human losses due to coronavirus disease 2019 (Covid-19) have led the World Health Organization to declare it a pandemic. Traditional epidemiological tools are being significantly complemented by recent innovations especially using artificial intelligence (AI) and machine learning. AI-based model systems could improve pattern recognition of disease spread in populations and predictions of outbreaks in different geographical locations. A variable and a minimal amount of data are available for the signs and symptoms of Covid-19, allowing a composite of maximum likelihood algorithms to be employed to enhance the accuracy of disease diagnosis and to identify potential drugs. AI-based forecasting and predictions are expected to complement traditional approaches by helping public health officials to select better response and preparedness measures against Covid-19 cases. AI-based approaches have helped address the key issues but a significant impact on the global healthcare industry is yet to be achieved. The capability of AI to address the challenges may make it a key player in the operation of healthcare systems in future. Here, we present an overview of the prospective applications of the AI model systems in healthcare settings during the ongoing Covid-19 pandemic.

Detection of coronaviruses in Pteropus & Rousettus species of bats from different States of India.

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.

Effect of piperine and quercetin alone or in combination with marbofloxacin on CYP3A37 and MDR1 mRNA expression levels in broiler chickens.

After oral route of administration, drug absorption is unpredictable and is governed by various factors such as multi drug resistance-1 (MDR1) an efflux transporter and drug metabolizing enzymes (like CYP3A4, CYP3A37, CYP2D6) at intestine and liver. Naturally available phyto chemicals like piperine and quercetin as well as some floroquinolones are known to inhibit MDR1 and CYP3A37 activity and increases bioavailability of co-administered drugs. This study was carried out to investigate the effect of piperine and quercetin alone or in combination with marbofloxacin on CYP3A37 and MDR1 mRNA expression levels in liver and intestine of broiler chicken. After oral administration of piperine and quercetin for 3 consecutive days followed by with or without oral administration of marbofloxacin for 5 days, CYP3A37 and MDR1 mRNA expression levels were determined using quantitative real-time PCR. Total of 36 broiler chickens in seven individual groups were treated with different regimen and the mRNA expression levels at duodenum and liver were analyzed with apt statistical tools. After piperine and quercetin combined treatment with marbofloxacin, CYP3A37 mRNA expression levels were significantly down regulated by 20.57 (p = .034) and 25.95 (p = .003) folds; and MDR1 mRNA expression levels were also significantly down regulated by 11.33 (p = .012) and 33.59 (p = .006) folds in liver and duodenum, respectively. Down regulation of CYP3A37 and MDR1 mRNA in liver and duodenum indicate the combined pretreatment of piperine and quercetin may be useful for improving the pharmacokinetics of orally administered drugs which are substrates for CYP3A37 and MDR1.

Isolation and phylogenetic characterization of haemagglutinin and neuraminidase genes of H9N2 low pathogenicity avian influenza virus isolated from commercial layers in India.

Avian influenza is a highly infectious and dynamically evolving disease of birds causing high morbidity and mortality. It is caused by avian influenza virus (AIV) that belongs to the family Orthomyxoviridae. Two types of AIV have been described based on their pathogenicity viz. highly pathogenic avian influenza virus that causes severe disease with high mortality and low pathogenic avian influenza virus (LPAI) that generally causes asymptomatic infection or a mild disease. The H9N2 subtype is the widely circulated LPAI type in the world. The H9N2 subtype of was first reported from northern India in March 2003. However, systematical surveillance information for the evolution of H9N2 viruses in poultry flocks of Southern India is lacking. The present study reports the isolation and characterization of H9N2 isolates from the southern parts of the country during the period between May 2010 and September 2011. Out of the 30 poultry flocks investigated, six were found to be positive for HA activity. Further, all the six samples conformed as AIV. Partial nucleotide sequencing of the HA and NA genes revealed that all were belonging to the H9N2 subtype. Phylogenetically, the HA and NA genes of the H9N2 viruses from India clustered with those isolated from Bangladesh, Pakistan and the Middle East, although we were not able to conclude on their exact geographic origin.

Detection and partial genetic characterisation of a novel variant of Avian nephritis virus in Indian poultry flocks showing diverse clinical signs.

Avian nephritis virus (ANV) infects poultry flocks worldwide, but no confirmed cases have been reported from India so far. In the current study, disease investigation was carried out in 21 broiler flocks at different parts of India with clinical signs of nephritis, uneven and stunted growth, diarrhoea, reduced body weight, and mortality up to 9.72%. Out of the 21 flocks screened, two were found positive for ANV in RT-PCR assay. BLAST analysis revealed that the ANV of Indian origin was closely related to ANV-1 strains reported from Japan, Hungary and China. However, comparison of a small portion (~12% of nucleotides, i.e. ~60 nts, common site for ANV-1 and ANV-3, position 2200-2260 of ORF 1a gene) of the Indian ANV sequence with ANV-3 sequences revealed 89-93% identities with different ANV-3 isolates. Phylogenetically, ANV-1 forms three clades, and the Indian ANV clustered under clade II. This study confirms the existence of ANV in Indian poultry flocks and is the first report on the molecular detection and genetic characterisation of ANV from India.

Molecular detection and characterization of infectious laryngotracheitis virus (Gallid herpesvirus-1) from clinical samples of commercial poultry flocks in India.

Although the existence of infectious laryngotracheitis virus (ILTV) in India was first reported in 1964, no reports are available regarding its molecular detection and characterization. The present study was aimed to detect and characterize ILTV from recent respiratory disease complex (RDC) outbreaks of commercial poultry flocks in different parts of the country by using envelope glycoprotein G gene (US4 gene) based PCR and sequencing. A total of thirty two flocks with a history of RDC were investigated. Overall, all the strains/breeds of birds and all ages of birds are equally susceptible and depending on the severity, the clinical signs and gross lesions were varied. Out of 32 flocks investigated 10 were found positive for ILTV infection by PCR. The phylogenetic analyses of eight representative sequences in the present study deciphered that Indian ILT viruses are closely related to chicken embryo origin vaccine strains of Italy, USA, China and Brazil.

Emergence of a novel lineage genetically divergent from the predominant Ind2001 lineage of serotype O foot-and-mouth disease virus in India.

In India, emergence of Ind2001 lineage of foot-and-mouth disease virus (FMDV) serotype O was recorded in the year 2001. After causing sporadic incidences, the Ind2001 lineage that re-surged in 2008 out-competed PanAsia from the field during 2009 and continued its dominance during 2010 and 2011 as well. The lineage has diversified in due course of time, leading to two sub-lineages (Ind2001a and Ind2001b). The sub-lineage Ind2001a include isolates collected during 2001-2002 and sub-lineage Ind2001b is constituted largely by isolates collected during 2008-2012. The nucleotide substitution rate of sub-lineage Ind2001b was estimated at 6.58×10⁻³ substitutions/site/year. The most stable PanAsia lineage is restricted only to few outbreaks. During 2011, emergence of a new genetic group with >9% nucleotide divergence from rest of the lineages circulating in the country was detected and named as lineage Ind2011. Two specific amino acid substitutions at positions VP1-36 (F) and VP2-133 (T) were observed in the Ind2011 lineage. The new lineage at present is restricted only to southern states of the country. It is uncertain whether the emergence was triggered by immune pressure or due to a bottleneck in transmission or selected for higher fitness value. Six sites (4, 68, 83, 135, 138 and 209) in VP1 protein were identified to undergo episodic diversifying selection in serotype O field isolates. Both emerging and re-emerging lineages had appropriate antigenic match with currently used vaccine strain, INDR2/1975. Irrespective of genetic variability, the field isolates showed remarkable conservation at antigenically critical residues that might contribute to the observed antigenic stability. With the emergence of a new genetic group after a span of 10 years, the overall epidemiological scenario in the region is expected to change in the coming years.

Differential expression of pro-inflammatory cytokines in endometrial tissue of buffaloes with clinical and sub-clinical endometritis.

The objective of this study was to investigate the endometrial expression of pro-inflammatory cytokines (IL1ß, IL6, IL8 and TNFα) along with TLR4 and CD14 in normal and endometritic buffaloes. The genitalia were collected in the abattoir and divided into three groups as normal (gr. I=12), clinical endometritis (CE, gr. II=12) based on positive color reaction to white side test of uterine discharge and sub-clinical endometritis (SCE, gr. III=12) based on endometrial cytology (presence of ≥5% PMNs) and histopathology. The equal numbers of genitalia were grouped into follicular and luteal stage in each group. Endometrial tissue scrapings were used for total RNA extraction and cDNA was transcribed and amplified by Real time PCR. The results showed several fold higher expression of all cytokine transcripts in CE (gr. II), whereas significant up-regulation of CD14 (1 to 2-fold), IL6 (15 to 36-fold), IL8 (8 to 14-fold) and TNFα (10 to 11-fold) mRNA was observed in SCE. This indicates that the evaluation of expression patterns of certain cytokines gene holds promise to diagnose the severity and degree of uterine inflammation.

Immunodiagnosis of foot-and-mouth disease using mutated recombinant 3ABC polyprotein in a competitive ELISA.

Differentiation of infected from vaccinated animals (DIVA) is essential for effective control of foot-and-mouth disease (FMD) by vaccination. The antibody response against FMD viral non-structural proteins (NSPs) has been used widely for this purpose. Among all the NSPs, the 3ABC polyprotein has been recognized as the most appropriate indicator for DIVA. In this study, mutated full-length 3ABC polyprotein was expressed in a prokaryotic system and monoclonal antibody against the recombinant protein was developed. A competitive ELISA (C-ELISA) for DIVA was standardized for different species of livestock animals using recombinant 3ABC and monoclonal antibodies. The diagnostic sensitivity and specificity of the assay were estimated by testing a panel of known serum samples consisting of sera from naive, vaccinated and infected animals as 86.9% with 66.4-97.2 (95%) confidence interval and 97% with 89.6-99.6 (95%) confidence interval respectively at 40% inhibition cut-off. The assay was validated further by testing sera from different livestock species collected at random from different parts of the country. The assay will provide a common method for testing sera from different species of livestock and wild animals. The C-ELISA is a sensitive and specific DIVA assay for FMD and can be used as a method for FMD control programme with vaccination.

Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion.

Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, beta-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.