The rapid evolution of SARS-CoV-2 variants, have posed significant public health challenges due to their heightened transmissibility and varying clinical outcomes. While the impact of these variants in humans has been extensively studied, their effect on domestic animals has not been explored thoroughly. Given the close contact between humans and pets, combined with documented cases of virus transmission from humans to domestic animals and the potential for these animals to act as viral reservoirs, highlights the importance of understanding the molecular dynamics of SARS-CoV-2 infection in them. Consequently, there is an increasing demand for infection models that closely mimic viral behavior in animal tissues. In vivo infection of cats or any other non-laboratory animal species is extremely resource intensive, time consuming, require ABSL3 facilities, and animal ethical clearances which are not practically possible for the majority of researchers. In this scenario the ex vivo cultures model can be an excellent alternative. Therefore, in this study, we have utilized an ex vivo lung explant culture model derived from domestic cats to examine the host’s response to Delta and Omicron variants of SARS-CoV-2 in cat lung explant culture. Comprehensive transcriptomic profiling at multiple time points post-infection revealed significant disruptions in genes associated with cell adhesion, structural components, extracellular matrix (ECM) organization and regulation, secreted and growth factors, and immune responses. Our study revealed that the Delta variant triggered early activation of genes associated with tissue damage as early as 12 hours post-infection (hpi). By 24 hpi, both the Delta and Omicron variants showed significant activation of these genes, with the Delta variant linked to the activation of a larger number of tissue damage-related genes. Notably, we identified several hub genes, including MMP9, CCL5, MCP-1/CCL2, VWF, HGF, ANGPT1, CD34, CD68, SPP1, IGF1, CSF1, and VCL, involved in critical signaling pathways such as focal adhesion, PI3K-Akt signaling, and TNF signaling. These hub genes hold potential as valuable biomarkers. This study provides key insights into the pathogenesis of SARS-CoV-2 in cats and highlights the utility of ex vivo lung explant cultures as a platform for studying viral infections.

The increasing resistance cases in influenza A viruses (IAVs) to different classes of antiviral drugs, necessitates for a detailed investigation of molecular interactions governing reduced susceptibility to these drugs. This study explores the molecular basis of the Highly Pathogenic Avian Influenza (HPAI) A H5N1 viruses’ resistance to neuraminidase inhibitors (NAIs), which we identified in our previous study. Using sophisticated protein modeling and docking methods, we investigated two HPAI H5N1 clade 2.2 viruses, A/chicken/India/85459/2008 (N294S) and A/chicken/WestBengal/142121/2008 (E119A + I117V). The N294S substitution provided oseltamivir resistance while retaining zanamivir susceptibility, but the E119A + I117V substitutions resulted in resistance to both zanamivir and oseltamivir. Molecular interactions analyses unveiled that N294S and E119A + I117V substitutions resulted in varying fitness levels, hydrogen bonding, and affinity transitions. It is evident that the impact of different substitutions within the neuraminidase (NA) protein of IAV varies widely depending on the subtype, clade, or the specific NAIs (oseltamivir and zanamivir). Given the variable susceptibility to NAIs caused by distinct substitutions in NA protein, it is critical to implement an integrated surveillance framework that incorporates the One Health approach, involving avian, environmental, mammalian and human’s sources, to monitor susceptibility or resistance profiles of IAVs. This study is the first to investigate the genetic basis for reduced NAIs susceptibility in HPAI H5N1 viruses having dual resistance molecular markers, emphasizing the significance for refining antiviral strategies and improving pandemic preparedness.

Domestic ducks (Anas platyrhynchos domesticus) are resistant to most of the highly pathogenic avian influenza virus (HPAIV) infections. In this study, we characterized the lung proteome and phosphoproteome of ducks infected with the HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala) at 12 h, 48 h, and 5 days post-infection. A total of 2082 proteins were differentially expressed and 320 phosphorylation sites mapping to 199 phosphopeptides, corresponding to 129 proteins were identified. The functional annotation of the proteome data analysis revealed the activation of the RIG-I-like receptor and Jak-STAT signaling pathways, which led to the induction of interferon-stimulated gene (ISG) expression. The pathway analysis of the phosphoproteome datasets also confirmed the activation of RIG-I, Jak-STAT signaling, NF-kappa B signaling, and MAPK signaling pathways in the lung tissues. The induction of ISG proteins (STAT1, STAT3, STAT5B, STAT6, IFIT5, and PKR) established a protective anti-viral immune response in duck lung tissue. Further, the protein–protein interaction network analysis identified proteins like AKT1, STAT3, JAK2, RAC1, STAT1, PTPN11, RPS27A, NFKB1, and MAPK1 as the main hub proteins that might play important roles in disease progression in ducks. Together, the functional annotation of the proteome and phosphoproteome datasets revealed the molecular basis of the disease progression and disease resistance mechanism in ducks infected with the HPAI H5N1 virus.

The increasing resistance cases in influenza viruses to different classes of antiviral drugs, necessitates the in-depth analysis of molecular interactions governing reduced susceptibility to these drugs. This study explores the molecular basis of neuraminidase inhibitors resistance in avian H5N1 influenza viruses identified in our previous research. Using comprehensive modeling and docking tools, we investigated two isolates—A/chicken/India/85459/2008 (N294S) and A/chicken/WestBengal/142121/2008 (E119A + I117V). The N294S mutation conferred oseltamivir resistance while retaining zanamivir susceptibility, whereas the E119A + I117V mutations led to zanamivir resistance while reducing oseltamivir susceptibility. Molecular interactions analysis unveiled varied fitness levels, hydrogen bonding, and affinity transitions associated with N294S and E119A + I117V mutations. This study provides crucial insights into molecular interactions responsible for reduced susceptibility to neuraminidase inhibitors, which is essential for optimizing antiviral strategies and pandemic preparedness.### Competing Interest StatementThe authors have declared no competing interest.

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to evolve carrying flexible amino acid substitutions in the spike protein's receptor binding domain (RBD). These substitutions modify the binding of the SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) receptor and have been implicated in altered host fitness, transmissibility, and efficacy against antibody therapeutics and vaccines. Reliably predicting the binding strength of SARS-CoV-2 variants RBD to hACE2 receptor and neutralizing antibodies (NAbs) can help assessing their fitness, and rapid deployment of effective antibody therapeutics, respectively. Here, we introduced a two-step computational framework with 3-fold validation that first identified dissociation constant as a reliable predictor of binding affinity in hetero- dimeric and trimeric protein complexes. The second step implements dissociation constant as descriptor of the binding strengths of SARS-CoV-2 variants RBD to hACE2 and NAbs. Then, we examined several variants of concerns (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron and demonstrated that these VOCs RBD bind to the hACE2 with enhanced affinity. Furthermore, the binding affinity of Omicron variant's RBD was reduced with majority of the RBD-directed NAbs, which is highly consistent with the experimental neutralization data. By studying the atomic contacts between RBD and NAbs, we revealed the molecular footprints of four NAbs (GH-12, P2B-1A1, Asarnow_3D11, and C118)-that may likely neutralize the recently emerged Omicron variant-facilitating enhanced binding affinity. Finally, our findings suggest a computational pathway that could aid researchers identify a range of current NAbs that may be effective against emerging SARS-CoV-2 variants.

• Highthrouput sequencing of small RNA of H5N1 infected and mock infected chicken lungs. • 297 miRNAs identified in mock-infected and 201 miRNAs identified in AIV infected chicken lungs. • 36 miRNAs were upregulated and 90 were downregulated during H5N1 infection. • Functional analysis and gene ontology of predicted target genes of expressed miRNAs. • MAPK pathway, NF-κB, IGF and gga-let-7b might play important role during H5N1 pathogenesis.

The current study aimed to develop broadly protective vaccines for avian influenza. In an earlier study, HA stalk (universal flu vaccine) was found to be broadly protective against different subtypes of influenza virus in mice. Hence, we were interested to know its breadth of protective efficacy either alone or combined with inactivated rgH5N2 (clade 2.3.2.1a) vaccine against challenge viruses of homologous H5N1, heterologous H5N8 (clade 2.3.4.4) and heterosubtypic H9N2 virus in specific pathogen-free chickens. The rgH5N2 vaccine alone or in combination with HA stalk elicited sufficient pre-challenge immunity in the form of haemagglutination inhibiting (HI) antibodies and neutralizing antibodies (MNT) against H5N1, H5N8, and H9N2 in chickens. The rgH5N2 vaccine alone or in combination with HA stalk also attenuated the shedding of H5N1, H5N8 and H9N2 in chickens and protected against the lethal challenge of H5N1 or H5N8. In contrast, all HA stalk immunised chickens died upon H5N1 or H5N8 challenge and H9N2 challenged chickens survived. Our study suggests that the rgH5N2 vaccine can provide clinical protection against H5N1, H5N8 and can attenuate the viral shedding of H9N2 in chickens.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has increased and become a serious concern worldwide, including India. Additionally, MRSA isolates are showing resistance to other chemotherapeutic agents. Isolated and valuable reports on the prevalence of MRSA are available in India. There is no systematic review on the prevalence of MRSA in one place; hence, this study was planned. The overall prevalence of MRSA in humans in India was evaluated state-wise, zone-wise, and year-wise. A systematic search from PubMed, Indian journals, Google Scholar, and J-Gate Plus was carried out and retrieved 98 eligible articles published from 2015 to 2020 in India. The statistical analysis of data was conducted using R software. The overall prevalence of MRSA was 37% (95% CI: 32–41) from 2015 to 2019. The pooled prevalence of MRSA zone-wise was 41% (95% CI: 33–50), 43% (95% CI: 20–68), 33% (95% CI: 24–43), 34% (95% CI: 26–42), 36% (95% CI: 25–47), and 40% (95% CI: 23–58) for north, east, west, south, central, and northeast region-zones, respectively. The state-wise stratified results showed a predominance of MRSA in Jammu and Kashmir with 55% (95% CI: 42–67) prevalence, and the lowest was 21% (95% CI: 11–34) in Maharashtra. The study indicated that the prevalence data would help in formulating and strict implementation of control measures in hospital areas to prevent the outbreak of MRSA infection and management of antibiotic usage.

We report here a targeted risk-based study to investigate the presence of influenza A viruses at the migratory-wild-domestic bird interface across the major wetlands of central India's Maharashtra state during the winter migration season. The H9N2 viruses have been isolated and confirmed in 3.86% (33/854) of the fecal samples of resident birds. To investigate the genetic pools of H9N2 circulating in resident birds, we sequenced two isolates of H9N2 from distant wetlands. Sequence and phylogenetic analyses have shown that these viruses are triple reassortants, with HA, NA, NP, and M genes belonging to G1 sub-lineage (A/quail/Hong Kong/G1/1997), PB2, PB1, and NS genes originating from the prototype Eurasian lineage (A/mallard/France/090360/2009) and PA gene deriving from Y439/Korean-like (A/duck/Hong Kong/Y439/97) sub-lineage. It was confirmed not only that four of their gene segments had a high genetic association with the zoonotic H9N2 virus, A/Human/India/TCM2581/2019, but also that they had many molecular markers associated with mammalian adaptation and enhanced virulence in mammals including the unique multiple basic amino acids, KSKR↓GLF at the HA cleavage site, and analog N-and O-glycosylation patterns on HA with that of the zoonotic H9N2 virus. Furthermore, future experiments would be to characterize these isolates biologically to address the public health concern. Importantly, due to the identification of these viruses at a strategic geographical location in India (a major stop-over point in the Central Asian flyway), these novel viruses also pose a possible threat to be exported to other regions via migratory/resident birds. Consequently, systematic investigation and active monitoring are a prerequisite for identifying and preventing the spread of viruses of zoonotic potential by enforcing strict biosecurity measures.

Many viruses that cause serious diseases in humans and animals, including the betacoronaviruses (beta-CoVs), such as SARS-CoV, MERS-CoV, and the recently identified SARS-CoV-2, have natural reservoirs in bats. Because these viruses rely entirely on the host cellular machinery for survival, their evolution is likely to be guided by the link between the codon usage of the virus and that of its host. As a result, specific cellular microenvironments of the diverse hosts and/or host tissues imprint peculiar molecular signatures in virus genomes. Our study is aimed at deciphering some of these signatures. Using a variety of genetic methods we demonstrated that trends in codon usage across chiroptera-hosted CoVs are collaboratively driven by geographically different host-species and temporal-spatial distribution. We not only found that chiroptera-hosted CoVs are the ancestors of SARS-CoV-2, but we also revealed that SARS-CoV-2 has the codon usage characteristics similar to those seen in CoVs infecting the Rhinolophus sp. Surprisingly, the envelope gene of beta-CoVs infecting Rhinolophus sp., including SARS-CoV-2, had extremely high CpG levels, which appears to be an evolutionarily conserved trait. The dissection of the furin cleavage site of various CoVs infecting hosts revealed host-specific preferences for arginine codons; however, arginine is encoded by a wider variety of synonymous codons in the murine CoV (MHV-A59) furin cleavage site. Our findings also highlight the latent diversity of CoVs in mammals that has yet to be fully explored.

The study was aimed to evaluate the elicitation of highly pathogenic avian influenza (HPAI) virus (AIV) M2e and HA2-specific immunity in chicken to develop broad protective influenza vaccine against HPAI H5N1. Based on the analysis of Indian AIV H5N1 sequences, the conserved regions of extracellular domain of M2 protein (M2e) and HA2 were identified. Synthetic gene construct coding for M2e and two immunodominant HA2 conserved regions was designed and synthesized after codon optimization. The fusion recombinant protein (~38 kDa) was expressed in a prokaryotic system and characterized by Western blotting with anti-His antibody and anti-AIV polyclonal chicken serum. The M2e–HA2 fusion protein was found to be highly reactive with known AIV-positive and -negative chicken sera by ELISA. Two groups of specific pathogen-free (SPF) chickens were immunized (i/m) with M2e synthetic peptide and M2e–HA2 recombinant protein along with one control group with booster on the 14th day and 28th day with the same dose and route. Pre-immunization sera and whole blood were collected on day 0 followed by 3, 7, 14, 21, and 28 days and 2 weeks after the second booster (42 day). Lymphocyte proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method revealed that the stimulation index (SI) was increased gradually from days 0 to 14 in the immunized group ( p < 0.05) than that in control chicken. Toll-like receptor (TLR) mRNA analysis by RT-qPCR showed maximum upregulation in the M2e–HA2-vaccinated group compared to M2e- and sham-vaccinated groups. M2e–HA2 recombinant protein-based indirect ELISA revealed that M2e–HA2 recombinant fusion protein has induced strong M2e and HA2-specific antibody responses from 7 days post-primary immunization, and then the titer gradually increased after booster dose. Similarly, M2e peptide ELISA revealed that M2e–HA2 recombinant fusion protein elicited M2e-specific antibody from day 14 onward. In contrast, no antibody response was detected in the chicken immunized with synthetic peptide M2e alone or control group. Findings of this study will be very useful in future development of broad protective H5N1 influenza vaccine targeting M2e and HA2.

Elucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.

Natural infections with HoBi-like pestivirus (HoBiPeV) have been detected in cattle in South America, Europe and Asia with a range of clinical manifestations including fatal mucosal disease (MD). In India, although HoBiPeV in cattle has been reported, there is no information on currently circulating HoBiPeV strains and associated severe clinical disease. Between September 2018 and December 2019, suspected cases of bovine viral diarrhoea with severe disease in cattle were noticed in farmers' small holdings in Tamil Nadu State. To determine the extent of pestivirus infection, blood, serum, nasal or oral swab samples of 46 cattle from 18 villages were tested. Based on the real-time RT-PCR, antigen ELISA and nucleotide sequencing results, pestivirus was detected in nine cattle from eight villages in two districts and all pestiviruses were identified as HoBiPeV. All nine HoBiPeV-infected cattle displayed clinical signs resembling MD and HoBiPeV isolates (n = 9) obtained were characterized at genetic and antigenic level. Phylogenetic analyses based on 5 '-untranslated regions (5 '-UTR), N-pro and combined 5 '-UTR-N-pro gene sequences revealed that eight HoBiPeV isolates clustered into a clade, distinct from all reported HoBiPeV clades (a-d), whereas one belonged to HoBiPeV-d clade, thus providing evidence of emergence of a novel HoBiPeV clade (e). This was also supported by HoBiPeV-e clade-specific amino acid substitutions in N-pro and the antigenic reactivity patterns. The study demonstrates the existence and independent evolution of five HoBiPeV clades (four main clades) globally and surprisingly three exclusive to India. Also we confirm first HoBiPeV occurrence in southern India with predominant prevalence of HoBiPeV-e strains. Besides demonstrating increased HoBiPeV genetic diversity, here we show association of HoBiPeV with severe clinical disease involving fatalities highlighting impact of HoBiPeV on cattle health. The emergence of a novel HoBiPeV lineage provides new insights on global HoBiPeV epidemiology and genetic diversity reiterating the need for continuous monitoring of HoBiPeV in India.

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a highly fatal disease syndrome that predominantly affects susceptible hosts of the order Artiodactyla. In this study, an in-depth clinico-molecular investigation of SA-MCF disease in a morbid 50-days-old cattle calf (Bos taurus indicus) and asymptomatic infection in the in-contact reservoir hosts, sheep (Ovis aries), and goat (Capra hircus) housed on a farm located in the Southern India is reported. An OIE recommended SA-MCF type-specific PCR confirmed the etiological agent as OvHV-2. The genetic characterization and phylogenetic analyses based on the glycoprotein B (gB) gene indicate that three genetic variants of OvHV-2 had infected the animal cluster of this study. As the OvHV-2 infection eventually lead to the death of the cattle calf, and the fact that its gB sequence carried four unique amino acid substitutions (N169S, L594P, I645V, and V730A), an investigation of these substitutions impact on its stability and molecular flexibility was carried out. The mapping of these amino acid substitutions on the three-dimensional structure of gB coupled with supplementary investigations showed that these substitutions conveyed the molecular flexibility to the gB, at the cost of its stability. Future studies would be to investigate whether these gB substitutions have any impact on membrane fusion activity using a virus-free cell-to-cell membrane fusion assay. The study also highlights the importance of adopting stringent biosecurity measures where mixed animal farming is a common practice.

Lumpy skin disease (LSD) caused by lumpy skin disease virus (LSDV) inflicts significant economic losses in cattle production with impact on livelihoods of smallholders. This study reports the first occurrence of LSD in cattle in India and analyses epidemiological and genetic characterization data from LSD outbreaks in five districts of Odisha state in August 2019. In all, 182 of 2,539 cattle were affected with an apparent morbidity rate of 7.1% and no mortality. Out of 102 samples from 60 LSD suspected and 17 asymptomatic in-contact cattle tested, 29.87% cattle were positive by capripoxvirus generic PCR and 37.66% were positive by LSDV real-time PCR. All the in-contact cattle tested were negative for LSDV. Among affected cattle, LSDV genome was detected more in scabs (79.16%) than blood (31.81%) and frozen bull semen (20.45%). Differential diagnosis by PCR was negative for pseudo-LSD, buffalopox, cowpox, pseudo-cowpox and bovine papular stomatitis. Five selected PCR and real-time PCR-positive LSDV DNA were sequenced in three genomic regions, P32 (LSDV074), F (LSDV117) and RPO30 (LSDV036). Phylogenetic analysis based on partial P32 and F gene sequences and complete RPO30 gene sequences showed that all the five Indian LSDV strains were identical and clustered with other field strains of LSDV circulating globally. However, the F and RPO30 gene sequence analyses revealed that Indian LSDV strains are genetically closer to the South African NI2490/KSGP-like strains than the strains detected in Europe, which was rather surprising. The present study established the existence of LSDV in India and involvement of LSDV field strains in the outbreaks. Additionally, we provided evidence of LSDV shedding in semen of naturally infected bulls. Further studies are required to determine the source of LSD introduction, extent of spread, modes of transmission and impact on dairy cattle production in India and effective control measures must be undertaken urgently.

Accurate and rapid diagnosis of Influenza A viruses (IAVs) is challenging because of multiple strains circulating in humans and animal populations, and the emergence of new strains. In this study, we demonstrate a simple and rapid strategy for visual detection of multiple strains of IAVs (H1 to H16 subtypes) using peptide nucleic acid (PNA) as a biosensor and unmodified gold nanoparticles (AuNPs) as a reporter. The design principle of the assay is based on the color change on account of free PNA-induced aggregation of AuNPs in the presence of non-complementary viral RNA sequence and vice-versa. The assay could detect IAV RNA with a visual limit of detection of 2.3 ng. The quantification of RNA with a considerable accuracy on a simple spectrophotometer was achieved on plotting the PNA-induced colorimetric changes (absorption ratio of A640/A520) in the presence of a varying concentration of complementary RNA. As a proof-of-concept, the visual assay was validated on 419 avian clinical samples and receiver operating characteristic (ROC) curve analysis showed a high diagnostic specificity (96.46%, 95% CI = 93.8 to 98.2) and sensitivity (82.41%, 95% CI = 73.9 to 89.1) when RT-qPCR was used as reference test. Hence, the simplicity, rapidity, and universality of this strategy make it a potential candidate visual assay for clinical diagnosis and surveillance of IAVs, especially in the resource-limited settings. The proposed strategy establishes new avenues for developing a simple and rapid diagnostic system for viral infections and biomolecules.

One of the most challenging research areas in biology is to understand the complex immune system and the computational tools have played a pivotal role in increasing the pace of research in the field of immunology. Thus, applications of computational methods and tools in unravelling the complex immune system and thereof translating the understanding in solving the immunological problems have given rise of a new field, immunoinformatics. Among the diverse areas of research, prediction of B-and T-cell epitopes is being considered one of the major and potential translational applications of immunoinformatics. It is now possible to identify and characterise an individual's MHC allotype based on the whole genome sequencing, which is an essential and a preliminary component in designing effective vaccines and therapeutics. The potential in silico epitopes prediction methods have been developed that have made epitope mapping an easy task by decreasing the list of potential epitope candidates for experimental testing. Here, we review a range of immunoinformatics tools developed and available online for public use freely, with an emphasis on B-and T-cell epitopes prediction. We also highlight the various algorithms and methods that formed the basis of important immunoinformatics tools and discuss their strengths and weakness.

Bovine leukaemia virus (BLV) causes enzootic leucosis in cattle and is prevalent worldwide. Although recent studies have shown that BLV strains can be classified into 10 distinct genotypes, no information is available regarding the BLV genotype prevalent in cattle in India. To determine the genetic variability in BLV, in this study, 118 adult dairy cows from three states of India were screened for BLV infection by env gp51-specific ELISA and nested PCR. Of the 33 cows found positive by both PCR and ELISA, 10 selected BLV strains were subjected to molecular characterization. Phylogenetic analyses of partial and full-length env gp51 gene sequences of Indian BLV strains and other geographical diverse BLV strains representing all the 10 genotypes revealed that Indian strains belonged to BLV genotype 6. Although Indian strains showed close genetic proximity with the strains circulating in South America, they were classified into a new subgenotype within genotype 6. Alignment of deduced amino acid sequences in gp51 demonstrated substitutions mainly in conformational epitope G, neutralizing domain 2 and linear epitope D, with a novel mutation (threonine to alanine at residue 252) found in D-epitope of all the Indian BLV strains. Although serological evidence of BLV infection in India has been reported earlier, this study on molecular characterization of BLV strains established the existence of BLV genotype 6 in India. Additionally, the results of this study highlight the importance of genetic analysis of geographically diverse BLV strains to understand BLV global genetic diversity and further studies are required to determine BLV genetic diversity and extent of BLV infection in cattle in India.

The present study was aimed at generating a reassortant vaccine candidate virus with Glade 2.3.2.1 Hemagglutinin (HA) and its evaluation in a challenge study for protection against homologous (2.3.2.1 clade) and heterologous (2.2 Glade) highly pathogenic avian influenza (HPAI) H5N1 viruses. Plasmid-based reverse genetics technique was used to rescue a 5 + 3 reassortant H5N2 strain containing the modified HA of H5N1 (clade 2.3.2.1), the Neuraminidase (NA) of H9N2, the Matrix (M) of H5N1 and the internal genes of A/WSN/33 H1N1. In addition, another 6 + 2 reassortant virus containing modified HA from H5N1 (clade 2.3.2.1), the NA from H9N2 and the internal genes of A/WSN/33 H1N1 was also rescued. The 5 + 3 reassortant H5N2 virus could grow to a higher titer in both MDCK cells and chicken eggs compared to the 6 + 2 reassortant H5N2 virus. The vaccine containing the inactivated 5 + 3 reassortant H5N2 virus was used in a two-dose immunization regime which protected specific pathogen free (SPF) chickens against two repeated challenges with homologous 2.3.2.1 clade and heterologous 2.2 clade HPAI H5N1 viruses. The 5 + 3 reassortant H5N2 virus based on clade 2.3.2.1 generated in this study can be effective in protecting chickens in the case of an outbreak caused by antigenically different clade 2.2 HPAI H5N1 viruses and opens the way to explore its applicability as potential vaccine candidate especially in the Asian countries reporting these clades frequently. The study also indicates that sequential immunization can broaden protection level against antigenically diverse strains of H5N1 viruses.