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The cross-species transmissibility of SARS-CoV-2 infection has necessitated development of specific reagents for detecting infection in various animal species. The spike glycoprotein of SARS-CoV-2, which is involved in viral entry, is a highly immunogenic protein. To develop assays targeting this protein, we generated eight monoclonal antibodies (mAbs) against the S1 and seven against the S1/S2 protein (ectodomain) of SARS CoV-2. Based on neutralization capability and reactivity profile observed in ELISA, the mAbs generated against the S1/S2 antigen exhibited a broader spectrum of epitope specificity than those produced against the S1 domain alone. The full-length ectodomain induced antibodies that could neutralize the two most important variants of the virus encountered during the pandemic, namely Delta and Omicron. The availability of these reagents could greatly enhance the development of precise diagnostics for detecting COVID-19 infections in various host species and contribute to the advancement of mAb-based therapeutics.

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Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening.

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Lumpy skin disease (LSD), caused by the lumpy skin disease virus of the genus Capripoxvirus, is rapidly emerging across most countries in Asia. Recently, LSD has been linked to very high morbidity and mortality rates. Until 2019, India remained free of LSD, resulting in a lack of locally developed diagnostic kits, biologicals, and other tools necessary for managing the disease in a country with such a large livestock population. Therefore, this study aimed to design and validate an indigenous and cost-effective in-house ELISA for large-scale screening of cattle samples for antibodies to LSDV. The viral major open reading frames ORF 095 and ORF 103 encoding virion core proteins were expressed in a prokaryotic system and the recombinant antigen cocktail was used for optimization and validation of an indirect ELISA (iELISA). The calculated relative diagnostic sensitivity and diagnostic specificity of the iELISA were 96.6 % and 95.1 %, respectively at the cut-off percent positivity (PP≥50 %). The in-house designed double-antigen iELISA was found effective to investigate the seroprevalence of LSDV in various geographical regions of India.

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Elephant endotheliotropic herpesviruses (EEHV) belong to the family Herpesviridae and cause a highly fatal hemorrhagic infection in elephants. EEHV poses a global threat to the already endangered elephant population. Since EEHV is a non-cultivable virus, there is a scarcity of specific diagnostics, therapeutics, and vaccines. In this study, our objective was to develop biologicals for diagnosis and pathological studies against the most prevalent EEHV1A/1B. We expressed two truncated fragments of the DNA polymerase, glycoprotein B (gB), and glycoprotein (gL) of EEHV in the prokaryotic system. Hyperimmune serum against the purified antigens was raised in rabbits and guinea pigs. We validated the reactivity of this hyperimmune serum using western blotting, ELISA, and immune-histochemistry on known positive infected tissues. Samples collected from 270 animals across various states in India were evaluated with these biologicals. The raised antibodies successfully demonstrated virus in immune-cytochemistry. Additionally, all known positive samples consistently exhibited significant inhibition in the OD values when used in the competitive format of ELISA across all four antigens when compared to the serum collected from known negative animals. An apparent sero-prevalence of 10 % was observed in the randomly collected samples. In summary, our study successfully developed and validated biologicals that will be invaluable for EEHV diagnosis and control.

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The present study focuses on the pathological and molecular characterization of African swine fever virus (ASFV) associated with an outbreak in wild boars in two national parks in southern India in 2022-2023. Significant mortality was observed among free-ranging wild boars at Bandipur National Park, Karnataka, and Mudumalai National Park, Tamil Nadu. Extensive combing operations were undertaken in both national parks, spanning an area of around 100 km2, originating from the reported epicenter, to estimate the mortality rate. Recovered carcasses were pathologically examined, and ASFV isolates was genetically characterized. Our findings suggested spillover infection of ASFV from nearby domestic pigs, and the virus was equally pathogenic in wild boars and domestic pigs. ASFV intrusion was reported in the Northeastern region of the country, which borders China and Myanmar, whereas the current outbreak is very distantly located, in southern India. Molecular data will help in tracing the spread of the virus in the country.

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Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

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The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50 ) values ranging from 1.42 to 32.7 µM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half-maximum effective concentration (EC50 ) values of 21.73 and 31.19 µM, respectively. The anti-inflammatory roles of azadirachtin and withanolide_A when assessed using HEK293T cells, were found to significantly reduce the levels of CXCL10, TNFα, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-α1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2-specific enzymes and also host immune pathways involved in virus-mediated inflammation.

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Bovine herpes virus-1 (BoHV-1) is responsible for production losses through decreased milk yields, abortions, infertility, and trade restrictions in the bovine population. The disease is endemic in many countries including India. As the virus harbors a unique feature of latency animals once infected with the virus remain sero-positive for lifetime and can re-excrete the virus when exposed to stressful conditions. Hence, identification and culling of infected animals is only the means to minimize infection-associated losses. In this study, an economical indigenous assay for the detection of BoHV-1 specific antibodies was developed to cater to the huge bovine population of the country. The viral structural gD protein, expressed in the prokaryotic system was used for optimization of an indirect ELISA for bovines followed by statistical validation of the assay. The diagnostic sensitivity and specificity of the indirect ELISA were 82.9% and 91.3% respectively. Systematically collected serum samples representing organized, unorganized and breeding farms of India were tested with the indigenously developed assay for further validation.

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The nucleocapsid (N) protein of SARS-CoV-2 plays a pivotal role in encapsulating the viral genome. Developing antiviral treatments for SARS-CoV-2 is imperative due to the diminishing immunity of the available vaccines. This study targets the RNA-binding site located in the N-terminal domain (NTD) of the N-protein to identify the potential antiviral molecules against SARS-CoV-2. A structure-based repurposing approach identified the twelve high-affinity molecules from FDA-approved drugs, natural products, and the LOPAC1280 compound libraries that precisely bind to the RNA binding site within the NTD. The interaction of these potential antiviral agents with the purified NTD protein was thermodynamically characterized using isothermal titration calorimetry (ITC). A fluorescence-based plate assay to assess the RNA binding inhibitory activity of small molecules against the NTD has been employed, and the selected compounds exhibited significant RNA binding inhibition with calculated IC50 values ranging from 8.8 µM to 15.7 µM. Furthermore, the antiviral efficacy of these compounds was evaluated using in vitro cell-based assays targeting the replication of SARS-CoV-2. Remarkably, two compounds, Telmisartan and BMS-189453, displayed potential antiviral activity against SARS-CoV-2, with EC50 values of approximately 1.02 µM and 0.98 µM, and a notable selective index of >98 and > 102, respectively. This study gives valuable insight into developing therapeutic interventions against SARS-CoV-2 by targeting the N-protein, a significant effort given the global public health concern posed due to the virus re-emergence and long COVID-19 disease.

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We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of seropositivity was positively correlated with that of the incidence in humans, suggesting the occurrence of large spillover events.

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Lumpy skin disease (LSD) is a highly infectious and economically important viral disease, which is currently emerging in the Indian subcontinent. LSD is caused by Lumpy Skin Disease Virus (LSDV) under the genus Capripoxvirus and the family Poxviridae. Since its first incursion in India in the year 2019, the virus is rapidly disseminating through different means like direct contact, fomites and mainly by blood-feeding insects. As the disease has never been reported from India or neighbouring countries, there is a lack of planning and preparatory measures in terms of diagnostics and vaccines to control the disease. In the absence of any homologous vaccine, a live attenuated heterologous goat pox vaccine (Uttarkashi strain) is now being widely used in the country for the prevention of LSDV infection. Use of live attenuated goat pox virus vaccine necessitates the availability of an assay which could specifically detect and differentiate LSDV from goat pox virus. In this study, nucleotide sequences of LSDV126 gene encoding extracellular enveloped virus protein of circulating LSDV and goat pox virus were determined and analyzed. Deletion of 27 nt tandem repeats was observed in LSDV in comparison to goat pox and LSDV vaccine viruses. The deletion region was targeted for designing primers specific to LSDV, but not goat pox virus. A novel isothermal polymerase spiral reaction (PSR) was optimized as pen side diagnostic for prompt and sensitive detection of genomic DNA of LSDV. The assay was found to be highly sensitive and specific when compared to the real-time PCR. The assay was found to be specifically detecting only LSDV but not the goat pox virus. The limit of detection was identified as 9 × 10-6 ng of positive DNA. The assay will provide a point of care tool that will be a boon for the successful control of LSD in India.

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We report an incidence of natural infection of SARS-CoV-2 in free-ranging Indian leopard (Panthera pardus fusca). The case was detected during routine screening. Post-mortem and laboratory examination suggested virus-induced interstitial pneumonia. Viral genome could be detected in various organs including brain, lung, spleen, and lymph nodes by real-time PCR. Whole-genome sequence analysis confirmed infection of Pango lineage B.1.617.2 of SARS-CoV-2. Till now, only Asiatic lions have been reported to be infected by SARS-CoV-2 in India. Infections in animals were detected during peak phase of pandemic and all the cases were captive with close contacts with humans, whereas the present case was observed when human cases were significantly low. No tangible evidence linked to widespread infection in the wild population and the incidence seems to be isolated case. High nucleotide sequence homology with prevailing viruses in humans suggested spillover infection to the animal. This report underlines the need for intensive screening of wild animals for keeping track of the virus evolution and development of carrier status of SARS-CoV-2 among wildlife species. Supplementary Information: The online version contains supplementary material available at 10.1007/s10344-022-01608-4.

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Molecular characterization of Indian isolates of duck enteritis virus (DEV) so far has been limited to a few selected genomic regions. Here, we report the complete genome sequence of an isolate, DEV/India/IVRI-2016, from southern India that is 158,091 bp in length.

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This paper investigates analytically the effect of dissimilarity of mass flow rate [Formula: see text] and number of collectors (N) on exergo-enviro-economic parameters for solar still of single slope type integrated with N similar photovoltaic thermal flat plate collectors having series connection (NPVTFPC-SS) keeping water depth as 0.14 m. All four kinds of weather conditions for New Delhi have been taken for the computation of different parameters. All relevant equations obtained using energy balance equations for all components of the system have been fed to a computer code inscribed in MATLAB-2015a for computing different parameters. The computation of different relevant parameters has been performed for various values of [Formula: see text] and N while keeping water depth as constant to know the effect of variation of [Formula: see text] and N on exergo-enviro-economic parameters for NPVTFPC-SS. It has been concluded that the value of carbon credit earned, enviroeconomic and exegoeconomic parameters, and productivity diminishes with the enhancement in [Formula: see text] at given N. The optimum value of N for given value of [Formula: see text] has been found to be 10 from exergoeconomic parameter viewpoint and 6 from productivity viewpoint.

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Canine parvovirus-2 (CPV-2) is ubiquitously distributed in dog population worldwide causing a severe and often fatal gastroenteritis. Owing to its highly contagious nature, rapid detection of CPV is crucial in effective control of the disease. Aptamers have emerged as potential alternative to antibodies as affinity reagents in diagnostic field. Present study was aimed to select and validate ssDNA aptamers specific to CPV. Systematic evolution of ligands through exponential enrichment (SELEX) method was employed for selection of CPV structural protein (VP2) specific DNA aptamers. SELEX was performed using a pool of ssDNA library comprising 30 random nucleotide region. A total of seven rounds of SELEX were performed using VP2 protein as target antigen which yielded ten aptamers (1A-10A) with distinct sequences. Target binding of all ten aptamers was assessed by dot blot and enzyme-linked oligonucleotide assay (ELONA) which revealed that 5A, 6A, 9A, and 10A were superior binders. In silico analysis of the aptamers revealed the existence of binding site on VP2 protein, and binding pattern was similar to in vitro findings. The affinity (KD) of all these four binders against CPV was evaluated by ELONA indicating relatively higher affinity of 6A and 10A than remaining two DNA sequences. Out of which, aptamer 6A displayed cross-reactivity with canine distemper virus and canine corona virus. Hence, aptamer 10A was considered as better binding sequence having high specificity and affinity for CPV. The study confirms the future utility of selected aptamers in development of a reliable diagnostic for rapid detection of CPV. KEY POINTS: • Canine parvovirus-specific ssDNA aptamers were identified with nanomolar affinity (100-150 nM). • Three aptamers displayed negligible cross-reactivity with other related viruses. • Aptamer 10A displayed high binding affinity and specificity to CPV.

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The foot-and-mouth disease virus (FMDV) causes a highly infectious disease of all cloven-footed animals. The RNA genome of the virus continuously evolves, leading to the generation of new strains; this necessitates the selection of new vaccine strains to ensure complete protection. Infection with one FMDV serotype does not provide cross-protection against the other FMDV serotypes. Many of the recovered animals may become carriers of the FMDV, but they still remain susceptible to the other serotypes. Coinfection with multiple FMDV serotypes has been reported and studied to understand the virus evolution. Isolation and characterization of all the involved serotypes in the mixed infection case is essential to understand the molecular evolution of the virus. In this study, two cases of coinfection were studied by selective isolation of each of the FMDV serotypes under the cross-serotype-specific immune pressure. It was estimated that the virus present in a minimum of 10-0.92 TCID50 could be isolated from the mixed population containing other serotypes in infective doses of 100.25 TCID50 or less. All involved serotypes present in the mixed infection cases were isolated, without any cross-contamination. Virus characterization revealed that genotype 2 was of serotype A virus from a sample collected in 1995, which was last reported in 1986, indicating a possible subdued prevalence of the genetic group even after vanishing from the field.

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Salmonella enterica serovar Gallinarum is a host-restricted bacterial pathogen that causes a serious systemic disease exclusively in birds of all ages. Salmonella enterica serovar Typhimurium is a host-generalist serovar. Dendritic cells (DCs) are key antigen-presenting cells that play an important part in Salmonella host-restriction. We evaluated the differential response of chicken blood monocyte-derived dendritic cells (chMoDCs) exposed to S. Gallinarum or S. Typhimurium. S. Typhimurium was found to be more invasive while S. Gallinarum was more cytotoxic at the early phase of infection and later showed higher resistance against chMoDCs killing. S. Typhimurium promoted relatively higher upregulation of costimulatory and other immune function genes on chMoDCs in comparison to S. Gallinarum during early phase of infection (6 h) as analyzed by real-time PCR. Both Salmonella serovars strongly upregulated the proinflammatory transcripts, however, quantum was relatively narrower with S. Gallinarum. S. Typhimurium-infected chMoDCs promoted relatively higher proliferation of naïve T-cells in comparison to S. Gallinarum as assessed by mixed lymphocyte reaction. Our findings indicated that host restriction of S. Gallinarum to chicken is linked with its profound ability to interfere the DCs function. Present findings provide a valuable roadmap for future work aimed at improved vaccine strategies against this pathogen.

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Coronavirus disease 2019 (COVID-19) has turned out as one of the worst medical and economic misfortunes across the globe. The etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the Coronaviridae family and represents a disease manifestation from asymptomatic to severe respiratory damage. High transmissibility and contagious nature of the virus helps it to flourish in a large population. The immune system aids to retain the virus, but with accelerated cytokine secretion, it could transform into double edge sword resulting in unrestrained systemic inflammation which might become life-threatening. SARS-CoV-2 sets substantial impact on T-lymphocytes during its course of infection. The number of CD4+ T, CD8+ T, and Treg cells tend to decrease profoundly in case of severe illness. Besides, the virus modulates the CD4+ T/ CD8+ T and Treg/Th17 cells ratio and induces the functional exhaustion of T cells to make them inefficient. T cells define the pathogenesis of severe cases and provide major contributions in antiviral defense. Therefore, the apprehension of T-lymphocytes in SARS-CoV-2 infection would implicate in developing antivirals, disease control, and would broaden the way for vaccine formulation. Thus, the review depicts the significance of T-lymphocytes interaction with SARS-CoV-2. Keywords: SARS-CoV-2; COVID-19; T-lymphocytes; cytokine; inflammation; immune response.

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Bovine tuberculosis is an economically important disease with very high zoonotic potential. Single intradermal cervical tuberculin test (SICT) is considered a gold standard assay for the diagnosis of bovine tuberculosis. However, bovines especially buffaloes may produce a false negative result when the animal becomes cell-mediated immune (CMI) anergic in the advanced stage of the disease. In the present study, ELISA and PCR assays were successfully demonstrated to be useful in diagnosing tuberculosis especially in the CMI anergic buffaloes infected with Mycobacterium bovis. ELISA and PCR assays are able to detect 8.94% and 8.13%, respectively, more animals as positive in comparison to standard SICT assay in a selected population of 123 buffaloes. The moderate agreement between SICT and ELISA (k: 0.528; 0.249-0.807), a substantial agreement between SICT and PCR (k: 0.648; 0.364-0.931), and high agreement between ELISA and PCR (k: 0.856; 0.697-1.0) highlight that ELISA and PCR, if used in parallel with SICT, will provide better sensitivity over single assay. Reduction of false negative reactors may help in minimizing the zoonotic threat from bovine tuberculosis especially in disease endemic region where human and livestock interface is quite high.

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The 2C protein of foot-and-mouth disease virus (FMDV) is reported to play a critical role in the virus replication complex and modulating the host's immune response. However, the underlying molecular intricacies of subversion of cellular machinery remains poorly understood, thus emphasizing the need to study 2C-host interactions. In this study, we identified the host proteins interacting with the 2C using yeast-two hybrid (Y2H) approach, which is one of the most recognized, high-throughput tools to study protein-protein interactions. The FMDV-2C bait was characterized for auto-activation, toxicity, and expression and was found to be suitable for mating with cDNA library. On preliminary screening a total of 32 interacting host proteins were identified which were reduced to 22 on subsequent confirmation with alternative yeast based assays. Amongst these, NMI/2C interaction has been reported earlier by Wang et al. (2012) and remaining 21 are novel interactions. The Reactome analysis has revealed the role of the identified host proteins in cellular pathways exploited by 2C during FMDV replication. We also confirmed interaction of MARCH7, an E3 ubiquitin ligase with 2C using mammalian two-hybrid system and co-immunoprecipitation. This study leads to the identification of novel 2C interacting host proteins which enhance our understanding of 2C-host interface and may provide checkpoints for development of potential therapeutics against FMDV.

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