Lumpy skin disease (LSD) remains a major threat to Indian cattle, causing significant economic and production losses. After a widespread outbreak in 2022, LSD incidence declined in 2023–2024, but by mid-2025, new outbreaks emerged across several states following the monsoon season, highlighting the continued vulnerability of Indian cattle. Since 2021-22, India has implemented annual mass vaccination with the heterologous goatpox vaccine for LSD control; however, frequent reports of LSD cases prompted an evaluation of its field effectiveness. Field investigations were conducted in October 2025 in Rajasthan across 18 farms in five districts, covering 8930 cattle. Overall, the Uttarkashi strain–based goatpox vaccine demonstrated low field effectiveness (21.7

Currently available SARS-CoV-2 neutralisation tests continue to face trade-offs between rapid turnaround and large-scale applicability. They either fail to capture the complete spectrum of functional neutralizing antibodies, or difficult to scale up for large-cohort studies. Therefore, we present a live-virus immunocolorimetric neutralization test (LVINT) that overcomes these limitations. A total of 403 human sera collected during the year 2021 to 2025 were employed for evaluation of diagnostic performance of LVINT against surrogate virus neutralization test (sVNT) and Plaque reduction neutralization test (PRNT50) for screening of neutralizing antibodies (nABs). We also evaluated independent clinical utility of LVINT to assess nABs against Wuhan-like, Delta and predominantly circulating, XFG variants. Precision and analytical sensitivity were also measured. The LVINT exhibited a strong correlation with both the sVNT (rS = 1.000, p < 0.0001), and PRNT50 (rS = 0.910, p < 0.0001). Moreover, it showed a perfect diagnostic concordance with PRNT50 (weighted kappa = 0.90650, SE = 0.06, 95 %CI = 0.779 to 1.000), validating the use of intracellular nucleocapsid protein quantification for determining the neutralizing capacity of antibodies. The clinical utility of LVINT was further evaluated against Delta and XFG variants, revealing a declining trend of nABs titers against Delta (56.14 %) and XFG (47.36 %) variants compared with Wuhan-like variant (80.59 %). These findings prove the clinical utility of LVINT for measuring nABs against both existing and newly emerging SARS-CoV-2 variants. Collectively, the LVINT offers a rapid and scalable alternative to currently available neutralization tests.

Orthoflavivirus are single-stranded positive-sense RNA viruses that pose a significant threat to human and animal health globally, accounting for more than 400 million infections annually. They are predominantly transmitted by arthropods and cause a plethora of clinical manifestations, ranging from mild self-limiting infections to lethal encephalitis, with the potential to cause epidemics and pandemics. The complication in effectively treating Orthoflavivirus infections stems from the intricate interactions between virus and host proteins. A prominent hallmark of Orthoflavivirus infection is the use of sophisticated strategies to cleverly evade and dampen the host's innate immune response, which is a vital defence mechanism against viral invasion. This review provides substantial information on the mechanism of virus hijacking key components of the host immune response, featuring physical interactions between viral and host protein components, in the light of the innate immune system. Understanding and targeting these complex interactions would be instrumental in discovering novel antivirals against these viruses.

We generated a recombinant lumpy skin disease virus (LSDV) using the Ranchi strain as backbone by targeted insertion of an enhanced green fluorescent protein (EGFP) expression cassette into the ORF130 locus. A pET28-derived donor plasmid carrying EGFP flanked by ORF130 homology arms was assembled by Gibson cloning and transfected into LSDV-infected Vero cells to mediate homology-directed recombination. Recombinant virus (LSDV-GFP) was isolated by fluorescence-guided cell sorting. Conventional PCR, quantitative RT-PCR, and whole-genome sequencing confirmed precise, single-copy integration of EGFP at the intended locus with no detectable off-target alterations. Serial passaging demonstrated stable EGFP expression and complete elimination of parental virus, indicating genetic stability and purity. A fluorescence-based neutralization assay using LSDV-GFP detected anti-LSDV neutralizing antibodies with accuracy comparable to the standard virus neutralization test while reducing assay turnaround from 72 to 12 hours. This study establishes an in-house Ranchi strain-based LSDV reporter system that provides a genetically stable, rapid, and reliable platform for the detection and quantification of LSDV-neutralizing antibodies. In addition, it identifies ORF130 as a suitable locus for heterologous gene insertion and support LSDV-GFP as a potential versatile platform for viral vector development.

Emerging evidence highlights the role of epigenetic modification in virus infection. In this study, inhibition of H3K27-methylation (H3K27-me3) by UNC1999 (H3K27-methyltransferase inhibitor) was demonstrated to inhibit SARS-CoV-2 replication, as evidenced by reduced levels of viral RNA/protein. The m6A modifications of SARS-CoV-2 RNA were predominantly present on the 3' end, particularly the "N" gene. The methylated RNA immunoprecipitation (MeRIP) and western-blot analysis revealed a negative correlation between levels of cellular H3K27-me3 and m6A-modifications on the SARS-CoV-2 "N" gene. Moreover, m6A-modifications of the SARS-CoV-2 "N" gene were shown to promote the recruitment of YTHDF2, which eventually resulted in decay of the viral transcripts. The application of the H3K27-demethyltransferase or KDM6A/B inhibitor GSK-J4 can restore H3K27-me3 levels and mitigating the decay of viral mRNA in UNC1999-treated SARS-CoV-2-infected cells. Furthermore, long-term sequential passage (P = 50) of the virus in the presence of UNC1999 did not yield any UNC1999-resistant SARS-CoV-2 mutants. In conclusion, by integrating transcriptomics, molecular virology and functional analyses, we for the first time demonstrated that inhibition of H3K27-me3 induces m6A-mediated decay of SARS-CoV-2 transcripts, highlighting UNC1999 as novel antiviral candidate against SARS-CoV-2.

Excessive growth of granulation tissue (EGT) is a common complication during wound healing in horses. Wounds involving lower limbs are especially prone to the development of EGT. Often increased expression of transforming growth factor-beta (TGF-beta) is found in the EGT. In the present report, resveratrol was used as TGF-beta modifier. Topical application of an aqueous solution of resveratrol at 0.01 mg/mL once daily for 5-7 weeks depending on EGT size, successfully suppressed the EGT growth in all seven horses in the present case report.

In January 2025, the highly pathogenic avian influenza A(H5N1) virus clade 2.3.2.1a infection was detected in domestic cats and whole-genome sequencing of two cat H5N1 isolates was performed using the Oxford Nanopore MinION sequencing platform. Phylogenetic analysis revealed the circulation of triple reassortant viruses in cats. Although cat viruses lacked classic mammalian adaptation markers they carried mutations associated with enhanced polymerase activity in mammalian cells and increased affinity for α2-6 sialic acid receptor suggesting their potential role in facilitating infection in cats. The identification of reassortant HPAI H5N1 clade 2.3.2.1a viruses in domestic cats in India highlights the urgent need for enhanced surveillance in domestic poultry, wild birds, and mammals, including humans, to track genomic diversity and molecular evolution of circulating strains. ### Competing Interest Statement The authors have declared no competing interest.

Newcastle disease (ND) is a highly pathogenic infectious disease of poultry, causing significant economic losses globally. Wild birds act as natural reservoir of Newcastle disease virus (NDV) and spread the virus to domestic poultry. Frequent exposure of backyard poultry to wild birds andor their excretions increases the risk of acquiring infection from wild birds. In the present study, tracheal andor cloacal swabs were collected from a total of 242 apparently healthy chicken and ducks from the backyards. The samples were tested for the presence of NDV using matrix (M) gene based quantitative real time polymerase chain reaction (RT-qPCR), where all the samples were found negative. Virus isolation was attempted using specific pathogen free- embryonated chicken eggs (SPF-ECEs). Out of 242 samples, 22 samples (9.09) showed haemagglutination (HA) across different passages. Genome of NDV could not be amplified in any of these HA-positive samples by using M gene-based RT-qPCR, and L- and F- genes based conventional reverse transcription polymerase chain reaction (RT-PCR). This indicates the presence of haemagglutinating agent other than NDV in the samples. Further studies are needed to identify the haemagglutinating agent detected in this study. All the samples in the present study were negative for NDV which might be due to absence of active infection during sample collection and vaccination status of the birds.

SUMMARY:The wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may provide information on the potential transmission of COVID-19 infection when mass clinical testing declines. In the present study, a total of 1110 wastewater samples were collected from Hisar, Haryana (India), from August 2022 to June 2023. The SARS-CoV-2 detection in the wastewater correlated with clinical cases of COVID-19 in Hisar, suggesting that wastewater-based epidemiology can potentially complement classical testing of COVID-19 and may serve as an early warning system to prevent potential disease transmission and consequential outbreaks.

The coronavirus disease 2019 (COVID-19) pandemic highlighted the critical need for broad-spectrum antivirals with high resistance barriers. Here, we demonstrate that SB431542, a selective TGF-β receptor I (ALK5) inhibitor, exhibits potent antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through unprecedented multitargeted mechanisms. Through comprehensive in vitro, isothermal titration calorimetry, and in silico analyses, we identified that SB431542 directly binds to SARS-CoV-2 ORF3a and disrupts its canonical function in inhibiting autophagosome-lysosome fusion. This interaction restored lysosomal acidification and normalized perinuclear LAMP-1 localization, significantly impairing virion assembly as evidenced by disrupted nucleocapsid-RNA association and reduced intracellular viral titers. Additionally, SB431542 downregulated the CLEAR network genes responsible for lysosomal biogenesis, further restricting viral egress pathways. Our temporal analyses revealed that at later infection stages (36-48 hours post-infection [hpi]), SARS-CoV-2 exploits TGF-β-induced lysosomal membrane permeabilization (LMP) and apoptosis for viral release-processes effectively inhibited by SB431542 through suppression of GADD45b and BAX expression. These multiple mechanisms resulted in an exceptional EC50 of 751.8 nM against SARS-CoV-2. In vivo efficacy was demonstrated in embryonated chicken eggs, where SB431542 conferred dose-dependent protection against lethal infectious bronchitis virus (IBV) challenge, with a favorable therapeutic index of 34.54. Remarkably, sequential passaging of SARS-CoV-2 for 50 generations under SB431542 selection pressure failed to generate resistant variants, contrasting sharply with the rapid resistance emergence typical of direct-acting antivirals. These findings establish SB431542 as a promising broad-spectrum coronavirus inhibitor with a unique triple-mechanism approach that simultaneously targets viral entry via TGF-β/Smad modulation, disrupts ORF3a-mediated lysosomal dysfunction affecting assembly, and attenuates TGF-β-induced apoptosis during late-stage infection, collectively imposing multiple selective constraints that impede escape mutation development. IMPORTANCE:The COVID-19 pandemic highlighted the urgent need for antiviral drugs with high barriers to resistance. This study reveals that SB431542, a drug previously developed to inhibit TGF-β signaling, exhibits remarkable effectiveness against SARS-CoV-2 through an unprecedented triple-mechanism approach. Unlike conventional antivirals that target a single viral component, SB431542 simultaneously disrupts viral entry, assembly, and release by binding to the viral ORF3a protein and modulating host cellular processes. Most importantly, SARS-CoV-2 failed to develop resistance against SB431542 even after 50 generations of exposure-a significant advantage over current therapeutics that quickly lose effectiveness due to viral mutations. Our findings also uncover that coronaviruses exploit both lysosomal dysfunction and programmed cell death to spread efficiently, providing new targets for therapeutic intervention. This research establishes SB431542 as a promising broad-spectrum coronavirus inhibitor and demonstrates the value of targeting host-virus interactions to overcome antiviral resistance.

Mass vaccination to achieve disease-free status requires a serological test to differentiate infected and vaccinated animals (DIVA) towards the end of the control program. The lumpy skin disease (LSD) vaccines currently in use (licensed) are not DIVA-compatible. India recently approved a new live-attenuated LSD vaccine derived from the local LSD virus (LSDV) strain (Ranchi). Unlike field LSDV strains, the Ranchi strain has a distinct 801-nucleotide deletion in its inverted terminal repeat (ITR) region, affecting ORF003/ORF154. In this study, we successfully cloned and expressed LSDV ORF154 into pET28a and purified the His-tagged protein using Ni-NTA affinity chromatography. SDS-PAGE and Western blot analyses confirmed the presence of a ∼28 kDa protein, consistent with the predicted molecular weight. An optimized antigen concentration of 500 ng/well and serum dilution of 1:50, and at a positivity cut-off of 22.63 %, the assay showed high sensitivity (95.77 %) and specificity (95.42 %), effectively distinguishing infected from vaccinated cattle. These findings demonstrate the potential of an ORF154-based ELISA as a reliable serological diagnostic tool for LSDV surveillance and disease control programs, making the Ranchi strain-based LSDV vaccine the first DIVA-compatible vaccine.

Despite the recognized significance of intrinsically disordered proteins (IDPs) in cellular processes and diseases, the landscape of intrinsic disorder within bat proteomes remains unexplored. Here, we systematically analyzed the prevalence and functional implications of IDPs across the complete proteomes of fourteen bat species and compared these to the human proteome. We first assessed ten widely used IDP predictors using a dataset of experimentally resolved disordered regions, identifying fIDPnn as the most suitable among the predictors benchmarked here for large-scale analyses of mammalian proteins. The results showed that the bat proteomes exhibit significantly lower IDP content (ranging from 12.6 % to 18.6 %) compared to humans (32.8 %). Correlation analyses revealed an inverse relationship between protein size and disorder content across both bats and humans. By integrating functional annotations with disorder contents, we found that intrinsic disorder enrichment or depletion in bat proteins aids in the species-specific immune gene regulation to coexist with a diverse array of pathogens. Our findings provide the first comprehensive map of intrinsic disorder in bat proteomes, highlighting distinct evolutionary and functional patterns relative to humans, and offer valuable insights into the molecular underpinnings of bat biology and their role as viral reservoirs.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne flavivirus endemic to the Western Ghats region of India, with increasing reports of geographic expansion. This study employs phylogenetic analysis and spatial diffusion modeling to understand the evolutionary dynamics and transmission patterns of KFDV. Whole genome and E-gene sequences were analysed to identify major phylogenetic clusters, transmission velocity, and environmental factors influencing viral spread. The analysis revealed two primary phylogenetic clusters: Cluster A, originating in Karnataka, and linked to initial outbreaks (1957-1972) and subsequent re-emergence post-2010 in Karnataka, Kerala, and Goa; and Cluster B, which expanded from Maharashtra in the late 1970s into Tamil Nadu, Karnataka, and Kerala. Phylogenetic findings indicated a slow mutation rate, indicative of long-term viral persistence in sylvatic reservoirs rather than sustained human-human transmission. Spatial diffusion analysis estimated a median transmission velocity of 59.67 km/year. Environmental factors such as deforestation, land cover change, and livestock density acted as facilitators of viral spread, while urbanization, open water bodies, and precipitation served as resistance factors. The findings underscore the need for enhanced surveillance, ecological monitoring, and public health interventions to mitigate the increasing risk of KFD outbreaks. This study provides a comprehensive framework for understanding KFDV transmission and evolution, integrating phylogenetic and ecological data to improve risk assessment and guide control strategies in both endemic and emerging regions.

In recent times, the understanding of the human microbiome and its impact on health and disease has undergone a paradigm shift, leading to ground-breaking discoveries in the field of probiotics. Probiotics, live microorganisms known for conferring health benefits when administered adequately, have garnered significant interest for their potential to modulate the immune system's response to viral infections in both humans and animals. The emergence of enteric and respiratory viruses as significant global health threats has prompted intensive research efforts to identify novel therapeutic strategies. Traditional antiviral therapies often face challenges such as drug resistance, limited efficacy and adverse effects, underscoring the urgent need for alternative approaches. In this context, probiotics have emerged as a promising avenue for the prevention and treatment of viral infections due to their ability to modulate the host immune response, enhance mucosal barrier function and exert direct antiviral effects. This review aims to provide a comprehensive overview of the therapeutic landscape of probiotics against enteric and respiratory viruses. Based on latest findings from preclinical and clinical studies, we have explored the mechanisms underlying the antiviral activity of probiotics and their potential role in mitigating viral infections. Furthermore, promising avenues for harnessing probiotics as adjunctive or standalone interventions against enteric and respiratory viral infections have been discussed here.

Lumpy skin disease (LSD), which was confined to the Africa for many decades, has expanded its geographical distribution to numerous countries across Asia and Europe in recent years. The LSD virus (LSDV) is a relatively poorly studied virus. Its 151 Kb genome encodes 156 open reading frames (ORF); however, the exact number of the proteins encoded by the viral genome and their specific functions remain largely unknown. Arthropod vectors primarily transmit the LSDV mechanically, but the precise nature of these vectors in different regions and their role in transmission is not fully understood. Homologous live-attenuated vaccines prepared using LSDV have proven to be highly efficacious compared to heterologous vaccines based on sheep pox virus or goatpox virus, in protecting cattle against LSD. This review offers the latest insights into the molecular biology and transmission of LSDV and discusses the safety and efficacy of available vaccines, along with the challenges faced in controlling and eradicating the disease in endemic regions.

Hesperetin, a natural flavonoid, has been investigated for its potential therapeutic properties. In this study, we delve into its antimicrobial and antiviral activities against clinically relevant bacterial and fungal strains. Our investigation unveiled substantial antimicrobial and antioxidant properties of hesperetin against a diverse array of pathogens, encompassing both Gram-positive as well as Gram-negative bacteria. Furthermore, notable antifungal activities were observed, particularly against resistant fungal strains. The findings from our study underscore the potential of hesperetin as a promising candidate for the development of broad-spectrum antimicrobial agents. Overall, hesperetin exhibits versatility with implications for combating infectious diseases. These insights pave the way for further exploration of hesperetin’s therapeutic applications and its potential utility in addressing the challenges posed by antimicrobial resistance.

Poxviruses encode two highly conserved S-adenosylmethionine (SAM)-dependent methyltransferases for mRNA capping. While D1L facilitates translation via cap-0 formation, the role of VP39-mediated cap-1 modification has been canonically restricted to shield viral mRNA from cytoplasmic pattern-recognition receptors (PRRs). Here, we uncover a novel function of VP39 by demonstrating that prolonged exposure to the SAM cycle inhibitor DZNep selected a virus escape mutant harbouring a point mutation (M236I) in VP39. VP39\_mut bypasses S-adenosylhomocysteine (SAH)-mediated feedback inhibition, enhancing 2'-O-methylation of viral transcripts and promoting their association with active translation machinery. Consequently, VP39\_mut significantly improves viral protein translation, replicative fitness, and yields across species. Notably, VP39\_mut-capped in vitro transcribed (IVT) mRNA exhibited ~2.7-fold higher protein expression compared to VP39\_wt, the standard tool for IVT applications. These findings reveal a unique resistance mechanism and provide critical insights into poxvirus mRNA biology while underscoring the potential of optimized VP39 for advancing mRNA-based therapeutics and vaccine production. ### Competing Interest Statement N.K. and A.V. are inventors of patent application related to this work. The remaining authors declare no competing interests.