Search Results (96)

Bovine viral diarrhea (BVD) is a globally significant disease that adversely affects cattle health and productivity, including in India. It is caused by three bovine pestiviruses: bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, and HoBi-like pestivirus (HoBiPeV), which belong to the Pestivirus genus within the Flaviviridae family. Despite the prevalence of all three pestivirus species in India, no commercial vaccine based on the local circulating strain is currently available. This study evaluates the safety, immunogenicity, and protective efficacy of an inactivated whole-virus BVD vaccine, based on an Indian BVDV-1 strain. The virus was propagated in MDBK cells, inactivated using 3 mM binary ethylenimine (BEI) for 24 h at 37 °C, and formulated with Montanide ISA 61 VG (SEPPIC) in a 50:50 water-in-oil emulsion. Vaccine safety was confirmed in both guinea pigs and bovine calves, with no adverse effects observed. Immunogenicity testing in guinea pigs (n = 6) showed neutralizing antibody titres up to 9 log₂ (1/512). In calves aged 9–12 months (n = 3), the vaccine elicited strong humoral and cell-mediated immune responses, with mean neutralizing antibody titres against the homologous BVDV-1 strain reaching 14 log₂ (1/16,384). Neutralizing antibody levels remained detectable for up to 12 months post vaccination with sustained mean titres of 7 log₂ (1/128). Notably, titres reported to be adequate for fetal protection (≥9 log₂ or ≥1/512 were maintained for five months following vaccination. Challenge studies demonstrated complete protection of vaccinated calves against homologous BVDV-1 acute infection. In addition, the vaccine conferred partial cross-protection against heterologous strains including BVDV-2 and HoBiPeV. In a field trial involving 125 cattle, 74% of animals developed protective neutralizing titres (≥7 log₂ or ≥1/128), while 48% achieved titres reported to be adequate for fetal protection (9 log₂ or 1/512). Furthermore, 92% of vaccinated cattle maintained neutralizing antibody titres of at least 6 log₂ (≥1/64) for up to six months post-booster vaccination. A strong positive correlation was observed between guinea pig and bovine antibody responses (R² = 0.6809; p < 0.0001), indicating the potential of guinea pigs as a predictive model. Vaccine stability was confirmed for up to 8 months when stored at 4 °C, as demonstrated by the immunogenicity in guinea pigs. Collectively, these findings demonstrate that the locally developed inactivated BVDV-1 vaccine is safe, highly immunogenic, and capable of providing protective immunity against BVDV-1 infection, supporting its potential use in BVD control programs in India. Full article

Background: Lumpy skin disease (LSD) is caused by a Capripoxvirus. Live attenuated vaccines, which are commercially available, could be not safe because of the side effects. The aim of this study was the evaluation of the safety, immunogenicity, and effects on the qualitative and quantitative parameters of milk. The feasibility of identifying vaccinated animals using our inactivated vaccine in dairy cows was analysed. The vaccine was tested in guinea pigs as an immunogenicity predictive model. Methods: LSD virus was propagated on Madin–Darby Bovine Kidney (MDBK) cells, then inactivated and supplemented with keyhole limpet hemocyanin (KLH) protein, obtaining a positive marker vaccine. This was inoculated in guinea pigs and in dairy cows, and animal sera were analysed using enzyme-linked immunosorbent assay (ELISA) and a serum neutralisation (SN) test. Quantitative and qualitative analyses were performed on milk. Results: The vaccine was previously tested for efficacy in vaccinated calves, showing a pronounced reduction in clinical symptoms after challenge. The safety and immunogenicity obtained in calves were also confirmed in dairy cows in this study. In fact, high values of the SN test (1:20 to 1:80) and ELISA (90 and 240 S/P%) were obtained after vaccination. Moreover, high immunogenicity of the vaccine was also assessed in guinea pigs. In addition, the results of the milk analyses did not show any differences between vaccinated and control groups. The KLH was able to elicit an immune response detectable using an ELISA (3.0 and 3.5 optical density values). Finally, our vaccine could be used to reduce LSD symptoms and identify vaccinated animals. Full article

During infection in vitro with the strain 438/06 of bovine coronavirus (BCoV), a β-coronavirus similar to severe acute respiratory syndrome (SARS) CoV-2, treatment with 6-pentyl-α-pyrone (6PP), a fungal metabolite obtained from Trichoderma atroviride, was recently shown to influence viral load by reducing viral entry. Herein, the ability of 6PP to counteract the BCoV infection was further investigated both in vitro and in silico. Following the BCoV (strain 282/23) infection in bovine (MDBK) cells, the 6PP in co-treatment increased cell viability, reduced morphological signs of cell death, and significantly inhibited viral yield, by lessening the expression of the viral spike (S) protein, as well as the gene transcription of the viral nucleocapsid (NP) protein. In addition, a noticeable down-regulation in the expression of aryl hydrocarbon receptor (AhR) signaling, a strategic modulator of CoVs infection, was found. Molecular docking studies were performed to evaluate the potential interaction between 6PP and AhR involved in the BCoV infection. The docking 3D structural model showed that 6PP fits into a binding pocket positioned between the PASB and TAD domains of bovine AhR (bAhR), where the ligand is stabilized through hydrophobic interactions. In addition, the obtained computational data strongly suggest that the bAhR binding mechanism of 6PP is principally mediated by a well-conserved hydrophobic cavity playing a key role in the modulation of the receptor functions. Overall, our findings showed an antiviral action of 6PP versus BCoV infection in vitro and in silico. Full article

Caprine herpesvirus 1 (CpHV-1) is responsible for significant economic losses in goat farming. The CpHV-1 genital infection in goats has been used as a homologous animal model for the study of human herpes simplex virus type 2 (HSV-2). This study aimed to investigate the in vitro virucidal and antiviral effect of lemon juice (LJ) and its main component, citric acid (CA), against CpHV-1 on Madin-Darby Bovine Kidney (MDBK) cells. Cytotoxicity was assessed using an XTT assay, while viral titers were determined by the Reed–Muench method and viral DNA was quantified via qPCR. Pure LJ (pH 2.3) and its corresponding CA solution demonstrated potent and rapid virucidal activity, reducing the viral titer by over 5.0 log10 TCID₅₀/50 µL within 1 min. When applied after viral entry, a non-cytotoxic dilution of LJ (pH 4.32) significantly inhibited viral replication, causing a 2.5 log10 TCID₅₀/50 µL reduction in viral titer and a corresponding decrease in viral DNA. The antiviral effects were minimal at a near-neutral pH of 6.67, probably interacting with envelope structures. These results suggest that LJ could be a potential low-cost topical agent or disinfectant for controlling CpHV-1 in goat populations and offer a basis for translational research on human herpesviruses. Full article

Mycotoxins are the most frequently occurring natural contaminant in food and feed products. Through the deployment of diverse agricultural strategies or biological, chemical, or physical treatments of crop products, mycotoxin contamination remains a persistent issue for the agricultural sector and food/feed industry. We previously suggested that halophytes, thanks to their high antioxidant activity, could protect animal cell lines from mycotoxin contamination. Here, a hydroalcoholic extract of Calystegia soldanella L. leaves was evaluated for in vitro total antioxidant capacity (TAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-quenching bioassays, as well as anti-inflammatory (ELISA measurement of IL-8 secretion), ROS-inhibiting production (CellROX Green assay), and calcium influx restoration (fluorescent probe Fura2-QBT assay) activities in two animal cells upon mycotoxin intoxication. C. soldanella extract displayed high antioxidant activities (DPPH IC₅₀ < 80 μg·mL⁻¹ and TAC of 90 mg AAE·g⁻¹ DW. Moreover, it exhibited a significant protective action on renal (MDBK) and intestinal (IPEC-J2) cells against zearalenone (ZEA) or T2-toxin contamination, restoring about 75% of cell viability (MTS bioassay) at 1 μg·mL⁻¹. This effect was accompanied by strong anti-inflammatory, ROS-inhibition, and membrane integrity restoration activities. A bio-guided study revealed that the fraction of C. soldanella extract eluted from C18-bound silica with 60% methanol was the most active one. Upon HPLC and 1D- and 2D-NMR analyses, major compounds identified in this fraction were flavonol-type flavonoids, including quercetin-3-O-glucose (X1), quercetin-3-O-rutinoside (X2), and quercetin-3-O-glucose-6″-acetate (X3). Enriched sub-fractions containing these compounds largely contributed to the cytoprotective effects of C. soldanella, supporting its potential use as a food/feed ingredient. Full article

Bovine Viral Diarrhea Virus (BVDV) poses a significant threat to the global cattle industry, causing substantial economic losses. Long non-coding RNAs (lncRNAs) play crucial regulatory roles in various biological processes, including viral infections. However, the specific lncRNAs influencing BVDV replication remain poorly characterized. This study identified lncSMIM14 as a key host factor upregulated during BVDV infection in MDBK cells. Functional analyses demonstrated that lncSMIM14 overexpression significantly enhanced BVDV replication, evidenced by increased viral mRNA levels, progeny virus titers, cytopathic effects, and dsRNA abundance, while its knockdown exerted the opposite effect. Mechanistically, we revealed that lncSMIM14 specifically targets and positively regulates the expression of the endocytosis-related GTPase Rab3a. Importantly, Rab3a itself was shown to be essential for efficient BVDV replication, as its overexpression promoted viral replication, and its knockdown inhibited it. Furthermore, Rab3a co-localized with key endocytic regulators Rab5a and Rab7a, and both lncSMIM14 overexpression and Rab3a overexpression promoted the formation of endocytic vesicles, particularly post-BVDV infection. Our findings unveil a novel mechanism wherein BVDV exploits the host lncRNA lncSMIM14 to hijack Rab3a-mediated endocytosis, facilitating its own replication. This study identifies the lncSMIM14-Rab3a axis as a critical host pathway subverted by BVDV, providing new potential targets for antiviral intervention. Full article

Background: Fetuin (Ft) is the most abundant protein in fetal bovine serum (FBS) and is considered one of its essential components. This acidic glycoprotein plays a key role in cell adhesion and proliferation and is vital for maintaining in vitro cultures of animal and human cells, tissues, and organs. FBS is a natural source for Ft purification. However, the high demand for FBS as a standard reagent in cell culture severely limits its availability for use as a raw material for protein purification. Furthermore, the industrial production of FBS results in a significant amount of contaminated FBS. This contaminated fraction can thus be utilized for Ft recovery. Methods: In this work, we present a novel method for Ft recovery from FBS using a single chromatographic step based on anion exchange chromatography under acidic conditions. Results: Optimal adsorption conditions for Ft were studied using response surface methodology (RSM), which suggested a buffer pH of 4.2 and an FBS dilution of 40%. However, increasing the pH to 5 resulted in a 28% increase in Ft recovery, although with a slight reduction in Ft purity to 88%. A scale-up to half a liter of FBS was performed using a 400 mL column. A single-step elution with 0.3 M NaCl was employed, yielding an Ft recovery of 90% with a purity greater than 82%. Conclusions: The purified Ft demonstrated biological activity as a growth promoter in MDBK cell culture when utilized in a serum-free culture medium. Full article

Mycoplasmopsis bovis is a significant pathogen causing substantial economic losses in cattle, yet its within-herd microevolution remains poorly understood. This study aimed to characterize phenotypic and genomic variations within a dominant ST-52 clone circulating in a closed dairy herd. We isolated M. bovis from respiratory (n = 11) and milk (n = 5) samples. Phenotypic characterization included biofilm formation, antimicrobial susceptibility testing, and cellular invasion assays. Whole-genome sequencing was performed on four representative isolates to identify genetic variations. All isolates were genetically identical according to MLST (ST-52). However, significant phenotypic diversity was observed. Biofilm formation capacity varied significantly (OD595 from 0.25 to 1.10), and resistance to doxycycline was higher in nose swabs (100%) than milk isolates (20%). Cellular invasion assays demonstrated that all isolates could invade bovine-derived cells (MDBK, MAC-T, EBL, and PBMC), but the invasion efficiency differed by strain and cell type. These findings confirm the circulation of a single genetic lineage within a closed herd while highlighting significant phenotypic diversification in biofilm formation, antibiotic resistance, and cellular invasiveness. The results provide evidence consistent with microevolution and underscore the adaptive potential of M. bovis. This study underscores the adaptive potential of M. bovis during within-host colonization and cross-tissue transmission, providing critical insights for optimizing herd management and treatment strategies. Full article

Lumpy Skin Disease (LSD) is a transboundary bovine viral disease. It has a significant economic impact and is caused by the Lumpy Skin Disease Virus (LSDV). Effective surveillance tools are essential for the early detection of infection, outbreak control, and assessment of vaccination coverage in endemic regions such as India. In this study, an in-house ELISA based on inactivated whole-virus antigen (WVA) was developed, optimized, and validated for the detection of LSDV antibodies in cattle. Its field applicability was assessed through a cross-sectional seroprevalence survey conducted across five Indian states. A local field isolate of LSDV (strain 5-Chitra) was cultured in MDBK cells, inactivated using binary ethylenimine (BEI), and used as the antigen source. The assay was optimized by checkerboard titration and evaluated against the Serum Neutralization Test (SNT). Diagnostic sensitivity and specificity were evaluated using the receiver operating characteristic (ROC) curve and area under the curve (AUC) analyses, while cross-reactivity was assessed using sera positive for HS, IBR, BQ, MCF, GTP, SPP, CE, FMD, and Brucellosis. Assay reproducibility was confirmed through inter- and intra-laboratory validation. For the seroprevalence study, 3230 cattle serum samples were collected using a stratified random sampling design across five Indian states, and logistic regression analysis of a subset of 1302 samples was performed to assess the influence of age and sex on LSDV seropositivity. Checkerboard titration identified optimal ELISA conditions at 50 ng of antigen per well, a 1:150 serum dilution, and a 1:10,000 dilution of anti-bovine HRP-conjugated secondary antibody. The WVA-ELISA demonstrated excellent diagnostic performance, with 100% sensitivity, 95% specificity, and no cross-reactivity with other ruminant bacterial or viral pathogens, and showed high laboratory reproducibility (κ > 0.96). Seroprevalence ranged from 50.6% to 71.1% across the five states, indicating widespread exposure to LSDV. Risk factor analysis revealed significantly higher odds of seropositivity among calves (≤1 year old) and female cattle, suggesting age- and sex-dependent susceptibility. Full article

Coronaviruses are worldwide-distributed RNA viruses with zoonotic potential and the ability to jump from one host species to another, including humans. Even after the COVID-19 pandemic, the search for new, biologically active substances with anti-coronavirus activity continues to be a critical milestone for human health protection. In the framework of a complex engineering strategy, we cultivated the microalgal species Scenedesmus acutus in two different innovative types of flat-plate photobioreactors (PBR1 and K1) for CO₂ utilization and biomass production with special features. Isolated extracts from the microalgal biomass of each one were compared for their anti-coronavirus potential. The design of both PBRs allows a hydrodynamic regime to achieve best fluid flow distribution in their sections, therefore providing the optimal so-called flashing light effect. Of course, this is achieved under well-controlled operational conditions. A strain of beta coronavirus 1 (BCoV, bovine coronavirus) replicated in MDBK cells was used as an in vitro model for the evaluation of the antiviral activity of both extracts. The cell viability, number of survived BCoV particles, and cytopathic effect were evaluated after pre-incubation of the virus with the extracts or direct treatment. The extracts’ samples exhibited evident antiviral activity—extract 1 (from PBR1) in concentrations ≥ 200 µg/mL and extract 2 (from K1) in concentrations ≥150 µg/mL. The ddPCR result revealed significant diminishment of the BCoV particles in samples treated with higher concentrations of the extracts. The phytochemical analysis for certain main groups of compounds (flavonoids, polyphenols, carotenoids, and lipids) showed some differences for both extracts, which could be a possible reason for the observed difference in the antiviral activity. In conclusion, the innovative PBRs are a good platform for studying microalgal growth kinetics by applying different stress conditions from hydrodynamics and mass transfer subsystems. Both extracts showed promising potential for the isolation of metabolites with antiviral activity against BCoV and could be an object for future pharmacological investigations. Full article

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic orthoflavivirus that poses a significant global health threat. It causes severe neuroinflammatory disease in humans and reproductive failure in swine. Because of the broad host range and cell tropism of JEV, identifying animal cell lines resistant to infection has been a persistent challenge. In this study, we demonstrate that Madin–Darby bovine kidney (MDBK) cells are resistant to JEV infection yet remain fully permissive to viral replication when transfected with viral genomic RNA. Using immunoblotting, immunofluorescence, and flow cytometry, we show that MDBK cells, unlike the highly susceptible baby hamster kidney (BHK-21) cells used as controls, do not support viral entry but sustain all post-entry stages of the replication cycle. Further investigation confirmed that MDBK cells possess a functional clathrin-mediated endocytic pathway, as evidenced by their susceptibility to bovine viral diarrhea virus, which relies on clathrin-dependent endocytosis for host cell entry. These findings establish MDBK cells as a nonsusceptible cell line for JEV entry despite intact endocytic function, providing a valuable platform for studying virus–host cell interactions and for identifying and validating host cell entry factors, a major challenge in JEV research. Full article

Bovine viral diarrhea virus (BVDV) causes severe mucosal inflammation in cattle, and effective treatment options remain limited. Dysregulated activation of the NLRP3 inflammasome, driven by NF-κB and STAT3 signaling, may exacerbate disease pathogenesis, highlighting this axis as a potential therapeutic target. Although traditional Chinese medicine has shown promise in antiviral and anti-inflammatory applications, it remains unclear whether it can inhibit BVDV replication via the NF-κB/STAT3-NLRP3 pathway. The present study aimed to clarify the inhibitory effect of luteolin on bovine viral diarrhea virus (BVDV) replication, and to elucidate its underlying mechanisms from two perspectives: interference with viral internalization and replication processes, as well as regulation of the NF-κB/STAT3-NLRP3 inflammasome pathway. Collectively, this work intended to provide experimental evidence and theoretical support for the development of luteolin as a natural anti-BVDV agent. To this end, BVDV-infected MDBK cells were treated with gradient concentrations of luteolin, followed by quantification of viral load using qRT-PCR and Western blot assays. Meanwhile, the activation status of the NF-κB/STAT3-NLRP3 signaling pathway was evaluated via immunofluorescence staining and luciferase reporter gene assays. Our results demonstrate that luteolin exhibits potent dual antiviral activity against cytopathic BVDV-1m in MDBK (Madin-Darby Bovine Kidney) cells, effectively suppressing both viral replication and inflammatory responses. At non-cytotoxic concentrations, luteolin specifically inhibited the internalization and replication stages of the viral lifecycle, accompanied by reduced NS5B polymerase activity. Importantly, luteolin disrupted the NF-κB/STAT3-NLRP3 axis by suppressing phosphorylation of p65 (Ser536) and STAT3 (Ser727), downregulating NLRP3 and pro-caspase-1 expression, and inhibiting caspase-1 cleavage (p20) as well as maturation of IL-1β and IL-18. Consequently, it attenuated the overexpression of TNF-α and IL-8. To our knowledge, this is the first report of a single compound simultaneously targeting multiple stages of the BVDV lifecycle and counteracting NLRP3-mediated immunopathology, offering a strategic basis for developing flavonoid-based therapies against Flavivirus infections. Full article

Bovine viral diarrhea virus (BVDV) is a major pathogen responsible for significant economic losses in the global cattle industry. The diverse transmission routes and the characteristics of asymptomatic infections make it difficult to contain the spread; there is an urgent need to develop new effective antiviral strategies. Nanobodies (Nbs) have become a promising new type of antiviral agent due to their advantages, including small molecular size, stable structure, high specificity, and ease of production. This study successfully screened a specific nanobody, Nb7, targeting the key functional protein NS5A of BVDV using phage display technology. Furthermore, the nanobody was effectively delivered into Madin–Darby bovine kidney (MDBK) cells by fusing it with the cell-penetrating peptide TAT. The results demonstrate that TAT-Nb7, specifically targeting the non-structural protein NS5A of BVDV, significantly inhibits viral replication in MDBK cells. In conclusion, this study indicates that TAT-Nb7 holds promise as a therapeutic candidate for the prevention and control of BVDV infection. Full article

The highly pathogenic avian influenza (HPAI) H5N1 virus poses a growing global health threat, particularly following its unprecedented spillover into dairy cattle and subsequent transmission to more than 1000 dairy farms in 18 states. This study investigates the host cell responses to distinct H5N1 strains (bovine- and mink-derived H5N1) in the presence and absence of raw milk across diverse mammalian cell lines (MDCK, MDBK, A549, Vero, MV1). Our findings reveal that the bovine-derived H5N1 strain exhibits more robust replication than the mink-derived H5N1 and demonstrates intra-host viral evolution with emerging amino acid substitutions detectable by deep sequencing. Although raw milk supplementation did not directly enhance viral replication in vitro, it significantly modulated host gene expression, often dampening key antiviral interferon-stimulated gene (ISG) responses and disrupting essential host cellular processes like intracellular trafficking and sialic acid biosynthesis. These host gene modulations are cell-type- and strain-specific, suggesting a complex interplay that may theoretically influence virus–host dynamics, though the biological significance of these in vitro observations requires validation through infectious virus assays and in vivo studies. This hypothesis-generating work provides preliminary insights into H5N1-milk interactions, highlighting the need for further mechanistic investigation to assess potential implications for viral transmission in dairy environments. Full article

Background: Classical swine fever virus (CSFV) is a highly contagious and fatal disease in pigs and wild boars. While hunting and bait vaccination are effective for CSFV eradication, additional strategies are needed to control wild boar populations. This study aimed to develop an oral vaccine, Flc-LOM-GnRHx3, by inserting gonadotropin-releasing hormone (GnRH) epitopes into the Flc-LOM clone. Methods: The Flc-LOM-GnRHx3 strain was rescued from CPK cells and propagated to high titers in MDBK cells. Male boars (20 weeks old) received three doses (10^5.0 TCID₅₀/ml/dose) of Flc-LOM-GnRHx3 either orally or intramuscularly at 2-week intervals. Anti-CSFV E2 antibodies were detected via immunofluorescence and Western blotting. Results: Both vaccination routes induced anti-GnRH antibodies and reduced testosterone levels. Testis size and weight were slightly lower than controls, with seminiferous tubule and spermatid deformities observed in 52.5% of intramuscularly vaccinated pigs and 20.8% of orally vaccinated pigs. Conclusions: Flc-LOM-GnRHx3 demonstrates potential as a dual-function oral vaccine that can eradicate CSFV and impair reproductive capacity in wild boars, offering a novel approach for integrated disease control and population management. Full article