Search Results (81)

Lumpy skin disease (LSD) is an invasive infectious disease caused by the lumpy skin disease virus (LSDV), which is detrimental to the production of cattle. LSDV encodes about 156 proteins, most of whose functions are still unknown. In this study, we found that the ORF137 protein was identified as one of the strongest inhibitors of IFN-β and ISG expression, determining LSDV ORF137 as a negative regulator of interferon (IFN) β signaling. Further evidence suggests that ORF137 interacts with the signal transduction factor IRF3 and inhibits the activation of IFN-β signaling by reducing Phospho-IRF3 (p-IRF3). Further investigation indicated that overexpression of ORF137 in BMEC could significantly inhibit the transcription of IFN-β and ISGs, thereby promoting the replication of LSDV. More importantly, through homologous recombination, we deleted the ORF137 gene from the LSDV/FJ/CHA/2021 strain and constructed the recombinant strain LSDV-ΔORF137-EGFP. Compared with the parental strain, LSDV-ΔORF137-EGFP showed a weakened effect on inhibiting the transcription of IFN-β and ISGs and a reduced replication level in infected MDBK cells. In summary, ORF137 facilitates LSDV replication by targeting IRF3 to inhibit IFN-β signaling. Our findings reveal a new mechanism by which LSDV suppresses the host antiviral response, which may facilitate the development of attenuated live vaccines for LSDV. Full article

The scientific community’s interest in natural compounds with antiviral properties has considerably increased after the emergence of the severe acute respiratory syndrome coronavirus (SARS-CoV-2), especially for their potential use in the treatment of the COVID-19 infection. From this perspective, bovine coronavirus (BCoV), member of the genus β-CoV, represents a valuable virus model to study human β-CoVs, bypassing the risks of handling highly pathogenic and contagious viruses. Pimarane diterpenes are a significant group of secondary metabolites produced by phytopathogenic fungi, including several Diplodia species. Among the members of this class of natural products, sphaeropsidin A (SphA) and its analog sphaeropsidin B (SphB) are well known for their bioactivities, such as antimicrobial, insecticidal, herbicidal, and anticancer. In this study, the antiviral effects of SphA and SphB were evaluated for the first time on bovine (MDBK) cells infected with BCoV. Our findings showed that both sphaeropsidins significantly increased cell viability in infected cells. These substances also caused substantial declines in the virus yield and in the levels of the viral spike S protein. Interestingly, during the treatment, a cellular defense mechanism was detected in the downregulation of the aryl hydrocarbon receptor (AhR) signaling, which is affected by BCoV infection. We also observed that the presence of SphA and SphB determined the deacidification of the lysosomal environment in infected cells, which may be related to their antiviral activities. In addition, in silico investigations have been performed to elucidate the molecular mechanism governing the recognition of bovine AhR (bAhR) by Sphs. Molecular docking studies revealed significant insights into the structural determinants driving the bAhR binding by the examined compounds. Hence, in vitro and in silico results demonstrated that SphA and SphB are promising drug candidates for the development of efficient therapies able to fight a β-CoV-like BCoV during infection. Full article

The recent COVID-19 pandemic has prompted the scientific community to prioritize the discovery of preventive methods and new therapeutics, including the investigation of natural compounds with antiviral potential. Fungal secondary metabolites (SMs) represent a promising source of antiviral drugs due to their structural diversity and intrinsic biocompatibility. Herein, the antiviral activity of 6-pentyl-α-pyrone (6PP) against bovine coronavirus (BCoV) has been evaluated in vitro. Considering that BCoV and SARS-CoV-2 are both members of the Betacoronavirus genus and share several key features, BCoV represents a valuable reference model for human coronavirus research. A non-cytotoxic dose of 6PP was used on MDBK cells to evaluate its antiviral activity against BCoV. Different experimental conditions were employed to examine cell monolayer protection both pre- and post-infection, as well as the potential inhibition of viral internalization. Overall, post-infection 6PP treatment reduced viral load and decreased viral internalization. Results were analyzed using viral titration and quantitative PCR, while data interpretation was performed by statistical software tools. This study presents a novel fluorescence quantification approach with high confidence demonstrated by its significant concordance with RT-qPCR results. These data suggest that 6PP could be an effective antiviral agent for BCoV, warranting further investigation of its role in coronavirus inhibition. Full article

The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin–Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of TRIM5α, a restriction factor in HIV-1 infection, can dramatically enhance HIV-1 infection in MDBK cells. Furthermore, we generated a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9) suitable for CRISPR/Cas9-mediated editing. On this basis, we created a TRIM5α knock-out MDBK-iCas9 cell line MDBK-iCas9^{TRIM5α−/−} without additional genome insertions by combining sgRNA transfection and single-cell cloning. We found that MDBK-iCas9^{TRIM5α−/−} displayed greater permissiveness to lentivirus infection compared with MDBK-WT cells. Notably, we found that treatment with the chemical compound cyclosporine A, which directly interacts with cell factor cyclophilin A (CypA), could markedly increase the infectivity of lentivirus in both MDBK-iCas9^{TRIM5α−/−} and MDBK-WT cell lines, suggesting that CypA functions independently with TRIM5α as an inhibitor of the lentivirus in bovine cells. Therefore, combining bovine TRIM5α and CypA targeting could remarkably enhance lentivirus infection. In conclusion, our findings highlight a promising gene engineering strategy for bovine cells that can surmount the significant barriers to investigating the interplay between bovine viruses and their host cells. Full article

Dihydroartemisinin (DHA) is a bioactive phytopharmaceutical with diverse pharmacological potential, predominantly because of its established antiplasmodial efficacy. Here, we investigated the effects of DHA on metabolic homeostasis in Madin-Darby bovine kidney cells in the context of dose-specific adaptation of metabolism and regulation of biochemistry pathway changes. According to our findings, extensive changes in metabolism were revealed by PCA, accounting for a variability of 59.4% to distinguish contrasting metabolic signatures from normal cells. Metabolomic characterization demonstrated 67 constituting metabolites of baseline cellular processes, while 32 and 44 metabolites have demonstrated differential abundance in low- and high-dose treatments, respectively. Impaired metabolism of glycerophospholipid, amino acid, and nucleotide biosynthesis was reported with implications such as regulation of membrane reorganization, nitrogen metabolism, and cellular bioenergetics. Bioindicators of high-volume lysophosphatidylcholine (18:0) and choline phosphate revealed a lipid homeostatic change, in addition to imbalances in glutamic acid and proline levels. Pathway regulation further modulated ABC transporters and arachidonic acid signaling as implications of systemic phytopharmaceutical-modulated reorganization of metabolism. Hence, the study gives mechanistic insight into DHA-initiated modulation of cellular metabolism in MDBK cells, elucidating its status as a cellular metabolism regulator. Findings confirm the potential of DHA as a phytopharmaceutical in modulating diseases of metabolism, further solidifying its relevance in evidence-based traditional herbal remedies and natural compound therapeutics. Full article

A precision genome edit in the bovine CD46 gene (A₈₂LPTFS₈₇) dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned heifer. However, pathogen evolution threatens the long-term efficacy of such interventions. Here, our aim is two-fold: first, to determine whether BVDV can adapt in vitro to use the edited CD46 receptor to infect Madin–Darby bovine kidney (MDBK) cells, and second, to evaluate the ex vivo infectivity of culture-adapted viruses in cells from the CD46-edited heifer. Serial passage of BVDV on CD46-edited MDBK cells selected for virus variants capable of CD46-independent infection. Virus genome sequencing revealed mutations in the viral E^RNS gene predicted to enhance HS-mediated entry. HS adaptation was confirmed by inhibiting virus infection with heparin or Heparinase I/III treatment. A naturally occurring HS-adapted field isolate from a persistently infected calf showed similar results. However, when tested on primary cells from the CD46-edited heifer, HS-adapted viruses showed reduced infectivity in skin fibroblasts, monocytes, and lymphocytes in a manner that correlated with HS expression. Thus, although BVDV can adapt to use HS as an alternative entry receptor, HS adaptation does not overcome the protection conferred by the CD46 edit in all relevant cell types. Full article

Oregano oil was successfully encapsulated into chitosan–albumin nanogel via emulsification and electrostatic gelation. The system was characterized with a mean diameter around 26 nm, narrow size distribution (PDI = 0.242) and approximately 40% encapsulation efficiency. The incorporation of the oil into the nanogel was confirmed by XRD and FTIR analyses, and the dissolution of the oil was enhanced after the encapsulation. Furthermore, the treatment of Betacoronavirus 1 infected bovine kidney MDBK cells with the oregano oil-loaded nanogel (25 µg/mL) showed more than 50% protection against the infection, as compared to the non-treated virus infected control. The cytopathic effect (CPE) of the virus was inhibited in a concentration-dependent manner. The system inhibited the virus replication, resulting in a decrease of the viral particles by more than half, as shown by the cytotoxicity and CPE assays. The virus titer in treated and non-treated samples was determined by digital droplet PCR and revealed Δ3 log diminishment of the virus particles in samples treated with 25 µg/mL encapsulated oregano oil. This study is a basis for further investigations on the pharmacodynamics of the nanogel and its possible combinations with clinically applied chemotherapeutics. Full article

Objectives: This article presents a study evaluating the antibody levels against infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD) in Kazakh Whiteheaded calves born to dams immunized with an experimental inactivated combined vaccine against these infections. The vaccine formulation includes the strains “R-93” (IBR) and “Oregon C₂₄V” (BVD), which are preserved in the microorganism collection of the Research Institute for Biological Safety Problems. Methods: To assess the immune response in newborn calves, blood serum samples were collected before the first intake of colostrum, followed by weekly sampling for 28 weeks post-birth. The antibody response was determined using a virus neutralization assay on MDBK cell cultures and lamb testicle cell cultures. Results: The results demonstrated that the protective antibody level against the IBR virus (≥2 log₂) persisted for up to 25 weeks, while the protective level against the BVD virus (≥3 log₂) remained for 23 weeks. Based on these findings, the vaccine was deemed safe, as it did not induce abortions or clinical manifestations of the diseases. The overall duration of the colostral immunity in calves against the IBR and BVD viruses reached 23 weeks. Conclusions: Therefore, it is recommended that Kazakh Whiteheaded calves be vaccinated with the associated inactivated vaccine against infectious bovine rhinotracheitis and bovine viral diarrhea no earlier than 23 weeks of age. Full article

Orthoherpesviridae is a large family of enveloped DNA virus. Among the most significant animal-infecting viruses are bovine alphaherpesvirus 1 (BoAHV1), caprine alphaherpesvirus 1 (CpAHV1) and equid alphaherpesvirus 1 (EqAHV1). Research into new methods to combat herpesvirus infections is ongoing. The aim of this study was to evaluate the antiviral activity of three extracts of the Helleborus bocconei roots against BoAHV1, CpAHV1 and EqAHV1. The roots were air-dried, extracted with methanol (MeOH) and then partitioned between n-butanol (n-BuOH) and water. All three extracts were tested for cytotoxicity on MDBK and RK-13 cells, and for antiviral activity. Two non-cytotoxic concentrations were assessed for their anti-BoAHV1, anti-CpAHV1 and anti-EqAHV1effects. Cells were incubated with the extracts for 72 h under three experimental conditions: pretreatment before viral infection, treatment post virus infection and simultaneous viral infection and treatment with extracts. The n-BuOH extract (BE) at 0.62 µg/mL inhibited the cytopathic effects of all three viruses in the simultaneous assay. Additionally, no cytopathic effect was observed in MDBK cells infected with CpAHV1and treated with 0.31 µg/mL BE post virus infection. Therefore, the BE contains molecules or groups of molecules potentially useful for developing an alternative therapy against herpesvirus (HV) infection. Full article

In February 2024, while conducting surveillance for novel respiratory viruses, we studied four beef cattle farms near Monterrey, Mexico. Nasal swabs were collected from sick and healthy beef cattle along with 3 h aerosol samples. None of the samples had molecular evidence of influenza A viruses. Three (8%) of thirty-six nasal swabs collected from the four farms and four (33.3%) of the twelve bioaerosol specimens had molecular evidence of influenza D virus. Five sick cow nasal swabs and one bioaerosol sample on a single farm had molecular evidence of rodent coronavirus-like (RCoV), an alphacoronavirus. Three (60%) of the five RCoV-positive cattle nasal swabs also had molecular evidence of influenza D. Attempts to isolate the RCoV in Vero-E6, LLC-MK2, MDBK, and L-2 cells were unsuccessful. However, we were able to assemble ~60% of the RCoV genome using next-generation sequencing. The six RCoV-positive samples clustered with RCoV strains identified in China in 2021. During the last 12 months, we have studied an estimated 478 dairy and beef cattle nasal swabs on 11 farms in the US and Mexico, and these RCoV detections are the first we have encountered. While feed contamination cannot be ruled out, given the propensity of CoVs to jump species and that we detected RCoV only in the noses of sick cows on this one farm, we are concerned that these findings could represent an isolated RCoV spillover event. With this report, we are alerting veterinarians and cattle farm owners of our observations that RCoV may be a new cause of bovine respiratory disease. Full article

Mycoplasma bovis is the most common mycoplasma associated with cattle diseases worldwide. However, other seemingly less virulent Mycoplasma spp. such as M. bovigenitalium and M. bovirhinis have also been associated with mycoplasmosis. The study objective was to compare the adhesion and cellular invasion characteristics of these bovine Mycoplasma spp. using Madin–Darby Bovine Kidney (MDBK) epithelial cells. MDBK cells were separately infected with 12 M. bovis strains and one strain each of M. bovigenitalium and M. bovirhinis. Following infection, a gentamicin protection assay was performed and the cells lysed at 6 and 54 h post-infection. The MDBK cell lysates were cultured for Mycoplasma spp. and qPCR was used to estimate the average number of Mycoplasma bacterial cells that infected each MDBK cell (Myc/Cell ratio). Confocal and electron microscopy studies using M. bovis mNeonGreen strain were also performed. All 14 Mycoplasma strains multiplied within the MDBK cells, a finding confirmed by microscopy studies of the M. bovis mNeonGreen strain. Unexpectedly, the M. bovis strains, obtained from diseased and asymptomatic cattle and bison, had lower Myc/Cell ratios than M. bovirhinis and M. bovigenitalium strains. These findings suggest that the ability for mycoplasmas to invade and replicate within host cells does not account for the differences in virulence between species. Full article

It is well known that the host response to different human and animal coronaviruses infection is regulated by the aryl hydrocarbon receptor, a ligand-activated transcription factor. The present study investigates the expression of the aryl hydrocarbon receptor during bovine coronavirus infection, through in vitro and in silico investigations. The in vitro studies demonstrate that the aryl hydrocarbon receptor and as well as its targets, CYP1A1 and CYP1B1, were significantly activated by bovine coronavirus infection in bovine cells (MDBK). During infection, the pretreatment of cells with non-cytotoxic doses of CH223191, a selective inhibitor of the aryl hydrocarbon receptor, resulted in a significant reduction in virus yield and a downregulation in the viral spike protein expression. These findings occurred in the presence of the inhibition of aryl hydrocarbon receptor signaling. Our results reveal that the bovine coronavirus acts on viral replication, upregulating the aryl hydrocarbon receptor and its downstream target proteins, CYP1A1 and CYP1B1. In addition, following the in silico studies, the three-dimensional structural model of the bovine aryl hydrocarbon receptor in complex with the antagonist CH223191 indicates that the molecular mechanism, by which the PASB and TAD domains of the receptor interact with the inhibitor, is mainly driven by an extensive network of hydrophobic interactions, with a series of hydrogen bonds contributing to stabilizing the complex. Interestingly, bioinformatic analyses revealed that the PASB and TAD domains in the human and bovine aryl hydrocarbon receptor present high similarity at the primary sequence and three-dimensional structure levels. Taken together, these findings represent a fundamental step for the development of innovative drugs targeting AhR as a potential object for CoVs therapy. Full article

Herpes viruses are highly contagious agents affecting all classes of vertebrates, thus causing serious health, social, and economic losses. Within the One Health concept, novel therapeutics are extensively studied for both veterinary and human control and management of the infection, but the optimal strategy has not been invented yet. Lactic acid bacteria are key components of the microbiome that are known to play a protective role against pathogens as one of the proposed mechanisms involves compounds released from their metabolic activity. Previously, we reported the anti-herpes effect of postmetabolites isolated from Lactobacilli, and here, we confirm the inhibitory properties of another nine products against the phylogenetically distant human Herpes simplex virus-1 (HSV-1) and fish Koi Herpes virus (KHV) in cell cultures. Cytotoxicity, cytopathic effect inhibition, virucidal effect, the influence on the adsorption stage of the virus to the cells, as well as the protective effect of the postmetabolites on healthy cells were evaluated. The inhibitory effect was more pronounced against HSV-1 than against KHV at all studied viral cycle stages. Regarding the intracellular replicative steps, samples S7, S8, and S9 (Mix group) isolated from Ligilactobacillus salivarius (vaginal strain) demonstrated the most distinct effect with calculated selective indices (SIs) in the range between 69.4 and 77.8 against HSV-1, and from 62.2 to 68.4 against KHV. Bioactive metabolites from various LAB species significantly inhibit extracellular HSV-1 and, to a lesser extent, KHV virions. The blockage of viral adsorption to the host cells was remarkable, as recorded by a decrease in the viral titer with Δlg ≥ 5 in the Mix group for both herpes viruses. The remaining postmetabolites also significantly inhibited viral adsorption to varying degrees with Δlg ≥ 3. Most metabolites also exerted a protective effect on healthy MDBK and CCB cells to subsequent experimental viral infection. Our results reveal new horizons for the application of LAB and their postbiotic products in the prevention and treatment of herpes diseases. Full article

Photodynamic inactivation (PDI) has been revealed as a valuable approach against viral infections because of the fast therapeutic effect and low possibility of resistance development. The photodynamic inhibition of the infectivity of human herpes simplex virus type 1 (HSV-1) strain Victoria at different stages of its reproduction was studied. PDI activity was determined on extracellular virions, on the stage of their adsorption to the Madin-Darby bovine kidney (MDBK) cell line and inhibition of the viral replication stage by application of two tetra-methylpyridiloxy substituted gallium and zinc phthalocyanines (ZnPcMe and GaPcMe) upon 660 nm light exposure with a light-emitting diode (LED 660 nm). The PDI effect was evaluated on extracellular virions and virus adsorption by the terminal dilution method and the change in viral infectivity, which was compared to the untreated control group. The decrease in viral titer (Δlgs) was determined. The effect on the replicative cycle of the virus was determined using the cytopathic effect inhibition (CPE) assay. The direct influence on the virions showed a remarkable effect with a decrease in the viral titer more than 4 (Δlg > 4). The influence of the virus to the cell on the stage of adsorption was also significantly affected by the exposure time and the concentration of applied photosensitizers. A distinct inhibition was evaluated for ZnPcMe at the viral replication stage, which demonstrated a high photoinactivation index (PII = 33.0). This study suggested the high efficacy of PDI with phthalocyanines on HSV-1 virus, with full inhibition caused by the mechanism of singlet oxygen generation. These promising data are a good basis for further investigations on the PDI application against pathogenic viruses. Full article

Background: Lumpy skin disease virus (Poxviridae family—Capripoxvirus genus) is the aetiological agent of LSD, a disease primarily transmitted by hematophagous biting, affecting principally cattle. Currently, only live attenuated vaccines are commercially available, but their use is limited to endemic areas. There is a need for safer vaccines, especially in LSD-free countries. This research aims to develop and test a safe and efficacious inactivated vaccine. Moreover, in this study, we used keyhole limpet hemocyanin (KLH) as a positive marker to distinguish infected from vaccinated animals (DIVA). Methods: Lumpy skin disease virus was propagated on primary lamb testis cells and Madin–Darby bovine kidney cells (PLT and MDBK, respectively), and four inactivated vaccines were produced. The vaccines differed from each other with the addition or not of KLH and in cells used for virus propagation. To evaluate the safety and immunogenicity, the vaccines and two placebos were administered to six groups comprising six male calves each, and antibody response was investigated using both an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization (SN) test. In addition, the LSD/γ-interferon test and KLH (IgM-IgG) ELISA were performed on the collected samples. Furthermore, the use of KLH allowed us to distinguish vaccinated animals in the ELISA results, without any interference on the strength of the immune response against the LSDV. Finally, the efficacy of one of four vaccines was investigated through a challenge, in which one group of vaccinated animals and one animal control group were infected with a live field strain of LSDV. Results: Four out of the six control animals showed severe clinical signs suggestive of LSD, and, therefore, were euthanized for overcoming the predetermined limit of clinical score. By contrast, the vaccinated animals showed only mild symptoms, suggesting a reduction in severe disease notwithstanding the incapability of the vaccine in reducing the virus shedding. Conclusion: The vaccines produced were safe and able to elicit both a humoral and a cellular immune response, characteristics that, together with the demonstrated efficacy, make our vaccine a good candidate for countering the LSD spread in disease-free countries, thus also facilitating disease containment throughout the application of a DIVA strategy. Full article