ABSTRACT Bovine parainfluenza virus type 3 (BPIV-3) is a major pathogen associated with the bovine respiratory disease complex. However, the limited understanding of host factors crucial for BPIV-3 replication has hindered the development of effective preventive and therapeutic strategies. To tackle this critical issue, we constructed a bovine genome-wide CRISPR/Cas9 knockout library in Madin-Darby bovine kidney cells, which was then used to systematically identify and characterize the host genes essential for BPIV-3a replication. Subsequently, 10 genes were validated using both RT-qPCR and viral titration assays. Furthermore, through gene knockout or knockdown and rescue experiments, we identified three key genes required for BPIV-3a replication: Wnt family member 5A (WNT5A), solute carrier family 16 member 13 (SLC16A13), and selenoprotein N (SELENON). However, their effects on viral adhesion and internalization varied. WNT5A was involved in both processes, SLC16A13 participated solely in internalization, while SELENON had no significant impact on either. Beyond BPIV-3a, these three genes were also found to be essential for the infection of BPIV-3c and Bovine enterovirus. In conclusion, this study offers novel insights into the molecular mechanisms governing the replication and pathogenesis of BPIV-3a, BPIV-3c, and bovine enterovirus within host cells, thereby providing a foundation for identifying potential targets in the development of novel antiviral strategies.

First isolation and characterization of caprine parainfluenza virus type 3 from cattle in China.

This study successfully isolated a novel bovine enterovirus strain from a bovine fecal sample, which was designated as Sichuan/SQ/20. The isolate showed typical enterovirus morphology under electron microscopy. Phylogenetic analysis showed that this strain exhibits the closest genetic relationship with the HeN-YR91 and JPN/TottoriU-31 strains, and all three belong to the BEV-F1 genosubtype. Subsequently, comprehensive investigations were conducted on the biological characteristics of this virus, both in vivo and in vitro. In vitro characterization revealed that viral replication commenced at 3 h post-infection (hpi) in Madin-Darby bovine kidney cells, reaching peak at 48 hpi with a virus titer of 1 × 108.73 TCID50/0.1 mL. Cytopathic effects initially appeared at 12 hpi. A 12-minute treatment at 55°C was sufficient to completely inactivate the virus. In vivo analysis revealed that significant pathological changes were specifically observed in the spleen, with no lesions observed in other organs. Immunofluorescence assay detected specific fluorescent signals in the liver, spleen, and small intestine, which were consistent with the PCR results. These findings provide a scientific foundation for vaccine design and antiviral drug screening, as well as for the development of effective prevention and control strategies.IMPORTANCEBovine enterovirus (BEV) is an important pathogen causing calf diarrhea and has been detected in the feces of calves with diarrhea, although its pathogenicity remains unclear. This study systematically established an isolation and identification protocol for BEV, characterized its physicochemical properties, and further investigated the pathogenicity and tissue tropism of the isolated strain in mice. These findings establish crucial baseline data for future vaccine development and therapeutic intervention strategies.

A comprehensive understanding of host-virus interactions during persistent foot-and-mouth disease virus (FMDV) infection is essential for elucidating the mechanisms that underpin disease causation by this highly contagious pathogen. This understanding necessitates the development of stable in vitro models. In this study, we established a model of persistent FMDV serotype O infection in Madin-Darby bovine kidney epithelial cells followed by integrated multiomics analyses. These analyses revealed that host cells adapt to persistent viral infection by reprogramming mitochondrial metabolism. This reprogramming is accompanied by alterations in mitochondrial structure and function, as well as the suppression of the apoptotic response in host cells. In particular, bystander cells, which are devoid of active viral replication, display enhanced proliferative capacity and possess a distinct microenvironmental signature that potentially increases viral susceptibility, facilitating sustained virus persistence within the cell population. Moreover, persistent viruses have evolved enhanced replicative fitness. Our findings elucidate the biological characteristics of FMDV-infected cell populations that persistently harbour the virus, reveal host-virus coadaptation, and highlight the critical role of bystander cells in sustaining persistent FMDV infection. These discoveries establish the foundation for further mechanistic studies of FMDV persistence maintenance.

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.

Lumpy skin disease virus ORF142 suppresses the cGAS/STING-mediated IFN-I pathway through NBR1-mediated STING autophagic degradation.

Bovine viral diarrhea virus E2 targets SLC3A2 to modulate lipid peroxidation for replication advantage.

Modulatory effects of platelet-rich plasma on viral kinetics of BoAHV-1.1, BoGHV-4, and BVDV in bovine cell cultures: A proof-of-concept study

BackgroundInfectious bovine rhinotracheitis virus (IBRV), a member of the Herpesviridae family, causes infectious bovine rhinotracheitis (IBR) and induces mitochondrial dysfunction in host cells. Circular RNAs (circRNAs)—a novel class of non-coding RNAs—have been implicated in various biological processes and pathologies related to mitochondrial damage. However, their role in IBRV-induced mitochondrial damage in Madin-Darby bovine kidney (MDBK) cells remains unclear.ResultsTransmission electron microscopy(TEM), laser confocal microscopy, and flow cytometry confirmed that IBRV infection causes mitochondrial damage in MDBK cells. High-throughput sequencing revealed 144 differentially expressed (DE) circRNAs, 725 messenger RNAs (mRNAs), and 160 microRNAs (miRNAs) in IBRV-infected cells. We predicted that DE circRNAs regulate mitochondrial damage via source genes of circRNA, circRNA-miRNA-mRNA networks, and RNA-binding proteins (RBPs). Source genes of circRNA were enriched in mitochondria-related pathways, such as the mammalian target of rapamycin (mTOR), thyroid hormone, and Hippo signalling; 11 genes were localized to mitochondria. CircRNA-miRNA-mRNA network target genes were associated with cellular senescence, mitophagy, and ubiquitin-mediated proteolysis; 471 genes were linked to mitochondria. Additionally, 961 RBPs were enriched in pathways, such as nucleocytoplasmic transport and RNA degradation; 107 RBPs were localized to mitochondria. Functional validation revealed knockdown of circ_002584 reduced reactive oxygen species (ROS) accumulation (p < 0.05) and mitochondrial membrane potential depolarization (p < 0.05). Knockdown of circ_004326 increased both (p < 0.01).ConclusionsCircRNAs play a regulatory role in IBRV-induced mitochondrial damage within MDBK cells. This finding is significant for virus-associated mitochondrial damage research, forming a theoretical foundation for utilizing circRNAs as diagnostic biomarkers and potential therapeutic targets for IBR.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12158-9.

Heat-stable peptide markers for bovine heart, kidney, liver, lung, and spleen for authenticity testing of minced beef products.

ABSTRACTBackground and Aim:Lumpy skin disease (LSD), caused by the LSD virus (LSDV), results in severe economic losses, reduced productivity, and restricted livestock trade. Although live attenuated vaccines are available, they pose risks such as viral shedding, recombination, and reversion to virulence. Inactivated vaccines, being safer alternatives, are particularly suitable for disease-free regions. This study aimed to develop an inactivated oil-adjuvanted vaccine using a local LSDV isolate and evaluate its immunogenicity and protective efficacy in rabbits.Materials and Methods:Scab samples were collected from clinically suspected LSD cases, and LSDV was isolated through the chorioallantoic membrane route in embryonated chicken eggs. The virus was adapted to Madin-Darby bovine kidney (MDBK) cells, inactivated with binary ethyleneimine, and formulated with Montanide Immune System Activator 50 V2 adjuvant. Sterility and safety were evaluated in laboratory animals. Twenty-four rabbits were divided into three groups: Group A received the experimental inactivated vaccine intramuscularly, Group B received a commercial live attenuated vaccine subcutaneously, and Group C served as controls. Antibody responses were assessed using enzyme-linked immunosorbent assay (ELISA) and virus neutralization tests. A challenge study with a virulent local LSDV strain was conducted to evaluate protective efficacy.Results:The inactivated vaccine elicited robust antibody responses, with ELISA sample-to-positive ratios increasing from 4.3% at baseline to 166.6% on day 42, compared with 210.1% in the live vaccine group and 6% in controls. Neutralizing antibody titers ranged from 1:32 to 1:128 (mean 1:80) in the inactivated group, compared with 1:32–1:256 (mean 1:148) in the live vaccine group, both surpassing the protective threshold (≥1:16). Post-challenge, the inactivated vaccine conferred 86% vaccine efficacy, with only mild clinical signs observed in one rabbit, while the control group developed typical LSD symptoms. No adverse reactions were recorded in vaccinated animals.Conclusion:The experimental inactivated oil-adjuvanted vaccine induced strong protective immunity in rabbits, comparable to the live attenuated vaccine but with an improved safety profile. Its inability to revert to virulence or transmit between animals makes it a promising candidate for large-scale use, especially in regions aiming to maintain disease-free status. Further evaluation in cattle under field conditions is warranted to confirm its long-term protective efficacy and potential for inclusion in control strategies.

ABSTRACTBackground and Aim:Bovine viral diarrhea virus (BVDV) is an economically significant pathogen of cattle, causing reproductive disorders, immunosuppression, and production losses worldwide. In Indonesia, BVDV-1a is among the most prevalent subgenotypes; however, field diagnosis still relies heavily on imported kits developed using non-local strains, which can lead to potential gaps in sensitivity and specificity. Locally tailored immunological reagents could enhance diagnostic accuracy and support future control strategies. This study aimed to produce and characterize polyclonal antisera against a local BVDV-1a isolate from Indonesia.Materials and Methods:Four New Zealand white rabbits were immunized with inactivated BVDV-1a antigen propagated in Madin–Darby bovine kidney (MDBK) cells. Booster immunizations were administered on days 14 and 28. Ten days after the final booster, sera were collected, pooled, and purified using ammonium sulfate precipitation and dialysis. Purified antisera were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Antibody titers were assessed using indirect enzyme-linked immunosorbent assay (ELISA), and specificity was validated by immunoperoxidase monolayer assay (IPMA) in infected MDBK cells.Results:The purification process yielded polyclonal antisera with a protein concentration of 40.33 mg/mL. SDS-PAGE revealed characteristic bands at approximately 53, 75, and 100 kDa, consistent with immunoglobulin components. Indirect ELISA showed strong antibody titers, with positive reactivity sustained up to 1:100. IPMA confirmed specific recognition of BVDV antigens, as infected MDBK cells exhibited distinct cytoplasmic staining, whereas uninfected controls remained negative.Conclusion:This preliminary study successfully generated high-titer polyclonal antisera against a local Indonesian BVDV-1a strain. The antibodies demonstrated robust reactivity and specificity, highlighting their potential utility as foundational reagents for developing regionally relevant diagnostic assays. While limited by a small sample size and pooled sera, these findings represent an important first step toward establishing locally adapted immunodiagnostic resources for BVDV. The development of local diagnostic tools not only strengthens veterinary disease surveillance but also safeguards livestock-dependent livelihoods, enhances food security, and reduces reliance on imported kits. Improved BVDV control in cattle contributes to One Health by minimizing economic losses, ensuring the safety of animal-derived food products, and reducing the risk of viral persistence in mixed livestock populations.

ABSTRACTCardiolipin (CL), a mitochondria‐specific non‐bilayer phospholipid, plays an essential role in the assembly and structural dynamics of the respiratory chain, affecting the membrane morphology and functional activity of inner mitochondria membrane (IMM)‐embedded proteins. CL forms CL‐rich domains on the IMM where negative curvature is required to increase the stability of cristae. However, CL constantly transitions between lamellar bilayer and non‐bilayer phases, such as inverted CL hexagonal phases and inverted CL micelles. Non‐bilayer phases of CL promote mitochondrial fission and fragmentation, transition of CL to the outer mitochondrial membrane (OMM), and mitophagy. In addition, non‐bilayer phases of CL can increase proton leakage, which leads to mitochondrial depolarization and decreased mitochondrial ATP synthesis. Thus, therapeutic applications for minimizing non‐bilayer CL phases should be able to optimize mitochondrial stability during various stresses. We have developed a novel, high‐density aromatic peptide (HDAP2) that targets CL and enhances the stability of CL within the lipid core of bilayers in CL‐POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) liposomes. We also demonstrated that HDAP2 interacts with inverted CL micelles, forming HDAP2‐CL micelles. This suggests that HDAP2 interacts with the non‐bilayer phase of CL, thereby stabilizing CL in the bilayer configuration. Scanning electron microscopy confirmed that HDAP2 assembles into spherical micelles approximately 1–3 μm in diameter. We have also demonstrated that this novel, water‐soluble peptide is cell‐permeable and targets mitochondria without causing cell toxicity. Furthermore, we used a well‐known mitochondrial toxicity model of serum starvation to demonstrate that HDAP2 significantly promoted cell survival in a dose‐dependent manner in mitochondria‐dependent Madin‐Darby bovine kidney (MDBK) cells. Importantly, HDAP2 preserved mitochondrial membrane potential and mitigated oxidative stress during serum deprivation. These protective effects suggest that, through its unique mechanism of action, HDAP2 can enhance cellular homeostasis, which would offer broad therapeutic potential for the prevention, recovery, and reversal of many acute and chronic disease conditions, including neurodegeneration, ischemia–reperfusion injury, and inflammation.

Understanding the optical properties of biological tissues is essential for optimizing light-based medical applications such as phototherapy, laser surgery, and biomedical imaging. In this study, the absorption coefficient (μₐ) and reduced scattering coefficient (μₛ′) of bovine muscle, heart, brain, kidney and fat tissues were determined at 635 nm using an integrating sphere system and Inverse Adding-Doubling (IAD) method. The experimental results were then used as input parameters for Monte Carlo (MCML) simulations to model light propagation and determine fluence rate distributions within the tissue models. The results demonstrated significant variations in optical properties across different types of tissue, with brain and fat tissues exhibiting lower penetration depths due to higher scattering coefficients. The findings align with previous literature while providing a more comprehensive evaluation through the integration of experimental and computational approaches.

Bovine viral diarrhea virus (BVDV) is commonly detected in biological products such as tissues and serum. This study identified BVDV contamination in a commercially available fetal bovine serum (FBS) sample. To determine whether the detected virus was infectious or merely genetic material, the FBS was inoculated into Madin-Darby bovine kidney (MDBK) cells. Following six serial passages, both indirect immunofluorescence assay results and electron microscopic visualization of viral particles confirmed the presence of infectious BVDV. The isolated strain, designated BI-2023, had a complete genome length of 12,273 nucleotides. Comparative sequence analysis showed that the 5′ untranslated region (UTR) and full genome of BI-2023 shared 87.4–97.1% and 92.4–95.2% nucleotide identity, respectively, with reference strains of BVDV1 (Pestivirus bovis) genotype 1b. Phylogenetic analyses based on the 5′UTR and whole genome placed BI-2023 within the genotype 1b cluster of Pestivirus bovis. Several amino acid substitutions were identified in the E2 and Erns proteins of the BI-2023 regions involved in immune evasion and viral secretion. This suggests this strain may represent a distinct variant within the genotype 1b group. These results highlight the critical need for routine viral screening in commercial FBS preparations.

In the South American Pampean Plain, high arsenic (As) concentrations are present in groundwater, exceeding, in some locations, 4 mg/L. Also, the concentrations of other elements, like selenium (Se), vanadium (V), molybdenum (Mo) or uranium (U), are elevated in this region. The elements could be taken up by crops, forages and animals, may be accumulated in tissues and have a negative impact on the health of the animals. Consumption of meat with elevated arsenic concentrations could be also a risk for human health. The aim of the present study was to determine the concentrations of As, Se, V, Mo and U in bovine liver and kidney samples and analyze the human health risk from its consumption. We found that the concentration of the elements present in liver and kidney samples were generally in accordance with the quality standards for food security set by national and international guidelines. Our results indicate that there is no health risk for consumers from the investigated elements, neither from kidneys nor from livers from the investigated regions.

The lumpy skin disease (LSD), caused by the lumpy skin disease virus (LSDV), represents an emerging infectious disease that poses substantial economic losses to the cattle industries in China. This study aimed to investigate the epidemiological characteristics of LSDV in Yunnan Province, Southwest China, from 2019 to 2023. A Taqman-probe-based real-time PCR (qPCR) assay was developed for the molecular detection of LSDV nucleotides. In total, 2495 samples were collected and tested using the established method. LSDV-positive samples were further analyzed by amplifying and sequencing the GPCR and p32 genes. In addition, Viral isolation was performed to explore the biological characterisitics of the isolate. The developed qPCR assay demonstrated high sensitivity, with a limit of detection of 4.83 copies/µL. Six samples (0.24%) tested positive for LSDV, all originating from skin scabs. Molecular and phylogenetic analyses of the GPCR and p32 genes revealed that these six LSDV strains were genetically related to strains previously reported from China, Russia, and Thailand. One LSDV strain, designated YUN-LSDV, was successfully isolated in primary sheep testicular (PST) cells; while the isolate exhibited insensitivity to Madin-Darby bovine kidney cells. Collectively, this study represents the first report on the epidemiological characteristics of LSDV in cattle in Yunnan Province and contributes to the development of effective prevention and control strategies for LSD.

Anthelmintic evaluation of triterpenes derived from conifers (family Pinaceae) on the digenean parasite Fasciola hepatica.

Mycoplasma bovis is a highly infectious pathogenic microorganism that causes various clinical signs in cattle, including pneumonia, arthritis, and mastitis, often resulting in significant economic losses. The adhesion of M. bovis to host cells is a pivotal step in the infection process, which is a complex process involving multiple pathogenic and host proteins. Molecules involved in M. bovis adhesion and colonization are widely recognized as important virulence factors and often implicated in the infection and pathogenesis. In this study, the Mbov_0510 gene of M. bovis was cloned, and the protein encoded by this gene was expressed and purified. The rabbit polyclonal antibody against this protein was also produced. This protein was shown to be a surface protein and to react with the M. bovis-positive serum. The ability of this protein to adhere to host cells was verified using embryonic bovine lung (EBL) and Madin-Darby bovine kidney (MDBK) cells. Furthermore, the adhesion function of this protein was found to be achieved through interaction with heparin on the host cell surface, and the key region of the protein involved in heparin binding was identified. The conservation of the protein encoded by the Mbov_0510 gene was also analyzed. The results of this study suggest that the protein encoded by the Mbov_0510 gene is a heparin-binding surface membrane protein of M. bovis associated with infection.IMPORTANCEMycoplasmas lack a cell wall, and the membrane proteins interacting with host cells play essential roles in their infection and proliferation processes. In this study, we identified a membrane protein encoded by Mycoplasma bovis that interacts with heparin on the surface of host cells. Heparin is widely distributed in various cells and tissues of the host and serves as a receptor for the infection and invasion of many pathogenic microorganisms. The ability of M. bovis to invade multiple tissues may be related to its heparin-binding capacity. The heparin-binding protein identified in this study is valuable for further research on the infection and invasion mechanisms of M. bovis.

Bisphenol P (BPP) is a recognized endocrine disruptor with detrimental effects on human health. This study aimed to evaluate BPP's cytotoxic and genotoxic effects on Madin-Darby bovine kidney (MDBK) cells by examining changes in gene expression, genotoxicity, and cell survival. Various assays were employed, including the MTT assay, comet assay, micronucleus assay, and real-time PCR for gene expression analysis. Among the series of concentrations (0.5µM, 1µM, 2µM, 4µM, 8µM, 16µM, 32µM, 64µM, 128µM, and 256µM), the treatment with 32µM BPP (LC50) resulted in 50% cell viability after 24h via MTT assay. The comet assay revealed a significant increase in comet tail length in BPP-treated groups compared to controls, indicating DNA with the highest damage at the 3xLC50/2 dose concentration of BPP. The frequency of micronuclei (MNi) was higher than binuclei. A significantly higher level of cytokinesis-block proliferation index (CBPI) was also observed at higher doses than in the negative control group. Gene expression analysis indicated increased levels of OGG1 and HPRT1 in BPP-treated cells compared to untreated controls, with a dose-dependent elevation in OGG1 expression involved in DNA damage response. This study concluded that BPP exhibits both cytotoxic and genotoxic effects on MDBK cells. Expression of DNA repair genes (OGG1, HPRT1) served as biomarkers for genotoxicity. Furthermore, it is recommended that additional studies on BPP's molecular toxicity and its cross-species effects should be explored further to combat its harmful effects.