PI3K/AKT mediated De novo fatty acid synthesis regulates RIG-1/MDA-5-dependent type I IFN responses in BVDV-infected CD8+T cells.

Bovine viral diarrhea virus (BVDV) has caused massive economic losses in the cattle business worldwide. Fatty acid synthase (FASN), a key enzyme of the fatty acid synthesis (FAS) pathway, has been shown to support virus replication. To investigate the role of fatty acids (FAs) in BVDV infection, we infected CD8+T lymphocytes obtained from healthy cattle with BVDV in vitro. During early cytopathic (CP) and noncytopathic (NCP) BVDV infection in CD8+ T cells, there is an increase in de novo lipid biosynthesis, resulting in elevated levels of free fatty acids (FFAs) and triglycerides (TG). BVDV infection promotes de novo lipid biosynthesis in a dose-dependent manner. Treatment with the FASN inhibitor C75 significantly reduces the phosphorylation of PI3K and AKT in BVDV-infected CD8+ T cells, while inhibition of PI3K with LY294002 decreases FASN expression. Both CP and NCP BVDV strains promote de novo fatty acid synthesis by activating the PI3K/AKT pathway. Further investigation shows that pharmacological inhibitors targeting FASN and PI3K concurrently reduce FFAs, TG levels, and ATP production, effectively inhibiting BVDV replication. Conversely, the in vitro supplementation of oleic acid (OA) to replace fatty acids successfully restored BVDV replication, underscoring the impact of abnormal de novo fatty acid metabolism on BVDV replication. Intriguingly, during BVDV infection of CD8+T cells, the use of FASN inhibitors prompted the production of IFN-α and IFN-ß, as well as the expression of interferon-stimulated genes (ISGs). Moreover, FASN inhibitors induce TBK-1 phosphorylation through the activation of RIG-1 and MDA-5, subsequently activating IRF-3 and ultimately enhancing the IFN-1 response. In conclusion, our study demonstrates that BVDV infection activates the PI3K/AKT pathway to boost de novo fatty acid synthesis, and inhibition of FASN suppresses BVDV replication by activating the RIG-1/MDA-5-dependent IFN response.

The gut microbiota contributes to the infection of bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide.

Mitochondria-mediated ferroptosis contributes to the inflammatory responses of bovine viral diarrhea virus (BVDV) in vitro.

Bovine viral diarrhea virus (BVDV) belongs to the family Flaviviridae and includes two biotypes in cell culture: cytopathic (CP) or non-cytopathic (NCP) effects. Ferroptosis is a non-apoptotic form of programmed cell death that contributes to inflammatory diseases. However, whether BVDV induces ferroptosis and the role of ferroptosis in viral infection remain unclear. Here, we provide evidence that both CP and NCP BVDV can induce ferroptosis in Madin-Darby bovine kidney cells at similar rate. Mechanistically, biotypes of BVDV infection downregulate cytoplasmic and mitochondrial GPX4 via Nrf2-GPX4 pathway, thereby resulting in lethal lipid peroxidation and promoting ferroptosis. In parallel, BVDV can degrade ferritin heavy chain and mitochondrial ferritin via NCOA4-mediated ferritinophagy to promote the accumulation of Fe2+ and initiate ferroptosis. Importantly, CP BVDV-induced ferroptosis is tightly associated with serious damage of mitochondria and hyperactivation of inflammatory responses. In contrast, mild or unapparent damage of mitochondria and slight inflammatory responses were detected in NCP BVDV-infected cells. More importantly, different mitophagy pathways in response to mitochondria damage by both biotypes of BVDV are involved in inflammatory responses. Overall, this study is the first to show that mitochondria may play key roles in mediating ferroptosis and inflammatory responses induced by biotypes of BVDV in vitro.IMPORTANCEBovine viral diarrhea virus (BVDV) threatens a wide range of domestic and wild cattle population worldwide. BVDV causes great economic loss in cattle industry through its immunosuppression and persistent infection. Despite extensive research, the mechanism underlying the pathogenesis of BVDV remains elusive. Our data provide the first direct evidence that mitochondria-mediated ferroptosis and mitophagy are involved in inflammatory responses in both biotypes of BVDV-infected cells. Importantly, we demonstrate that the different degrees of injury of mitochondria and inflammatory responses may attribute to different mitophagy pathways induced by biotypes of BVDV. Overall, our findings uncover the interaction between BVDV infection and mitochondria-mediated ferroptosis, which shed novel light on the physiological impacts of ferroptosis on the pathogenesis of BVDV infection, and provide a promising therapeutic strategy to treat this important infectious disease with a worldwide distribution.

Replication of standard bovine viral diarrhea strain OregonC24Va induces endoplasmic reticulum stress-mediated apoptosis of bovine trophoblast cells.

Bovine viral diarrhea (BVD) is a worldwide infectious disease caused by bovine viral diarrhea virus (BVDV) infection, which invades the placenta, causes abortion, produces immune tolerance and continuously infects calves, and causes huge economic losses to the cattle industry. The endoplasmic reticulum (ER) is an important organelle in cells, which is prone to ER stress after being stimulated by pathogens, thus activating the ER stress-related apoptosis. Studies have confirmed that BVDV can utilize the ER of its host to complete its own proliferation and stimulate ER stress to a certain extent. However, the role of ER stress in BVDV infecting bovine placental trophoblast cells (BTCs) and inducing apoptosis is still unclear. We are using the cytopathic strain of BVDV (OregonC24Va), which can cause apoptosis of BTCs, as a model system to determine how ER stress induced by BVDV affects placental toxicity. We show that OregonC24Va can infect BTCs and proliferate in it. With the proliferation of BVDV in BTCs, ER stress-related apoptosis is triggered. The ER stress inhibitor 4-PBA was used to inhibit the ER stress of BTCs, which not only inhibited the proliferation of BVDV, but also reduced the apoptosis of BTCs. The ER stress activator Tg can activate ER stress-related apoptosis, but the proliferation of BVDV does not change in BTCs. Therefore, BVDV utilizes the UPR of activated ER stress to promote the proliferation of BVDV in the early stage of infection, and activates the ER stress-related apoptosis of BTCs in the later stage with the virus proliferation to promote the cell apoptosis and further spread of the virus. Our research provides a new theoretical basis for exploring the placental infection and vertical transmission of BVDV.

Augmentation of 3ß-hydroxysteroid-Δ24 Reductase (DHCR24) Expression Induced by Bovine Viral Diarrhea Virus Infection Facilitates Viral Replication via Promoting Cholesterol Synthesis.

Bovine viral diarrhea virus (BVDV) is the etiologic agent of bovine viral diarrhea-mucosal disease, one of the most important viral diseases of cattle, leading to numerous losses to the cattle rearing industry worldwide. The pathogenicity of BVDV is extremely complex, and many underlying mechanisms involved in BVDV-host interactions are poorly understood, especially how BVDV utilizes host metabolism pathway for efficient viral replication and spread. In our previous study, using an integrative analysis of transcriptomics and proteomics, we found that DHCR24 (3ß-hydroxysteroid-Δ24 reductase), a key enzyme in regulating cholesterol synthesis, was significantly upregulated at both gene and protein levels in the BVDV-infected bovine cells, indicating that cholesterol is important for BVDV replication. In the present study, the effects of DHCR24-mediated cholesterol synthesis on BVDV replication was explored. Our results showed that overexpression of the DHCR24 effectively promoted cholesterol synthesis, as well as BVDV replication, while acute cholesterol depletion in the bovine cells by treating cells with methyl-ß-cyclodextrin (MßCD) obviously inhibited BVDV replication. In addition, knockdown of DHCR24 (gene silencing with siRNA targeting DHCR24, siDHCR24) or chemical inhibition (treating bovine cells with U18666A, an inhibitor of DHCR24 activity and cholesterol synthesis) significantly suppressed BVDV replication, whereas supplementation with exogenous cholesterol to the siDHCR24-transfected or U18666A-treated bovine cells remarkably restored viral replication. We further confirmed that BVDV nonstructural protein NS5A contributed to the augmentation of DHCR24 expression. Conclusively, augmentation of the DHCR24 induced by BVDV infection plays an important role in BVDV replication via promoting cholesterol production. IMPORTANCE Bovine viral diarrhea virus (BVDV), an important pathogen of cattle, is the causative agent of bovine viral diarrhea-mucosal disease, which causes extensive economic losses in both cow- and beef-rearing industry worldwide. The molecular interactions between BVDV and its host are extremely complex. In our previous study, we found that an essential host factor 3ß-hydroxysteroid-δ24 reductase (DHCR24), a key enzyme involved in cholesterol synthesis, was significantly upregulated at both gene and protein levels in BVDV-infected bovine cells. Here, we experimentally explored the function of the DHCR24-mediated cholesterol synthesis in regulating BVDV replication. We elucidated that the augmentation of the DHCR24 induced by BVDV infection played a significant role in viral replication via promoting cholesterol synthesis. Our data provide evidence that BVDV utilizes a host metabolism pathway to facilitate its replication and spread.

bta-miR-2904 inhibits bovine viral diarrhea virus replication by targeting viral-infection-induced autophagy via ATG13.

MicroRNAs (miRNAs) are endogenous small and noncoding RNA molecules (18-25 nt) that can regulate expression of their target genes post-transcriptionally. Previously, using high-throughput sequencing data obtained on a Solexa platform, we found that Bos taurus bta-miR-2904 (miR-2904) was significantly upregulated in Madin-Darby bovine kidney (MDBK) cells infected with bovine viral diarrhea virus (BVDV) strain NADL at 2, 6, and 18 h postinfection (hpi) compared to uninfected MDBK cells. Moreover, miR-2904 overexpression significantly reduced BVDV replication. However, the mechanism by which miR-2904 inhibits viral replication remains unclear. In this study, we used electron microscopy, laser confocal microscopy, dual-luciferase reporter analysis, real-time PCR, and Western blot assays to investigate the effect of the miR-2904 expression on BVDV NADL replication and virus-infection-induced autophagy. The results indicate that miR-2904 inhibits autophagy of MDBK cells by targeting autophagy-related gene 13 (ATG13), and overexpression of miR-2904 inhibited the replication of BVDV NADL.

Forsythiaside A Improves the Inhibitory Efficiency of Recombinant Protein Vaccines against Bovine Viral Diarrhea Virus Infection.

Bovine viral diarrhea virus (BVDV) is a critical animal pathogen that leads to cattle production losses associated with acute disease, immune dysregulation, reproductive failure, and respiratory disease. Due to the monotonous control technique and neglect of BVDV, increasing prevalence of BVDV has caused significant economic losses in the cattle industry worldwide. Therefore, novel anti-BVDV drugs are essential to prevent and control BVDV. Our previous studies have found that Forsythoside A (FTA) could inhibit the replication of BVDV via TRAF2-dependent CD28-4-1BB signaling in bovine peripheral blood mononuclear cells (PBMCs), but whether they can directly inhibit the BVDV remains unclear. Here, we further investigated the effects of FTA on BVDV and its underlying mechanisms of action. We found that FTA significantly inhibited the replication of BVDV in the MDBK cell directly. The results demonstrated that FTA could reduce the functional activation of Caspase-1 to inhibit the inflammatory response caused by BVDV infection and increase the expression of type I interferon (IFN-I) to clear the virus in vitro. The animal experiment was performed to evaluate the antiviral effect of FTA in vivo. Notably, after challenged with BVDV, mice with FTA + Erns-E2 protein displayed alleviated pathological damage and decreased the viral load in the spleen compared with mice inoculated with Erns-E2 protein. Furthermore, treatment with FTA enhanced body defense and delayed infection by the BVDV. Our results reveal that FTA suppresses BVDV replication both in vitro and in vivo and therefore shows promise as an anti-BVDV agent.

Research advances on interferon (IFN) response during BVDV infection.

Bovine viral diarrhea virus (BVDV) is an important pathogen responsible for significant economic loss to cattle. BVDV infection in pregnant cattle leads to fetal infection and reproductive losses, including early embryonic death, abortion, and stillbirth. Importantly, vaccinated heifers could not provide fetal protection against BVDV. It can be divided into two genotypes (BVDV-1 and BVDV-2) and two biotypes (cytopathic (CP) and non-cytopathic (NCP)). Infection with NCP-BVDV during gestation, the fetus becomes persistently infected (PI) and sheds BVDV throughout life, serving as the main source of infection for other cattle. BVDV potentially induces immunosuppression and aggravates bovine respiratory disease (BRD). Accordingly, BVDV infection results in a heterogeneous range of clinical signs and immune responses. Interferon (IFN) plays a vital role by mediating the innate immune response against antiviral infection through the Janus Kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. BVDV infection can reportedly exert variable degrees of influence on IFN response. Interestingly, reports have suggested that IFN can exert a significant inhibitory effect on various viruses. Human IFN-α was used to restrain BVDV in vitro. In this article, we summarized the latest researches on IFN response during BVDV infection.

ADAM17 Is an Essential Factor for the Infection of Bovine Cells with Pestiviruses.

The entry of BVDV into bovine cells was studied using CRIB cells (cells resistant to infection with bovine viral diarrhea virus [BVDV]) that have evolved from MDBK cells by a spontaneous loss of susceptibility to BVDV. Recently, larger genetic deletions were reported but no correlation of the affected genes and the resistance to BVDV infection could be established. The metalloprotease ADAM17 was reported as an essential attachment factor for the related classical swine fever virus (CSFV). To assess whether ADAM17 might be involved in the resistance of CRIB-1 cells to pestiviruses, we analyzed its expression in CRIB-1 and MDBK cells. While ADAM17 protein was detectable in MBDK cells, it was absent from CRIB-1 cells. No functional full-length ADAM17 mRNA could be detected in CRIB cells and genetic analysis revealed the presence of two defective alleles. Transcomplementation of functional ADAM17 derived from MDBK cells in CRIB-1 cells resulted in a nearly complete reversion of their resistance to pestiviral infection. Our results demonstrate that ADAM17 is a key cellular factor for the pestivirus resistance of CRIB-1 cells and establishes its essential role for a broader range of pestiviruses.

Different impact of bovine complement regulatory protein 46 (CD46bov) as a cellular receptor for members of the species Pestivirus H and Pestivirus G.

The genus Pestivirus within the family Flaviviridae comprises highly relevant animal pathogens such as bovine viral diarrhoea virus 1 and 2 (BVDV-1 and -2) classified into the two species Pestivirus A and Pestivirus B, respectively. First described in 2004, HoBi-like pestiviruses (HoBiPeV) represent emerging bovine pathogens that belong to a separate species (Pestivirus H), but share many similarities with BVDV-1 and -2. Additionally, two giraffe pestivirus (GPeV) strains both originating from Kenya represent another distinct species (Pestivirus G), whose members replicate very efficiently in bovine cells. In this study, we investigated the role of bovine complement regulatory protein 46 (CD46bov), the receptor of BVDV-1 and -2, in the entry of HoBiPeV and GPeV. For this purpose, bovine CD46-knockout and CD46-rescue cell lines were generated by CRISPR/Cas9 technology and subsequent trans-complementation, respectively. Our results provide strong evidence that the impact of CD46bov differs between viruses belonging to Pestivirus H and viruses representing Pestivirus G: CD46bov revealed to be a major cellular entry factor for HoBiPeV strain HaVi-20. In contrast, GPeV strain PG-2 presented as largely independent of CD46bov, suggesting a different entry mechanism involving other molecular determinants which remain to be identified. In addition, we demonstrated that, similar to BVDV-1 and -2, virus isolates of both Pestivirus H and Pestivirus G are able to adapt to cell culture conditions by using heparan sulfate to enter the host cell. In conclusion, our findings show that different bovine pestiviruses use diverse mechanisms of host cell entry.

Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance.

Bovine viral diarrhea virus's (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Novel Potent IFN-γ-Inducing CD8+ T Cell Epitopes Conserved among Diverse Bovine Viral Diarrhea Virus Strains.

Studies of immune responses elicited by bovine viral diarrhea virus (BVDV) vaccines have primarily focused on the characterization of neutralizing B cell and CD4+ T cell epitopes. Despite the availability of commercial vaccines for decades, BVDV prevalence in cattle has remained largely unaffected. There is limited knowledge regarding the role of BVDV-specific CD8+ T cells in immune protection, and indirect evidence suggests that they play a crucial role during BVDV infection. In this study, the presence of BVDV-specific CD8+ T cells that are highly cross-reactive in cattle was demonstrated. Most importantly, novel potent IFN-γ-inducing CD8+ T cell epitopes were identified from different regions of BVDV polyprotein. Eight CD8+ T cell epitopes were identified from the following structural BVDV Ags: Erns, E1, and E2 glycoproteins. In addition, from nonstructural BVDV Ags Npro, NS2-3, NS4A-B, and NS5A-B, 20 CD8+ T cell epitopes were identified. The majority of these IFN-γ-inducing CD8+ T cell epitopes were found to be highly conserved among more than 200 strains from BVDV-1 and -2 genotypes. These conserved epitopes were also validated as cross-reactive because they induced high recall IFN-γ+CD8+ T cell responses ex vivo in purified bovine CD8+ T cells isolated from BVDV-1- and -2-immunized cattle. Altogether, 28 bovine MHC class I-binding epitopes were identified from key BVDV Ags that can elicit broadly reactive CD8+ T cells against diverse BVDV strains. The data presented in this study will lay the groundwork for the development of a contemporary CD8+ T cell-based BVDV vaccine capable of addressing BVDV heterogeneity more effectively than current vaccines.

Quantitative risk assessment of introduction of BVDV and BoHV-1 through indirect contacts based on implemented biosecurity measures in dairy farms of Spain.

A stochastic quantitative risk assessment model was developed to estimate the annual probability of introduction of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) on 127 dairy farms through indirect contacts. Vehicles transporting calves, cattle to slaughterhouse, dead animals, and mixture of feed, as well as visits by veterinarians and hoof trimmers, farm workers and contacts with neighbors were considered in the model. Data from biosecurity questionnaires of each farm, scientific literature and expert opinion from field veterinarians, animal vehicle drivers, hoof trimmers and personnel from rendering transport companies were used to estimate values for input parameters. Results showed that the annual probability of introducing BVDV or BoHV-1 through indirect contacts was very heterogeneous. The overall distribution of median values for each farm ranged from 0.5 to 14.6% and from 1.0 to 24.9% for BVDV and BoHV-1, respectively. The model identified that providing protective clothing and boots to visits, not allowing the animal vehicle driver to come into contact with animals present on the farm and ensuring that calf vehicles arrived empty, were the measures with the highest impact on the probability of infection for most farms. This model could be a useful tool to show the impact of the measures to farmers and veterinarians, thus increasing their awareness on biosecurity. In addition, it could support decision making on which measures should be prioritized in dairy cattle herds to reduce the probability of introduction of diseases.

Modelling the economics of bovine viral diarrhoea virus control in pastoral dairy and beef cattle herds.

Bovine viral diarrhoea virus (BVDV) is a prevalent pathogen in the New Zealand cattle industries, yet few studies to date have evaluated the economics of BVDV in pastoral dairy and beef herds to help inform management decisions. To address this knowledge gap, we developed stochastic individual-based simulation models to represent the transmission dynamics of BVDV in typical spring-calving dairy and beef farms in New Zealand. The models conservatively estimated the direct losses due to a BVDV outbreak at NZ$ 22.22 and NZ$ 41.19 per mixed-age cow per year for a naïve dairy and beef farm, respectively, over a 5-year period. The greatest economic impacts for the dairy farm occurred when persistently infected replacement heifers joined the lactating cow group and caused transient infection of cows to drop in milk production, whereas the greatest impacts for the beef farm was through the loss of fattening stock for sale due to lowered pregnancy rates. Various combinations of diagnostic testing, vaccination, and biosecurity measures were then explored to evaluate the cost-efficiency of different strategies for controlling BVDV at the farm-level. Providing farmers with the estimates of economic impacts of BVDV in their herds may further encourage the uptake of control measures, but close collaboration with a veterinarian to determine the optimal strategy for their unique farm circumstances is still required.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy.

BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Herd-level factors associated with detection of calves persistently infected with bovine viral diarrhoea virus (BVDV) in Irish cattle herds with negative herd status (NHS) during 2017.

A compulsory national BVD eradication programme commenced in Ireland in 2013. Since then considerable progress has been made, with the animal-level prevalence of calves born persistently infected (PI) falling from 0.67 % in 2013 to 0.06 % in 2018. The herd-level prevalence fell from 11.3 % in 2013 to 1.1 % in 2018. In the Irish programme, herds in which all animals have a known negative status and which have not contained any PI animals for 12 months or more are assigned a negative herd status (NHS). While considerable progress towards eradication has been made, PI calves have been identified in a small proportion of herds that had previously been assigned NHS. Given this context, a case-control study was conducted to investigate potential risk factors associated with loss of NHS in 2017. 546 herds which had NHS on 1 January 2017 and lost that status during 2017 (case herds) were matched with 2191 herds (control herds) that retained their NHS status throughout 2017. Previous history of BVD infection, herd size, herd expansion, the purchase of cattle including potential Trojan cattle and the density of BVD infection within 10 km of the herd emerged as significant factors in a multivariable logistic regression model. This work adds to the evidence base in support of the BVD eradication programme, particularly establishing why BVD re-emerged in herds which had been free of BVD for at least the previous 12 months prior to the identification of a BVD positive calf. This information will be especially important in the context of identifying herds which may be more likely to contain BVD positive animals once the programme moves to herd-based serology status for trading purposes in the post-eradication phase.

PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4+ and CD8+ T Cells During BVDV Infection in vitro.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocyte (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the immunomodulatory effects of PD-1 pathway on major PBL subsets are unclear and their underlying molecular mechanisms need to be further studied. Therefore, in this study, we examined PD-1 expression in bovine PBL subsets after BVDV infection in vitro and analyzed the effects of PD-1 blockade on the apoptosis and proliferation of CD4+ and CD8+ T cells and expression of PD-1 downstream signaling molecules. The results showed that PD-1 expression was enhanced on CD4+ and CD8+ T cells, but not on CD21+ B cells after cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection in vitro and PD-1 blockade significantly reduced the apoptosis of CD4+ and CD8+ T cells after these two strains infection. Remarkably, PD-1 blockade significantly increased the proliferation of CD4+ and CD8+ T cells after CP BVDV infection, but only significantly increased the proliferation of CD4+ T cells after NCP BVDV infection. In addition, we confirmed that PD-1-mediated PI3K/Akt/mTOR, caspase 9/caspase 3 and ERK pathways are involved in regulating the apoptosis and proliferation of CD4+ and CD8+ T cells during BVDV infection in vitro. Notably, ERK is involved in the regulation mechanism PD-1 mediated only when the cells are infected with CP BVDV. Our findings provide a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

A description and qualitative comparison of the elements of heterogeneous bovine viral diarrhea control programs that influence confidence of freedom.

For endemic infections in cattle that are not regulated at the European Union level, such as bovine viral diarrhea virus (BVDV), European Member States have implemented control or eradication programs (CEP) tailored to their specific situations. Different methods are used to assign infection-free status in CEP; therefore, the confidence of freedom associated with the "free" status generated by different CEP are difficult to compare, creating problems for the safe trade of cattle between territories. Safe trade would be facilitated with an output-based framework that enables a transparent and standardized comparison of confidence of freedom for CEP across herds, regions, or countries. The current paper represents the first step toward development of such a framework by seeking to describe and qualitatively compare elements of CEP that contribute to confidence of freedom. For this work, BVDV was used as a case study. We qualitatively compared heterogeneous BVDV CEP in 6 European countries: Germany, France, Ireland, the Netherlands, Sweden, and Scotland. Information about BVDV CEP that were in place in 2017 and factors influencing the risk of introduction and transmission of BVDV (the context) were collected using an existing tool, with modifications to collect information about aspects of control and context. For the 6 participating countries, we ranked all individual elements of the CEP and their contexts that could influence the probability that cattle from a herd categorized as BVDV-free are truly free from infection. Many differences in the context and design of BVDV CEP were found. As examples, CEP were either mandatory or voluntary, resulting in variation in risks from neighboring herds, and risk factors such as cattle density and the number of imported cattle varied greatly between territories. Differences were also found in both testing protocols and definitions of freedom from disease. The observed heterogeneity in both the context and CEP design will create difficulties when comparing different CEP in terms of confidence of freedom from infection. These results highlight the need for a standardized practical methodology to objectively and quantitatively determine confidence of freedom resulting from different CEP around the world.

Seroprevalence and risk factors assessment of the three main infectious agents associated with abortion in dairy cattle in Isfahan province, Iran.

This study aimed to determine the seroprevalence and identify the risk factors associated with Neospora caninum, Bovine herpesvirus type 1 (BHV-1), and Bovine viral diarrhea virus (BVDV) infection on industrial Holstein dairy cattle farms in Isfahan province, Central Iran. Blood samples were taken from 216 apparently healthy cattle from 16 randomly selected Holstein dairy farms in the North, South, East, and West of Isfahan in the summer of 2017. The antibodies to N. caninum, BHV-1, and BVDV were detected using a commercially available ELISA kit. The overall seroprevalence for N. caninum, BHV-1, and BVDV was 19%, 72.2%, and 52.8%, respectively. The significant major risk factors of BHV-1 in cattle were identified as farm direction, age groups, parity, and milk yield by the univariate analysis (p < 0.05). The significant major risk factors of BVDV in cattle were identified as age groups, parity, milk yield, and stage of pregnancy (p < 0.05). The only significant major risk factor of N. caninum was farm direction (p < 0.05). A significant association of concurrent infection with BVDV and BHV-1 has shown in the current study (p < 0.05). This study is the first to report the risk factors for N. caninum, BHV-1, and BVDV infection in the central part of Iran and allows us to conclude that these agents are widely distributed in this region.

Real Time Analysis of Bovine Viral Diarrhea Virus (BVDV) Infection and Its Dependence on Bovine CD46.

Virus attachment and entry is a complex interplay of viral and cellular interaction partners. Employing bovine viral diarrhea virus (BVDV) encoding an mCherry-E2 fusion protein (BVDVE2-mCherry), being the first genetically labelled member of the family Flaviviridae applicable for the analysis of virus particles, the early events of infection-attachment, particle surface transport, and endocytosis-were monitored to better understand the mechanisms underlying virus entry and their dependence on the virus receptor, bovine CD46. The analysis of 801 tracks on the surface of SK6 cells inducibly expressing fluorophore labelled bovine CD46 (CD46fluo) demonstrated the presence of directed, diffusive, and confined motion. 26 entry events could be identified, with the majority being associated with a CD46fluo positive structure during endocytosis and occurring more than 20 min after virus addition. Deletion of the CD46fluo E2 binding domain (CD46fluo∆E2bind) did not affect the types of motions observed on the cell surface but resulted in a decreased number of observable entry events (2 out of 1081 tracks). Mean squared displacement analysis revealed a significantly increased velocity of particle transport for directed motions on CD46fluo∆E2bind expressing cells in comparison to CD46fluo. These results indicate that the presence of bovine CD46 is only affecting the speed of directed transport, but otherwise not influencing BVDV cell surface motility. Instead, bovine CD46 seems to be an important factor during uptake, suggesting the presence of additional cellular proteins interacting with the virus which are able to support its transport on the virus surface.