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.

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.

Spatio-temporal distribution and international context of bovine viral diarrhoea virus genetic diversity in France.

Bovine viral diarrhoea (BVD) is one of the most economically damaging livestock enzootic diseases in the world. BVD aetiological agents are three pestiviruses (BVDV-1, -2 and HoBi-like pestivirus), which exhibit high genetic diversity and complex transmission cycles. This considerably hampers the management of the disease, which is why eradication plans have been implemented in several countries. In France, a national plan has been in place since 2019. Our understanding of its impact on the distribution of BVDV genotypes is limited by the availability of French genetic data. Here, we conducted a molecular epidemiology study to refine our knowledge of BVDV genetic diversity in France, characterise its international relationships, and analyse national spatio-temporal genotypic distribution. We collated 1037 BVDV-positive samples throughout France between 2011 and 2023, with a greater sampling effort in two major cattle production areas. We developed a high-throughput sequencing protocol which we used to complete the 5'UTR genotyping of this collection. We show that two main BVDV-1 genotypes, 1e and 1b, account for 88% of genotyped sequences. We also identified seven other BVDV-1 genotypes occurring at low frequencies and three BVDV-2 samples (genotype 2c). Phylogenetic analyses indicate different worldwide distribution patterns between the two main BVDV-1 genotypes. Their relative frequencies present no major changes in France since the 1990s and few variations at the national scale. We also found some degree of local spatial structuring in western France. Overall, our results demonstrate the potential of large-scale sequence-based surveillance to monitor changes in the epidemiological situation of enzootic diseases.

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.

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.

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.

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.

Estimation of the within-herd transmission rates of bovine viral diarrhoea virus in extensively grazed beef cattle herds.

Many research groups have developed mathematical models to simulate the dynamics of BVDV infections in cattle herds. However, most models use estimates for within-herd BVDV transmission rates that are either based on expert opinion or adapted from other dairy herd simulation models presented in the literature. There is currently little information on the transmission rates for BVDV in extensively grazed beef herds partly due to the logistical challenges in obtaining longitudinal data of individual animal's seroconversion, and it may not be appropriate to apply the same transmission rates from intensive dairy herds given the significant differences in herd demographics and management. To address this knowledge gap, we measured BVDV antibody levels in 15 replacement heifers in each of 75 New Zealand beef breeding farms after their first calving and again at pregnancy scanning or weaning to check for seroconversion. Among these, data from 9 farms were used to infer the within-herd BVDV transmission rate with an approximate Bayesian computation method. The most probable within-herd BVDV transmission rate was estimated as 0.11 per persistently infected (PI) animal per day with a 95% highest posterior density interval between 0.03 and 0.34. This suggests that BVDV transmission in extensively grazed beef herds is generally slower than in dairy herds where the transmission rate has been estimated at 0.50 per PI animal per day and therefore may not be sufficient to ensure that all susceptible breeding females gain adequate immunity to the virus before the risk period of early pregnancy for generating new PI calves.

Neighbourhood contacts and trade movements drive the regional spread of bovine viral diarrhoea virus (BVDV).

To explore the regional spread of endemic pathogens, investigations are required both at within and between population levels. The bovine viral diarrhoea virus (BVDV) is such a pathogen, spreading among cattle herds mainly due to trade movements and neighbourhood contacts, and causing an endemic disease with economic consequences. To assess the contribution of both transmission routes on BVDV regional and local spread, we developed an original epidemiological model combining data-driven and mechanistic approaches, accounting for heterogeneous within-herd dynamics, animal movements and neighbourhood contacts. Extensive simulations were performed over 9 years in an endemic context in a French region with high cattle density. The most uncertain model parameters were calibrated on summary statistics of epidemiological data, highlighting that neighbourhood contacts and within-herd transmission should be high. We showed that neighbourhood contacts and trade movements complementarily contribute to BVDV spread on a regional scale in endemically infected and densely populated areas, leading to intense fade-out/colonization events: neighbourhood contacts generate the vast majority of outbreaks (72%) but mostly in low immunity herds and correlated to a rather short presence of persistently infected animals (P); trade movements generate fewer infections but could affect herds with higher immunity and generate a prolonged presence of P. Both movements and neighbourhood contacts should be considered when designing control or eradication strategies for densely populated region.

The evaluation of an immunoperoxidase assay applicable in antiviral drug screening.

Bovine viral diarrhea virus (BVDV) fall into cytopathic (CP) and noncytopathic (NCP) biotypes, based on their ability to kill cultured cells. NCP-BVDV can not be titrated by conventional means as used for CP-BVDV, which has impeded the identification of antiviral drugs targeting NCP-BVDV virus strains. In this study, the application of an immunoperoxidase assay in the screening of antiviral drugs was tested using two known BVDV inhibitors, ribavirin and ammonium chloride (NH4Cl). Phospholipase C inhibitor U73122 was identified to affect BVDV infection by using this immunoperoxidase assay. In addition, the results of immunoperoxidase assay were validated by real-time PCR. Taken together, the immunoperoxidase assay is a useful and versatile method suitable for antiviral drug screening targeting NCP-BVDV.

Seroprevalence and risk factors associated with bovine viral diarrhea virus and bovine herpes virus-1 in Zanjan Province, Iran.

Bovine viral diarrhea virus (BVDV) and bovine herpes virus-1 (BHV-1) remain as the major pathogens with heavy economic consequences in Iran. The prevalence of antibodies against BVDV and BHV-1, the rate of BVDV persistently infected (PI) animals, and associated risk factors were evaluated in a cross-sectional study carried out in Zanjan Province, Northwest Iran, in December 2011. A total number of 562 cattle in 10 herds and five cities were randomly selected, and their serum samples were tested to detect antibodies to these viruses and also BVDV antigen-positive (PI) animals. The data were analyzed with Pearson's correlation coefficient, chi-square, and logistic regression test. In total, nine and eight of the selected herds were seropositive to BVDV and BHV-1, respectively. The overall seroprevalence of these infections were estimated at 28.6 and 10.7% for BVDV and BHV-1, respectively, and 0.53% of the samples were detected as persistently infected. Statistical analysis revealed that sex, age, and farming system are risk factors for both infections (P < 0.05), while breed was determined as a strong risk factor only for BVDV (P < 0.001). In addition, the present study certainly identifies that infection with BVDV is associated with infection to BHV-1 (OR = 4.52, 95% CI: 2.60-7.80; P Ë‚ 0.001). The results add our knowledge about the prevalence and associated risk factors of BVDV and BHV-1 in Iran and imply that the prophylactic and surveillance strategies need to be implemented to reduce the risk of spread of these viruses.

BVDV Npro protein mediates the BVDV induced immunosuppression through interaction with cellular S100A9 protein.

The innate immune response is a vital part of the body's antiviral defense system. The innate immune response is initiated by various receptor interactions, including danger associated molecular patterns (DAMPs). The S100A9 is a member of the DAMPs protein family and, is released by activated phagocytic cells such as neutrophils, monocytes, macrophages or endothelial cells, and S100A9 induces its effect through TLR4/MyD88 pathway. Bovine viral diarrhea virus (BVDV) is one of the major devastating disease in the cattle industry worldwide. It shows its effect through immunosuppression and develops persistent infection in calves born from infected cows. The current study revealed that BVDV potentially induced immunosuppression by the interaction of BVDV Npro protein with cellular S100A9 protein. The Inhibition of S100A9 protein expression by small interfering RNA (siRNA) enhanced the virus replication in infected cells. Overexpression of bovine S100A9 enhanced the ncpBVDV2a 1373 mediated Type-I interferon production. A co-immunoprecipitation experiment demonstrated a strong interaction between ncp BVDV2a 1373 Npro protein and cellular S100A9 protein. This suggested that BVDV Npro reduced the S100A9 protein availability/activity in infected cells, resulting in reduced Type-I interferon production. A further study of S100A9-BVDV interaction will be need for better understanding of BVDV pathophysiology.

Mycoplasma bovis co-infection with bovine viral diarrhea virus in bovine macrophages.

Several studies suggest that synergisms between Mycoplasma bovis and other microorganisms might exacerbate disease outcome of bovine mycoplasmosis. Screening several bovine cell types to assess their potential use as in vitro infection models for M. bovis, it was observed that a widely used cell line of bovine macrophages (Bomac cells) is in fact persistently infected with bovine viral diarrhea virus (BVDV). The cell line was first cured of this virus allowing comparative studies between both cell lines. Subsequently, uptake and co-culture of two M. bovis strains of different clonal complexes with Bomac cells contaminated with BVDV and in BVDV-free Bomac cells were assessed. Additionally, cell viability, cytotoxicity and induction of apoptosis after infection with M. bovis were evaluated. No differences in the levels of uptake and growth in co-culture were observed between the two Bomac cell types and both M. bovis strains. Cytotoxicity was increased after infection of BVDV-free cells with one of the two strains, while apoptotic cell death was slightly induced by this strain in both cell lines. Overall, the presence or absence of BVDV in Bomac cells did not grossly change the parameters tested upon infection with M. bovis. Nevertheless, this cell model is very useful when studying viral co-infections with bacteria and could also be used for multiple co-infections. Considering the broad contamination of cell cultures with BVDV, careful screening for this virus should routinely be performed as its presence might be relevant depending on the molecular mechanisms being investigated.

Complete genome sequences of a cytophatic/noncytophatic pair of bovine viral diarrhea virus subtype 1a viruses.

The complete genome sequences of both biotypes of a pair of bovine viral diarrhea viruses isolated from a bovid affected by mucosal disease were determined by next generation sequencing. The cytopathic virus possessed a 423-base insertion derived from bovine poly ubiquitin in the NS2/3 coding region and one nucleotide change. Both biotypes showed an additional glycosylation site in their N-terminus.

9-Aminoacridine-based agents impair the bovine viral diarrhea virus (BVDV) replication targeting the RNA-dependent RNA polymerase (RdRp).

Bovine viral diarrhea virus (BVDV) infection is still a plague that causes important livestock pandemics. Despite the availability of vaccines against BVDV, and the implementation of massive eradication or control programs, this virus still constitutes a serious agronomic burden. Therefore, the alternative approach to combat Pestivirus infections, based on the development of antiviral agents that specifically inhibit the replication of these viruses, is of preeminent actuality and importance. Capitalizing from a long-standing experience in antiviral drug design and development, in this work we present and characterize a series of small molecules based on the 9-aminoacridine scaffold that exhibit potent anti-BVDV activity coupled with low cytotoxicity. The relevant viral protein target - the RNA-dependent RNA polymerase - the binding mode, and the mechanism of action of these new antivirals have been determined by a combination of in vitro (i.e., enzymatic inhibition, isothermal titration calorimetry and site-directed mutagenesis assays) and computational experiments. The overall results obtained confirm that these acridine-based derivatives are promising compounds in the treatment of BVDV infections and, based on the reported structure-activity relationship, can be selected as a starting point for the design of a new generation of improved, safe and selective anti-BVDV agents.

Quantitative and rapid interference of bovine viral diarrhea virus BVDV/END- and BVDV/END+ strains.

Homologous interference in vitro is defined as the ability of primary viral infection to prevent secondary homologous superinfection. Non-cytopathic bovine viral diarrhea virus (ncp BVDV) has been classified according to the exaltation of Newcastle disease phenomenon (END) as END positive (E+) and END negative (E-) strains. Simultaneous inoculation of MDBK-SY cell monolayers with BVDV/E- virus and a three log higher amount of BVDV RK13/E+ virus, leads to acquisition of the BVDV/E- feature of blocking Newcastle disease virus (NDV) infection in cells. BVDV/E- strains, particularly at a high titre and MOI ≥1.25, can exert and impose their effects in BVDV/E+ infected cells; however, if BVDV/E- MOI is reduced to MOI below 0.625, the BVDV/E+ effect can be restored leading to cytopathic effects (CPE) induction by NDV reciprocal to the titre of the BVDV RK13/E+ strain. Moreover, blocking and prevention of induced CPE by NDV or vesicular stomatitis virus (VSV) occurs even when BVDV/E- superinfects primary BVDV/E+ infected cells, indicating a defective homologous interference between BVDV/E+ and BVDV/E- strains. Taken together, BVDV/E- strains have a strong competitive potency and mediate a fast acting (i.e. within 60 min) influence against BVDV/E+ activity. This may be relevant in vivo where BVDV/E- and BVDV/E+ combinations are frequently isolated from infected individuals.

Acute bovine viral diarrhea virus infection inhibits expression of interferon tau-stimulated genes in bovine endometrium.

Bovine viral diarrhea virus (BVDV) can evade host detection by downregulation of interferon signaling pathways. Infection of cows with noncytopathic (ncp) BVDV can cause early embryonic mortality. Upregulation of type I interferon stimulated genes (ISGs) by blastocyst-secreted interferon tau (IFNT) is a crucial component of the maternal recognition of pregnancy (MRP) in ruminants. This study investigated the potential of acute BVDV infection to disrupt MRP by modulating endometrial ISG expression. Endometrial cells from 10 BVDV-free cows were cultured and treated with 0 or 100 ng/ml IFNT for 24 h in the absence or presence of ncpBVDV infection to yield four treatment groups: CONT, ncpBVDV, IFNT, or ncpBVDV+IFNT. ncpBVDV infection alone only upregulated TRIM56, but reduced mRNA expression of ISG15, MX2, BST2, and the proinflammatory cytokine IL1B. As anticipated, IFNT treatment alone significantly increased expression of all 17 ISGs tested. In contrast to the limited effect of ncpBVDV alone, the virus markedly inhibited IFNT-stimulated expression of 15 ISGs tested (ISG15, HERC5, USP18, DDX58, IFIH1, IFIT1, IFIT3, BST2, MX1, MX2, RSAD2, OAS1Y, SAMD9, GBP4, and PLAC8), together with ISG15 secreted protein. Only TRIM56 and IFI27 expression was unaltered. IL1B expression was reduced by the combined treatment. These results indicate that acute ncpBVDV infection may decrease uterine immunity and lead to MRP failure through inhibition of IFNT-stimulated endometrial ISG production. This in turn could reduce fertility and predispose cows to uterine disease, while evasion of the normal uterine immune response by ncpBVDV may contribute to maintenance and spreading of this economically important disease.

Autophagy induced by bovine viral diarrhea virus infection counteracts apoptosis and innate immune activation.

Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle that plays a complex role in disease. There are two biotypes of BVDV: non-cytopathic (NCP) and cytopathic (CP). One strategy that has been used to treat or prevent virus-associated diseases is the modulation of autophagy, which is used by the innate immune system to defend against viral infection; however, at present, the interplay between autophagy and BVDV remains unclear. Madin-Darby bovine kidney cells stably expressing microtubule-associated protein 1 light chain 3B (LC3B) with green fluorescent protein (GFP) (GFP-LC3-MDBK cells) and autophagy-deficient MDBKs (shBCN1-MDBK cells) were constructed. Then MDBK, GFP-LC3-MDBK and shBCN1-MDBK cells were infected with CP or NCP BVDV strains. The LC3-II turnover rate was estimated by western blot, autophagosomes were visualized by confocal microscopy, and ultrastructural analysis was performed using electron microscopy. Autophagy flux was observed using chloroquine as an inhibitor of the autophagic process. The influence of autophagy on BVDV replication and release was investigated using virus titration, and its effect on cell viability was also studied. The effect of BVDV-induced autophagy on the survival of BVDV-infected host cell, cell apoptosis, and interferon (IFN) signalling was studied by flow cytometric analysis and quantitative RT-(q)PCR using shBCN1-MDBK cells. we found that infection with either CP or NCP BVDV strains induced steady-state autophagy in MDBK cells, as evident by the increased number of double- or single-membrane vesicles, the accumulation of GFP- microtubule-associated protein 1 light chain 3 (LC3) dots, and the conversion of LC3-I (cytosolic) to LC3-II (membrane-bound) forms. The complete autophagic process was verified by monitoring the LC3-II turnover ratio, lysosomal delivery, and proteolysis. In addition, we found that CP and NCP BVDV growth was inhibited in MDBK cells treated with high levels of an autophagy inducer or inhibitor, or in autophagy deficient-MDBK cells. Furthermore, our studies also suggested that CP and NCP BVDV infection in autophagy-knockdown MDBK cells increased apoptotic cell death and enhanced the expression of the mRNAs for IFN-α, Mx1, IFN-ß, and OAS-1 as compared with control MDBK cells. Our study provides strong evidence that BVDV infection induces autophagy, which facilitates BVDV replication in MDBK cells and impairs the innate immune response. These findings might help to illustrate the pathogenesis of persistent infection caused by BVDV.