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In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2–96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5′-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56–61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.

Bovine viral vaccines contain both live or inactivated/killed formulations, but few studies have evaluated the impact of vaccinating with either live or killed antigens and re-vaccinating with the reciprocal. Commercial dairy heifers were utilized for the study and randomly assigned to three treatment groups. Treatment groups received a commercially available modified-live viral (MLV) vaccine containing BVDV and were revaccinated with a commercially available killed viral (KV) vaccine containing BVDV, another group received the same KV vaccine and was revaccinated with the same MLV vaccine, and yet another group served as negative controls and did not receive any viral vaccines. Heifers in KV/MLV had higher virus neutralizing titers (VNT) at the end of the vaccination period than heifers in MLV/KV and control groups. The frequency of IFN-γ mRNA positive CD4+, CD8+, and CD335+ populations, as well as increased mean fluorescent intensity of CD25+ cells was increased for the MLV/KV heifers as compared to KV/MLV and controls. The data from this study would suggest that differences in initial antigen presentation such as live versus killed could augment CMI and humoral responses and could be useful in determining vaccination programs for optimizing protective responses, which is critical for promoting lifetime immunity.

Background and Aim: Globally, pestiviruses are among the most economically important viral pathogens of livestock. The genus Pestivirus comprises four species, including bovine viral diarrhea virus type 1 and 2 (BVDV-1 and BVDV-2), which infect cattle, border disease virus and classical swine fever virus which infect small ruminants and pigs, respectively. Accumulating evidence suggests that pestiviruses are no longer species-specific, creating new challenges for disease control. In Indonesia, investigations related to pestiviruses remain focused on cattle as the primary host and no research has been conducted on small ruminants (sheep and goats). Therefore, the present study aimed to study the possible occurrence of pestivirus (BVDV or BVD) infections in small ruminants in Indonesia, particularly in Central Java. Materials and Methods: We used 46 blood samples consisting of 26 sheep's blood and 20 goat's blood. Samples were selected from 247 small ruminant blood collected between July and October 2020 in Central Java, Indonesia, which met the following criteria: Female, local species, approximately 1-2 years old, never been pregnant, raised in the backyard, and had no close contact with cattle in either shelter or grazing area. We tested plasma samples from sheep and goats using competitive antibody enzyme-linked immunosorbent assay to detect specific antibodies against pestivirus followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis for all positive samples to differentiate the species of pestivirus. Results: Two of the 20 samples collected from goats were positive for pestivirus at the serological and molecular levels, whereas 2 of 26 samples collected from sheep were doubtful but tested negative by RT-PCR. The genotyping test results obtained using nested PCR revealed that the positive samples collected from goats had a BVDV-1 genotype. Conclusion: The results of the present study demonstrated that BVDV-1 can infect species other than bovines, in Central Java, Indonesia. Further studies involving a larger number of samples are required to: (1) Determine the actual seroprevalence of pestiviruses in small ruminants and (2) Determine the potency of small ruminants as reservoirs for pestiviruses, both of which are important for the identification of the appropriate control program for pestiviruses in Indonesia.

Background Bovine viral diarrhea virus (BVDV) is a major economic disease that has been spread in most countries. In addition to vaccination, one of the main ways to control the disease and prevent it from spreading is to detect and cull infected animals, especially those with persistent infection (PI). We developed and compared two colorimetric biosensor assays based on probe-modified gold nanoparticles (AuNPs) to detect BVDV. Specific probes were designed to detect the 5′ untranslated region of BVDV-RNA. The thiolated probes were immobilized on the surface of the AuNPs. Two methods of cross-linking (CL) and non-crosslinking (NCL) probe-AuNPs hybridization were developed and compared. Results The hybridization of positive targets with the two probe-AuNPs formed a polymeric network between the AuNPs which led to the aggregation of nanoparticles and color change from red to blue. Alternatively, in the NCL mode, the hybridization of complementary targets with the probe-AuNPs resulted in the increased electrostatic repulsion in nanoparticles and the increased stabilization against salt-induced aggregation. The CL and NCL assays had detection limits of 6.83 and 44.36 ng/reaction, respectively. Conclusion The CL assay showed a higher sensitivity and specificity; in contrast, the NCL assay did not require optimizing and controlling of hybridization temperature and showed a higher response speed. However, both the developed methods are cost-effective and easy to perform and also could be implemented on-site or in local laboratories in low-resource countries.

There has been a slight shift in animal disease surveillance from input-based standards towards output-based standards, meaning that it is not prescribed what needs to be done, but rather what must be achieved. An objective and standardized assessment of the outputs of differently designed control programmes (CPs) is needed and therefore the aim of this thesis was to develop and test a generic output-based framework to determine the probability of freedom from Bovine Viral Diarrhea virus (BVDV) infection in cattle herds. The in this project developed STOC free framework (Surveillance analysis Tool for Outcome-based Comparison of the confidence of FREEdom), consists of a data collection tool (STOC free DATA) and a model to estimate herd-level freedom from infection (STOC free MODEL). Elements of BVDV CPs in six European countries that contribute to confidence of freedom from BVDV were described and qualitatively compared. Many differences in the context and design of BVDV CPs were found. CPs were either mandatory or voluntary, resulting in variation in risks from (in)direct animal contacts. Risk factors such as cattle density and the number of imported cattle varied greatly between countries. Differences were also found in testing protocols and definitions of freedom from infection. A systematic search and meta-analysis of risk factors for the presence of BVDV in cattle herds in Europe was performed to obtain generic estimates that could be used as default input data for STOC free MODEL. Data from 18 observational studies were analyzed by a random effects meta-analysis and significant higher odds were found for dairy herds compared to beef herds, for larger herds, for herds that participated in shows or markets, for herds that introduced cattle and for herds that had contact with cattle of other herds at pasture. STOC free DATA was developed to evaluate data availability and quality and to collect actual input data required for STOC free MODEL. Initially, the tool was developed for assessment of freedom from BVDV in six Western European countries and was then further generalized to enable inclusion of data for other cattle diseases and for use throughout Europe. STOC free DATA includes a wide range of variables that could reasonably influence confidence of freedom from infection, including those relating to cattle demographics, risk factors for introduction and characteristics of CPs. STOC free MODEL, a Bayesian Hidden Markov model, was applied to BVDV field data from CPs based on ear notch samples from newborn calves in four study regions to estimate the probability of herd level freedom from BVDV. The probability of freedom from BVDV for dairy herds that are free according to each study region’s CP was predicted to be very high ranging from 0.98 to 1.00, regardless of the use of default or country-specific priors. The STOC free model can be used to evaluate and improve BVDV CPs and to determine whether they comply with output-based regulations of the EU. The tool is currently evaluated by other research groups for application to other infectious cattle diseases such as Johne’s disease and salmonella.

There has been a slight shift in animal disease surveillance from input-based standards towards output-based standards, meaning that it is not prescribed what needs to be done, but rather what must be achieved. An objective and standardized assessment of the outputs of differently designed control programmes (CPs) is needed and therefore the aim of this thesis was to develop and test a generic output-based framework to determine the probability of freedom from Bovine Viral Diarrhea virus (BVDV) infection in cattle herds. The in this project developed STOC free framework (Surveillance analysis Tool for Outcome-based Comparison of the confidence of FREEdom), consists of a data collection tool (STOC free DATA) and a model to estimate herd-level freedom from infection (STOC free MODEL). Elements of BVDV CPs in six European countries that contribute to confidence of freedom from BVDV were described and qualitatively compared. Many differences in the context and design of BVDV CPs were found. CPs were either mandatory or voluntary, resulting in variation in risks from (in)direct animal contacts. Risk factors such as cattle density and the number of imported cattle varied greatly between countries. Differences were also found in testing protocols and definitions of freedom from infection. A systematic search and meta-analysis of risk factors for the presence of BVDV in cattle herds in Europe was performed to obtain generic estimates that could be used as default input data for STOC free MODEL. Data from 18 observational studies were analyzed by a random effects meta-analysis and significant higher odds were found for dairy herds compared to beef herds, for larger herds, for herds that participated in shows or markets, for herds that introduced cattle and for herds that had contact with cattle of other herds at pasture. STOC free DATA was developed to evaluate data availability and quality and to collect actual input data required for STOC free MODEL. Initially, the tool was developed for assessment of freedom from BVDV in six Western European countries and was then further generalized to enable inclusion of data for other cattle diseases and for use throughout Europe. STOC free DATA includes a wide range of variables that could reasonably influence confidence of freedom from infection, including those relating to cattle demographics, risk factors for introduction and characteristics of CPs. STOC free MODEL, a Bayesian Hidden Markov model, was applied to BVDV field data from CPs based on ear notch samples from newborn calves in four study regions to estimate the probability of herd level freedom from BVDV. The probability of freedom from BVDV for dairy herds that are free according to each study region’s CP was predicted to be very high ranging from 0.98 to 1.00, regardless of the use of default or country-specific priors. The STOC free model can be used to evaluate and improve BVDV CPs and to determine whether they comply with output-based regulations of the EU. The tool is currently evaluated by other research groups for application to other infectious cattle diseases such as Johne’s disease and salmonella.

There has been a slight shift in animal disease surveillance from input-based standards towards output-based standards, meaning that it is not prescribed what needs to be done, but rather what must be achieved. An objective and standardized assessment of the outputs of differently designed control programmes (CPs) is needed and therefore the aim of this thesis was to develop and test a generic output-based framework to determine the probability of freedom from Bovine Viral Diarrhea virus (BVDV) infection in cattle herds. The in this project developed STOC free framework (Surveillance analysis Tool for Outcome-based Comparison of the confidence of FREEdom), consists of a data collection tool (STOC free DATA) and a model to estimate herd-level freedom from infection (STOC free MODEL). Elements of BVDV CPs in six European countries that contribute to confidence of freedom from BVDV were described and qualitatively compared. Many differences in the context and design of BVDV CPs were found. CPs were either mandatory or voluntary, resulting in variation in risks from (in)direct animal contacts. Risk factors such as cattle density and the number of imported cattle varied greatly between countries. Differences were also found in testing protocols and definitions of freedom from infection. A systematic search and meta-analysis of risk factors for the presence of BVDV in cattle herds in Europe was performed to obtain generic estimates that could be used as default input data for STOC free MODEL. Data from 18 observational studies were analyzed by a random effects meta-analysis and significant higher odds were found for dairy herds compared to beef herds, for larger herds, for herds that participated in shows or markets, for herds that introduced cattle and for herds that had contact with cattle of other herds at pasture. STOC free DATA was developed to evaluate data availability and quality and to collect actual input data required for STOC free MODEL. Initially, the tool was developed for assessment of freedom from BVDV in six Western European countries and was then further generalized to enable inclusion of data for other cattle diseases and for use throughout Europe. STOC free DATA includes a wide range of variables that could reasonably influence confidence of freedom from infection, including those relating to cattle demographics, risk factors for introduction and characteristics of CPs. STOC free MODEL, a Bayesian Hidden Markov model, was applied to BVDV field data from CPs based on ear notch samples from newborn calves in four study regions to estimate the probability of herd level freedom from BVDV. The probability of freedom from BVDV for dairy herds that are free according to each study region’s CP was predicted to be very high ranging from 0.98 to 1.00, regardless of the use of default or country-specific priors. The STOC free model can be used to evaluate and improve BVDV CPs and to determine whether they comply with output-based regulations of the EU. The tool is currently evaluated by other research groups for application to other infectious cattle diseases such as Johne’s disease and salmonella.

Reproductive tests in cattle are of great economic importance, given the impact it can have on the production system and may be caused by agents. Neospora caninum and Bovine Viral Diarrhea virus (BVDV) are considered of great importance as reproductive and should be considered responsible for keeping animals persistently infected. The present study included 479 calf serum samples for export in the state of Rio Grande do Sul (RS). All samples were screened for BVDV by an ELISA antigen. BVDV antigen-positive ELISA samples were isolated from BVDV in cell culture. An indirect immunofluorescence (IFT) technique was used to detect anti-N. caninum antibodies. Of the 479 export-treated serum samples, 361 were positive for BVDV antigens by ELISA and/or viral isolation test (361/479-75.36%), and 109 IFT-positive samples for N. caninum (109/479-22.75%). Despite detection of antibodies anti-N. caninum did not differ statistically between naturally infected BVDV and non-BVDV infected animals suggesting that there is no interference of BVDV infection on infection or detection rate of animals with N. caninum, positive animals in viral isolation and high DO in BVDV-Ag ELISA. may present active disease and consequent immunosuppression, contributing to a potential reactivation of N. caninum.

Background and Aim: Bovine viral diarrhea virus (BVDV) is present in most cattle-raising countries around the world, and it has a negative economic impact in cattle herds. In Colombia, previous studies have estimated the prevalence of BVDV in specific locations. The aim of this study was to estimate the prevalence of BVDV in cattle herds located at several municipalities across the country and to identify the associated risk and protective factors. Materials and Methods: A cross-sectional study was carried out to investigate the prevalence of BVDV in Colombian cattle populations at farm and animal-levels. A total of 387 herds and 8110 animals located in seven different departments were included in this study. Results: An animal- and farm-level prevalence of 36% and 69%, respectively, were estimated. A high variation for the farm-level prevalence was found among the municipalities studied. Moreover, seropositive cattle to the infectious bovine rhinotracheitis virus (odds ratio (OR)=2.38, p=0.0479) and Neospora caninum (OR=3.15, p=0.0122) were more likely to be seropositive for BVDV, while the practice of burning dead animals at the farm was identified as a protective factor (OR=0.17, p=0.014). Conclusion: The prevalence of BVDV varied more at farm-level compared to animal-level. Two risk factors and one protective factor were identified. The results of the current study are essential to understand the epidemiology of BVDV in Colombia, and to formulate strategies in the region to mitigate the impact of this virus on the productive and reproductive indicators of cattle farms at the regional level.

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.

Background Bovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures. Bovine enterovirus (BEV) has a broad host range with low virulence and is a good candidate as a viral vaccine vector. In this study, we explored new insertion sites for the expression of exogenous genes in BEV, and developed a recombinant infectious cDNA clone for BEV BJ101 strain expressing BVDV E0 protein. Methods A recognition site for the viral proteinase 3Cpro was inserted in the GpBSK-BEV plasmid at the 2C/3A junction by overlapping PCR. Subsequently, the optimized full-length BVDV E0 gene was inserted to obtain the recombinant infectious plasmid GpBSK-BEV-E0. The rescued recombinant virus was obtained by transfection with linearized plasmid. Expression of BVDV E0 in the recombinant virus was confirmed by PCR, western blotting, and immunofluorescence analysis, and the genetic stability was tested in MDBK cells over 10 passages. We further tested the ability of the recombinant virus to induce an antibody response in mice infected with BVDV and immunized them with the recombinant virus and parental strain. Results The rescued recombinant virus rBEV-E0 was identified and confirmed by western blot and indirect immunofluorescence. The sequencing results showed that the recombinant virus remained stable for 10 passages without genetic changes. There was also no significant difference in growth dynamics and plaque morphology between the recombinant virus and parental virus. Mice infected with both recombinant and parental viruses produced antibodies against BEV VP1, while the recombinant virus also induced antibodies against BVDV E0. Conclusion A new insertion site in the BEV vector can be used for the prevention and control of both BEV and BVDV, providing a useful tool for future research on the development of viral vector vaccines.

A quantitative risk assessment model was developed to estimate the annual probability of introducing bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) at the farm level through animal movements. Data from 2017 official animal movements, biosecurity questionnaires, scientific literature, and expert opinion from field veterinarians were taken into consideration for model input parameters. Purchasing or introducing cattle, rearing replacement heifers offsite, showing cattle at competitions, sharing transport vehicles with other herds, and transporting cattle in vehicles that have not been cleaned and disinfected were considered in the model. The annual probability of introducing BVDV or BoHV-1 through infected animals was very heterogeneous between farms. The median likelihoods of BVDV and BoHV-1introduction were 12 and 9%, respectively. Farms that purchased cattle from within their region (i.e., local movements) and shared transport with other farms had a higher probability for BVDV and BoHV-1 introduction. This model can be a useful tool to support decision-making on biosecurity measures that should be prioritized to reduce the probability of introduction of these 2 diseases in dairy herds This work was supported by a Grant from the Ministerio de Ciencia e Innovación of Spain (AGL2016-77269-C2-1-R and AGL2016-77269-C2-2-R). The Universidad de Nariño (Pasto, Colombia) provided funding to the first author (BBB) SI

Pestivirus infections are important in the livestock industries, with infection occurring in cattle, sheep and pigs. The Pestivirus genus of the family Flaviviridae, includes four recognized species: bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2), border disease virus (BDV), and classical swine fever virus (CSFV). All pestivirus species can infect pigs, therefore accurate and specific pestivirus detection and differentiation is of great importance to assure control measures in swine populations. The aim of the study was the molecular detection of different pestiviruses in domestic and feral pigs. A total of 527 samples (92 pigs and 435 wild boars) were tested for pestiviruses detection using molecular assays. Eleven positive samples (6 wild boars and 5 domestic pigs) were identified using panpestivirus primers targeting the 5’- UTR region of the pestivirus RNA genome. Further all the positive samples were sequentially tested for detection of CSFV, BVDV-1 and BVDV-2 using specific primers. All RNAs were identified as positives for BVDV-1 and no amplification signals were obtained from BVDV-2 and CSFV. The current detection of BVDV-1 in clinical swine specimens highlights the important risk factor of swine population as reservoir and consequently carrier for BVDV.

Bovine viral diarrhea virus (BVDV) is a pathogen that affects ruminants worldwide and is one of the most economically important diseases of cattle. Although BVDV infections have been increasingly reported in China, the pathogenesis and genetic characteristics of these BVDV isolates have not been thoroughly investigated. Here, we report the identification and characterization of a novel BVDV isolate, designated LC, which was isolated from the feces of a cattle with diarrhea. The complete genome of isolate LC was 12.271 nucleotides and contained a 5’-UTR of 389 nucleotides, a 3’-UTR of 189 nucleotides, and a large ORF encoding a polyprotein consisting of 3898 amino acids. Genomic comparisons and phylogenetic analyses of the complete genomic sequence clearly showed that the isolate was a BVDV-1q subtype. Experimental infection of calves with isolate LC resulted in the development of clinical signs including elevated rectal temperatures, nasal discharge and decreased leucopenia. Viral antigen was detected in infected animal tissues using immunohistochemistry. This is the first report of the genomic sequence of a BVDV-1q virus isolated from cattle. The virus strain was moderately pathogenic in calves and could potentially be used as a BVDV challenge virus to evaluate the efficacy of BVDV vaccines.

Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus of the Flaviviridae family and has worldwide distribution, being one of the main causes of economic losses in cattle raising. The genome of pestiviruses is a single strand of positive-sense RNA with a length of 12.3 kb, which encodes one open reading frame flanked by untranslated regions. E2 glycoprotein is required for binding to cell-surface receptors and it also contains major antigenic determinants. The nucleotide sequence coding E2 is the most variable part of the viral genome. The heterogeneity that exists among circulating strains causes problems in the development of effective vaccines and reliable diagnostics. In this study, and for the first time analysis was made of the E2 glycoprotein coding sequences of 14 Polish BVDV-1 strains which belong to four subtypes: 1b (n = 7), 1f (n = 3), 1s (n = 3), and 1r (n = 1). These sequences showed evidence of strong purifying (negative) selection. However, we also identified positively selected sites. The availability of E2 sequences of Polish BVDV strains for reference, knowledge gained through epitope prediction attempts, and information on protein glycosylation sites can afford a better understanding of host–pathogen interactions.

Bovine viral diarrhea virus (BVDV) belongs to the Flaviviridae family and the Pestivirus genus. Infection with BVDV causes a disease with a wide spectrum of clinical symptoms, most often mild, although infections with this virus constitute a serious economic problem all over the world. The virus is characterized by a high genetic variability, while the accumulation of single mutations leads to the formation of its new variants. The aim of this study was to better understand the complicated pathogenesis of this disease at the molecular level via the analysis of the transcriptome of cells infected with this virus. The bovine kidney cell line (MDBK), the cytopathic (cp) reference strain, and two non-cytopathic (ncp) BVD virus field strains were used in transcriptomic studies. The cell transcriptome was tested 24 and 72 h after infection. The results of the microarray analysis revealed changes in the expression levels of numerous genes. Genes with changed expression as a result of infection with the cp strain caused changes in the expression levels of a large number of genes and enriched a number of pathways. Genes with increased expression levels were enriched among other pathways involved in the cell cycle, while genes with reduced expression levels enriched pathways mostly related to metabolism. Genes with increased expression levels as a result of infection with ncp strains enriched a much smaller number of pathways, among them, pathways related to signaling activity 24 h post-infection and serine biosynthetic pathways both 24 and 72 h post-infection. Pathways enriched by genes with reduced expression levels were related to the innate immune response (72 h post-infection) or metabolism (24 and 72 h post-infection). The results of microarray studies can help us to better understand the host’s response to BVDV infection.

Bovine viral diarrhea virus (BVDV) infection during early gestation results in persistently infected (PI) immunotolerant calves that are the primary reservoirs of the virus. Pathologies observed in PI cattle include congenital defects of the brain, heart, and bone as well as marked functional defects in their immune system. It was hypothesized that fetal BVDV infection alters T cell activation and signaling genes by epigenetic mechanisms. To test this, PI and control fetal splenic tissues were collected on day 245 of gestation, 170 days post maternal infection. DNA was isolated for reduced representation bisulfite sequencing, protein was isolated for proteomics, both were analyzed with appropriate bioinformatic methods. Within set parameters, 1951 hypermethylated and 691 hypomethylated DNA regions were identified in PI compared to control fetuses. Pathways associated with immune system, neural, cardiac, and bone development were associated with heavily methylated DNA. The proteomic analysis revealed 12 differentially expressed proteins in PI vs. control animals. Upregulated proteins were associated with protein processing, whereas downregulated proteins were associated with lymphocyte migration and development in PI compared to control fetal spleens. The epigenetic changes in DNA may explain the immune dysfunctions, abnormal bone formation, and brain and heart defects observed in PI animals.

Viruses 14(2), 381 (2022). doi:10.3390/v14020381 special issue: "Veterinary Infectious Diseases / Topic Editors: Dr. Chao-Nan Lin, Dr. Peck Toung Ooi", Published by MDPI, Basel

Bovine viral diarrhea virus (BVDV) comprises two species, BVDV-1 and BVDV-2. But given the genetic diversity among pestiviruses, at least 22 subgenotypes are described for BVDV-1 and 3-4 for BVDV-2. Genetic characterization is generally accomplished through complete or partial sequencing and phylogeny, but it is not a reliable method to define antigenic relationships. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to define antigenic relatedness can be difficult to discern for BVDV isolates within the same BVDV species. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between US vaccine strains and field isolates from Switzerland, Italy, Brazil, and the UK. Polyclonal sera were generated against six BVDV strains currently contained in vaccine formulations, and each serum was used in VNs to measure the titers against seven vaccine strains (including the six homologous strains) and 23 BVDV field isolates. Principal component analysis (PCA) was performed using VN titers, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among various isolates suggesting antigenic relatedness. As expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. Notably, a number of clusters representing antigenically related BVDV-1 subgroups contain isolates of different subgenotypes. The multivariate analysis may be a method to better characterize antigenic relationships among BVDV isolates that belong to the same BVDV species and do not have distinct antigenic differences. This might be an invaluable tool to ameliorate the composition of current vaccines, which might well be important for the success of any BVDV control program that includes vaccination in its scheme.

More and more European countries have implemented a bovine viral diarrhea virus (BVDV) control program. The economic effects of such programs have been evaluated in simulations, but empirical studies are lacking, especially in the final stage of the program. We investigated the economic (gross margin) and production effects (milk yield, somatic cell count, and calving interval) of the herds obtaining BVDV-free certification based on longitudinal annual accounting and herd performance data from Dutch dairy herds between 2014 and 2019, the final stages of the Dutch national BVDV-free program. This study was designed as a case-control study: two types of case herds were defined for two analyses. The case herds in the first analysis are herds where the BVDV status changed from “BVDV not free” to “BVDV free” during the study period. The not-free status refers to a herd that participated in the BVDV-free program but had not yet obtained the BVDV-free certification. In the second analysis, the case herds started participating in the Dutch BVDV-free program during the study period and obtained the BVDV-free certification. Control herds in both analyses were BVDV-free during the entire study period. Potential bias between the covariates of the two herd groups was reduced by matching case and control herds using the propensity score matching method. To compare the differences between case and control herds before and after BVDV-free certification, we used the time-varying Difference-in-Differences estimation (DID) methodology. The results indicate that there was no significant change in milk yield, somatic cell count, calving interval, and gross margin upon BVDV-free certification. There are several possible explanations for the non-significant effects observed in our study, such as the final stage of the BVDV control program, not knowing the true BVDV infection situation in case herds and not knowing if control measures were implemented in case herds prior to participating in the BVDV-free program. In our study, the effects of BVDV-free certification might have been underestimated, given that the Dutch BVDV control program became mandatory during the study period, and some of the case herds might have never experienced any BVDV infection. The results of this study suggest that in the final stage of the BVDV control program, the program may no longer have a clear benefit to the herd performance of participating dairy herds. When designing national programs to eradicate BVDV, it is therefore important to include incentives for such farms to motivate them to join the program.

The role of bovine CD46 in the host cell entry of BVDV has been established for more than a decade. By generating novel MDBK CD46 knock-out clones, we confirm previously reported data on the CD46 motives important for BVDV binding and the importance of the G479R exchange within BVDV Erns to gain independence of bovine CD46 during entry. The comparison of different knock-out genotypes revealed a high variability of cellular susceptibility for a BVDV encoding the G479R exchange. These data highlight the effect of clonal selection of knock-outs on virus susceptibility, which should be considered when planning knock-out experiments.

Highlights • There is a high distribution of BVDV in the evaluated área. • Significant statistical association was found for breed, age, management practices. • Age group > 4 years, Normande breed, PI3 and grazing lease were risk factors., Worldwide distributed Bovine Viral Diarrhea Virus (BVDV) represents a high risk of infection in most bovine farms, in which it is associated with gastrointestinal, respiratory, and reproductive diseases. The purpose of this research was to establish the seroprevalence and the main risk factors associated with the presentation of BVDV in the municipality of Sotaquirá, Colombia. Samples were taken from 1000 cattle of Holstein, Ayrshire, Jersey, Normande Gyr and Holstein x Gyr. Epidemiological surveys were implemented, reproductive and management variables were taken into consideration. Indirect ELISA was performed to detect specific antibodies against BVDV using the commercial kit SERELISA® BVD p80 Ab Mono Blocking. The overall seroprevalence of antibodies against BVDV was 42.5% (425/1000), where the Gyr breed (59.1% apparent prevalence (AP); 60.3% real prevalence (PR)) and the age group > 4 years (53.0% PA; 54.4% PR) presented the highest seroprevalences. A significant statistical association was found for the breed, age, management practices evaluated and the presentation of PI3 (p ≤ 0.05). Age group > 4 years, Normande breed, presentation of PI3 and grazing lease were established as risk factors associated with BVDV in the herds. These infections are mainly associated with dairy cattle and herds with many animals, so it is important to consider vaccination plans as a preventive system and follow up on the most common diseases., Graphical abstract Image, graphical abstract

Bovine viral diarrhea virus (BVDV) infection results in a wide variety of clinical manifestations and is a pathogen that is able to cause huge economic losses in the cattle industry worldwide. It is important to identify cattle that are persistently infected (PI) by BVDV within the herd as early as possible because PI animals are the main reservoir of the virus. In contrast, cattle who are acutely infected (AI) with BVDV show various clinical signs, but most cattle show either mild symptoms or are asymptomatic. In general, AI and PI animals can be distinguished by repeat testing within an interval of at least 21 days. However, we found a rare case of a BVDV2-infected AI animal with long-term viral presence, making it indistinguishable from PI through two tests within an interval of 21 days. As a result, we diagnosed one infected animal as AI after 35 days from the initial sample collection via multiple analyses. Our findings recommend performing an additional test using samples that have been collected after 14–21 days from the second sample collection in cases where it is difficult to accurately differentiate an AI diagnosis from a PI diagnosis after only two tests. Additionally, our analysis exhibits that monitoring the number of copies of viruses with similar genomes in the sera by means of quantitative real-time RT-PCR through several sample collections periods might be useful to distinguish AI from PI. Furthermore, our data suggest that the AI animals with a long-term viral presence who show test results similar to those of PI animals might be the result of a coincidental combination of various factors that are present in cattle fields. These findings provide useful information that can be used to improve the diagnosis of BVDV in the field.

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 lymphocytes (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the situation in vivo remains to be further studied and confirmed. Therefore, in this study, we established a BALB/c mouse model of acute BVDV infection with cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain NY-1). Then, we examined the mRNA and protein levels of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC) from BVDV-infected mice and analyzed the effects of PD-1 blockade on the proportions of CD3+, CD4+, and CD8+ T cell subsets, the apoptosis and proliferation of PBL, and the production of IL-2 and IFN-γ. We found that leukopenia, lymphocytopenia, and thrombocytopenia were developed in both CP and NCP BVDV-infected mice at day 7 of post-infection. The mRNA and protein expression of PD-1 and PD-L1 were significantly upregulated in CP and NCP BVDV-infected mice. Moreover, PD-1/PD-L1 upregulation was accompanied by leukopenia and lymphopenia. Additionally, PD-1 blockade inhibited PBL apoptosis and virus replication, restored the proportions of CD3+, CD4+, and CD8+ T cell subsets, and increased IFN-γ production and p-ERK expression in BVDV-infected mice. However, blocking PD-1 did not significantly affect PBL proliferation and IL-2 production in NCP BVDV-infected mice. Our findings further confirmed the immunomodulatory role of PD-1 in peripheral blood lymphocytopenia in vivo and provided a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

Bovine viral diarrhea virus (BVDV) infection has a major effect on the health of cows and consequently on herd performance. Many countries have implemented control or eradication programs to mitigate BVDV infection and its negative effects. These negative effects of BVDV infection on dairy herds are well documented, but there is much less information about the effects of new introduction of BVDV on dairy herds already participating in a BVDV control program. The objective of our study was to investigate the effect of a new BVDV introduction in BVDV-free herds participating in the Dutch BVDV-free program on herd performance. Longitudinal herd-level surveillance data were combined with herd information data to create 4 unique data sets, including a monthly test-day somatic cell count (SCC) data set, annual calving interval (CIV) and culling risk (CR) data sets, and a quarterly calf mortality rate (CMR) data set. Each database contained 2 types of herds: herds that remained BVDV free during the whole study period (defined as free herds), and herds that lost their BVDV-free status during the study period (defined as breakdown herds). The date of losing the BVDV-free status was defined as breakdown date. To compare breakdown herds with free herds, a random breakdown date was artificially generated for free herds by simple random sampling from the distribution of the breakdown month of the breakdown herds. The SCC and CIV before and after a new introduction of BVDV were compared through linear mixed-effects models with a Gaussian distribution, and the CR and CMR were modeled using a negative binomial distribution in generalized linear mixed-effects models. The explanatory variables for all models included herd type, BVDV status, year, and a random herd effect. Herd size was included as an explanatory variable in the SCC, CIV, and CMR model. Season was included as an explanatory variable in the SCC and CMR model. Results showed that free herds have lower SCC, CR, CMR, and shorter CIV than the breakdown herds. Within the breakdown herds, the new BVDV introduction affected the SCC and CMR. In the year after BVDV introduction, the SCC was higher than that in the year before BVDV introduction, with a factor of 1.011 [2.5th to 97.5th percentile (95% PCTL): 1.002, 1.020]. Compared with the year before BVDV breakdown, the CMR in the year of breakdown and the year after breakdown was higher, with factors of 1.170 (95% PCTL: 1.120; 1.218) and 1.096 (95% PCTL: 1.048; 1.153), respectively. This study reveals that a new introduction of BVDV had a negative but on average relatively small effect on herd performance in herds participating in a BVDV control program.

Bovine viral diarrhea virus (BVDV) causes a severe threat to the cattle industry due to ineffective control measures. Gypenoside is the primary component of Gynostemma pentaphyllum, which has potential medicinal value and has been widely applied as a food additive and herbal supplement. However, little is known about the antiviral effects of gypenoside. The present study aimed to explore the antiviral activities of gypenoside against BVDV infection. The inhibitory activity of gypenoside against BVDV was assessed by using virus titration and performing Western blotting, quantitative reverse transcription PCR (RT-qPCR), and immunofluorescence assays in MDBK cells. We found that gypenoside exhibited high anti-BVDV activity by interfering with the viral attachment to and internalization in cells. The study showed that BVDV infection inhibits apoptosis of infected cells from escaping the innate defense of host cells. Our data further demonstrated that gypenoside inhibited BVDV infection by electively activating the apoptosis of BVDV-infected cells for execution, as evidenced by the regulation of the expression of the apoptosis-related protein, promotion of caspase-3 activation, and display of positive TUNEL staining, no toxicity was observed in non-infected cells. Collectively, the data identified that gypenoside exerts an anti-BVDV-infection role by inhibiting viral attachment and internalization and selectively purging virally infected cells. Therefore, our study will contribute to the development of a novel prophylactic and therapeutic strategy against BVDV infection.

The LFBK-αvβ6 cell line is highly sensitive for the isolation of foot-and-mouth disease virus (FMDV) and porcinophilic vesicular viruses. However, LFBK-αvβ6 cells are contaminated with a non-cytopathic bovine viral diarrhea virus (BVDV), which complicates handling procedures in areas where other cell lines are maintained, as well downstream use of viral isolates. In this study, we used an aromatic cationic compound (DB772) to treat LFBK-αvβ6 cells using an approach that has been previously used to eliminate persistent BVDV from fetal fibroblast cell lines. After three cell passages with 4 μM DB772, BVDV could no longer be detected in unclarified cell suspensions using a pan-pestivirus real-time RT-PCR assay, and remained undetectable after treatment was stopped (nine passages) for an additional 28 passages. The analytical sensitivity of the DB772-treated LFBK-αvβ6 cultures (renamed WRL-LFBK-αvβ6) to titrations of FMDV and other vesicular virus isolates was comparable to untreated LFBK-αvβ6 cells. These new BVDV-free cells can be handled without the risk of cross-contaminating other cells lines or reagents, and used for routine diagnostics, in vivo studies and/or preparation of new vaccine strains.

Bovine viral diarrhea (BVD) is an infectious disease, globally-distributed, caused by bovine Pestiviruses, endemic of cattle and other ruminant populations. BVD leads to significant economic losses to the cattle industry due to the wide range of clinical manifestations, including respiratory and gastrointestinal diseases and reproductive disorders. Within the Pestivirus genus of the family Flaviviridae three viral species are associated with BVD; Pestivirus A (Bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (Bovine viral diarrhea virus 2, BVDV-2), and Pestivirus H (HoBi-like pestivirus, atypical ruminant pestivirus). These species are subdivided into subgenotypes based on phylogenetic analysis. The extensive genetic diversity of BVDV has been reported for several countries, where the incidence and genetic variation are more developed in Europe than in the Americas. The first report of BVDV in Mexico was in 1975; this study revealed seropositivity of 75% in cows with a clinical history of infertility, abortions, and respiratory disease. Other studies have demonstrated the presence of antibodies against BVDV with a seroprevalence ranging from 7.4 to 100%. Recently, endemic BVDV strains affecting cattle populations started to be analyzed, providing evidence of the BVDV diversity in several states of the country, revealing that at least four subgenotypes (BVDV-1a, 1b, 1c, and 2a) are circulating in animal populations in Mexico. Little information regarding BVD epidemiological current status in Mexico is available. This review summarizes available information regarding the prevalence and genetic diversity viruses associated with BVD in cattle from Mexico.

This study examined the effects of multivalent respiratory vaccine treatment (VT) and animal temperament classification on feeding behavior traits, feed intake and animal performance in response to a bovine viral diarrhea virus (BVDV) challenge. Nellore–Angus crossbred steers (n = 360, initial body weight (BW) 330 ± 48 kg) were assigned to one of three vaccine treatments: non-vaccinated (NON), modified live (MLV) and killed (KV) regarding respiratory viral pathogens, and inoculated intranasally with the same BVDV1b strain. Cattle temperament categories were based on exit velocity. Overt clinical signs of respiratory disease were not observed, yet the frequency and duration of bunk visit events as well as traditional performance traits decreased (p &lt, 0.01) following BVDV challenge and then rebounded in compensatory fashion. The reduction in dry matter intake (DMI) was less (p &lt, 0.05) for MLV-vaccinated steers, and MLV-vaccinated steers had longer (p &lt, 0.01) durations of bunk visit and meal events and slower (p &lt, 0.01) eating rates compared with KV- and non-vaccinated steers following BVDV challenge. Greater differences in most feeding behavior traits due to VT existed within calm vs. excitable steers. Respiratory vaccination can reduce the sub-clinical feeding behavior and performance effects of BVDV in cattle, and the same impacts may not occur across all temperament categories.

International audience; Border Disease (BD) is a major sheep disease characterized by immunosuppression, congenital disorders, abortion, and birth of lambs persistently infected (PI) by Border Disease Virus (BDV). Control measures are based on the elimination of PI lambs, biosecurity, and frequent vaccination which aims to prevent fetal infection and birth of PI. As there are no vaccines against BDV, farmers use vaccines directed against the related Bovine Viral Diarrhea Virus (BVDV). To date, there is no published evidence of cross-effectiveness of BVDV vaccination against BDV infection in sheep. We tested three commonly used BVDV vaccines, at half the dose used in cattle, for their efficacy of protection against a BDV challenge of ewes at 52 days of gestation. Vaccination limits the duration of virus-induced leukopenia after challenge, suggesting partial protection in transient infection. Despite the presence of BDV neutralizing antibodies in vaccinated ewes on the day of the challenge, fetuses of vaccinated and unvaccinated sheep were, two months after, highly positive for BDV RNA loads and seronegative for antibodies. Therefore, BVDV vaccination at half dose was not sufficient to prevent ovine fetal infection by BDV in a severe challenge model and can only be reconsidered as a complementary mean in BD control.

Bovine viral diarrhea (BVD) caused by BVD virus (BVDV) leads to economic loss worldwide. Cattle that are persistently infected (PI) with BVDV are known to play an important role in viral transmission in association with the animal movement, as they shed the virus during their lifetime. In this research, the “hot spot” for BVD transmission was estimated by combining phylogenetic and epidemiological analyses for PI cattle and cattle that lived together on BVDV affected farms in Tokachi district, Hokkaido prefecture, Japan. Viral isolates were genetically categorized into BVDV-1a, 1b, and 2a, based on the nucleotide sequence of the entire E2 region. In BVDV genotype 1, subgenotype b (BVDV-1b), cluster I was identified as the majority in Tokachi district. Network analysis indicated that 12 of the 15 affected farms had cattle movements from other facilities (PI-network) and farms affected with BVDV-1b cluster I consisted of a large network. It was implied that the number of cattle movements themselves would be a risk of BVD transmission, using the PageRank algorithm. Therefore, these results demonstrate that cattle movements would contribute to disease spread and the combination of virological and epidemiological analysis methods would be beneficial in determining possible virus transmission routes.

Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%). For BVDV-1 Singer, it was also possible to detect Abs production in G1 (log2=5.8, 100% seroconversion rate) and G3 (log2=3.5, seroconversion rate = 60%), only after the booster dose (D42). Neutralizing Abs to BVDV-2 (SV253) were detected only in G3, observing 90% seroconversion associated with high titers of Abs (log2=6.7) after the 2nd dose of vaccine (D42). Heifers from G1 and G3 responded to BoHV-1 after the first dose (D21): G1 (log2=2.5, seroconversion rate = 67%) and G3 (log2=0.7, seroconversion rate = 80%). In D42, a higher magnitude response was observed in the heifers from G3 (log2=6.1, 100%) compared with G1 (log2=4.3, 100%) and G2 (log2=2.7, 60%). Based on the data obtained, it can be concluded that the commercial vaccine contained aluminum hydroxide (G1) was most effective in the induction of antibodies against BVDV-1. On the other hand, this vaccine did not induce the production of neutralizing Abs against BVDV-2. Only the heifers from G3 (Quil A, amphigen and cholesterol) generated neutralizing Abs against BVDV-2. The animals that received commercial vaccine containing oil emulsion as adjuvant (G2) had a weak/undetectable response against BVDV-1 and BVDV-2. The best protective response against BoHV-1 was observed in heifers vaccinated with the live modified thermosensitive virus. RESUMO: A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%). Para o BVDV-1 Singer, somente após D42 foi observada a produção de Acs no G1 (log2=5,8; taxa de soroconversão de 100%) e G3 (log2=3,5; taxa de soroconversão = 60%). Os anticorpos contra BVDV-2 (SV253) foram detectados apenas nas novilhas do G3, observando-se taxa de soroconversão de 90% com altos títulos de anticorpos neutralizantes (log2=6,7) em D42. Novilhas G1 e G3 responderam ao BoHV-1 após a primeira dose (D21): G1 (log2=2,5; taxa de seroconversão = 67%) e G3 (log2=0,7; taxa de seroconversão = 80%). Em contrapartida, foi observada uma maior magnitude de resposta para as novilhas G3 (log2=6,1; 100%) em D42, em relação aos animais G1 (log2=4,3; 100%) e G2 (log2=2,7; 60%). Com base nos dados obtidos, foi possível concluir que a vacina composta por hidróxido de alumínio (G1) foi mais eficaz na produção de anticorpos contra o BVDV-1, em contrapartida esse produto não induziu anticorpos contra o BVDV-2. Apenas as novilhas do G3 (Quil A, amphigen e colesterol) geraram Acs neutralizantes contra o BVDV-2. Os animais que receberam a vacina em emulsão oleosa (G2) como adjuvante apresentaram uma resposta fraca/indetectável contra o BVDV-1 e BVDV-2. A melhor resposta protetiva contra o BoHV-1 foi observada nas novilhas vacinadas com a vacina viva modificada termosensível.

Bovine viral diarrhea virus (BVDV, Pestivirus) causes significant economic losses to the livestock industry worldwide. Although serological surveys show that BVDV exposure is widespread in cattle in Uruguay, BVDV-associated diseases are greatly underreported. The aim of this work is to describe the epidemiological, clinical, pathological, and virological findings from spontaneous outbreaks of BVDV-associated diseases in cattle in Uruguay. Diagnostic investigations were performed during 6 spontaneous disease outbreaks on beef and dairy cattle farms in the departments of Colonia, Rio Negro, and Soriano between November 2016 and April 2018. Carcasses of 8 naturally deceased cattle from these outbreaks were necropsied and subjected to histological examination and immunohistochemistry to detect BVDV antigen in the tissues. Reverse transcription real-time PCR and genomic sequencing were also performed to identify BVDV at the species and subtype levels. Other ancillary diagnostic tests, including bacterial cultures, were performed on a case-by-case basis to rule in/out differential diagnoses based on initial clinicopathological presumptive diagnoses. BVDV-associated conditions that were diagnosed in the 8 cases included mucosal disease, transient postnatal BVDV infections associated with digestive/septicemic salmonellosis by Salmonella serovar typhimurium, Histophilus somni bronchopneumonia, urinary tract coinfections with Escherichia coli and Streptococcus sp., enteric coinfection with coccidia, and transplacental fetal infections and abortions with Neospora caninum coinfection. BVDV-1a and BVDV-2b were each identified in four of the eight cases. We conclude that BVDV-1a and BVDV-2b contribute significantly to disease and mortality in cattle in Uruguay. Future research should estimate the economic impact of BVDV in the Uruguayan livestock sector. Electronic supplementary material The online version of this article (10.1007/s42770-019-00170-7) contains supplementary material, which is available to authorized users.

Background Bovine viral diarrhea virus (BVDV) causes severe economic losses and is one of the most important viral pathogens of ruminants worldwide. The infection manifests itself in a variety of clinical symptoms. Phylogenetic studies based mainly on 5’UTR of its genome, identified many different subtypes of BVDV. Previous study indicated the predominance of BVDV-1b and BVDV-1d in Poland. The aim of this study was to genotype BVDV isolates currently circulating in Polish dairy herds. Results BVDV was detected in 30 herds. Viral subtypes were identified using sequences of the 5’UTR fragment and they were confirmed within a fragment of the Npro region. Seven subtypes of BVDV-1 species have been identified: 1b, 1 g, 1f, 1d, 1r, 1 s and 1e. Conclusion The number of subtypes of BVDV in Poland evolves and 2 new subtypes have been identified for the first time. Such studies may have a positive impact on successful eradication of the virus using effective vaccines and diagnostic tests. Electronic supplementary material The online version of this article (10.1186/s12917-019-2029-z) contains supplementary material, which is available to authorized users.

Bovine viral diarrhea virus (BVDV) footprint has spread across the globe and is responsible for one of the most economically important diseases in cattle. In Japan, some regional surveillance and preventive measures to control bovine viral diarrhea (BVD) have been implemented. However, BVDV infection is poorly understood in cattle industries, and there is no systematic BVD surveillance system and control program. Kyushu is the center for raising beef cattle in Japan. Therefore, this study aimed to determine the BVDV infection using a slaughterhouse survey among beef cattle in Kyushu, Japan. A total of 1,075 blood samples were collected at two regional slaughterhouses in Miyazaki prefecture from December 2015 to June 2016. Antigen ELISA was used for detection of BVDV antigen in blood samples. Two samples showed positive results (2/1,075; 0.18%). BVDV RNA was extracted from positive blood samples; the sequence was determined and analyzed by the neighbor-joining method for construction of the phylogenetic tree. Phylogenetic analysis based on the 5'-UTR revealed that the two positive samples were grouped into the same subtype BVDV-1b in the BVDV-1 genotype, but the infected cattle belonged to two different farms. In conclusion, this is the first study to identify the presence of BVDV in a slaughterhouse survey in Kyushu. These findings suggest that a slaughterhouse survey is a useful tool for developing a surveillance system for monitoring infectious diseases in cattle.

Background and Aim: A previous study divided Indonesian bovine viral diarrhea virus (BVDV)-1 into subgenotypes BVDV-1a to BVDV-1d based on the partial NS5B gene using strain Bega as reference for BVDV-1a. In fact, it is clustered into BVDV-1c with strain Bega-like Australia. BVDV genotyping has been done on isolates from Jakarta, West and Central Java, but East Java isolates have not been genotyped. This study aimed to analyze genetic variability and amino acid residues in the nucleotide-binding pocket of the NS5B gene from infected cattle. Materials and Methods: Samples were obtained from the Sera Bank originating from active and passive surveillance of cattle that had been tested for BVDV antigen from 2013 to 2017. Detection of the p80 antibody and BVDV genotyping was carried out using ELISA and nested-multiplex-polymerase chain reaction (PCR), respectively. We defined 15 nested PCR products for partial sequencing of NS5B. Those field samples were selected from each location and year using proportional calculation as a representative sample. Homological and phylogenetic analyses of the partial NS5B gene were performed using BLAST and MEGA version 6. Results: Based on the phylogenetic tree analysis using 360 nucleotides as the partial NS5B gene, Indonesian BVDV-1 isolates from Central and East Java were subdivided to BVDV-1a (n=9), BVDV-1b (n=1), and BVDV-1c (n=5). In the present study, the homology of BVDV subgenotype -1a, -1b, and -1c was compared to the BVDV GenBank data and found 90-93%, 93%, and 92-95% respectively with the average pairwise distance of 0.207. A point mutation was shown at R283K of all BVDV isolates based on the sequence of three amino acid residues R283, R285, and I287 in the nucleotide-binding pocket as a part of the encoded RNA-dependent RNA polymerase. Conclusion: This study revealed the genetic variability of BVDV infecting cattle in Central Java and East Java, Indonesia, the subtypes BVDV-1a, BVDV-1b, BVDV-1c, and a point mutation at the R283K residue.

The etiology and pathologic findings of bovine respiratory disease (BRD) in adult dairy cows (n = 35) from a commercial dairy herd in Southern Brazil were investigated. Pulmonary samples were examined for histopathologic patterns and specific features within these patterns, while immunohistochemical (IHC) assays were designed to detect the intralesional antigens of viral infectious disease agents and Mycoplasma bovis. Pneumonia was diagnosed in 91.4% (32/35) of these cases; neither pneumonia nor any of the infectious disease pathogens evaluated occurred in three cows. The presence of multiple respiratory pathogens in 75% (24/32) of these cases indicated the complex origin of pneumonia in cattle. Interstitial pneumonia, necrosuppurative bronchopneumonia and suppurative bronchopneumonia were the principal patterns of pulmonary disease identified by histopathology. The most frequent pathogens identified by IHC were bovine viral diarrhea virus (BVDV; n = 18), M. bovis (n = 16) and bovine alphaherpesvirus type 1 (BoHV‐1; n = 14), followed by bovine respiratory syncytial virus (BRSV; n = 11) and bovine parainfluenza virus type 3 (BPIV‐3; n = 5). Obliterative bronchiolitis and peribronchial lymphocytic cuffings were the characteristic histopathologic features associated with M. bovis. Necrohemorrhagic bronchitis with bronchial angiogenesis was associated with BoHV‐1. Necrotizing bronchitis and bronchiolitis were associated with BVDV, BoHV‐1 and BRSV. Ballooning degeneration of the bronchial and bronchiolar epithelia was associated with BRSV and BoHV‐1. This is the first report from Brazil that correlated the histopathologic findings of BRD with the associated infectious disease agents by immunohistochemistry. M. bovis was frequently detected in the tissues of cows with fatal pulmonary disease during this study and may be a possible primary disease pathogen associated with the development of BRD in dairy cows. Additionally, the histopathologic features identified within patterns of pulmonary disease during this investigation may be an efficient diagnostic tool to associate histopathologic findings with specific agents of BRD in dairy cows.

Background Bovine viral diarrhea virus (BVDV) is an economically important viral pathogen of domestic and wild ruminants. Apart from cattle, small ruminants (goats and sheep) are also the susceptible hosts for BVDV. BVDV infection could interfere both of the innate and adaptive immunity of the host, while the genes and mechanisms responsible for these effects have not yet been fully understood. Peripheral blood mononuclear cells (PBMCs) play a pivotal role in the immune responses to viral infection, and these cells were the target of BVDV infection. In the present study, the transcriptome of goat peripheral blood mononuclear cells (PBMCs) infected with BVDV-2 was explored by using RNA-Seq technology. Results Goat PBMCs were successfully infected by BVDV-2, as determined by RT-PCR and quantitative real-time RT-PCR (qRT-PCR). RNA-Seq analysis results at 12 h post-infection (hpi) revealed 499 differentially expressed genes (DEGs, fold-change ≥ ± 2, p

Acute infections of bovine viral diarrhea virus (BVDV) lead to a range of clinical presentations. Laboratory tests for detection depend on collection of samples during a short viremia. Acutely infected animals remain largely undiagnosed. Transfer RNA halves (tsRNAs) are hypothesized to function like microRNAs to regulate gene expression during an immune response. The objective of this study was to identify tsRNAs in cattle that had been challenged with a non-cytopathic field strain of BVDV. Colostrum-deprived neonatal Holstein calves were either challenged with BVDV (n=5) or mock challenged (n=4). Sera was collected prior to challenge and days 4, 9, and 16 post challenge. RNA was extracted and read counts of small non-coding RNAs were assessed using next-generation sequencing. A total of 87,838,207 reads identified 41 different tsRNAs. Two 5’ tsRNAs, tsRNAProAGG and tsRNAValAAC, differed across time. Two 5’ tsRNAs, tsRNAGlyCCC and tsRNAGlyGCC, differed between treatment groups across time. Four days post challenge, 5’ tsRNAGlyCCC and tsRNAGlyGCC were significantly lower in the challenged group than the control group. Further studies are needed to identify the importance and function of 5’ tsRNAGlyCCC and tsRNAGlyGCC in serum samples of cattle challenged with BVDV.

Background: Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea. It can infect cattle, sheep, pigs, and other animals, causing diarrhea, miscarriage, and stillbirth, among other symptoms, and it can result in huge economic losses to animal husbandry. There are reports on BVDV infection rates in sheep and goat herds from all over the world and this meta-analysis aimed to evaluate the prevalence of and risk factors for BVDV in sheep and goats.Results: Using the data of 41,297 sheep and goats in 24 countries/regions to calculate a comprehensive prevalence rate for BVDV. The overall prevalence of BVDV infection in sheep and goats was estimated to be 8.6% (95% CI: 5.2–12.7) by immunological methods and 7.3% (95% CI: 2.7–13.7) by molecular methods. Analysis by national income level revealed that prevalence is higher in middle-income countries than in high-income countries (P < 0.05). The study also compared prevalence rates by species of BVDV, sampling year, and test species, but did not find significant differences.Conclusion: This systematic review and meta-analysis is the first to determine the global prevalence of BVDV in ovine and caprine flocks. The prevalence of BVDV in sheep and goat populations varies from region to region, and the situation is not optimistic in some countries.

Antigenic differences between bovine viral diarrhea virus (BVDV) vaccine strains and field isolates can lead to reduced vaccine efficacy. Historically, antigenic differences among BVDV strains were evaluated using techniques based on polyclonal and monoclonal antibody activity. The most common method for antigenic comparison among BVDV isolates is determination of virus neutralization titer (VNT). BVDV antigenic comparisons using VNT only account for the humoral component of the adaptive immune response, and not cell mediated immunity (CMI) giving an incomplete picture of protective responses. Currently, little data is available regarding potential antigenic differences between BVDV vaccine strains and field isolates as measured by CMI responses. The goal of the current paper is to evaluate two groups of cattle that differed in the frequency they were vaccinated, to determine if similar trends in CMI responses exist within each respective group when stimulated with antigenically different BVDV strains. Data from the current study demonstrated variability in the CMI response is associated with the viral strain used for stimulation. Variability in IFN-γ mRNA expression was most pronounced in the CD4+ population, this was observed between the viruses within each respective BVDV subgenotype in the Group 1 calves. The increase in frequency of CD25+ cells and IFN-γ mRNA expression in the CD8+ and CD335+ populations were not as variable between BVDV strains used for stimulation in the Group 1 calves. Additionally, an inverse relationship between VNT and IFN-γ mRNA expression was observed, as the lowest VNT and highest IFN-γ mRNA expression was observed and vice versa, the highest VNT and lowest IFN-γ mRNA expression was observed. A similar trend regardless of vaccination status was observed between the two groups of calves, as the BVDV-1b strain had lower IFN-γ mRNA expression. Collectively, data from the current study and previous data support, conferring protection against BVDV as a method for control of BVDV in cattle populations is still a complex issue and requires a multifactorial approach to understand factors associated with vaccine efficacy or conversely vaccine failure. Although, there does appear to be an antigenic component associated with CMI responses as well as with humoral responses as determined by VNT.

The aim of this pilot study was to determine viral loads and distribution over the total length, at short distances, and in the separate layers of the intestine of virus-infected animals for future inactivation studies. Two calves, two pigs, and two goats were infected with bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV), and peste des petits ruminants virus (PPRV), respectively. Homogenously distributed maximum BVDV viral loads were detected in the ileum of both calves, with a mean titer of 6.0 log10 TCID50-eq/g. The viral loads in colon and caecum were not distributed homogenously. In one pig, evenly distributed CSFV mean viral loads of 4.5 and 4.2 log10 TCID50-eq/g were found in the small and large intestines, respectively. Mucosa, submucosa, and muscular layer/serosa showed mean viral loads of 5.3, 3.4, and 4.0 log10 TCID50-eq/g, respectively. Homogenous distribution of PPRV was shown in the ileum of both goats, with a mean viral load of 4.6 log10 TCID50-eq/g. Mean mucosa, submucosa, and muscular layer/serosa viral loads were 3.5, 2.8, and 1.7 log10 TCID50-eq/g, respectively. This pilot study provides essential data for setting up inactivation experiments with intestines derived from experimentally infected animals, in which the level and the homogeneous distribution of intestinal viral loads are required.

As the worldwide population has been experiencing since 2020, viruses represent a serious threat to global well-being. To avoid viral transmission through surgery or medical examination, sterilization of medical material is needed. From emerging sterilization processes, the use of non-thermal plasma (NTP) arises as a promising technique to efficiently reduce microbial burden on medical devices, including new complex polymers as thermosensitive ones. Thus, we evaluated the antiviral efficacy of a low-pressure NTP process taking place in a sealed bag. For this purpose, two different plasmas, O2 100% plasma and Ar 80%–O2 20% plasma, were tested against two viruses: the bovine viral diarrhea virus and the porcine parvovirus, surrogates of human hepatitis C virus and human parvovirus B19, respectively. The efficacy of both NTP treatments on viral load can be detected after only five minutes. Moreover, the longer the NTP treatments last, the more the load decreases. The most effective load reduction was obtained with a 120-min O2 plasma treatment inducing a minimum of four-log viral load reduction. So, this process demonstrated strong virucidal capacity inside a sealed bag and represents a very interesting opportunity in the field of fragile medical devices sterilization or disinfection.

Supplemental material for the paper titled "The effect of new bovine viral diarrhea virus (BVDV) introduction on somatic cell count, calving interval, culling rate and calf mortality of dairy herds in the Dutch BVDV-free program".

Dairy cows are negatively affected by the introduction of bovine viral diarrhea virus (BVDV), and consequently, produce less milk. Existing literature on potential milk production losses is based on relatively outdated data and hardly evaluates milk production loss in relation to a new BVDV infection in a surveillance system. This study determined the annual and quarterly loss in milk production of BVDV introduction in 3,126 dairy herds participating in the Dutch BVDV-free program between 2007 and 2017. Among these herds, 640 were "breakdown-herds" that obtained and subsequently lost their BVDV-free status during the study period, and 2,486 herds obtained and retained their BVDV-free status during the study period. Milk yields before and after BVDV introduction were compared through annual and quarterly linear mixed models. The fixed variables for both models included herd type (breakdown-herd or free-herd), bovine viral diarrhea status (on an annual and quarterly basis), year, season, and a random herd effect. The dependent variable was the average daily milk yield on the test day. To define the possible BVDV-introduction dates, 4 scenarios were developed. In the default scenario, the date of breakdown (i.e., loss of the BVDV-free status) was assumed as the BVDV-introduction date. For the other 3 scenarios, the BVDV-introduction dates were set at 4, 6, and 9 mo before the date of breakdown, based on the estimated birth date of a persistently infected calf. In the default scenario, the loss in milk yield due to BVDV introduction occurred mainly in the first year after breakdown, with a reduction in yield of 0.08 kg/cow per day compared with the last year before breakdown. For the other 3 scenarios, the greatest yield reduction occurred in the second year after BVDV introduction, with a loss of 0.09, 0.09, and 0.1 kg/cow per day, respectively. For the first 4 quarters after BVDV introduction in the default scenario, milk yield loss was 0.14, 0.09, 0.02, and 0.08 kg/cow per day, respectively. These quarterly results indicated that milk yield loss was greatest in the first quarter after BVDV introduction. Overall, BVDV introduction had a negative, but on average a relatively small, effect on milk yield for herds participating in the BVDV-free program. This study will enable dairy farmers and policymakers to have a clearer understanding of the quantitative milk production effect of BVDV on dairy farms in a control program.

Bovines Virusdiarrhoe-Virus (BVDV, Familie Flaviviridae, Gattung Pestivirus) ist ein wirtschaftlich bedeutendes Pathogen in Rindern. Die zellulären Faktoren, die für den Eintritt von BVDV in die Wirtszelle benötigt werden, konnten bisher nicht vollends bestimmt werden. Um essenzielle Faktoren der Wirtszelle für diesen Prozess zu identifizieren, wurde ein CRISPR-Cas9 vermitteltes Screening einer Library von 362 sgRNAs für den Knockout von 73 verschiedenen Genen (Fünf sgRNAs für die meisten Gene) durchgeführt. Für die Klonierung der Library konnte ein Klonierungsprotokoll mit hoher Durchsatzmenge erfolgreich erstellt werden. SgRNA exprimierende Vektoren wurden mittels HIV basierten Pseudotypen in MDBK Zellen genomisch integriert, mit einer Transduktionseffizienz von 50 bis 70 %. 17 Tage nach der Transduktion wurden die Zellen mit Fluoreszenz-markierten BVDV-1 Viren infiziert und anschließend durch Fluoreszenzmikroskopie auf die Anwesenheit transduzierter, proliferierender Zellen ohne Anzeichen einer Virusinfektion überprüft. Basierend auf dieser Selektion konnten 14 Gene identifiziert werden, die möglicherweise eine Rolle im Entry von BVDV spielen. Es handelt sich hierbei um CLDN16, CLDN22, CLDN24, IFI30, TMEM114, TMEM204, TMEM47, TSPAN1, TSPAN10, TSPAN11, CLTC and Rab7a. Diese Studie stellt die Grundlage für weitergehende Experimente dar, um wichtige Wirtsfaktoren im Entry von BVDV klar zu identifizieren. Bovine viral diarrhea virus (BVDV, family Flaviviridae, genus Pestivirus) is an economically important pathogen in cattle. The cellular factors required for the entry of BVDV into its host cell are only partially known to date. To identify additional host factors involved in this process, a CRISPR-Cas9 mediated knockout screening with a library of 362 sgRNAs targeting 73 different genes (five sgRNAs for most of the genes) was conducted. For the cloning of the library, a high throughput cloning protocol was successfully established. SgRNA expressing vectors were genomically integrated into MDBK cells by HIV based pseudotypes. The efficiency of transduction ranged from 50-70 %. 17 days after the transduction, the cells were infected with fluorophore labelled BVDV-1 and subsequently monitored for the presence of transduced and proliferating cells showing no signs of virus infection by fluorescence microscopy. Based on this read-out, 14 genes, whose knockout inhibited virus infection, could be identified. These included CLDN16, CLDN22, CLDN24, IFI30, TMEM114, TMEM204, TMEM47, TSPAN1, TSPAN10, TSPAN11, CLTC and Rab7a. This thesis provides the basis for further experiments to distinctly identify important host factors for BVDV entry.

Bovine Viral Diarrhea Virus (BVDV) is an important pathogen that plays a significant role in initiating Bovine Respiratory Disease Complex (BRDC) in cattle. The disease causes multi-billion dollar losses globally due to high calf mortality and increased morbidity leading to heavy use of antibiotics. Current commercial vaccines provide limited cross-protection with several drawbacks such as safety, immunosuppression, potential reversion to virulence, and induction of neonatal pancytopenia. This study evaluates two prototype vaccines containing multiple rationally designed recombinant mosaic BVDV antigens for their potential to confer cross-protection against diverse BVDV strains. Genes encoding three novel mosaic antigens, designated E2123, NS2-31, and NS2-32, were designed in silico and expressed in mammalian cells for the formulation of a prototype protein-based vaccine. The mosaic antigens contain highly conserved protective epitopes from BVDV-1a, -1b, and -2, and included unique neutralizing epitopes from disparate strains to broaden coverage. We tested immunogenicity and protective efficacy of Expi293TM-expressed mosaic antigens (293F-E2123, 293F-NS2-31, and 293F-NS2-32), and baculovirus-expressed E2123 (Bac-E2123) mosaic antigen in calves. The Expi293TM-expressed antigen cocktail induced robust BVDV-specific cross-reactive IFN-γ responses, broadly neutralizing antibodies, and following challenge with a BVDV-1b strain, the calves had significantly (p < 0.05) reduced viremia and clinical BVD disease compared to the calves vaccinated with a commercial killed vaccine. The Bac-E2123 antigen was not as effective as the Expi293TM-expressed antigen cocktail, but it protected calves from BVD disease better than the commercial killed vaccine. The findings support feasibility for development of a broadly protective subunit BVDV vaccine for safe and effective management of BRD.

Background and Aim: Globally, pestiviruses are among the most economically important viral pathogens of livestock. The genus Pestivirus comprises four species, including bovine viral diarrhea virus type 1 and 2 (BVDV-1 and BVDV-2), which infect cattle, border disease virus and classical swine fever virus which infect small ruminants and pigs, respectively. Accumulating evidence suggests that pestiviruses are no longer species-specific, creating new challenges for disease control. In Indonesia, investigations related to pestiviruses remain focused on cattle as the primary host and no research has been conducted on small ruminants (sheep and goats). Therefore, the present study aimed to study the possible occurrence of pestivirus (BVDV or BVD) infections in small ruminants in Indonesia, particularly in Central Java. Materials and Methods: We used 46 blood samples consisting of 26 sheep’s blood and 20 goat’s blood. Samples were selected from 247 small ruminant blood collected between July and October 2020 in Central Java, Indonesia, which met the following criteria: Female, local species, approximately 1-2 years old, never been pregnant, raised in the backyard, and had no close contact with cattle in either shelter or grazing area. We tested plasma samples from sheep and goats using competitive antibody enzyme-linked immunosorbent assay to detect specific antibodies against pestivirus followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis for all positive samples to differentiate the species of pestivirus. Results: Two of the 20 samples collected from goats were positive for pestivirus at the serological and molecular levels, whereas 2 of 26 samples collected from sheep were doubtful but tested negative by RT-PCR. The genotyping test results obtained using nested PCR revealed that the positive samples collected from goats had a BVDV-1 genotype. Conclusion: The results of the present study demonstrated that BVDV-1 can infect species other than bovines, in Central Java, Indonesia. Further studies involving a larger number of samples are required to: (1) Determine the actual seroprevalence of pestiviruses in small ruminants and (2) Determine the potency of small ruminants as reservoirs for pestiviruses, both of which are important for the identification of the appropriate control program for pestiviruses in Indonesia.

Reproductive tests in cattle are of great economic importance, given the impact it can have on the production system and may be caused by agents. Neospora caninum and Bovine Viral Diarrhea virus (BVDV) are considered of great importance as reproductive and should be considered responsible for keeping animals persistently infected. The present study included 479 calf serum samples for export in the state of Rio Grande do Sul (RS). All samples were screened for BVDV by an ELISA antigen. BVDV antigen-positive ELISA samples were isolated from BVDV in cell culture. An indirect immunofluorescence (IFT) technique was used to detect anti-N. caninum antibodies. Of the 479 export-treated serum samples, 361 were positive for BVDV antigens by ELISA and/or viral isolation test (361/479-75.36%), and 109 IFT-positive samples for N. caninum (109/479-22.75%). Despite detection of antibodies anti-N. caninum did not differ statistically between naturally infected BVDV and non-BVDV infected animals suggesting that there is no interference of BVDV infection on infection or detection rate of animals with N. caninum, positive animals in viral isolation and high DO in BVDV-Ag ELISA. may present active disease and consequent immunosuppression, contributing to a potential reactivation of N. caninum. RESUMO: Testes reprodutivos em bovinos são de grande importância econômica, dado o impacto que podem ter no sistema de produção e podem ser causados por agentes. O Neospora caninum e o vírus da Diarreia Viral Bovina (BVDV) são considerados de grande importância como reprodutivos e devem ser considerados responsáveis por manter os animais persistentemente infectados. O presente estudo incluiu 479 amostras de soro de bezerro para exportação no estado do Rio Grande do Sul (RS). Todas as amostras foram rastreadas para BVDV por um antígeno ELISA. As amostras de ELISA positivas para o antigénio BVDV foram isoladas a partir de BVDV em cultura de células. Uma técnica de imunofluorescência indireta (IFT) foi utilizada para detectar anticorpos anti-N caninum. Das 479 amostras de soro tratadas para exportação, 361 foram positivas para antígenos de BVDV por ELISA e/ou teste de isolamento viral (361/479-75,36%) e 109 amostras positivas para IFT para N. caninum (109/479-22,75%). Apesar da detecção de anticorpos anti-N. caninum não diferiu estatisticamente entre animais infectados naturalmente BVDV e não BVDV sugerindo que não há interferência da infecção pelo BVDV na infecção ou taxa de detecção de animais com N. caninum, animais positivos em isolamento viral e alta DO em BVDV-Ag ELISA, pode apresentar doença ativa e consequente imunossupressão, contribuindo para uma potencial reativação de N. caninum.

Bovine Viral Diarrhea Virus (BVDV) fetal infections occur in two forms, persistent infection (PI) or transient infection (TI), depending on what stage of gestation the fetus is infected. Examination of lymphoid organs from both PI and TI fetuses reveals drastically different fetal responses, dependent upon the developmental stage of the fetal immune system. Total RNA was extracted from the thymuses and spleens of uninfected control, PI, and TI fetuses collected on day 190 of gestation to test the hypothesis that BVDV infection impairs the innate and adaptive immune response in the fetal thymus and spleen of both infection types. Transcripts of genes representing the innate immune response and adaptive immune response genes were assayed by Reverse Transcription quatitative PCR (RT-qPCR) (2&minus, &Delta, Cq, fold change). Genes of the innate immune response, interferon (IFN) inducible genes, antigen presentation to lymphocytes, and activation of B cells were downregulated in day 190 fetal PI thymuses compared to controls. In contrast, innate immune response genes were upregulated in TI fetal thymuses compared to controls and tended to be upregulated in TI fetal spleens. Genes associated with the innate immune system were not different in PI fetal spleens, however, adaptive immune system genes were downregulated, indicating that PI fetal BVDV infection has profound inhibitory effects on the expression of genes involved in the innate and adaptive immune response. The downregulation of these genes in lymphocytes and antigen-presenting cells in the developing thymus and spleen may explain the incomplete clearance of BVDV and the persistence of the virus in PI animals while the upregulation of the TI innate immune response indicates a more mature immune system, able to clear the virus.