Bovine Viral Diarrhea Virus Strains Research Articles

Genetic features of bovine viral diarrhea virus subgenotype 1c in newborn calves at nucleotide and synonymous codon usages.

Bovine viral diarrhea virus (BVDV), serving as an important pathogen for newborn calves, poses threat to reproductive and economic losses in the cattle industry. To survey the infection rate and genetic diversity of BVDV in newborn calves in northern China, a total of 676 sera samples of newborn calves were collected from four provinces between 2021 and 2022. All sera samples were individually detected for BVDV infection by RT-PCR and ELISA. Our results showed that the overall serological rate was 9.76% (66/676) and the average positive rate of BVDV RNA was 8.14% (55/676) in the newborn calves. Eight BVDV strains were successfully isolated from RT-PCR positive sera samples, and four isolates displayed the cytopathic effect (CPE). Based on phylogenetic tree at the genome level, the eight strains were classified into subgenotype 1c. Moreover, the BVDV isolates had a close genetic relationship with the GSTZ strain at either nucleotide or codon usage level. Interestingly, in comparison of synonymous codon usage patterns between the BVDV isolates with CPE and ones without CPE, there were four synonymous codons (UCG, CCC, GCA, and AAC) which displayed the significant differences (p < 0.05) at codon usage pattern, suggesting that synonymous codon usage bias might play a role in BVDV-1c biotypes. In addition, the usage of synonymous codons containing CpG dinucleotides was suppressed by the BVDV-1c isolates, reflecting one of strategies of immune evasion of BVDV to its host. Taken together, our study provided data for monitoring and vaccination strategies of BVDV for newborn calves in northern China.

Development of an in-house Indirect ELISA for detection of bovine viral diarrhoea virus antibodies in bovine sera

Bovine viral diarrhoea virus (BVDV) infection is a worldwide distributed animal disease. BVDV is the causal agent of congenital defects, diarrhea, reproductive failure, and contaminating biological products. Virus Neutralization test (VNT) as a gold standard method is used for detection of BVDV. Although this assay is very sensitive and specific, it has disadvantages including requires to an experienced person and cell culture facilities. VNT is time-consuming. It is important to design a method that does not have the mentioned disadvantages. So, in-house indirect enzyme linked immunosorbent assay (i-ELISA) was developed for laboratories where it is not possible to perform VNT. The system was made using NADL strain of BVDV and MDBK cell line. This ELISA system was compared with a commercial ELISA kit using 99 bovine sera. Coefficient of variation (CV) was calculated 3.9% and 4.8% for the positive and negative control, respectively for the designed i-ELISA system. The sensitivity, specificity, and accuracy of i-ELISA system was 88%, 53.6%, and 70.7% respectively. Based on our result correlation between in- house and commercial ELISA kit for detection of antibody against BVDV in bovine sera was significant (Kappa coefficient =0.41, p < 0.05). Results of the present study suggested that an in-house ELISA as an affordable and confident system for primary screening of the sera used for biological product.

Molecular Diversity and Histopathological Findings of Novel Bovine Viral Diarrhea Virus Strains Isolated from Bull Semen

Bovine viral diarrhea virus (BVDV) is one of the most common viral pathogens affecting the cattle industry worldwide. The present study aimed to molecularly characterize BVDV isolates that are currently circulating in breeding bulls farmed with cattle suffering from reproductive disorders, and also to assess the consequences of BVDV infection on bulls’ semen quality and conception, and its pathological effects on the structure of testicular tissue and spermatozoa. For this purpose, semen, serum, and testicular samples were collected from four breeding bulls in four private dairy farms in the governorates of Kafr-El Sheik, Beni-Suef, Giza, and Assuit, in Egypt from April 2019 to May 2020. An evaluation of sperm abnormalities was carried out by assessing the integrity of the plasma and acrosomal membranes where severe damage and abnormalities were found. Ultrastructure analysis of the spermatozoa by transmission electron microscopy revealed the presence of a swollen plasma membrane with segmented outer acrosomal membrane of spermatozoa and vacuolar degenerated mitochondria. Histopathological examination of testicular and epididymal tissues indicated moderate to severe degenerative effects of virus infection on seminiferous tubules with hypospermatogenesis. By detection of virus antigen in the serum samples using ELISA, bulls were identified as persistently infected with BVDV. Virus isolation revealed four noncytopathic (NCP-BVDV) strains that were confirmed by fluorescent antibody technique (FAT) and amplification of the 5′ untranslated genomic region (5’UTR) and molecularly typed by amplification of the Erns glycoprotein region. Isolates’ Phylogenetic analysis revealed two subgenotypes: BVDV-1b (Genbank accession numbers; LC634512, LC634513, LC634515) and BVDV-1d (LC634516). According to the knowledge of the authors of the present study, the circulation of the BVDV-1d subgenotype is not reported in Egypt. Therefore, it would be of great importance to track circulating strains in specific countries for successful vaccination programs or accurate diagnostic tests, and this necessitates regular updates.

Prevalence of Bovine Viral Diarrhea Virus Infections in Pigs on Jeju Island, South Korea, from 2009-2019 and Experimental Infection of Pigs with BVDV Strains Isolated from Cattle.

On Jeju Island, South Korea, pigs have not been vaccinated against classical swine fever (CSF) since 1999. Analysis of bovine viral diarrhea virus (BVDV) isolated from pigs on Jeju Island between 2009 and 2019 identified five BVDV-1a strains and one BVDV-1b strain. These BVDV types were shown to be the same types as BVDV strains isolated from neighboring cow farms. BVDV antibody-positive pigs (both BVDV-1 and -2) were also detected at 54 of 168 pig farms during this period. In pig infection experiments using BVDV-1a and -2a strains isolated from neighboring cow farms, BVDV-1a was detected in the blood of one of four pigs infected at both 6 and 35 days post-infection (dpi) and in the blood of two of the four pigs at 28 dpi. Pigs showed higher anti-BVDV-1 titers (5.5 ± 1.5 log2) at 35 dpi. BVDV-2a was detected in the blood of one of four pigs infected with this virus at 28 dpi only, and lower antibody titers (2.75 ± 0.75 log2) were seen in these pigs at 35 dpi. While BVDV infection is not particularly pathogenic in pigs, it is still important to monitor porcine BVDV infections due to a differential diagnosis of CSFV.

Preparation and field study of combined vaccine against Clostridium perfringens type A and bovine viral diarrhea virus in camels.

PurposeThe key objective of this study was to formulate a local combined inactivated gel adjuvanted vaccine containing bovine viral diarrhea virus (BVDV)-1, BVDV-2 viruses and Clostridium perfringens type A toxoid. The study evaluated its ability to enhance protective active immune response in camels’ calves against these infectious pathogens under field conditions.Materials and MethodsThe local BVDV cytopathic strains and a local strain of toxigenic C. perfringens type A were used in vaccines formulation. Vaccines A and B were monovalent vaccines against C. perfringens and both strains of BVDVs, respectively. While the vaccine C was the combined vaccine used against the three agents. All vaccines were adjuvanted with Montanide gel. Sterility, safety, and potency tests were applied on the formulated vaccines. Virus neutralization and toxin anti-toxin neutralization tests were used to evaluate the immune responses.ResultsBoth monovalent (vaccine A) and combined vaccines (vaccine C) showed a protective level (4.5 and 3 IU/mL, respectively) against C. perfringens from the 2nd-week post-vaccination. The titer declined to 3 and 2 IU/mL, respectively at the 5th-month post-vaccination. The titer against BVDV, the monovalent vaccine (vaccine B) reached the beak (1.95 IU/mL) at the 1st-month post-vaccination and lasted till 6th-month post-vaccination (0.92 and 0.94 IU/mL) for BVDV-1a and BVDV-2, respectively.ConclusionVaccination of camels with the combined vaccine adjuvanted by Montanide gel containing C. perfringens type A toxoid and BVDV strains with 6-month intervals is recommended to protect camels safely and efficiently against such infections in the field.

Viral RNA extraction using an automatic nucleic acid extractor with magnetic particles and genetic characterization of bovine viral diarrhea virus in Tokachi Province, Japan, in 2016-2017.

In this study, the viral genome extraction performance of automatic nucleic acid extractors and manual nucleic acid extraction kits was compared. We showed that compared with manual kits, the automatic extractors showed superior genome extraction performance using bovine viral diarrhea virus (BVDV) genome-positive cattle sera and bovine coronavirus/infectious bovine rhinotracheitis virus-spiked cattle nasal swabs. In addition, the subgenotyping of BVDV strains detected in Tokachi Province in Japan during 2016-2017 was performed. Results showed that most of these BVDV strains belonged to subgenotype 1b, while few strains belonged to subgenotypes 1a and 2a. This study showed the high applicability of automatic nucleic acid extractors in extracting multiple viral genomes and the dominant subgenotype of BVDV in Tokachi.

Antiviral Effect of Cerium Dioxide Nanoparticles on the Model of the Causative Agent of Bovine Viral Diarrhea

Cerium dioxide nanoparticles (CeO2) have a wide range of biological properties, including antiviral activity. Preparations based on cerium oxide nanoparticles can be effective against animal RNA viruses, which have the greatest epizootic significance for Ukraine and potentially against human viruses, including SARS-CoV-2. In our studies, we determined the effect of cerium dioxide nanoparticles on bovine viral diarrhea virus (BVDV), a pestivirus that is the etiological agent of bovine viral diarrhea. To determine the antiviral efficacy of CeO2, a cytopathogenic strain of BVDV “BK-1” was used as an etiological agent of bovine viral diarrhea, which induces a cytopathic effect on cow embryo lungs (CEL) cell culture. When evaluating the antiviral efficacy of CeO2, it was determined that the MPC for CEL cell culture is 0.057 μg/cm3, and the CC50 is 0.077 μg/cm3. The antiviral activity of CeO2 was evaluated by the IC50 index, which was 0.036 μg/cm3 and the selectivity index, which after co-administration of CeO2 with BVDV was 2.14. There was a decrease in the titer of infectious activity of the virus during treatment with CeO2 by 2.09 lg TCD50/cm3.

Endemic infections of bovine viral diarrhea virus genotypes 1b and 2a isolated from cattle in Japan between 2014 and 2020.

Bovine viral diarrhea virus (BVDV) is a causative agent of bovine viral diarrhea. In Japan, a previous study reported that subgenotype 1b viruses were predominant until 2014. Because there is little information regarding the recent epidemiological status of BVDV circulating in Japan, we performed genetic characterization of 909 BVDV isolates obtained between 2014 and 2020. We found that 657 and 252 isolates were classified as BVDV-1 and BVDV-2, respectively, and that they were further subdivided into 1a (35 isolates, 3.9%), 1b (588, 64.7%), 1c (34, 3.7%), and 2a (252, 27.7%). Phylogenetic analysis using entire E2 coding sequence revealed that a major domestic cluster in Japan among BVDV-1b and 2a viruses were unchanged from a previous study conducted from 2006 to 2014. These results provide updated information concerning the epidemic strain of BVDV in Japan, which would be helpful for appropriate vaccine selection.

Use of multivariate analysis to evaluate antigenic relationships between US BVDV vaccine strains and non-US genetically divergent isolates

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.

Vaccination Failure in Eradication and Control Programs for Bovine Viral Diarrhea Infection.

Vaccination against bovine viral diarrhea (BVD) is one of the key elements to protect cattle herds from this economically important disorder. Bovine viral diarrhea virus (BVDV) is a pestivirus infecting animals at all ages with significant impact on reproductive, digestive, and respiratory systems. Financial burden caused by this pathogen prompts many farmers to introduce vaccination as the control and prophylactic measure especially when persistently infected (PI) individuals, being the main source of the virus in the herd, are removed after test-and-cull approach. The aim of the study was to compare the serological response in cattle herds where new PI calves were identified without prior removal of PI animals or despite their removal and after the introduction of whole herd vaccination against BVDV infection. Overall seroprevalence in 5 vaccinated herds was 91.7 and 83.3% using ELISA and virus neutralization test, respectively. Despite high titers for both vaccine and field strains of BVDV in analyzed herds the analysis of comparative strength of neutralization indicated that 41.4% of positive samples did not have a predominant titer against one specific subtype of BVDV. In 3 herds BVDV-1b subtype was identified while in 2 others it was BVDV-1d, while the vaccine used was based on BVDV-1a which was never identified in Poland so far. To increase the success of the BVDV eradication program, a careful approach is suggested when planning herd vaccination. Comparison of existing field strains and their similarity with vaccine strains at antigenic and genetic levels can be a useful approach to increase the effectiveness of vaccination and efficient protection of fetuses from persistent infection.

Characterization of a Cytopathogenic Reporter CSFV.

Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.

Bovine viral diarrhea virus subgenotype 1a in a mummified fetus from a Brazilian dairy cattle herd

We describe the molecular analysis of a wild-type field strain of bovine viral diarrhea virus (BVDV) identified in a mummified fetus from a small Brazilian dairy cattle herd. Nucleic acids extracted from samples of the lung, liver, heart, spleen, and kidney were tested by PCR assays for bovine alphaherpesvirus 1, Neospora caninum, Leptospira spp., Histophilus somni, and Brucella abortus, a nested PCR assay for Mycoplasma bovigenitalium and Ureaplasma diversum, and a RT-PCR assay for BVDV. Amplicons were only obtained in the RT-PCR assay for the partial amplification of the BVDV 5'UTR (288 bp) in kidney and spleen samples and the Npro (438 bp) gene in the kidney sample. Nucleotide sequencing of the amplified products and phylogenetic analyses based on the 2 BVDV genomic regions enabled the BVDV strain to be classified as subgenotype 1a.

Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1.

ABSTRACTPestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2.IMPORTANCE The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

Recent Advances on the Bovine Viral Diarrhea Virus Molecular Pathogenesis, Immune Response, and Vaccines Development.

The bovine viral diarrhea virus (BVDV) consists of two species and various subspecies of closely related viruses of varying antigenicity, cytopathology, and virulence-induced pathogenesis. Despite the great ongoing efforts to control and prevent BVDV outbreaks and the emergence of new variants, outbreaks still reported throughout the world. In this review, we are focusing on the molecular biology of BVDV, its molecular pathogenesis, and the immune response of the host against the viral infection. Special attention was paid to discuss some immune evasion strategies adopted by the BVDV to hijack the host immune system to ensure the success of virus replication. Vaccination is one of the main strategies for prophylaxis and contributes to the control and eradication of many viral diseases including BVDV. We discussed the recent advances of various types of currently available classical and modern BVDV vaccines. However, with the emergence of new strains and variants of the virus, it is urgent to find some other novel targets for BVDV vaccines that may overcome the drawbacks of some of the currently used vaccines. Effective vaccination strategy mainly based on the preparation of vaccines from the homologous circulating strains. The BVDV-E2 protein plays important role in viral infection and pathogenesis. We mapped some important potential neutralizing epitopes among some BVDV genomes especially the E2 protein. These novel epitopes could be promising targets against the currently circulating strains of BVDV. More research is needed to further explore the actual roles of these epitopes as novel targets for the development of novel vaccines against BVDV. These potential vaccines may contribute to the global eradication campaign of the BVDV.

Novel potent IFN-γ-inducing CD8+ T cell epitopes conserved among diverse Bovine Viral Diarrhea Virus strains

Abstract 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 availability of vaccines for decades, BVDV prevalence in cattle remains 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, 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 structural BVDV antigens: Erns, E1, and E2 glycoproteins. In addition, from nonstructural BVDV antigens: Npro, NS2-3, NS4A-B, and NS5A-B, twenty CD8+ T cell epitopes were identified. Majority of these IFN-γ-inducing CD8+ T cell epitopes were found to be highly conserved among more than two hundred strains from BVDV-1 and -2 genotypes. These conserved epitopes were also validated as cross-reactive since they induced high recall IFN-γ+ CD8+ T cell responses, ex vivo, in purified CD8+ T cells from BVDV-1- and -2-immunized cattle. Altogether, twenty-eight bovine MHC I-binding epitopes were identified from key BVDV antigens that can elicit broadly reactive CD8+ T cells against diverse BVDV strains. The data presented here 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.

Immunization with recombinant Erns-LTB fusion protein elicits protective immune responses against bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV), a major infectious pathogen and is associated with major economic losses and significant impact on animal welfare worldwide. Here, recombinant Erns-LTB protein vaccine containing MF59 adjuvant was prepared and assessed using a mouse model. The recombinant plasmid (pET32a-Erns-LTB) was constructed and transformed into BL21 (DE3) cells to produce Erns-LTB protein. The Erns-LTB protein was formulated with MF59 adjuvant, when delivered intraperitoneally in mice, exhibited higher immunogenic and induced superior levels of anti-BVDV IgG compared with the MF59 adjuvanted Erns protein. Importantly, after challenged with different BVDV BJ175170 and BJ1305 isolate strains, mice inoculated with Erns-LTB protein displayed alleviated pathological damage and decreased plasma virus shedding compared with mice inoculated with Erns protein. The enhanced protection from Erns-LTB protein is mediated by T cell immunity and primarily based on CD4+ T helper (Th) and CD8+ cytotoxic T lymphocyte (CTL), these results suggest that Erns-LTB protein has potential to protect against a broad range of BVDV strains thereby providing a novel direction for developing broadly protective vaccines.

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.

Effect of bovine viral diarrhea virus on subsequent infectivity of bovine gammaherpesvirus 4 in endometrial cells in primary culture: An in vitro model of viral co-infection

Bovine viral diarrhea virus (BVDV) and bovine gammaherpesvirus 4 (BoHV-4) infect the uterus of cattle, being responsible for huge economic losses. Most of the pathogenesis of BoHV-4 in the bovine reproductive tract has been elucidated by conducting tests on primary cultures. Thus, it is important to have optimal in vitro conditions, avoiding the presence of other pathogens that can alter the results. BVDV is one of the most frequent viral contaminants of cell cultures. Considering that non-cytopathic (NCP) BVDV biotype can generate persistently infected (PI) cattle, which are the major source for virus transmission in susceptible herds, it is important to check products derived from cattle that are intended to be used in research laboratories. The aim of this work was to evaluate how the natural infection of bovine endometrial cells (BEC) with a NCP BVDV strain (BEC + BVDV) affects BoHV-4 replication. We have demonstrated a delay in BoHV-4 gene expression and a decrease in viral load in the extracellular environment in BEC + BDVD cells compared to BEC (BVDV-free) cells. These results confirm that replication of BoHV-4 in BEC primary cultures is affected by previous infection with BVDV. This finding highlights the importance of ruling out BVDV infection in bovine primary cell cultures to avoid biological interference or misinterpretation of results at the time of performing in vitro studies with BoHV-4.

Identification and genotyping of a new subtype of bovine viral diarrhea virus 1 isolated from cattle with diarrhea

In 2019, diarrhea cases occurred on cattle farms in Qionglai and Guang'an, Sichuan Province. Two out of 20 (10%) serum and nasal swab samples were positive when tested using a bovine viral diarrhea virus (BVDV) antigen-capture ELISA kit. Two non-cytopathic strains of BVDV were isolated and named QL1903 and GA190608, respectively. The nucleotide sequences of the genomes of the two isolates were 89.52% identical. Phylogenetic analysis based on the 5'-UTR sequence revealed that the BVDV isolate QL1903 belonged to BVDV subtype 1b, whereas isolate GA190608 clustered with strains HN1814, EN-19, and BJ09_26 in a separate branch, which has tentatively been classified as a new genetic subtype, "1v".

Is contamination of bovine-sourced material with bovine viral diarrhea virus still a problem in countries with ongoing eradication campaigns?

Abstract In this report, we describe the detection of bovine viral diarrhea virus (BVDV) contamination in commercial animal-derived sera and vaccines against animal viral pathogens on the market in Poland. Antibodies against BVDV were detected in 4/45 sera samples (8.9%) using an ELISA test. The presence of BVDV antigen was found using ELISA in 3/45 serum samples (6.6%) and 18/172 vaccine samples (10.5%). An RT -PCR was conducted using primers targeting two genome regions, the five prime untranslated region (5’UTR) and N-terminal protease (Npro). BVDV RNA was detected in 33/45 (73.3%) of sera, and 11/172 samples (6.4%) of collected vaccines, of which one vaccine did not declare BVDV strain in its composition. A single serum showed the presence of an infectious virus and only one was contaminated with all 3 species of BVDV. The most frequent species in sera was BVDV-3 (75.5%), whereas in vaccines only BVDV-1 was identified. Sequence analysis showed that the tested commercial sera and one vaccine were contaminated by six genotypes of BVDV: -1a, -1b, -1c, -1d, -2a, and -3. Identification of BVDV and its genetic material in animal-derived products is important due to the possibility of pestivirus transmission as well as the chance of falsifying the results of a diagnostic test. It also demonstrates the necessity of rigorous monitoring of the bioproducts used at the laboratory and industry level.