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Bovine viral diarrhea (BVD) is a highly impactful disease in the cattle industry worldwide. The disease is caused by the Bovine Viral Diarrhea Virus (BVDV), a member of the genus Pestivirus within the Flaviviridae family. Higher seroprevalence of the disease has been recorded in Ethiopia; however, the virus’s genetic diversity has not yet been studied in this country. This study aimed to detect BVDV infection in dairy animals and identify viral species based on the analysis of the viral 5′untranslated region (5′ UTR). Between 2022 and 2023, 576 swab samples were collected from animals showing clinical signs. BVDV viruses were isolated and detected using the Immunofluorescent antibody technique. A one-step reverse transcriptase polymerase chain reaction technique (targeting a conserved 5’UTR region of BVDV 1 and BVDV 2) was used to identify BVDV sub-genotypes. Eighteen viral isolates were obtained from samples collected from dairy farms and one breeding center. Isolates were identified as ncp biotypes, and nucleotide sequence analysis revealed BVDV 2a and 1b, predominantly circulating in livestock populations. As far as we are aware, this represents the first identification and characterization of the BVDV sub-genotypes within Ethiopia’s cattle population. Consequently, our findings provide a valuable foundation for developing and implementing effective BVD prevention and control strategies in Ethiopia.

A chimeric pestivirus KD26_E2LOM strain can induce antibodies that can be partially distinguished from antibodies from classical swine fever virus (CSFV) infection. The chimeric pestivirus vaccine strain was created using bovine viral diarrhea virus as the backbone; however, the entire BVDV E2 gene region was replaced with the E2 gene, which encodes the major target for neutralizing antibodies against CSFV. Pigs were vaccinated once or twice with the chimeric pestivirus KD26_E2LOM strain, and protective efficacy was evaluated after subsequent challenge with virulent CSFV. Pigs inoculated with the chimeric pestivirus KD26_E2LOM strain did not have a high temperature or leukopenia, and CSFV neutralizing antibodies (>64-fold) were observed from 28 days postvaccination (dpv). In addition, the level of anti-CSFV E2 antibody positivity was >0.8 (s/p value) from 30 dpv, and there were no antibody-positive individuals among the sentinel pigs. In control pigs, CSF antigen was detected in blood, nasal, and fecal samples at 5, 7, 10, 14, and 21 days postchallenge (dpc) and in several organs; however, no CSFV was detected in the organs of pigs vaccinated with the chimeric pestivirus KD26_E2LOM strain, and no virus shedding or CSF antigen was detected on any dpc. Thus, the chimeric pestivirus KD26_E2LOM strain protects pigs against horizontal transmission of virulent CSFV; however, this strain may have only partial potential for the differential detection of CSFV E[sup.rns] antibodies.

Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea-mucosal disease (BVD-MD), an important viral disease in cattle that is responsible for extensive economic losses to the cattle industry worldwide. Currently, several underlying mechanisms involved in viral replication, pathogenesis, and evading host innate immunity of BVDV remain to be elucidated, particularly during the early stage of virus infection. To further explore the mechanisms of BVDV-host interactions, the transcriptomics and proteomics profiles of BVDV-infected MDBK cells were sequenced using RNA-seq and iTRAQ techniques, respectively, and followed by an integrative analysis. Compared with mock-infected MDBK cells, a total of 665 differentially expressed genes (DEGs) (391 down-regulated, 274 up-regulated) and 725 differentially expressed proteins (DEPs) (461 down-regulated, 264 up-regulated) were identified. Among these, several DEGs and DEPs were further verified using quantitative RT-PCR and western blot. Following gene ontology (GO) annotation and KEGG enrichment analysis, we determined that these DEGs and DEPs were significantly enriched in multiple important cellular signaling pathways including NOD-like receptor, Toll-like receptor, TNF, NF-κB, MAPK, cAMP, lysosome, protein processing in endoplasmic reticulum, lipid metabolism, and apoptosis signaling pathways. Significantly, the down-regulated DEGs and DEPs were predominantly associated with apoptosis-regulated elements, inflammatory factors, and antiviral elements that were involved in innate immunity, thus, indicating that BVDV could inhibit apoptosis and the expression of host antiviral genes to facilitate viral replication. Meanwhile, up-regulated DEGs and DEPs were primarily involved in metabolism and autophagy signaling pathways, indicating that BVDV could utilize the host metabolic resources and cell autophagy to promote replication. However, the potential mechanisms BVDV-host interactions required further experimental validation. Our data provide an overview of changes in transcriptomics and proteomics profiles of BVDV-infected MDBK cells, thus, providing an important basis for further exploring the mechanisms of BVDV-host interactions.

•The Erns, E2, or Erns-E2 protein vaccines containing MF59 and CPG-ODN being exploited.•Adjuvanted recombinant Erns-E2 protein vaccine producing higher antibody levels.•Adjuvanted recombinant Erns-E2 protein vaccine stimulating intense cellular immunity.•A subunit vaccine-adjuvant combination as a vaccine strategy to protect against BVDV. To obtain an effective vaccine candidate against bovine viral diarrhea virus (BVDV) disease which causes great economical loss in cattle industries, recombinant Erns-E2 protein vaccine containing MF59 and CPG-ODN adjuvants was prepared and assessed in this study. The recombinant plasmid (pET32a-Erns-E2) was constructed and transformed into BL21 (DE3) cells to produce Erns-E2 protein. We immunized mice with the MF59–and CPG-ODN–adjuvanted recombinant Erns-E2 protein, E2 protein, or Erns protein, respectively. To evaluate immunogenicity and efficacy of a vaccine-adjuvant combination, mice were challenged with BVDV BJ175170 strain after immunization. All adjuvanted vaccines elicited detectable humoral and cellular immune responses, the BVDV-specific antibody titers as well as interleukin 4 (IL-4) levels in sera of mice immunized with the recombinant Erns-E2 protein were higher than in those of mice immunized with either the recombinant Erns or E2 protein. Besides, immunization with the Erns-E2 vaccines induced higher percentage of CD4+IFN-γ+, CD8+IFN-γ+ T cells and CD3+TNF-α+ T cells compared with the other vaccines. More protective efficacy against BVDV infection was acquired in the mice treated with the recombinant Erns-E2 protein, as shown by a reduction of viremia and slight pathological changes compared with both the control mice and the other vaccinated mice. Our findings suggest that the use of the recombinant Erns-E2 protein vaccine formulated with MF59 and CPG-ODN adjuvants enhances T cell responses and viral control, which warrants the Erns-E2 protein vaccine-adjuvant combination could be as a vaccine strategy to against BVDV.

Objective: This study focused on Bovine Viral Diarrhea Virus (BVDV) and aimed to investigate its presence in cattle herds from five provinces of the Casablanca–Settat region of Morocco (Benslimane, Settat, Berrechid, El Jadida, and Sidi Bennour) that were experiencing respiratory and reproductive disorders. Detection of BVDV was performed using antigen capture ELISA and real-time RT-PCR assays. Materials and methods: From January 2021 to June 2025, a total of 500 blood serum samples and 500 leukocyte samples were collected from cattle exhibiting respiratory and abortive symptoms. The serum samples were analyzed for BVDV antigens using antigen capture ELISA assay, while the leukocyte samples were tested for the BVDV genome using real-time RT-PCR. Results: Out of 500 cattle sampled, 9 (1.8%) were positive for BVDV antigens, all of which were confirmed by RT-PCR, indicating active infection. Positive cases were distributed across four provinces, with the highest proportion in Benslimane (44.4% of positives), followed by Settat and Berrechid (22.2% each), and El Jadida (11.1%). No cases were detected in Sidi Bennour. Conclusion: This study provides the first documented evidence of BVDV infection in Morocco, suggesting that the virus is likely endemic in the region. The findings underscore the need for further research to isolate and characterize circulating BVDV strains. This will be crucial for developing effective prevention and control strategies to mitigate the impact of BVDV on cattle health and productivity

A chimeric pestivirus (KD26_(E)2LOM) was prepared by inserting the E2 gene of the classical swine fever virus (CSFV) LOM strain into the backbone of the bovine viral diarrhea virus (BVDV) KD26 strain. KD26_(E)2LOM was obtained by transfecting the cDNA pACKD26_(E)2LOM into PK-15 cells. KD26_(E)2LOM chimeric pestivirus proliferated to titers of 106.5 TCID50/mL and 108.0 TCID50/mL at 96 h post-inoculation into PK-15 cells or MDBK cells, respectively. It also reacted with antibodies specific for CSFV E2 and BVDV Erns, but not with an anti-BVDV E2 antibody. Piglets (55–60 days old) inoculated with a high dose (107.0 TCID50/mL) of KD26_(E)2LOM produced high levels of CSFV E2 antibodies. In addition, no co-habiting pigs were infected with KD26_(E)2LOM; however, some inoculated pigs excreted the virus, and the virus was detected in some organs. When pregnant sows were inoculated during the first trimester (55–60 days) with a high dose (107.0 TCID50/mL) of KD26_(E)2LOM, anti-CSFV E2 antibodies were produced at high levels; chimeric pestivirus was detected in one fetus and in the ileum of one sow. When 5-day-old calves that did not consume colostrum received a high dose (107.0 TCID50/mL) of KD26_(E)2LOM, one calf secreted the virus in both feces and nasal fluid on Day 2. A high dose of KD26_(E)2LOM does not induce specific clinical signs in most animals, does not spread from animal to animal, and generates CSFV E2 antibodies with DVIA functions. Therefore, chimeric pestivirus KD26_(E)2LOM is a potential CSFV live marker vaccine.

Bovine viral diarrhea virus (BVDV), a positive-sense single-stranded RNA virus, causes significant economic losses in the cattle industry. Current diagnostic methods for BVDV exhibit variable sensitivity and specificity, underscoring the need for more rapid and accurate detection approaches. Here, we developed a novel competitive ELISA (cELISA) to detect antibodies against the BVDV E2 protein. We generated three monoclonal antibodies (mAbs)-3E6, 2D5, and 5B9-by immunizing mice with purified BVDV E2 protein expressed in Expi293F cells. Among these, mAb 3E6 displayed superior competitive binding abilities to the E2 protein, enabling effective differentiation between BVDV positive and negative sera. Remarkably, mAb 3E6 exhibited pan-genotypic recognition of various BVDV strains, including BVDV-1a, -1b, -1c, -1m, -1p, -1v, and -2a, while showing no cross-reactivity with the classical swine fever virus (CSFV). Computational modeling using AlphaFold 3 identified domain B of the E2 protein as the primary binding site for mAb 3E6. Building upon these findings, we established a cELISA employing mAb 3E6 and recombinant E2 protein. Receiver-operating characteristic (ROC) analysis revealed outstanding diagnostic performance, achieving a sensitivity of 99.26% and specificity of 98.99%. Further tests confirmed the cELISA's specificity for detecting BVDV-specific antibodies, with no cross-reactivity with antisera from animals infected or immunized against BCoV, BHV-1, BRV, AKAV, LSDV, BLV, and CSFV. Consistency was observed between results from the BVDV E2 cELISA and traditional virus neutralization test (VNT), demonstrating high sensitivity for monitoring antibody dynamics. In performance evaluations, the established cELISA exhibited high concordance with VNT in assessing 160 vaccinated sera and 190 clinical samples. The BVDV E2 cELISA, utilizing mAb 3E6 to target domain B of the BVDV E2 protein, represents a reliable and effective serological diagnostic tool for the detection of antibodies against both BVDV-1 and BVDV-2. This methodology holds significant promise for applications in clinical diagnosis and the evaluation of vaccine efficacy.

The development of potent non-nucleoside inhibitors (NNIs) could be an alternate strategy to combating infectious bovine viral diarrhea virus (BVDV), other than the traditional vaccination. RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral replication; therefore, it is one of the primary targets for countermeasures against infectious diseases. The reported NNIs, belonging to the classes of quinolines (2h: imidazo[4,5-g]quinolines and 5m: pyrido[2,3-g] quinoxalines), displayed activity in cell-based and enzyme-based assays. Nevertheless, the RdRp binding site and microscopic mechanistic action are still elusive, and can be explored at a molecular level. Here, we employed a varied computational arsenal, including conventional and accelerated methods, to identify quinoline compounds’ most likely binding sites. Our study revealed A392 and I261 as the mutations that can render RdRp resistant against quinoline compounds. In particular, for ligand 2h, mutation of A392E is the most probable mutation. The loop L1 and linker of the fingertip is recognized as a pivotal structural determinant for the stability and escape of quinoline compounds. Overall, this work demonstrates that the quinoline inhibitors bind at the template entrance channel, which is governed by conformational dynamics of interactions with loops and linker residues, and reveals structural and mechanistic insights into inhibition phenomena, for the discovery of improved antivirals.

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study, we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis, bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells, lymphocytes or BL-3 cells. By protein electrophoresis, several protein bands were unique for high-virulence, 1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta, IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages.

Pestiviruses incur significant economic and welfare burdens on global livestock production. We used gene editing to produce pigs that were fully resistant to the pestivirus classical swine fever virus.DNAJC14 is essential for replication of classical pestiviruses in cell lines in vitro, but its importance for these viruses in the context of an animal was unknown.We used CRISPR/Cas to edit pig DNAJC14 in zygotes, which developed to produce healthy animals.Primary cells isolated from DNAJC14-edited pigs were resistant to infection with two different pestiviruses: classical swine fever virus and bovine viral diarrhoea virus.No signs of infection were detected when young adult pigs with edited DNAJC14 were inoculated with classical swine fever virus, demonstrating gene editing as a viable option for control of these devastating pathogens. Infectious diseases remain a major impediment to livestock production, negatively impacting both productivity and welfare. Where key interactions between viruses and host proteins have been identified, it is possible to rationally devise intervention strategies. In vitro studies have identified the host protein DNAJC14 as a core component of the replicative cycle of classical pestiviruses. Outbreaks caused by this group of viruses cause enormous losses in stock farming due to culling and export restrictions. Using CRISPR/Cas9 gene editing, we produced a cohort of pigs with altered DNAJC14. Primary cells from these animals did not support replication of either classical swine fever virus (CSFV) or bovine viral diarrhoea virus (BVDV) in vitro. In vivo challenge with CSFV revealed that the edited pigs displayed complete resistance to infection. This establishes gene editing as an additional strategy that can contribute to the control of classical pestiviruses. [Display omitted] We demonstrate that pigs with altered DNAJC14 are resistant to infection with classical swine fever virus under laboratory conditions, placing this discovery at a Technology Readiness Level of 4. While no obvious phenotypic deficits were observed, further work is required to confirm that important welfare and production parameters have not been altered, before commercialization. The closest related prior example is editing of CD163 in pigs to produce animals that are resistant to porcine reproductive and respiratory syndrome virus (PRRSV), another major pathogen. Thus far, gene-edited pigs with PRRSV resistance have been approved in Colombia, Brazil, Dominican Republic, and, most recently, the USA. This reflects maturing regulatory pathways for such products in many jurisdictions. The pigs presented in the current work will provide a valuable additional example to aid regulatory discourse. CRISPR/Cas9 editing of pig DNAJC14 resulted in animals that were completely resistant to in vivo challenge with classical swine fever virus, highlighting the pivotal role of this host protein in classical pestivirus infections and providing a novel strategy to improve livestock health, enhance animal welfare, and reduce economic losses.