Search Results (186)

Bovine viral diarrhea virus (BVDV) is a pathogen of globally significance in cattle that has two biotypes: non-cytopathogenic (non-cp) and cytopathogenic (cp). The cp biotype arises from the non-cp through genome rearrangements, which frequently involve the insertion of host cellular RNA sequences, often accompanied by viral genome duplications. Here, we performed a systematic analysis of insertions across all complete BVDV genomes available in GenBank. Despite a 10-fold increase in available sequences over the past 10 years (from 59 to 670), the repertoire of known rearrangements associated with the cp phenotype has expanded only modestly, with insertions occurring predominantly at five conserved genomic hotspots. Notably, independent acquisitions of similar insertions—such as DNAJC14 (Jiv), ubiquitin-like sequences, the NS4B-NS5A cleavage site, and the PYPDPQTLG motif—in phylogenetically unrelated virus lineages reflect a limited number of permissive sites. Our analysis further demonstrates that many insertions are mosaic and suggests multiple recombination events that are poorly compatible with template switching by viral polymerase. Mechanistically, we propose that non-replicative recombination provides the most consistent explanation for the observed insertion patterns, particularly common coupling of cellular RNA acquisitions with viral sequence duplications. Both the steric proximity of virus replication to the endoplasmic reticulum and the RNA end requirements (2′-3′ cyclic phosphate and 5′-OH) suggest that non-replicative recombination is likely mediated by the endoplasmic reticulum-associated IRE1 RNase and RtcB ligase, which are responsible for the alternative cytoplasmic splicing of cellular mRNA. Full article

Bovine viral diarrhea virus (BVDV) is a major cause of economic losses in the global cattle industry. This study investigated the clinical involvement and genetic diversity of BVDV in clinically affected cattle from the Thrace region in Türkiye, a critical border area with the European Union. A total of 533 nasal and rectal swabs were collected from 26 farms exhibiting clinical respiratory disease or diarrhea and analyzed using real-time RT-PCR. Positive samples were further characterized by sequencing and phylogenetic analysis to determine viral genotypes and subgenotypes. BVDV RNA was detected in 9 out of 26 farms (34.6%), with an overall positivity rate of 17.8% (95/533). Statistical analysis revealed significant associations between PCR positivity and both sampling year (p < 0.05) and animal age (p < 0.05). Genetic analysis of partial 5′-UTR sequences identified nine Pestivirus bovis subgenotype 1a strains, one Pestivirus bovis subgenotype 1f, and one Pestivirus brazilense. The Pestivirus bovis 1a strains clustered distinctly from previously reported Turkish BVDV-1 isolates, showing 97.32–99.11% nucleotide similarity with strains from Türkiye, Germany, China, Japan, the USA, Iran, and Argentina. Among the 533 samples tested, one Pestivirus brazilense isolate was identified, indicating its rare occurrence in the studied population despite targeted screening. This study represents the first report of Pestivirus brazilense in the Thrace region of Türkiye, expanding its known geographic range. The epidemiological data and genetic diversity analysis of BVDV strains characterized in this study offer critical insights for refining BVDV control programs, informing regional risk assessment, and supporting vaccine development. Full article

To develop an indirect ELISA kit for detecting serum antibodies against bovine herpesvirus 1 (BoHV-1), the gD protein of BoHV-1 was expressed in CHO-S cells and purified for use as the coating antigen. The optimized conditions were as follows: a coating antigen concentration of 2 µg/mL; serum dilution at 1:40 with incubation at 37 °C for 60 min; enzyme-labeled antibody dilution at 1:5000 with incubation at 37 °C for 60 min; and color development with TMB substrate at 37 °C for 15 min. The absorbance was then measured with a microplate reader. The kit could detect BoHV-1-positive serum at a dilution of up to 1:3200, showing favorable analytical detection capacity. No cross-reactivity was observed with positive sera against bovine viral diarrhea virus (BVDV), Akabane virus (AKAV), bovine parainfluenza virus type 3 (BPIV3), or lumpy skin disease virus (LSDV), reflecting ideal analytical specificity. Based on 126 field serum samples tested against a commercial IDEXX gB-based ELISA kit, we further calculated the diagnostic sensitivity (83.54%; 95% CI: 73.22–90.17%) and diagnostic specificity (100%; 95% CI: 92.43–100%) to quantitatively characterize its clinical detection performance. The ROC curve analysis based on IDEXX gB serostatus rather than true VNT-confirmed infection generated an AUC of 0.912 (95% CI: 0.856–0.951), which only reflects inter-assay concordance instead of absolute diagnostic validity. This indirect ELISA can be applied to epidemiological investigation and evaluation of immunization efficacy. Full article

The genus Orthopestivirus, within the family Flaviviridae, includes important pathogens such as classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV-1 and BVDV-2), and border disease virus (BDV). Although Costa Rica is endemic for BVDV, it remains officially free of CSFV and BDV, highlighting the need for reliable molecular tools for pestivirus surveillance and differential diagnosis in swine populations. This study validated a conventional broad-range in-house RT-PCR assay targeting the 5′ untranslated region (5′UTR) of pestiviruses, a genomic region that is highly conserved among members of the genus Orthopestivirus. A wide variety of PCR-based assays have been developed for pestivirus detection; however, many are designed specifically for the diagnosis of bovine pestiviruses, such as bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2, and HoBi-like pestivirus (HoBiPeV) in cattle. In contrast, the assay described in this study was standardized and validated to detect multiple pestivirus species, thereby providing a useful tool for surveillance and diagnostic applications across different host species. A total of 66 reference samples were analyzed to determine diagnostic sensitivity and specificity, while analytical sensitivity, repeatability, reproducibility, and selectivity were also evaluated. Additionally, pestivirus surveillance was conducted from 2014 to 2025. The assay showed a limit of detection of 1.2 copies/µL for CSFV and 11.7 copies/µL for BVDV, with diagnostic sensitivity and specificity of 98% and 100%, respectively. A total of 1072 surveillance samples were tested, including 11 bovine and 1061 porcine samples. Two porcine samples collected in 2015 were positive for pestivirus and clustered within the BVDV-2a group, representing the first molecular detection of BVDV-2a in pigs in Costa Rica. Circulation of BVDV-1b in cattle was also confirmed. The assay demonstrated satisfactory performance as a broad-range pestivirus surveillance tool and may support differential diagnosis in CSFV-free countries when combined with sequencing confirmation for pestivirus species identification. Full article

Bovine viral diarrhea virus (BVDV) infection is associated with inflammatory responses, but the mechanisms underlying inflammasome activation remain unclear. In this study, Madin–Darby bovine kidney (MDBK) cells were used to compare the inflammatory responses induced by the cytopathogenic NADL strain and the non-cytopathogenic TC strain. Both strains significantly increased IL-1β and IL-18 production and promoted NLRP3 inflammasome assembly, indicating activation of upstream inflammasome signaling. However, only the NADL strain markedly induced Gasdermin D (GSDMD) cleavage, membrane pore formation, lactate dehydrogenase release, and pyroptotic cell death. Further mechanistic analyses demonstrated that the NADL core protein C interacted with the inflammasome adaptor ASC, thereby facilitating NLRP3 inflammasome assembly and enhancing inflammatory cytokine secretion. Silencing ASC significantly impaired inflammasome activation and pyroptosis induced by protein C, confirming its essential role in this process. Collectively, these findings demonstrate that the cytopathogenic BVDV NADL strain activates ASC-dependent NLRP3 inflammasome signaling and GSDMD-mediated pyroptosis through its core protein C, providing a molecular explanation for the distinct inflammatory responses and pathogenic outcomes associated with different BVDV biotypes. Full article

Bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1) are major pathogens affecting cattle worldwide, leading to significant economic losses in livestock production systems. This study aimed to estimate the seroprevalence and dual seropositivity of both viruses in dual-purpose cattle in Villavicencio, Colombia. A total of 1000 serum samples were collected from cattle across 30 farms and analyzed using ELISA. Seroprevalence for BVDV ranged from 0.0% to 78.7%, while BoHV-1 ranged from 0.0% to 100.0%. Dual seropositivity with both viruses was observed at the farm level, ranging from 0.0% to 52.2%. At the herd level, 26.7% (8/30) of the farms were seronegative for BVDV (95% CI: 14.2–44.5%), whereas only 3.3% (1/30) were seronegative for BoHV-1 (95% CI: 0.6–16.7%). Additionally, seven farms (23.3%) were simultaneously seronegative for both BVDV and BoHV-1. Overall, the findings demonstrate widespread exposure and non-random concurrent seropositivity of both viruses, highlighting the importance of herd management, biosecurity, and animal movement in shaping infection dynamics in dual-purpose cattle systems. Management practices, animal movement, and environmental factors were significantly associated with seropositivity. Full article

Bovine viral diarrhea (BVD) is a globally significant disease that adversely affects cattle health and productivity, including in India. It is caused by three bovine pestiviruses: bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, and HoBi-like pestivirus (HoBiPeV), which belong to the Pestivirus genus within the Flaviviridae family. Despite the prevalence of all three pestivirus species in India, no commercial vaccine based on the local circulating strain is currently available. This study evaluates the safety, immunogenicity, and protective efficacy of an inactivated whole-virus BVD vaccine, based on an Indian BVDV-1 strain. The virus was propagated in MDBK cells, inactivated using 3 mM binary ethylenimine (BEI) for 24 h at 37 °C, and formulated with Montanide ISA 61 VG (SEPPIC) in a 50:50 water-in-oil emulsion. Vaccine safety was confirmed in both guinea pigs and bovine calves, with no adverse effects observed. Immunogenicity testing in guinea pigs (n = 6) showed neutralizing antibody titres up to 9 log₂ (1/512). In calves aged 9–12 months (n = 3), the vaccine elicited strong humoral and cell-mediated immune responses, with mean neutralizing antibody titres against the homologous BVDV-1 strain reaching 14 log₂ (1/16,384). Neutralizing antibody levels remained detectable for up to 12 months post vaccination with sustained mean titres of 7 log₂ (1/128). Notably, titres reported to be adequate for fetal protection (≥9 log₂ or ≥1/512 were maintained for five months following vaccination. Challenge studies demonstrated complete protection of vaccinated calves against homologous BVDV-1 acute infection. In addition, the vaccine conferred partial cross-protection against heterologous strains including BVDV-2 and HoBiPeV. In a field trial involving 125 cattle, 74% of animals developed protective neutralizing titres (≥7 log₂ or ≥1/128), while 48% achieved titres reported to be adequate for fetal protection (9 log₂ or 1/512). Furthermore, 92% of vaccinated cattle maintained neutralizing antibody titres of at least 6 log₂ (≥1/64) for up to six months post-booster vaccination. A strong positive correlation was observed between guinea pig and bovine antibody responses (R² = 0.6809; p < 0.0001), indicating the potential of guinea pigs as a predictive model. Vaccine stability was confirmed for up to 8 months when stored at 4 °C, as demonstrated by the immunogenicity in guinea pigs. Collectively, these findings demonstrate that the locally developed inactivated BVDV-1 vaccine is safe, highly immunogenic, and capable of providing protective immunity against BVDV-1 infection, supporting its potential use in BVD control programs in India. Full article

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is an acute, febrile, and highly contagious disease that has led to significant economic losses in the global swine industry. Although the attenuated lapinized CSF vaccine (C-strain) has effectively controlled CSF outbreaks in China since the 1950s, it remains challenging to serologically differentiate infected from vaccinated animals (DIVA). Currently, the application of E2 subunit vaccines allows for DIVA by detecting antibodies against the E^rns protein. Therefore, this study aimed to develop a blocking ELISA for CSFV E^rns antibody detection using porcine monoclonal antibodies (mAbs) derived from single B cell technology. Peripheral blood mononuclear cells (PBMCs) were isolated from immunized pigs, and single CD21⁺IgM⁻E^rns-His tag⁺ B cells were sorted via flow cytometry. Using one-step PCR, full-length genes of porcine IgG heavy and light chains were amplified separately, yielding 11 porcine mAbs against the CSFV E^rns protein. Among these, three mAbs (E0S3, E0S5, and E0S10) exhibited broad reactivity, while two (E0S1, E0S4) showed no cross-reaction with bovine viral diarrhea virus (BVDV). Using mAb E0S4 as the blocking antibody, a blocking ELISA was established and optimized. The assay demonstrated a detection limit of 1:128, no cross-reactivity with other swine viruses or BVDV, and intra- and inter-assay coefficients of variation below 10%. ROC curve analysis determined an optimal cut-off value of 48.4%, with high sensitivity and specificity. In conclusion, the developed blocking ELISA provides a reliable tool for high-throughput serological surveillance, facilitating the DIVA strategy and contributing to CSF eradication programs. Full article

This study aimed to investigate the current epidemiology and genetic evolution of bovine viral diarrhea virus (BVDV) on cattle farms in Gansu Province, China, between 2021 and 2025. A total of 749 samples from 62 farms across 14 cities and prefectures in Gansu were tested. The overall BVDV positivity rate was 19.89%, determined by amplification of the 5′-UTR and N^pro regions. Seven subtypes were identified: BVDV-1a, -1b, -1d, -1m, -1v, -1u, and -2a. BVDV-1u was the predominant subtype (59.06%), followed by BVDV-1v (13.42%). The greatest subtype diversity was observed in Jinchang City and Gannan Tibetan Autonomous Prefecture. A non-cytopathic BVDV-1v strain, designated YC-2025-Gansu2 (GenBank accession no. PV945812.1), was isolated. This study expands the BVDV subtype database for Gansu and supports the development of subtype-specific prevention strategies in the region. Full article

Bovine viral diarrhea virus (BVDV), a significant global pathogen threatening cattle industries worldwide, presents substantial challenges for disease control. Its ability to infect cattle across all age groups, coupled with incompletely understood transmission mechanisms, complicates prevention and treatment strategies. We previously reported that BVDV induced tunneling nanotubes (TNTs)—F-actin-rich cytoplasmic connections between adjacent cells—and utilizes these structures for intercellular transmission. In this study, we used lentiviral transfection to express various structural and non-structural proteins of BVDV and identified NS5A as a critical viral protein that induces the formation of TNTs. RNA-seq analysis revealed that CKAP2, a host protein, plays a key role in TNT generation, with the PI3K/AKT/mTOR signaling pathway being essential for this process. Further investigation demonstrated that CKAP2 interacts with BVDV NS5A, triggering the activation of the PI3K/AKT/mTOR pathway, thereby promoting TNT formation and enhancing viral dissemination. Our data highlight a previously unknown mechanism of BVDV spreading and replication, which could have significant implications for within-host spread and immune evasion. Full article

Bovine viral diarrhea virus (BVDV) is a major cattle pathogen associated with significant economic losses worldwide. In Brazil, the high genetic diversity of circulating strains represents an additional challenge for disease control. To update the molecular epidemiology of BVDV in southern Brazil, 16,198 bovine serum samples collected in 2020 through a national surveillance program were screened for pestivirus RNA by RT-qPCR. Forty-nine samples (0.36%) were positive and subjected to partial sequencing of the 5′UTR and N^pro regions. Phylogenetic analysis identified BVDV-1a (25/49; 51%), BVDV-1b (1/49; 2%), BVDV-1d (7/49; 14%), and BVDV-2b (16/49; 33%), with no detection of HoBiPeV. When compared descriptively with data from 2010 in the same region, BVDV-1a remained the most frequent subgenotype, while BVDV-2b also represented a substantial proportion of detections, contrasting with other regions worldwide. Although the two datasets are not directly comparable, and no statistically significant differences were observed, these findings provide an updated overview of circulating BVDV subgenotypes in Rio Grande do Sul. The absence of HoBiPeV contrasts with reports from other regions of Brazil and suggests a distinct regional pattern of pestivirus circulation. Overall, the results reinforce the importance of continuous genomic surveillance to monitor changes in viral diversity and support control strategies in cattle populations. Full article

Background/Objectives: Bovine viral diarrhea (BVD) is a major infectious disease of cattle caused by bovine viral diarrhea virus genotypes 1 and 2 (BVDV-1 and BVDV-2). Current inactivated and live attenuated vaccines provide incomplete cross-genotype protection and may exhibit limitations related to durability of immunity or safety. This study evaluated whether co-expression of the BVDV envelope glycoproteins E1 and E2 in a Modified Vaccinia Ankara (MVA) vector could support antigen expression and induce immune responses in a proof-of-concept model. Methods: Recombinant Modified Vaccinia Ankara (MVA) viruses expressing BVDV-1 E1E2 or BVDV-2 E1E2 were generated by homologous recombination. Recombinant viruses were purified and characterized for antigen expression, genetic stability, and growth properties in vitro. Immunogenicity was evaluated in a BALB/c mouse model by measuring E2-specific antibody responses, virus-neutralizing antibodies, and antigen-responsive cellular immune responses. Results: Both recombinant MVA constructs showed detectable E2 expression when E1 and E2 were co-expressed, and exhibited growth characteristics comparable to parental MVA with stable maintenance after serial passage. In contrast, recombinant MVA expressing E2 alone did not yield detectable E2 protein under the same experimental conditions. Immunization induced detectable humoral and cellular immune responses, including E2-specific IgG antibodies, virus-neutralizing antibodies, and increased frequencies of antigen-responsive CD8⁺ T cells with a tendency toward a Th1-biased profile. Conclusions: These findings indicate that co-expression of BVDV E1 and E2 in an MVA vector can support detectable antigen expression and induce measurable immune responses in a mouse proof-of-concept model. Further studies in cattle, including challenge experiments, will be required to determine the protective efficacy and practical applicability of this platform for BVDV vaccine development. Full article

This study investigated the in vitro replication kinetics and molecular characteristics of five field isolates of bovine viral diarrhea virus (BVDV) representing subgenotypes 1b, 1d, and 1f, currently circulating in Hungary. We compared cytopathogenic (cp) and non-cytopathogenic (ncp) biotype pairs using digital PCR (dPCR) and virus titration. While dPCR showed higher genome copy numbers for cp isolates, virus titration revealed comparable or lower infectious titers, suggesting the accumulation of replication-incompetent viral particles during the infection cycle. Molecular analysis identified (novel) amino acid substitutions in Npro, capsid, and NS4B regions, although typical large-scale genome rearrangements were absent. These findings demonstrate that biotype differences are molecularly complex and subgenotype-dependent. Our results emphasize that relying on a few genetic markers is insufficient for biotype categorization, necessitating comprehensive characterization in BVDV surveillance programs. This complexity must be considered when designing vaccines or control programs, especially in regions with diverse circulating strains. Full article

Bovine viral diarrhea virus (BVDV), a globally persistent pathogen, causes bovine viral diarrhea-mucosal disease (BVD-MD), a contagious bovine disease posing significant pressures on both public health and economic development. Baicalin (BA), a flavonoid derived from Scutellaria baicalensis, exhibits broad antiviral activities but suffers from poor aqueous solubility and low bioavailability, limiting its therapeutic potential against BVDV. To address this limitation, we developed BA-loaded poly (ethylene gly-col)-poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (BA-PEG-PLGA NPs). While autophagy and NLRP3 inflammasome activation have been individually implicated in viral pathogenesis, their functional crosstalk during BVDV infection remains uncharacterized. Herein, we evaluated the antiviral efficacy of BA-PEG-PLGA NPs through integrated in vitro and in vivo experiments. We employed quantitative polymerase chain reaction (qPCR), transcriptome sequencing, Western blot analysis, immunofluorescence microscopy, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) to investigate the mechanisms by which BA and BA-PEG-PLGA NPs combat bovine viral diarrhea virus (BVDV) infection. We found that both free BA and BA-PEG-PLGA NPs effectively attenuated BVDV replication in vitro and in vivo; notably, the nano-formulation exhibited superior efficacy. Mechanistically, BA and its nano-formulation restored autophagy homeostasis, suppressed ROS overproduction, and blocked NLRP3 inflammasome activation and pyroptotic cell death effects comparable to the specific NLRP3 inhibitor MCC950. These findings establish the autophagy–NLRP3/pyroptosis axis as a critical pathogenic mechanism in BVDV infection and reveal that nano-formulated baicalin represents an antiviral strategy by coordinately targeting this axis. This work not only provides a translatable nanomedicine approach for BVDV control but also expands the mechanistic understanding of flavonoid-based interventions in viral inflammatory diseases. Full article

Bovine viral diarrhea (BVD) is a major cause of economic losses in the global cattle industry, particularly in countries characterized by intensive livestock production systems. Pestivirus tauri, formerly known as Bovine viral diarrhea virus type 2 (BVDV-2), is the current taxonomic designation according to the International Committee on Taxonomy of Viruses (ICTV). Between 2005 and 2018, Pestivirus tauri was detected in cattle herds in mainland Italy, particularly in the Lombardy region. Four viral strains were successfully isolated in cell cultures and subjected to whole-genome sequencing. Phylogenetic reconstruction placed all Italian isolates within the Pestivirus tauri subgenotype c, a lineage encompassing strains reported in Asia, Europe and the United States. Consistently, comparative sequence identity analyses indicated the highest similarity with the Parker strain (USA, 1991) and the Potsdam 1600 strain (Germany, 2000). These results contribute to a more detailed understanding of Pestivirus tauri genomic architecture and evolutionary dynamics, providing a valuable resource for comparative genomic studies. Such data are crucial for exploring viral diversity and evolution, optimizing the design of diagnostic primers and probes, and advancing insights into the molecular epidemiology of Pestivirus. Full article