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 Npro 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.

Bluetongue virus (BTV), a double-stranded RNA virus, comprises 36 serotypes and is one of the most widely distributed animal pathogens. VP2 is located on the outermost layer of BTV and contains the majority of epitopes that are recognized by neutralizing antibodies. To date, the B-cell epitopes on BTV-1 VP2 remain poorly characterized. In this study, the VP21-670aa was expressed in Escherichia coli and used to generate 10 monoclonal antibodies (mAbs) with hybridoma technology. Epitope mapping with truncated glutathione S-transferase fusion peptides identified two linear B-cell epitopes: 619TVTAQYT625, recognized by mAbs 4A2, 4A8, 4D4, 4D5, 4E6, 4E8, and 4F3, and 430RLNHSTREITYAQG443, recognized by mAbs 4B1, 4E3 and 4E1. A cross-reactivity analysis showed that these antibodies were specific to BTV-1 VP2. These findings identify two immunodominant epitopes on VP2 and provide a foundation for the development of epitope-based, BTV-specific diagnostics and subunit vaccines.

Capripoxviruses cause diseases (e.g., lumpy skin disease, sheep pox, and goat pox) that significantly hinder the growth of livestock production in endemic areas. Here, we systemically describe a B-cell monoclonal antibody (mAb) derived from the lumpy skin disease virus (LSDV) ORF123, which exhibits cross-reactivity with goat poxvirus (GTPV) and sheep poxvirus (SPPV). A novel continuous linear and conformational epitope, 85PYFLKN90, of LSDV and GTPV was first identified using bioinformatics, western blotting, and indirect immunofluorescence methods. Furthermore, the linear epitope recognition of SPPV by this LSDV ORF123 mAb was determined by the natural point mutation from P to Q at amino acid 85. Moreover, through alanine-scanning mutagenesis analysis, we demonstrated that the critical amino acid of this conserved linear and conformational epitope of LSDV ORF123 slightly differs from that of the GTPV homologue. Importantly, this mAb retained cross-neutralizing activity against LSDV, GTPV, and extracellular SPPV replication. The cross-binding and neutralizing activity of this LSDV ORF123 mAb strengthens our understanding of the pathogenic mechanisms and antigenic similarities among different members of the genus Capripoxviruses. These findings provide theoretical and experimental evidence for the development of universal pan-vaccines and therapeutic antibodies.

Although brucellosis is endemic to the Qinghai-Tibet Plateau, the molecular epidemiology of circulating Brucella abortus is still poorly characterized. Thus, in this study, we adopted an integrated approach of bacteriology and whole-genome sequencing (WGS) to genetically characterize B. abortus isolates from sheep, yak, and cattle in Qinghai, China. Conventional biotyping assays and multilocus sequence typing (MLST) show that the three strains were conclusively identified as B. abortus biovar 1, sequence type 2 (ST2). Furthermore, average nucleotide identity (ANI) analysis indicated that the three strains (BA0611, BAHYS, and BAQHM) showed ANI values above 99.99% and over 99.91% identity with the B. abortus reference strain, confirming their species assignment. The three B. abortus isolates BA0611, BAHYS, and BAQHM exhibited identical virulence gene profiles, harboring 69 virulence-related genes altogether and uniformly lacking three crucial virulence-associated genes, namely bmaA, btpB, and virB10. Core-genome SNP phylogenetic analysis revealed close genetic relatedness to Tibetan strain XZ19-1 and Russian isolates, distinguishing them from previously identified local human and marmot strains. The high genetic similarity observed among the three strains indicates a common source of infection, supporting the classification of these cases as a cluster infection. These data together confirm the prevalence of genetically divergent B. abortus lineages within the Qinghai-Tibet Plateau, which enriches current insights into the host spectrum and genomic diversity of this zoonotic pathogen in the region. These findings provide robust support for optimizing local genomic surveillance and formulating precise prevention and control strategies to reduce brucellosis prevalence in both animal and human populations.

To establish a rapid method for detecting antibodies against epizootic hemorrhagic disease virus (EHDV), the highly conserved group-specific protein VP7 was used as the target antigen in this study. The recombinant VP7 protein was expressed in Sf9 cells using a baculovirus expression system and subsequently purified. Polyclonal antibodies were generated by immunizing New Zealand white rabbits with the purified recombinant VP7 protein. Western blotting and cellular immunofluorescence assays confirmed the strong immunogenicity of the protein. A colloidal gold-based immunochromatographic test strip for detecting anti-EHDV antibodies was developed by conjugating recombinant streptococcal protein G with colloidal gold nanoparticles. The control line was coated with rabbit anti-streptococcal protein G antibody, while the test line was coated with the purified recombinant VP7 protein. Performance evaluation indicated that the test strip possessed desirable sensitivity, specificity, reproducibility, and stability. No cross-reactivity was observed with positive sera from animals infected with bluetongue virus, sheep pox virus, orf virus, peste des petits ruminants virus, foot-and-mouth disease virus, or lumpy skin disease virus. Testing of 200 clinical serum samples demonstrated a 97% coincidence rate between this test strip and a commercial competitive ELISA assay kit for EHDV antibody detection, with a Kappa value of 0.88. This study provides technical support for the rapid diagnosis of EHDV infection and contributes to disease surveillance and control.

Bluetongue virus (BTV) causes a vector-borne disease of considerable veterinary and economic importance. The virus is spread by biting midges (family Ceratopogonidae). BTV exposure has been reported in a wide range of ecological settings, but evidence from very high-altitude areas remains limited. To our knowledge, this is the first report of serological evidence of BTV exposure of domestic ruminants at extremely high altitudes (>4500 m) in Ali Prefecture, western Tibetan Plateau, China. Testing with a competitive enzyme-linked immunosorbent assay revealed that 31.1% of 633 blood samples collected from sheep, goats, yaks, cattle, and yak-cattle hybrids in Ali Prefecture in 2023 were positive for BTV-specific antibodies, with seroprevalence varying from 90.7% in goats to only 0.4% in yaks, confirming significant geographical and interspecies differences (p < 0.001). BTV-specific antibodies were detected in animals from locations above 4500 m, indicating previous exposure at elevations where evidence remains scarce. These findings provide baseline seroepidemiological data relevant to livestock health in alpine ecosystems, and support the need for strengthened entomological surveillance in high-altitude regions.

Brucellosis poses a severe threat to public health in Northwest China; however, the genome phylogeny and transmission pattern of Brucella melitensis from sheep and yaks in this region remain unclear. In this study, bacteriology, conventional biototyping, and whole-genome single-nucleotide polymorphism (WGS-SNP) were applied to depict the phylogenetic profiles of strains from Northwest China. A total of 46 Brucella strains were identified as B. melitensis bv. 3, which was isolated from at least three animal (livestock and wildlife) hosts, implying that B. melitensis infection is prevalent in the Northwest and suggesting that host diversity provides an optimal niche for the spread and maintenance of B. melitensis in this region. WGS-SNP analysis divided the 46 B. melitensis strains into four clades (C-I-IV) that harbored eight SNP genotypes (STs), implying that at least four lineages are prevalent in the Northwest. Global WGS-SNP phylogenetic analysis of strains revealed that all Northwest strains belong to genotype II. Strains from different clades presented high genetic similarity with strains previously collected from the Northwest. This study provides robust evidence supporting the notion that multiple similar B. melitensis lineages are persistently prevalent in human populations and animals in the Northwest. The economic development of animal husbandry has accelerated the cross-regional flow of livestock and livestock products, driving the spread and reach of the disease. Therefore, tailoring a targeted control strategy is necessary to counter the current serious epidemic trend.

Enzootic nasal tumor virus 2 (ENTV2), the etiologic agent of enzootic nasal adenocarcinoma (ENA) in goats, is highly prevalent in China and causes significant economic losses to the goat industry. Here we describe the occurrence of ENA on a Dazu black goat farm in Chongqing City. At autopsy, nasal cavity masses were observed within the nose of an affected goat; histologically, the tumor was a nasal adenocarcinoma. The qPCR results demonstrated unequivocally that ENTV2 was the primary pathogen responsible for the tumor in this goat. We also collected nasal swab samples from all 180 goats on the farm; 9 goats tested positive for ENTV2. We generated the sequence of the full-length genome of ENTV2 (named ENTV2CQ, GenBank OR024676) with 7,469 nucleotides from nasal tumors from our case. ENTV2CQ shared the highest nucleotide identity with a previously sequenced isolate, ENTV2FJ (GenBank MK559457.1). ENTV2CQ and ENTV2FJ are located in the same major phylogenetic branch, mainly related to isolates from China from 2015 to 2022, and their phylogeny may be clustered geographically.

Background: In recent years, enzootic nasal tumor virus 2 (ENTV-2) has become prevalent in China, resulting in substantial economic losses for the goat industry. In order to enrich the availability of detection methods for ENTV-2, this study developed an expedited and accurate reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay to facilitate the detection and quantification of ENTV-2. Methods: Specifically, a pair of primers and a TaqMan probe targeting conserved regions of the pro gene were designed to allow the specific amplification and detection of viral RNA in clinical samples. Moreover, modifying the method for use in a quantitative real-time PCR (qPCR) assay enables the detection of proviral DNA in tumor specimens. Results: Both methods exhibited a detection limit for the ENTV-2 standard plasmid at 100 copies/µL. The detection methods we established exhibited high specificity and sensitivity to ENTV-2, without cross-reactivity with other pathogens causing respiratory diseases or endogenous retroviruses (EBRVs). We performed an ENTV-2 analysis of clinical samples in goats via RT-qPCR using nasal swab samples (n = 558) collected from three geographically distinct flocks in Lingyou County, Baoji City, Shaanxi Province, China, and 58 positive samples were detected for a positivity rate of 10.4%. After euthanasia, the autopsy report showed nasal cavity masses. Histopathological analysis demonstrated an epithelial neoplasm, in compliance with the features of enzootic nasal adenocarcinoma (ENA). Three full-length genomes were sequenced to assess genomic sequence conservation and variation. Multiple-sequence alignment demonstrated the existence of sequence variations among strains. Phylogenetic analysis of the nucleotide sequences revealed that the ENTV-2 SX1~3 isolates were phylogenetically related to the Chinese ENTV-2 isolates, especially the JY strain. Furthermore, recombination analysis suggested that both ENTV-2 SX1 and ENTV-2 SX2 might be recombinant variants. Conclusions: In conclusion, both methods are highly specific for the pro gene of ENTV-2, and the development of this assay has been deemed crucial to the early identification and subsequent control of this viral infection. Our results provide valuable information for further research on the genetic variation and evolution of ENTV-2 in China.

Peste des petits ruminants virus (PPRV) is a highly contagious pathogen that severely impacts goats and sheep due to its high contagiousness and pathogenicity. Viruses rely on host proteins for their pathogenicity and replication, but the specific mechanisms facilitating PPRV replication by host proteins remain poorly understood. In this study, we identified goat growth arrest and DNA damage protein 45A (GADD45A) as a positive regulator of PPRV replication. Overexpression of GADD45A enhances PPRV replication, while its knockdown significantly inhibits PPRV replication. Furthermore, GADD45A suppresses SeV- or Poly(I:C)-induced IFN-β promoter and ISRE activation in a dose-dependent manner, as well as the transcription of interferon-stimulated genes (ISGs). We also demonstrate that goat GADD45A interacts with TANK-binding kinase 1 (TBK1), leading to the downregulation of TBK1 expression. Co-immunoprecipitation and confocal microscopy confirmed that GADD45A interacts with the PPRV V protein. Both GADD45A and V synergistically inhibit IFN-β promoter activation and TBK1 expression, thereby promoting PPRV replication. Our findings suggest that GADD45A promotes PPRV replication by downregulating TBK1, offering new insights into host proteins that counteract innate immune responses during PPRV infection. These findings offer valuable insights into the role of host proteins in viral replication and immune evasion, shedding new light on how PPRV antagonizes innate immunity.

Lumpy skin disease (LSD) is a viral disease caused by lumpy skin disease virus (LSDV), which mainly infects cattle and can cause huge economic losses. In May 2023, yaks, cattle-yaks, and cattle in Tibet (Xizang), China, developed fever, skin nodules, and severe discharges and were suspected to be cases of LSD. Samples from these animals were analyzed using molecular biology and serological methods. The RPO30, P32, and GPCR genes were amplified by PCR and sequenced, and the whole genome of the virus was determined using viral metagenomics technology. Sequencing results showed that it was indeed an LSDV infection, and enzyme-linked immunosorbent assay results confirmed the presence of LSDV antibodies. The whole genome phylogenetic tree shows that LSDV/CHINA/Tibet/2023 is different from the previous epidemic strains in China, but clusters with India 2022 strain. This is the first report of LSD in yaks, cattle-yaks, and cattle on the highest altitude plateau in the world.

ABSTRACT Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression. IMPORTANCE The role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Many epidemics are caused by negative-stranded RNA viruses, leading to serious disease outbreaks that threaten human life and health. These viruses also have a significant impact on animal husbandry, resulting in substantial economic losses and jeopardizing global food security and the sustainable livelihoods of farmers. However, the pathogenic and infection mechanism of most negative-stranded RNA viruses remain unclear. Reverse genetics systems are the most powerful tools for studying viral protein function, viral gene expression regulation, viral pathogenesis, and the generation of engineered vaccines. The reverse genetics of some negative-strand viruses have been successfully constructed, while others have not. In this review, we focus on representative viruses from the Orthomyxoviridae family (IAV), the Filoviridae family (EBOV), and the Paramyxoviridae family (PPRV) to compile and summarize the existing knowledge on reverse genetics techniques for negative-strand viruses. This will provide a theoretical foundation for developing reverse genetics techniques for some negative-strand viruses.

In order to diagnose the etiology causing abortion in pregnant ewes in Inner Mongolia sheep farms, this paper was conducted by antibody detection, pathological dissection, PCR amplification, nucleotide sequence comparison, homology analysis and construction of phylogenetic trees on samples of aborted ewes’ sera, aborted fetuses and fetal coats.The resμLts showed the highest detection rate of Brucella abortus（34.28%）,followed by Chlamydia abortus（28.57%）,and the lowest detection rate of mixed infection of Q fever and Brucella abortus and mixed infection of three pathogens（2.86%）.This indicates that Chlamydia abortus,Q fever and Brucella abortus are all present in the tested sheep farms as separate or mixed infections, and the conservativeness of the three pathogens is high, which provides a scientific basis for the detection of disease prevention and control in sheep farms and other surrounding sheep farms.

BACKGROUND:Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls. Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells.OBJECTIVES:PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response.METHODS:Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4+ and CD8+ T cells after PPRV infection.RESULTS:PPRV stimulated the differentiation of CD4+ and CD8+ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively. Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation.CONCLUSIONS:This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.

Primary sheep testicular Sertoli cells (STSCs) are ideal for investigating the molecular and pathogenic processes of capripoxvirus. However, the high cost of isolation and culture of primary STSCs, time-consuming operation, and short lifespan greatly limit their real-world application. In our study, the primary STSCs were isolated and immortalized by transfection of a lentiviral recombinant plasmid containing simian virus 40 (SV40) large T antigen. Androgen-binding protein (ABP) and vimentin (VIM) protein expression, SV40 large T antigen activity, proliferation assays, and apoptosis analysis results showed that immortalized large T antigen STSCs (TSTSCs) still had the same physiological characteristics and biological functions as primary STSCs. Moreover, immortalized TSTSCs had strong anti-apoptosis ability, extended lifespan, and enhanced proliferative activity compared to primary STSCs, which had not transformed in vitro and showed any signs of malignancy phenotype in nude mice. Besides, immortalized TSTSCs were susceptible to goatpox virus (GTPV), lumpy skin disease virus (LSDV), and Orf virus (ORFV). In conclusion, immortalized TSTSCs are useful in vitro models to study GTPV, LSDV, and ORFV in a wide range of ways, suggesting that it can be safely used in virus isolation, vaccine and drug screening studies in future.

猪痘(Swine pox,SWP)又称为猪天花,是一种猪的病毒性传染病,其可以由猪痘病毒(Swine pox virus,SWPV)或痘苗病毒(Vaccinia virus,VACV)感染引起.自19世纪40年代,在欧洲第一次被发现并报道后,相继在北美、南美和亚洲等地区也出现相同病例,并在非洲和澳大利亚的许多地区流行.现在该病在世界范围内均有分布.虽然该病在生产实际中对养猪业发展带来的损失与非洲猪瘟、高致病性猪蓝耳病或伪狂犬病等相比相对较轻,但是发病后的继发感染等问题会造成患病猪生产性能降低,抵抗力下降等影响.近年来,该病逐渐受到更多养殖者的重视.

The Orf virus (ORFV) is a member of the Parapoxvirus genus of the Poxviridae family and can cause contagious diseases in sheep, goats, and wild ungulates. In the present study, two ORFV isolates (ORFV-SC isolated from Sichuan province and ORFV-SC1 produced by 60 passages of ORFV-SC in cells) were sequenced and compared to multiple ORFVs. The two ORFV sequences had entire genome sizes of 14,0707 bp and 141,154 bp, respectively, containing 130 and 131 genes, with a G + C content of 63% for the ORFV-SC sequence and 63.9% for the ORFV-SC1 sequence. Alignment of ORFV-SC and ORFV-SC1 with five other ORFV isolates revealed that ORFV-SC, ORFV-SC1, and NA1/11 shared > 95% nucleotide identity with 109 genes. Five genes (ORF007, ORF20, ORF080, ORF112, ORF116) have low amino acids identity between ORFV-SC and ORFV-SC1. Mutations in amino acids result in changes in the secondary and tertiary structure of ORF007, ORF020, and ORF112 proteins. The phylogenetic tree based on the complete genome sequence and 37 single genes revealed that the two ORFV isolates originated from sheep. Finally, animal experiments demonstrated that ORFV-SC1 is less harmful to rabbits than ORFV-SC. The exploration of two full-length viral genome sequences provides valuable information in ORFV biology and epidemiology research. Furthermore, ORFV-SC1 demonstrated an acceptable safety profile following animal vaccination, indicating its potential as a live ORFV vaccine.