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.

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.

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.

Brucella canis is an important veterinary pathogen with growing public health concerns, but it remains neglected. In this study, a B. canis strain (BMNM19) was characterized using bacteriological culture, biotyping, whole-genome sequencing single nucleotide polymorphism analysis (WGS-SNP). Bacteriological examination revealed rough, grayish-white, sticky, dry, and opaque colonies, and conventional biotyping confirmed it as B. canis, which posing risks to humans in contact with infected dogs. Genomic analysis showed that BMNM19 shares 99.99 % average nucleotide identity (ANI) with the reference strain B. canis ATCC 23365. The strain was found to carry an mprF resistance gene and 69 virulence-associated genes, shared 2829 core genes with other reference strains, and displayed no unique genes. In silico MLST assigned BMNM19 to sequence type ST21. Phylogenetic based on core genome SNPs revealed that BMNM19 is closely related to B. canis strains from Zhejiang, Jiangsu Province (China), Japan and South Korea. These results expand our understanding of the genetic diversity of B. canis in northern, China and emphasize its persistent zoonotic threat to dogs and humans in close contact. This study provides critical molecular insights into the epidemiology of B. canis, to underscore the need for strengthened public health surveillance of this emerging zoonotic pathogen.

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.

Oseltamivir, a neuraminidase inhibitor, is widely used in the clinic for treating influenza virus infections. However, suboptimal efficacy and risk of drug resistance development remain major challenges. Molnupiravir, a ribonucleoside analog, was originally developed to treat influenza, but was repurposed and first approved for treating COVID-19 in 2021. Considering their complementary mode-of-actions, this study aimed to investigate the combinatorial activities of oseltamivir and molnupiravir against influenza virus infections. In cell culture models, we found that β-d-N4-hydroxycytidine (NHC), the active form of molnupiravir, exerted more potent antiviral activities against influenza A and B viruses, when compared to oseltamivir treatment. Combination of NHC with oseltamivir exhibited a synergistic antiviral effect against the influenza A/Puerto Rico/8/34 H1N1 strain, but not the influenza B/Washington/02/2019 strain. In a mouse model infected with the PR/8 virus strain, treatment with molnupiravir alone or in combination with oseltamivir effectively attenuated lung injury and reduced viral load in the tissue. Taken together, molnupiravir can be explored in combination with oseltamivir to treat influenza, especially for patients infected with the oseltamivir-resistant strains, whereas further research is warranted.

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.

BACKGROUND:Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure.RESULTS:In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility.CONCLUSIONS:Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.

Coxiella burnetii is the causative agent of zoonotic Q fever. Animals are the natural reservoirs of C. burnetii, and domestic livestock represent the major sources of human infection. C. burnetii infection in pregnant females may causes abortion during late pregnancy, whereby massive shedding of C. burnetii with abortion products becomes aerosolized and persists in the environment. Therefore, monitoring and surveillance of this infection in livestock is important for the prevention of the C. burnetii transmission. Previous serological surveys have shown that C. burnetii infection is endemic in livestock in China. However, few data are available on the diagnosis of C. burnetii as a cause of abortion by molecular methods in livestock. To get a better understanding of the impact of C. burnetii infection on domestic livestock in China, a real-time PCR investigation was carried out on collected samples from different domestic livestock suffering abortion during 2021-2023. A total of 338 samples collected from eight herds of five livestock species were elected. The results showed that 223 (66 %) of the collected samples were positive for C. burnetii DNA using real-time PCR. For the aborted samples, 82 % (128/15) of sheep, 81 % (34/42) of goats, 44 % (15/34) of cattle, 69 % (18/26) of camels, and 50 % (17/34) of donkeys were positive for C. burnetii. Besides, 44 % (8/18) and 4 % (1/25) of asymptomatic individuals of sheep and donkey were also positive for C. burnetii. In addition, the positive samples were further confirmed by amplification and sequencing of the C. burnetii-specific isocitrate dehydrogenase (icd) gene. Phylogenetic analysis based on specific gene fragments of icd genes revealed that the obtained sequences in this study were clustered into two different groups associated with different origin of hosts and geographic regions. This is the first report confirming that C. burnetii exists in aborted samples of sheep, goats, cattle, donkeys and camels in China. Further studies are needed to fully elucidate the epidemiology of this pathogen in livestock as well as the potential risks to public health.

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.

According to sequencing of the 5’ untranslated region (5’UTR) in viral genome, five strains of bovine viral diarrhea virus (BVDV) isolated from different provinces in China were genetically typed. The phylogenetic tree for BVDV 5’UTR displays that the isolated strain 22AH-1 is clustered into the genotype 1c clade, the strain 22-Gansu-F2 is clustered into the genotype 2a clade, the strains 22Anhui-7, 22-Sichuan-B8 and 22-Gansu-F3 are clustered into the genotype 2d clade. Depending on principal component analysis for synonymous codon usage pattern, BVDV strains generally exhibit the genotype-specific model. Although most synonymous codons are selected at the highly variable levels, the strongly suppressive usages focus on synonymous codons containing CG dinucleotides in all BVDV strains. Moreover, the plot of ENC value vs. GC3 content obviously shows that apart from nucleotide usage at highly variable patterns derived from mutation pressure, strong selective forces caused by natural selection mediate synonymous codon usage pattern of BVDV. Taken together, the related results give new insight into multiple selective forces acting on synonymous codon usage beyond nucleotide usage variation during BVDV evolutionary pathway.

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.

为了确定某养殖场奶山羊细菌性感染导致死亡的具体病原种类,从送检的肺和肝组织病料中分离纯化细菌.通过生化培养特性观察、16S rRNA和毒力基因的PCR扩增、血清型鉴定、药敏试验和致病性试验来鉴定病原种类.结果显示,从送检的组织中分离出2株革兰阴性球杆菌;16S rRNA和毒力基因LKT测序结果表明,所分离的病原菌为溶血性曼氏杆菌,血清型鉴定均为溶血性曼氏杆菌A2型,故可确定分离株为具有致病性的溶血性曼氏杆菌2型.药敏试验结果显示,分离株对羧苄西林、庆大霉素敏感,对氨苄西林、卡那霉素等中度敏感,而对复方新诺明具有耐药性.小鼠致病性试验表明,接种分离株后小鼠于24h内陆续死亡,并从剖检小鼠肺中也检测到溶血性曼氏杆菌,表明该菌有一定的致病力.结果表明,造成山羊死亡的主要病原是溶血性曼氏杆菌,这为溶血性曼氏杆菌病的临床用药提供了参考.

为建立绵羊产气荚膜梭菌ε毒素抗体间接ELISA检测方法,对D型产气荚膜梭菌(C60-1株)的ε毒素基因进行扩增,构建pET-32a-ε重组质粒,转入BL21(DE3)PlysS诱导蛋白表达,经Ni柱纯化后获得可溶性ε 毒素重组蛋白.以纯化的重组蛋白作为包被抗原,对ELISA反应条件进行优化,并对该方法的特异性、敏感性、重复性和临床检测中的应用进行验证.结果显示,重组蛋白包被浓度为0.5μg/mL,封闭液为1％BSA,待检绵羊血清1 ∶100稀释,家兔抗山羊HRP酶标二抗1∶ 5 000稀释,显色液避光显色20 min为最佳反应条件.测定45份阴性血清,计算其平均值(X)和标准差(SD),确定所建立的ε毒素抗体间接ELISA方法的阴阳性临界值((X)+3SD)为0.254.检测阳性血清敏感度为1∶ 3 200,批间和批内重复率显示,变异系数均小于10％,特异性试验显示,检测羊痘病毒(SPV)、蓝舌病病毒(BTV)、羊布鲁氏菌、沙门菌和绵羊肺炎支原体均没有特异性反应.检测90份临床羊血清样品,该间接ELISA方法与商品化试剂盒之间的阳性符合率为82.9％,阴性符合率为95％,总符合率为85.6％.结果表明,本试验建立的方法具有良好的特异性、敏感性和重复性,可初步用于绵羊产气荚膜梭菌8毒素抗体的检测.

Inflammatory responses play a central role in host defense against invading pathogens. Peste des petits ruminants virus (PPRV) causes highly contagious acute or subacute disease of small ruminants. However, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. Here, we revealed a novel mechanism by which PPRV induces inflammation. Our study showed that PPRV induced the secretion of interleukin 1β (IL-1β) by activating the NF-κB signaling pathway and the NLRP3 inflammasome. Moreover, PPRV replication and protein synthesis were essential for NLRP3 inflammasome activation. Importantly, PPRV N protein promoted NF-κB signaling pathway and NLRP3 inflammasome via direct binding of MyD88 and NLPR3, respectively, and induced caspase-1 cleavage and IL-1β maturation. Biochemically, N protein interacted with MyD88 to potentiate the assembly of MyD88 complex and interacted with NLPR3 to facilitate NLRP3 inflammasome complex assembly by forming an N-NLRP3-ASC ring-like structure, leading to IL-1β secretion. These findings demonstrate a new function of PPRV N protein as an important proinflammation factor and identify a novel underlying mechanism modulating inflammasome assembly and function induced by PPRV. IMPORTANCE An important part of the innate immune response is the activation of NF-κB signaling pathway and NLPR3 inflammasome, which is induced upon exposure to pathogens. Peste des petits ruminants virus (PPRV) is a highly contagious virus causing fever, stomatitis, and pneumoenteritis in goats by inducing many proinflammatory cytokines. Although the NF-κB signaling pathway and NLRP3 inflammasome play an important role in regulating host immunity and viral infection, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. This study demonstrates that PPRV induces inflammatory responses. Mechanistically, PPRV N protein facilitates the MyD88 complex assembly by directly binding to MyD88 and promotes the NLRP3 inflammasome complex assembly by directly binding to NLRP3 to form ring-like structures of N-NLRP3-ASC. These findings provide insights into the prevention and treatment of PPRV infection.

为了研究和探讨布鲁氏菌核糖体L7/L12蛋白对鼠源树突状细胞(BM-DCs)分化和成熟的影响,用布鲁氏菌S2疫苗株为模板扩增L7/L12基因,构建重组质粒pET30a-L7/L12,用大肠埃希菌原核表达系统进行诱导表达,并用Ni柱对表达的蛋白进行纯化.用IL-4和GM-CSF诱导培养鼠源DCs,用脂多糖(LPS)和L7/L12蛋白刺激DCs后检测其表面共刺激分子和炎症因子的变化.结果表明,重组蛋白在1 mmol·L-1的IPTG、16℃过夜的条件下以可溶性形式高效表达,其大小为18 ku,纯度达到93％以上并有一定的反应原性.流式细胞仪检测被刺激的BM-DCs细胞表面CD40、CD80等抗原分子的表达显著(P<0.05)高于空白对照组.qPCR结果显示,炎性细胞因子TNF-β、IL-1β、IL-12极显著(P<0.01)高于空白对照组.重组L7/L12蛋白具有刺激DCs细胞分化、成熟和促进炎症因子释放的功能.