Feline panleukopenia virus (FPV) is a major feline pathogen, but canine-derived FPV variants have recently been identified. Here, we compared the pathogenicity of a canine-derived FPV strain in cats with that of a lethal feline-derived FPV strain and evaluated the evolutionary significance of its NS1 mutations. Kittens infected with the canine-derived strain developed only mild, self-limiting diarrhea without fever or mortality, whereas those infected with the feline-derived strain developed severe disease and reached humane endpoints by 9 dpi. The canine-derived strain caused prolonged fecal shedding from 6 to 38 dpi but only low tissue viral loads (101-103 copies/g), while the feline-derived strain reached markedly higher loads (103-106 copies/g), particularly in the ileum, jejunum, and lungs. Viral DNA levels in the lungs, ileum, caecum, and rectum were significantly higher in the feline-derived group. Sequence analysis identified four NS1 mutations, 115I, 132L, 247Q, and 595Q, which showed stepwise evolutionary accumulation and signatures of positive selection. These findings indicate that canine-derived FPV retains infectivity in cats but exhibits attenuated pathogenicity and reduced replication fitness, highlighting NS1 as a potential determinant of host adaptation.

Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen associated with bovine respiratory disease. In this study, we report the isolation and genomic characterization of BPIV3 from a water buffalo with respiratory symptoms in China. Virus isolation was performed using susceptible cell cultures, followed by identification via RT-qPCR, transmission electron microscopy, and indirect immunofluorescence. Metagenomic sequencing of the near-complete genome showed that the isolate shared 89.9%-91.1% nucleotide identity with BPIV3 genotype A strains. Notably, several distinct mutations were identified in the structural protein genes, and phylogenetic analysis demonstrated that the isolate formed a separate cluster within genotype A, suggesting that it may represent a novel subtype within this genotype. To our knowledge, this is the first report describing the isolation and genomic characterization of BPIV3 from water buffaloes in China. These findings provide baseline molecular data for further studies on the genetic diversity and evolution of BPIV3.

Bovine viral diarrhea virus (BVDV) is a globally significant pestivirus currently classified into three species: BVDV-1 (Pestivirus A), BVDV-2 (Pestivirus B), and BVDV-3 (HoBi-like, Pestivirus H). Here, we report the isolation and identification of a potential new BVDV genotype associated with severe outbreaks in yaks in China. Since 2024, an epidemic characterized by respiratory and gastrointestinal symptoms has been spreading across Sichuan and Qinghai Provinces. From January 2024 to October 2025, we collected 260 samples from 167 affected yaks from 14 farms. Among these, 55.69% of the yaks and all sampled farms tested positive for BVDV. Sequencing of the 5' untranslated region (5' UTR) and Npro fragments from clinical samples revealed that the dominant epidemic strains may represent a novel BVDV lineage. We isolated four cytopathic strains and successfully sequenced two genomes of strains SC-H03 and QH-B04, which share less than 77.8% nucleotide identity across the complete coding sequence (CDS) with well-defined BVDV-1/2/3 strains. Phylogenetic analyses of the complete CDS and individual genes placed SC-H03 and QH-B04 in a distinct monophyletic clade with strong bootstrap support. Pairwise genetic distances confirmed substantial divergence, and cross-neutralization assays revealed significant antigenic differences from BVDV-1 and BVDV-2 reference strains. In accordance with ICTV Pestivirus classification standards, these strains satisfy the criteria for a new Pestivirus species, which we propose to name BVDV-4 (Pestivirus T). Given its wide prevalence in yaks and association with high morbidity and mortality, our findings underscore the urgent need for targeted prevention and control measures.

Bovine coronavirus (BCoV) is a major cause of respiratory and enteric diseases in cattle, resulting in substantial economic losses to the global cattle industry. Next-generation epitope-focused vaccines require conserved neutralizing determinants to achieve broad protective efficacy across circulating variants. Here, we applied a reverse-vaccinology and immunoinformatics-guided strategy to identify conserved linear B-cell epitope candidates on the BCoV spike protein. Using ABCPred (16 aa; threshold score 0.51), 139 putative linear B-cell epitopes were predicted, and 10 candidates (B1-B10) were selected based on antigenicity, allergenicity/toxicity filters, and Shannon-entropy-based conservation (≥95%). Each candidate was displayed on Helicobacter pylori ferritin and TEM confirmed the formation of self-assembled nanocages. BALB/c mice (n = 6 per group) were subcutaneously immunized with 50 μg of each epitope-ferritin fusion protein emulsified 1:1 with Montanide ISA 201 in a prime-boost regimen (at a 14-day interval), and sera were collected 14 days after the booster immunization. All 10 constructs induced BCoV-specific binding IgG (endpoint titers ranging from 1:2667 to 1:11,733), but only B1, B2, B4, B6, B7, and B8 elicited detectable in vitro neutralizing activity against both representative strains, whereas B3, B5, B9, and B10 remained below the detection limit (<1:8). Neutralizing titers were 1:24-1:99 for the enteric strain XHD4 and 1:27-1:88 for the respiratory strain HXD1, with no significant difference observed between the two strains (p > 0.05). Overall, these findings identify six conserved B-cell epitope candidates capable of eliciting neutralizing antibody responses against both representative enteric and respiratory field strains, providing experimental evidence to support epitope-focused BCoV vaccine design.

IntroductionBovine coronavirus (BCoV) is an important pathogen of enteric and respiratory disease in cattle, resulting in huge economic losses to the beef and dairy industries worldwide. A specific and sensitive detection assay for BCoV is critical to the early-stage disease prevention and control.MethodsWe established a specific, sensitive, and stable assay for BCoV nucleic acid detection based on CRISPR/Cas13a combined with reverse transcription recombinase-aided amplification (RT-RAA) technology. The specific primers for RT-RAA and CRISPR RNA (crRNA) were designed in the conserved region of the BCoV nucleocapsid (N) gene.ResultsThe detection limit of the RT-RAA CRISPR/Cas13a assays for BCoV detection was 1.72 copies/μl, and there were no cross-reactions with the other 10 common bovine enteric and respiratory disease-associated pathogens. The coefficient of variations (CVs) of within and between batches were less than 4.98 and 4.58%, respectively. The RT-RAA-CRISPR/Cas13a assays work well in clinical samples of cattle and yak, the BCoV positive rate of 84 clinical samples detected by RT-RAA-CRISPR/Cas13a assays was 58.3% (49/84), it was notably higher than that of RT-qPCR (2.4%, 2/84; p < 0.001). The 49 positive samples detected by RT-RAA-CRISPR/Cas13a assays were further confirmed as BCoV by Sanger sequencing.DiscussionA specific, sensitive, and stable assay based on RT-RAA-CRISPR/Cas13a assays for BCoV was developed, providing new technical support for the clinical detection and epidemiological monitoring of BCoV.

Bovine coronavirus (BCoV) is a major pathogen of bovine respiratory disease, causing respiratory and enteric infections in cattle and wild ruminants. It is responsible for economic losses and threatens the health and welfare of cattle industry. In this study, a BCoV isolate, BCoV/SUWN/XHD-5, had cytopathogenic effects in Madin Darby bovine kidney (MDBK) cells and showed a high viral titer at 24 and 48 h post infection (hpi). Gene expression profiling using RNA sequencing and protein network mapping using the Tandem Mass Tag-based quantitative proteomics approach were performed in MDBK cells with BCoV infection at 24 and 48 hpi, respectively. Compared with mock-infected MDBK cells, 8,720 differentially expressed genes (DEGs) and 296 differentially expressed proteins (DEPs) were identified in BCoV infection at 24 hpi, whereas 5,838 DEGs and 747 DEPs were identified in BCoV infection at 48 hpi. Following GO annotation and KEGG enrichment analysis, most DEGs and DEPs were significantly enriched in metabolic pathways, endocytosis, ribosome and protein processing in endoplasmic reticulum, apoptosis and immune response. A correlation analysis of the proteome and transcriptome revealed that the up-regulated DEGs and DEPs were predominantly associated with metabolic pathways, apoptosis and the MAPK/TNF/Ras signaling pathway, whereas the down-regulated DEGs and DEPs were involved in complement and coagulation cascades and the Wnt signaling pathway. Importantly, BCoV decreases the mRNA and protein levels of complement component C3 in MDBK cells. The findings of this study provide the first report of the integrative transcriptomic and proteomic analyses of BCoV infection in MDBK cells. It revealed a regulatory network for analyzing the mechanisms of BCoV-host interactions and provides valuable insights into the pathogenesis of BCoV.

Aichivirus D (AiV-D) is a newly emerging diarrhea-associated pathogen in cattle and sheep, and comprised two officially recognized genotypes (AiV-D1 and AiV-D2) and two potential novel genotypes (AiV-D3 and AiV-D4). This study aimed to establish a fluorescent RT-PCR assay for detecting AiV-D and to investigate its prevalence in yak diarrheic samples. A SYBR Green fluorescent RT-PCR assay was successfully established by targeting the conserved region of viral 3D genes, enabling detection of all known AiV-D genotypes. The established assay was specific for AiV-D, exhibiting no cross-reactivity with other common diarrhea-causing pathogens in ruminants, with coefficients of variation for reproducibility ranging from 0.23% to 2.16% and detection limits for the positive plasmids of AiV-D1 to AiV-D4 at 2.21, 4.78, 4.10, and 5.94 copies/mu L, respectively. Of the 166 diarrheic samples collected from calf yaks across nine farms in Sichuan province, China, between May and August 2023, 22.3% (37/166) tested positive for AiV-D, suggesting its substantial prevalence within the sampled region. Furthermore, seven complete 3D gene sequences were cloned from positive samples, and subsequent phylogenetic analysis revealed distinct evolutionary patterns among these isolates. In conclusion, this newly established assay represents a reliable diagnostic tool for AiV-D detection, while our findings provide significant insights into the epidemiological distribution and evolutionary dynamics of AiV-D in the yak population.

Canine respiratory coronavirus (CRCoV) is a prevalent pathogen implicated in canine infectious respiratory disease, yet information on its genomic characteristics and pathogenicity remains scarce. To address this situation, we investigated the genetic evolution and pathogenic potential of CRCoV strains circulating in China. Five complete CRCoV genomes (GenBank: PQ725948-PQ725952) were obtained from clinical samples, and phylogenetic analysis showed these strains formed a distinct genetic branch. The evolutionary trees for ORF1ab, HE, and S genes closely mirrored the full genome tree, indicating key roles for these genes in CRCoV evolution. Multiple unique amino acid mutations were identified in the ORF1ab, HE, S, M, and N proteins. Notably, molecular docking analysis suggests that mutations S158F and L161F in the HE lectin domain are associated with improved docking scores, indicating a potential increase in receptor-binding affinity. Consecutive nucleotide deletions in two non-coding regions between non-structural protein genes-which were also identified in strains of a Thai lineage (OQ621707.1-OQ621727.1)-were observed. A CRCoV strain (106 TCID50/mL) was isolated, and experimental infection confirmed its ability to induce pneumonia and tracheal cilia loss in dogs. These findings reveal the emergence and unique genetic diversity of a novel CRCoV variant in China, highlighting the need for ongoing epidemiological surveillance.

Bovine parainfluenza virus type 3 (BPIV3) is a significant pathogen responsible for bovine respiratory disease complex (BRDC) and reproductive disorders. Despite its importance, genomic studies of BPIV3 in abortion samples are currently lacking. We detected a BPIV3-positive sample via PCR in the lung sample from an aborted Holstein fetus; therefore, this study aimed to isolate and identify a BPIV3 strain from the aborted fetus sample of Holstein and analyze its genome. We successfully isolated and obtained a BPIV3 strain using MDBK cell lines, named LC1. Phylogenetic analysis based on complete genome sequences revealed that the LC1 strain belongs to genotype C and clusters into a branch with the Chinese strain SD2020. Notably, unique amino acid mutations were identified in the P (I297T) and M (P5N) proteins of the LC1 strain. To the best of our knowledge, this is the first BPIV3 genotype C strain to be isolated from a bovine abortion sample, as well as the first characterization of the abortion source BPIV3 genome reported. This finding lays the foundation for further studies on the genetic diversity of BPIV3.

Aichivirus D (AiV-D), a newly emerging member of the Kobuvirus genus, is associated with diarrhea in cattle. This study aimed to investigate the prevalence and molecular characteristics of AiV-D among dairy cattle in China. From October 2021 to August 2022, 279 fecal samples were collected from diarrheal dairy cattle across seven provinces in China. Among these, 37 samples (13.2%) tested positive for AiV-D by RT-PCR, indicating a wide geographical distribution of AiV-D in Chinese dairy cattle. Phylogenetic analysis based on the complete VP1 gene revealed that Chinese dairy cattle AiV-D strains belong to the AiV-D2 genotype, with unique amino acid changes in VP0, VP3, and VP1 that distinguish them from known AiV-D strains. Additionally, an AiV-D strain was successfully isolated, and its complete genome was sequenced. Phylogenetic analysis of the complete genome and individual genes confirmed the strain’s classification within the AiV-D2 genotype. This study reports the first detection of the AiV-D2 genotype outside Japan, highlighting the need for future surveillance to better understand the epidemiology and diversity of AiV-D in China.

ObjectivesThis study aimed to develop a sensitive detection method and investigate feline chaphamaparvovirus (FeChPV) in cats from southwestern China.MethodsA SYBR Green I-based qPCR assay targeting the VP1 gene was established and validated. It was then applied to 87 feline diarrhoeic faecal samples (2021-2023). Near-full-length genomes of positive samples were sequenced for phylogenetic, structural and selection analysis.ResultsThe qPCR assay showed high sensitivity (50.9 copies/μl) and reproducibility (coefficient of variation <4.0%). FeChPv was detected in 22/87 (25.3%) cats with diarrhoea. Four strains shared 97.6-99.5% identity with global isolates and formed a distinct clade within Asian lineages. A consistent valine-to-isoleucine mutation at VP1-340 was identified under positive selection, which can induce conformational changes.Conclusions and relevanceWe provide a reliable tool for the detection of FeChPV and reveal unique evolutionary features of local strains, supporting further research into its pathogenesis and spread.

Hybrid models that leverage both Convolutional Neural Networks (CNNs) and Transformers are gaining traction in medical image segmentation. However, conventional hybrid models often overlook two issues: firstly, the simplistic connections or interactions between CNN and Transformer architecture lead to underutilization of multi-level features, and secondly, the misalignment between global and local information in the spatial domain often hampers effective feature fusion and interaction. In this paper, we propose FreFormer, a hybrid CNN-Transformer architecture that employs frequency domain transform to harmonize multilevel encoded features. Specifically, FreFormer effectively harnesses the multi-level representations from both CNNs and Transformers, ensuring the preservation of global and local contexts. Our key innovation, the Frequency Bridging Transform (FBT) module, addresses feature misalignment by introducing frequency-based mechanisms that cohesively bridge heterogeneous CNN and Transformer layers. This block adeptly transforms multi-level features from spatial domains into a consistent frequency domain, promoting a more harmonious feature fusion. Experiments have confirmed the exceptional performance of Freformer, highlighting the remarkable potential of the FBT module.

Semi-supervised learning (SSL) has been a popular technique to resolve the annotation scarcity problem in image segmentation. Recently, contrastive learning (CL), which encourages intra-class compactness and inter-class dispersion, has shown great potential in helping SSL learn discriminative features. However, vanilla CL tends to focus on negative samples, while ignoring those hard positives (i.e., samples have dissimilar feature representations with respect to anchors) which could also deliver discriminative knowledge. In this paper, we propose to inject hard positives oriented contrastive (HPC) learning into SSL and present an effective three-stage framework for medical image segmentation. Specifically, the first and second stages pre-train an encoder-decoder architecture through bi-level HPC learning, including unsupervised image-level HPC (IHPC) learning and supervised pixel-level HPC ( PHPC) learning. Notably, the PHPC loss is implemented in a region-based manner so that the model is competent to capture both global contextual and local semantic information with less memory consumption. In the third stage, the well-pre-trained architecture is adapted to a semi-supervised segmentation framework. Experiments on two public datasets demonstrate that our proposed method surpasses the state-of-the-art methods.

Aim: This study explored the protective effect of Enterococcus faecium as a probiotic against Salmonella typhimurium infection. Materials & methods: The protective role of E. faecium against tissue damage by S. typhimurium infection and the expression of inflammatory cytokines and tight junction proteins were detected by histological observation, real-time quantitative PCR and immunohistochemical methods. Results: E. faecium demonstrated a regulatory function that affected the expression of Claudin-1 and enhanced tight junctions, suppressed the NF-kappa B/NLRP3/IL-1 beta signaling pathway and reduced the release of IL-6, TNF-alpha, IFN-gamma, TLR4 and MYD88 and inflammatory damage to tissues by S. typhimurium in the duodenum, cecum and colon of mice. Conclusion: E. faecium antagonized S. Typhimurium alleviating inflammatory injury in mice through the NF-kappa B/NLRP3/IL-1 beta signaling pathway.

This study identifies a novel Aichivirus D in goats for the first time, and a case-control study suggested that the virus is associated with diarrhea in goats. The goat Aichivirus D shared the highest gnomic sequence homology with ovine Aichivirus D strain, and may origin from ovine Aichivirus D through natural VP1 gene recombination. These findings contribute to a better understanding of the host range and genetic evolution of Aichivirus D.

鹅细小病毒(GPV)是一种主要侵害雏鹅和雏番鸭的病原,给养禽业带来了巨大的经济损失.利用GPV的VP3基因保守区域设计了一对引物和探针,经反应体系的优化,建立检测鹅细小病毒的恒温隔绝式荧光PCR方法(ii PCR方法),并与TaqMan荧光定量PCR方法比较.结果显示,该方法能特异性检出GPV,对其他常见无关病原不检出,特异性好;检测下限是38.1拷贝·μL-1,灵敏度高;批内、批间变异系数均小于5％,重复性好;该方法对31份临床疑似样本的检出率为93.54％,高于TaqMan荧光定量PCR方法的检出率(83.87％).本研究为鹅细小病毒的现场快速检测提供了新的技术手段.

牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV3)是引起牛呼吸道疾病综合征(bovine respiratory disease syndrome,BRDC)的重要病原,为建立一个基于恒温隔绝式 RT-PCR(insulated isothermal RT-PCR,iiRT-PCR)的方法实现现场检测BPIV3,本研究根据BPIV3 P基因的保守区域,设计合成引物和探针、优化检测体系和预混检测试剂,成功建立了能够检测BPIV3 A,B和C 3种基因型的iiRT-PCR方法.该方法只能特异性检出BPIV3,而对牛冠状病毒、牛呼吸道合胞体病毒、牛病毒性腹泻病毒、牛传染性鼻气管炎病毒、牛腺病毒3型、牛鼻病毒、多杀性巴氏杆菌、溶血性曼氏杆菌和牛支原体等无关病原不检出.敏感性试验结果显示该方法对构建的重组质粒阳性标准品的检测下限为4.23 copies/μL.重复性结果显示,批内和批间的变异系数分别为1.37％～1.73％和2.16％～2.58％.采用建立的iiRT-PCR方法和报道的2种TaqMan-MGB荧光定量RT-PCR方法对2020-2021年采集自我国内蒙古、河南、宁夏、山西、四川和福建6个省的141份患BRDC肉牛鼻腔棉拭子样本和50份患BRDC牦牛肺脏样本进行检测,iiRT-PCR方法自肉牛样本中检出55份BPIV3阳性(阳性率为39.01％),自牦牛样本中检出22份BPIV3阳性(阳性率为44.00％),而2种TaqMan-MGB荧光定量RT-PCR方法自肉牛样本中分别检出40份和48份BPIV3阳性(阳性率为28.39％和34.04％),自牦牛样本中分别检出15份和19份BPIV3阳性(阳性率为30.00％和38.00％),iiRT-PCR检测结果优于2种TaqMan-MGB荧光定量RT-PCR方法.将优化的各检测试剂预混后配合使用商品化PetNAD核酸萃取试剂盒,获得检测结果仅需1 h,可实现BPIV3的现场检测.本研究丰富了国内BPIV3的分子流行病学调查资料,并为BRDC的诊断和防控提供了有力的工具.

This study was conducted to investigate the infection status of BAdV-3 by TB Green Real-time PCR on 140 nasal swab samples of sick cattle characterized by runny nose, fever, cough and dyspnea from 9 large-scale cattle farms in 6 provinces in China（Sichuan Province, Shanxi Province, Henan Province, Hebei Province, Jiangsu Province and Inner Mongolia Autonomous Region）, and the positive samples were tested for the fiber shaft gene. The results showed that the BAdV-3 positive rate was 36.4% among 140 samples, and BAdV-3 was detected in all provinces except for Hebei Province, with the highest detection rates in Sichuan and Henan Provinces, reaching 82.9%（29/35） and 83.3%（10/12）, respectively. Among them, the traditional types accounted for 80.4% of the total positive ratio, and the strain with the deletion of the fiber shaft region accounted for 19.6% of the total positive samples. The position and number of deletions were consistent, with 237 consecutive base pairs missing in the shaft region, and the deletion type was only detected in Sichuan and Henan Provinces, with a detection rate of 24.1%（7/29） and 30%（3/10）, and a field group positive rate of 100%（2/2） and 100%（1/1）, respectively. The results indicate that BAdV-3 has a wide distribution in cattle farms in China, and the current epidemic virus is mainly the traditional strain of BAdV-3. The fiber shaft gene deletion strain has a unique geographical distribution in China and is mainly prevalent in Sichuan and Henan Provinces.

Bovine parainfluenza virus type 3 (BPIV3) is a common respiratory pathogen that causes respiratory illness in cattle and makes a major contribution to the bovine respiratory disease complex (BRDC); however, data on the prevalence and molecular features of BPIV3 are still scarce in China. To investigate the epidemiological characteristics of BPIV3 in China, between September 2020 and June 2022, 776 respiratory samples were received from 58 BRDC-affected farms located in 16 provinces and one municipality. Those were screened for BPIV3 using a reverse transcription insulated isothermal PCR (RT-iiPCR) assay. Meanwhile, the HN gene and complete genome sequence of strains from different provinces were amplified, sequenced, and analyzed. The tests showed that 18.17% (141/776) of samples tested were positive for BPIV3, which originated from 21 farms in 6 provinces. Moreover, 22 complete HN gene sequences and 9 nearly complete genome sequences were obtained from the positive samples. Phylogenetic analysis based on the HN gene and complete genome sequences revealed that the sequences were clustered in one large clade for all Chinese BPIV3 genotype C strains, while overseas strain sequences of BPIV3 genotype C clustered into other clades. Moving beyond the known complete genome sequences of BPIV3 in GenBank, a total of five unique amino acid mutations were found in N protein, F protein, and HN protein in Chinese BPIV3 genotype C strains. Taken together, this study reveals that BPIV3 genotype C strains, the dominant strains in China, have a broad geographical distribution and some unique genetic characteristics. These findings contribute to our understanding of the epidemiological characteristics and genetic evolution of BPIV3 in China.

本试验对1例2月龄中华田园犬进行病原诊断,该病犬主要症状为腹泻、血便并伴随流涕、打喷嚏、咳嗽等.采集其鼻拭子、肛门拭子,利用PCR方法分别对7种常见的犬腹泻病毒及呼吸道病毒进行检测,结果显示该病例为犬冠状病毒Ⅰ型与犬腺病毒Ⅱ型的混合感染.待病犬进入濒死期立即安乐死并进行剖检,组织观察可见肺部出现明显的出血斑,盲肠、直肠、结肠有明显的出血点.本试验为犬病毒性腹泻、呼吸道疾病的病毒诊断以及犬冠状病毒Ⅰ型、犬腺病毒Ⅱ型自然感染引起的组织病变提供了参考.