Background Intensive indoor pig production systems can foster high antimicrobial exposure and dense animal contact, potentially enriching multidrug-resistant (MDR) Escherichia coli (E. coli) that may enter wider One Health microbial networks.Objectives This study investigated grower-finisher Tibetan pigs managed under commercial conditions harbouring phylogenetically diverse E. coli lineages carrying extensive resistance and virulence determinants.Methods Between 2021 and 2023, 280 rectal swabs were collected from nine mechanically ventilated farms; the isolated E.coli strains were subjected to phylogrouping, as well as detection of 13 virulence genes and 45 resistance genes, using multiplex PCR. Antimicrobial susceptibility of the isolates to 22 antibiotics was determined by the disk diffusion method.Results Enteroaggregative E. coli accounted for 27.86% of isolates, and putative STEC/EHEC accounted for 7.14%, with astA being the most prevalent (27.86%). Phenotypically, 94.29% were resistant to amoxicillin and 68.57% to sulfonamides, while 82.86% satisfied the MDR definition; genotypically, tetA (70.71%) and floR (57.14%) dominated the resistance repertoire. Phylogroups A and B1 predominated, but resistance and virulence profiles were dispersed across all lineages, indicating horizontal gene flow rather than clonal expansion.Conclusion Tibetan pigs represent a substantial reservoir of MDR E. coli, strengthening the rationale for farm-level antimicrobial stewardship and integrated surveillance that links animal, environmental and human compartments. The study is limited by its cross-sectional design, focus on a single province and animal host and the absence of parallel human or environmental isolates, which prevent inference of causal zoonotic pathways and should be addressed in longitudinal One Health investigations.

Clostridium perfringens is a zoonotic pathogen widely distributed in environmental reservoirs including soil, sewage, and animal intestines. In this study, the complete XZ-E1 genome was sequenced using the PacBio RS II platform. De novo assembly was performed using Hifiasm, followed by error correction using Pilon v1.22. Comparative genomic analysis between the XZ-E1 strain and the reference strain ATCC13124 revealed distinct virulence-associated features, including the presence of strain-specific genes. Sequence analysis indicated that the XZ-E1 strain comprised a single circular chromosome with 3,840,795 bp and six plasmids: pXZ-E1_1 (345,615 bp), pXZ-E1_2 (54,930 bp), pXZ-E1_3 (49,338 bp), pXZ-E1_4 (15,591 bp), pXZ-E1_5 (13,216 bp), and pXZ-E1_6 (12,962 bp). Genome annotation predicted 3,445 protein-coding sequences, 30 rRNA genes, and 95 tRNA genes, with 28.12

Clostridium perfringens is a ubiquitous foodborne and zoonotic pathogen responsible for necrotizing enteritis, food poisoning, and gas gangrene in humans and livestock, with mortality reaching 100% in specific subtypes. The Qinghai-Tibetan Plateau, with its high altitude, hypoxia, large temperature fluctuations, and strong UV radiation, imposes a strong selective regime that drives distinctive, plateau-specific microbial evolution. We isolated and sequenced eight C. perfringens isolates from yaks and Tibetan pigs and analyzed them with 129 public genomes. Multilocus sequence typing (MLST) revealed extensive genetic heterogeneity, with 35 novel alleles and 18 novel sequence types (STs), all plateau isolates carrying novel alleles or STs. Toxinotyping identified six toxinotypes, dominated by A (43.0%) and F (42.3%), with virulence gene repertoires matching toxinotype assignments. Sixteen antimicrobial resistance (AMR) genes across eight drug classes were detected, with vancomycin- and defensin-like genes present in >96.0% of isolates; four were linked to mobile genetic elements (MGEs) or type IV secretion systems (T4SS). Environmental tolerance assays showed that three plateau isolates maintained growth at pH 7.5 under anaerobic conditions, while the growth of all isolates was inhibited at pH 7.0. Pan-genomic association analysis identified a 146-node, 3,390-edge network, with IS family transposases (ISCbt3/ISCpe4), sigma factors (sigL and sigA), and DNA polymerase I (polA) as central hubs potentially mediating stress adaptation. Genomic epidemiology of C. perfringens from the Tibetan Plateau reveals marked genetic diversity, broad resistance, toxinotype-defined virulence, and genetic signatures of high-altitude adaptation, underpinning evidence-based control in plateau ecosystems.IMPORTANCEClostridium perfringens is a widespread pathogen, but its adaptation to extreme environments like high-altitude plateaus remains a mystery. Our integrated genomic analysis of 137 strains, including 8 newly sequenced from the plateau, uncovers a startling reality: this region harbors a highly diverse population with near-universal resistance to critical antibiotics. We identified new sequence types (STs) and genetic "hubs" that may drive this adaptation. These findings have profound implications for One Health as they highlight an environmental niche where resistance can evolve and potentially spread, underscoring the urgent need for surveillance in unique ecosystems.

Although mixed viral infections are increasingly recognized as contributors to bovine diarrhea syndrome, diagnosing such co-infections remains challenging, particularly in high-altitude regions where surveillance is limited. In July 2024, a calf presenting with severe diarrhea and respiratory distress was identified on a cattle farm in Linzhi, Xizang, China. Using unbiased whole-genome sequencing (WGS) of the fecal sample, we assembled near-complete genomes of three distinct RNA viruses: two bovine viral diarrhea virus type 1 (BVDV-1) strains (subtypes 1v and 1q, designated BVDV-1/XZ87 and XZ87), one bovine enterovirus (genotype EV-E, designated BEV/XZ87), and one caprine parainfluenza virus type 3 (CPIV3/XZ87). The CPIV3/XZ87 genome exhibited 99.9% nucleotide identity to the goat-derived GS2017-2 strain from Jiangsu, China, raising the possibility of viral spread through livestock trade. Quantitative real-time PCR (RT-qPCR) confirmed the presence of all three pathogens (Ct values: 24.78 for BEV, 25.98 for CPIV3, and 31.28 for BVDV). This study provides the genomic evidence of a triple co-infection involving BVDV-1, BEV, and CPIV3 in Xizang. It illustrates the potential of WGS for unbiased pathogen detection in complex clinical specimens. The near-complete genomes generated here fill critical gaps in the virological surveillance of this epidemiologically under-sampled high-altitude region.

Selective autophagy is a critical host defense mechanism that eliminates viral components through lysosomal degradation during coronavirus infection. Coronaviruses (CoVs), however, deploy countermeasures that disrupt this process, and several underlying mechanisms remain unresolved. Here, we identify the autophagy receptor CCDC50 as a substrate of the coronavirus-encoded NSP5 protease. During porcine deltacoronavirus (PDCoV) infection, NSP5 cleaves CCDC50 at glutamine 171 (Q171), a conserved site also processed by NSP5 from PEDV, TGEV, and SARS-CoV-2. Functionally, CCDC50 restricts PDCoV replication by recognizing the envelope (E) protein when it is modified with K63-linked polyubiquitin at lysine 72 (K72) and routing it for autophagic degradation, independently of canonical receptors such as SQSTM1/p62 and NBR1. NSP5-mediated cleavage disrupts CCDC50 interaction with LC3 and ubiquitin, reduces its capacity to target E for degradation, and thereby compromises its antiviral activity. Taken together, our study identifies CCDC50 as a previously uncharacterized antiviral autophagy receptor in coronavirus infection and reveals that PDCoV circumvents this defense through NSP5-mediated cleavage to promote productive infection.IMPORTANCEIn our study, we investigated the interplay between host autophagy pathways and coronavirus infection. We identified the selective autophagy receptor CCDC50 as a potent antiviral factor that suppresses porcine deltacoronavirus (PDCoV) replication. We demonstrated that CCDC50 specifically recognizes the viral envelope (E) protein and targets it for autophagic degradation, thereby restricting the virus. However, we also uncovered a sophisticated viral escape mechanism. We found that PDCoV's main protease, NSP5, cleaves CCDC50 directly at a specific residue, glutamine 171. This proteolytic event impairs the ability of CCDC50 to interact with ubiquitin and the core autophagy machinery, effectively neutralizing its antiviral function and promoting viral replication. Significantly, we determined this to be a highly conserved strategy among coronaviruses. Our findings show that the NSP5 proteases of other divergent coronaviruses, including PEDV, TGEV, and even SARS-CoV-2, all target the same conserved site in CCDC50. These results reveal a common mechanism that coronaviruses use to subvert selective host autophagy.

IntroductionClostridium perfringens is the primary causative agent of enterotoxemia in yaks, resulting in substantial economic losses on the Qinghai-Tibet Plateau. Conventional vaccines exhibit limited protective breadth and suboptimal efficacy, highlighting the need for innovative strategies. Here, we aimed to construct a novel vaccine candidate incorporating epitopes from multiple prevalent toxinotypes (A, C, E) of C. perfringens affecting yaks, using immunoinformatics approach.MethodsA hierarchical immunoinformatics pipeline was implemented, encompassing subtractive genomics to identify core virulence factors, prediction and filtering of immunogenic T-cell and B-cell epitopes, rational multi-epitope vaccine design incorporating adjuvant and linkers, three-dimensional structure modeling and validation, molecular docking to evaluate interactions with TLR4, molecular dynamics simulations to confirm complex stability, and codon optimization to facilitate heterologous expression.Results and discussionFive core virulence proteins (Iap, CpsE, NanH, Plc, Pfo) were identified from genomic data, leading to the prediction and selection of ten cytotoxic T lymphocyte (CTL) epitopes, five helper T lymphocyte (HTL) epitopes, and five B-cell epitopes. The final 352-amino-acid multi-epitope vaccine (MEV) construct was assembled using the adjuvant human β-defensin-3 and specific linkers (AAY, GPGPG, KK). Computational evaluations confirmed the vaccine’s high antigenicity (VaxiJen score: 0.9092), non-allergenic nature, and structural stability. Molecular docking revealed strong binding affinity with TLR2 (-1024.6 kcal/mol) and TLR4 (-1104.4 kcal/mol). Molecular dynamics simulations over 100 ns confirmed stable TLR4 complex with an average RMSD of 0.1971 ± 0.0377 nm, while the TLR2 complex showed an average RMSD of 0.2692 ± 0.0420 nm. Immune simulation profiles predicted the induction of robust humoral and cellular immune responses, including elevated antibody titers, T-cell activation, and cytokine production. In silico cloning verified the potential for efficient expression in E. coli.ConclusionThis study designed a novel multi-epitope vaccine against C. perfringens in yaks using an immunoinformatics approach. The vaccine showed high antigenicity, stability, and broad allelic coverage in silico, providing a promising candidate that requires rigorous in vitro and in vivo experimental validation to confirm these computational predictions. This work offers a foundation for the development of effective vaccines against yak C. perfringens infections on the Qinghai-Tibet Plateau.

Porcine epidemic diarrhea virus (PEDV) is a reemerging swine enteric coronavirus that causes severe diarrhea and high mortality in neonatal piglets. Despite its significant impact on the global swine industry, no available vaccine provides complete protection, particularly in neonatal piglets. In this study, we employed a reverse genetics system based on the highly virulent PEDV strain YN17 to investigate the role of conserved regions within nonstructural protein 1 (NSP1) in viral replication and pathogenesis. Two recombinant PEDV mutants harboring targeted NSP1 deletions were successfully rescued: NSP1∆C (amino acids 59-67) and NSP1∆D (amino acids 87-107). While NSP1∆D replicated comparably to the wild-type virus, NSP1∆C exhibited significantly reduced replication both in vitro and in vivo. Notably, only NSP1∆C induced earlier and stronger expression of IFN-β, IFN-λ3, and ISGs compared to the wild-type strain. In piglets, NSP1∆C infection caused only mild clinical signs and minimal intestinal lesions, whereas NSP1∆D induced severe diarrhea and marked villus atrophy, similar to wild-type PEDV. Collectively, these findings identify the 59-67 region of NSP1 as critical for PEDV replication, immune evasion, and virulence, and support NSP1∆C as a promising candidate for live-attenuated vaccine development.

This study aimed to investigate the polymorphism of porcine-origin Ral guanine nucleotide dissociation stimulator (RalGDS) in LLC-PK1 cells using RT-PCR and sequencing. Our results revealed seven polymorphisms in the RalGDS gene, including insertions, deletions, and frameshift mutations. These variations may significantly alter the protein structure and function of RalGDS, potentially influencing its role in Ral GTPase-mediated signaling pathways. This work provides foundational insights into the genetic diversity of porcine RalGDS and its implications for pig physiology and economically important traits.

This study investigated the isoforms of porcine-origin Ral guanine nucleotide dissociation stimulator (RalGDS) in LLC-PK1 cells using reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Through segmented amplification, sequence assembly, and comparative genomics analysis, seven RalGDS isoforms were identified, characterized by insertions, deletions, and frameshift mutations. These genetic variations may significantly alter RalGDS’s protein structure and function, potentially impacting its role in Ral GTPase-mediated signaling pathways. This work provides foundational insights into the genetic diversity of porcine RalGDS and its implications for porcine physiology and economically significant traits.

Canine parvovirus (CPV) is a highly contagious and severe infectious disease that can lead to hemorrhagic enteritis and even acute death in dogs. Despite mouse monoclonal antibodies (mAbs) have been employed in clinical treatment, their application in non-murine species is restricted due to immune rejection. In this study, we screened a mouse mAb (5E7) with high neutralizing activity against CPV using hybridoma technology. Subsequently, the variable regions of the heavy (VH) and light (VL) chains of 5E7 were amplified by PCR and fused with the constant regions of canine IgG antibody to produce canine-mouse chimeric antibody (CM-5E7). The chimeric antibody was successfully expressed in HEK293 cells and exhibited high neutralizing activity against multiple CPV subtypes in vitro. Furthermore, CM-5E7 exhibited effective therapeutic potential in dogs subjected to lethal dose CPV-2c challenge in vivo. Overall, CM-5E7 demonstrated high neutralizing activity against CPV and showed significant efficacy in treating CPV-2c infections, positioning it as a promising candidate therapeutic antibody for the treatment of CPV infection.

ABSTRACTGenomic and evolutionary analysis of epidemic porcine hepatitis E virus (HEV) in the Tibetan Plateau was performed. Faecal samples were collected from 216 Tibetan pigs and 78 Tibetan Yorkshire (Large White) and 53 tissue samples from Yorkshire from the Linzhi City slaughterhouse. Total RNA was extracted from faeces and fragments of HEV open reading frame 2 (ORF2) detected by reverse transcription and nested polymerase chain reaction (RT‐nPCR) and cloned. Twenty‐three samples (23/347; 6.63%) were positive for the virus, including 6.94% (15/216) Tibetan pig and 6.11% (8/131) Yorkshire samples. No tissue samples tested positive for the virus. Cloned sequences were uploaded to GenBank (accession numbers: OR392679‐OR392685, OR355817‐OR355824 and OR909495‐OR909502) and a phylogenetic tree constructed. The entire viral genome was amplified using primers for the 5‐month‐old Tibetan pig sequence which confirmed that the strain belonged to HEV type 4, subtype d (GenBank accession number: OQ981960) and showed 93.30% homology with Sichuan Tibetan pig sequence, MK410044. Bayesian tree analysis showed that the earliest divergence was in 1999 and evidence of homologous recombination was found. Genomic and evolutionary analysis of HEV in the Tibetan Plateau is presented. The importance of continuous surveillance and genomic analysis of HEV is highlighted, especially in regions like the Tibetan Plateau where new strains may emerge. The findings contribute to our understanding of HEV's genetic diversity, evolutionary history and potential risks to animal and human health.

This study aimed to evaluate the therapeutic effect of Terminalia chebula (TC) on Tibetan yak-origin Salmonella-induced diarrhea and dysentery in mice. The levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α), anti-inflammatory cytokines (IL-4 and IL-10), and the oxidative stress markers malondialdehyde (MDA), superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), reduced glutathione (GSH-PX), and catalase (CAT) in the serum of mice were measured using ELISA kits. Using microbial diversity sequencing and non-targeted metabolomics detection techniques, the relevant mechanisms of TC treatment in a mouse Salmonella infection model were evaluated. The results showed the following: TC can effectively reduce the diarrhea rate; alleviate weight loss caused by Salmonella invasion; reduce the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in serum; and increase the concentrations of the anti-inflammatory cytokines IL-4 and IL-10. TC can improve the body’s antioxidant levels to heal the damage caused by oxidative stress and lipid peroxidation. The histological section results show that TC can significantly improve gastric and intestinal tissue lesions and has no toxic effects on the liver and kidneys. 16S rRNA and ITS sequencing analysis suggests that Lactobacillus, Enterorhabdus, Alistipes (bacterial community), Lodderomyces, Saccharomyces, and Penicillium (fungal community) may be key functional microbial communities in TC. Non-targeted metabolomics also suggests that the antibacterial treatment of dysentery with chebulic acid may be related to regulation of the Ras signaling pathway, long-term potentiation, the MAPK signaling pathway, metabolic pathways, and gut microbiome composition. Conclusion: TC has clear clinical efficacy in treating bacterial diarrhea, presenting anti-inflammatory and antioxidant effects. Its roles in regulating the gut microbiome and metabolic pathways and products were determined as the main reason for its therapeutic effect in a mouse gastroenteritis model caused by Salmonella infection.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes severe diarrhea in neonatal piglets worldwide and presents a significant public health threat due to its potential for cross-species transmission. Selective macroautophagy/autophagy, mediated by autophagy receptors such as NBR1 (NBR1 autophagy cargo receptor), plays a key role in restricting viral infection and modulating the host immune response. In this study, we revealed that overexpression of NBR1 inhibits PDCoV replication, while its knockdown increases viral titers. Further analysis demonstrated that NBR1 interacts with the PDCoV envelope (E) protein independently of ubiquitination, directing it to phagophores for autophagic degradation to limit viral proliferation. To counteract this defense, PDCoV 3C-like protease, encoded by NSP5, cleaves porcine NBR1 at glutamine 353 (Q353), impairing its selective autophagy function and antiviral activity. Additionally, we demonstrated that NSP5 proteases from other coronaviruses including PEDV, TGEV, and SARS-CoV-2 also cleave NBR1 at the same site, suggesting that coronaviruses employ a conserved strategy of NSP5-mediated cleavage of NBR1 to evade host antiviral responses and facilitate infection. Overall, our study underscores the importance of NBR1-mediated selective autophagy in the host's defense against PDCoV and reveals a strategy by which PDCoV evades autophagic mechanisms to promote successful infection.Abbreviation: Cas9: CRISPR-associated protein 9; CC1: coiled-coil 1; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; KO: knockout; LIR: MAP1LC3/LC3-interacting region; mAb: monoclonal antibody; NBR1: NBR1 autophagy cargo receptor; NBR1-C: C-terminal fragment of NBR1; NBR1-N: N-terminal fragment of NBR1; OPTN: optineurin; pAb: polyclonal antibody; PB1: Phox/BEM1 domain; PDCoV: porcine deltacoronavirus; PEDV: porcine epidemic diarrhea virus; Q353A: a NBR1 construct with the glutamine (Q) residue at position 353 replaced with glutamic acid (A); SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1: sequestosome 1; TCID50: 50% tissue culture infective dose; TGEV: porcine transmissible gastroenteritis virus; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild type; ZZ: ZZ-type zinc finger domain.

Mammalian orthoreoviruses (MRVs) have a wide geographic distribution worldwide and have been detected from humans and a variety of animal species. This study represents the first isolation of MRV from sheep rectal swabs in China, with analyses of its molecular and pathogenicity characteristics. MRV-positive samples were inoculated into Madin-Darby bovine kidney (MDBK) cells, resulting in stable cytopathic effects (CPEs) after three generations of blind passage. Two isolates were isolated and confirmed as MRV, named MRV-XJ23 and MRV-sheep/SY13, through reverse-transcription polymerase chain reaction (RT-PCR), transmission electron microscopy, and indirect immunofluorescence assay (IFA). The viruses exhibited broad cellular tropism. Whole-genome sequences were obtained and subjected to homology and evolutionary analyses, revealing that MRV-XJ23 and MRV-sheep/SY13 belong to the MRV-1 serotype. Phylogenetic analyses demonstrated that MRV-XJ23 is a reassortant virus containing gene segments from three MRVs that infected humans, bovines, and bats, with nucleotide homology exceeding 94.56%. The gene segments of MRV-sheep/SY13 were derived from five strains-Osaka2005, BatMRV-2/SNU1/Korea/2021, T1/human/Netherlands/1/84, IND/MZ/3013814/reo, and B/03-with nucleotide homology exceeding 95.47%. Animal experiments demonstrated that MRV-sheep/SY13 infection induced significant pathological changes in the respiratory and digestive tracts of mice. In sheep, MRV-sheep/SY13 caused respiratory infections, but no obvious lesion was observed from the digestive tract. This study expands our understanding of the MRV host range, reveals the potential public health risk of MRV transmission across species and zoonotic transmission, and underscores the necessity of further studies on epidemiology, reassortment patterns, and pathogenicity of MRV in sheep and domestic animals.

Structural variations (SVs) play crucial roles in the evolutionary adaptation of domesticated animals to natural and human-controlled environments, but SVs have not been explored in Tibetan cattle, which recently migrated and rapidly adapted to the high altitudes of the Qinghai-Tibetan Plateau (QTP). In this study, a de novo chromosome-level genome assembly for Tibetan cattle is constructed. It is found that using a lineage-specific reference genome significantly increased variant detection accuracy and completeness. Analysis of long-read sequencing data from 36 high-altitude QTP and 48 low-altitude cattle identified 222 528 SVs and 259 SV hotspot regions. Positively selected SVs in high-altitude cattle are related to energy metabolism erythropoiesis and angiogenesis, and peroxisomal metabolism. A 102-bp intronic deletion in GNPAT likely upregulated its expression. It is distinguished 7293 SVs that may be introgressed from yak, including variants upstream of the hypoxia-inducing gene EGLN1. Finally, a ≈2-Mb heterozygous inversion and two translocations on chromosome 6 are likely associated with the cattle gray coat via regulatory effects on the KIT gene. The results confirm the importance of SVs in evolutionary adaptation and the contribution yak-introgressed SVs to the rapid acclimatization of QTP cattle.

To determine the genotypes of the epidemic strains of Echinococcus granulosus in livestock in Tibet, samples of E. granulosus cysts were collected from 11 yaks and 62 sheep. Genomic DNA was extracted from these samples, and gene fragments of mitochondrial cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit I (nad1) were amplified by PCR and sequenced. DNASTAR and MAGA7.0 were employed for homology analysis and phylogenetic tree construction. Echinococcus granulosus cysts were detected in 56.2% (41/73) of the samples screened. Of these, 63.4% (26/41) were identified as E. granulosus G1 genotype (common sheep strain), 24.4% (10 /41) as G3 genotype (buffalo strain), and 12.2% (5/41) were G6 genotype (camel strain). The study concludes that yaks and sheep in Langkazi county, Tibet, carry three E. granulosus genotypes (G1, G3, and G6), with the G1 genotype the predominant genotype in the region. This study clarifies the distribution of E. granulosus genotypes, providing genetic data and insight for the surveillance and prevention of echinococcosis.

The Qinghai-Tibet Plateau (QTP), renowned for its exceptional biological diversity, is home to numerous endemic species. However, research on the virology of vulnerable vertebrates like yaks remains limited. In this study, our objective was to use metagenomics to provide a comprehensive understanding of the diversity and evolution of the gut virome in yak populations across different regions of the QTP. Our findings revealed a remarkably diverse array of viruses in the gut of yaks, including those associated with vertebrates and bacteriophages. Notably, some vertebrate-associated viruses, such as astrovirus and picornavirus, showed significant sequence identity across diverse yak populations. Additionally, we observed differences in the functional profiles of genes carried by the yak gut virome across different regions. Moreover, the virus-bacterium symbiotic network that we discovered holds potential significance in maintaining the health of yaks. Overall, this research expands our understanding of the viral communities in the gut of yaks and highlights the importance of further investigating the interactions between viruses and their hosts. These data will be beneficial for revealing the crucial role that viruses play in the yak gut ecology in future studies.

Canine parvovirus (CPV) is a significant threat to canines and is widely distributed worldwide. While vaccination is currently the most effective preventive measure, existing vaccines are not able to offer comprehensive and dependable protection against CPV infection. Hence, there is a need to explore alternative or complementary strategies to tackle this problem. In this study, we present an approach for the efficient screening of canine antibodies targeting CPV using a single B cell antibody technique. We sorted single IgM- IgG+ CPV+ B cells from canine peripheral blood mononuclear cells using fluorescence-activated cell sorting (FACS) and obtained the variable region genes of heavy and light chains (VH and VL) by nested PCR amplification. Canine monoclonal antibodies were expressed in HEK293 cells, and a total of 60 antibodies were obtained, five of which demonstrated neutralizing activity against CPV. Those findings demonstrate the effectiveness of the method for obtaining canine monoclonal antibodies, which in turn aids in the identification and screening of neutralizing antibodies against various canine pathogens.

[目的]筛选牦牛卵泡发育过程中可能对靶向Smad家族成员4(Smad family member 4,Smad4)基因的bta-miR-146a具有"海绵吸附"作用的长链非编码RNA(long non-coding RNA,lncRNA).[方法]使用miRanda和RNAhybrid数据库对靶向牦牛bta-miR-146a的lncRNA进行预测;采集牦牛卵巢,分离健康、闭锁卵泡,用Trizol法提取RNA并反转录为cDNA,利用PCR检测所预测lncRNA的表达情况;利用实时荧光定量PCR法检测所筛选lncRNA和bta-miR-146a/Smad4在牦牛健康、闭锁卵泡中的表达情况;构建lncRNA-ENSBGRT00000000387.1的野生型和突变型双荧光素酶载体,将其与bta-miR-146a-mimics、mimics NC共转染至HEK293T细胞,检测双荧光素酶活性.[结果]试验共筛选出7个可能对靶向Smad4基因的bta-miR-146a具有"海绵吸附"作用的lncRNAs,其中的lncRNA-ENSBGRT00000000387.1在牦牛卵泡中表达量极显著高于其他lncRNAs(P＜0.01).实时荧光定量 PCR 检测发现,lncRNA-ENSBGRT00000000387.1、bta-miR-146a 和 Smad4 基因 mRNA 在牦牛健康和闭锁卵泡中共表达,且lncRNA-ENSBGRT00000000387.1和Smad4基因mRNA在牦牛健康卵泡中的表达量均显著高于闭锁卵泡(P＜0.05),bta-miR-146a在牦牛健康卵泡中的表达量极显著低于闭锁卵泡(P＜0.01).试验成功构建牦牛lncRNA-ENSBGRT00000000387.1野生型及突变型pmirGLO双荧光素酶报告质粒,双荧光素酶活性结果显示,bta-miR-146a mimics 对牦牛 lncRNA-ENSBGRT00000000387.1-WT 具有极显著下调作用(P＜0.01).[结论]lncRNA-ENSBGRT00000000387.1、bta-miR-146a 和 Smad4 基因 mRNA 可能在牦牛卵泡发育或闭锁过程中存在调控机制,并在体外初步证实lncRNA-ENSBGRT00000000387.1与bta-miR-146a具有"海绵吸附"作用,这为进一步研究lncRNA-ENSBGRT00000000387.1在牦牛卵泡中的功能机制提供依据.

Swine H1N1/2009 influenza is a highly infectious respiratory disease in pigs, which poses a great threat to pig production and human health. In this study, we investigated the global expression profiling of swine-encoded genes in response to swine H1N1/2009 influenza A virus (SIV-H1N1/2009) in newborn pig trachea (NPTr) cells. In total, 166 genes were found to be differentially expressed (DE) according to the gene microarray. After analyzing the DE genes which might affect the SIV-H1N1/2009 replication, we focused on polo-like kinase 3 (PLK3). PLK3 is a member of the PLK family, which is a highly conserved serine/threonine kinase in eukaryotes and well known for its role in the regulation of cell cycle and cell division. We validated that the expression of PLK3 was upregulated after SIV-H1N1/2009 infection. Additionally, PLK3 was found to interact with viral nucleoprotein (NP), significantly increased NP phosphorylation and oligomerization, and promoted viral ribonucleoprotein assembly and replication. Furthermore, we identified serine 482 (S482) as the phosphorylated residue on NP by PLK3. The phosphorylation of S482 regulated NP oligomerization, viral polymerase activity and growth. Our findings provide further insights for understanding the replication of influenza A virus.