First Specific Detection of Mammalian Orthoreovirus from Goats Using TaqMan Real-Time RT-PCR Technology.

Mammalian orthoreovirus (MRV) infections are ubiquitous in multiple mammalian species including humans, and mainly causes gastroenteritis and respiratory disease. In this study, we developed a rapid and sensitive TaqMan qRT-PCR method for MRV detection based on the primers and probe designed within the conserved L1 gene. The qRT-PCR assay was evaluated for its sensitivity, specificity, efficiency and reproducibility. It was found that the detection sensitivity was equivalent to 10 DNA copies/µL, and the standard curves had a linear correlation of R2 = 0.998 with an amplification efficiency of 99.6%. The inter- and intra-assay coefficients of variation (CV%) were in the range of 0.29% to 2.16% and 1.60% to 3.60%, respectively. The primer sets specifically amplified their respective MRV segments and had the highest detection sensitivities of 100.25 TCID50/mL with amplification efficiencies of 99.5% (R2 = 0.999). qRT-PCR was used for MRV detection from samples of sheep, goats, and calves from four regions in China, and the overall MRV prevalence was 8.2% (35/429), whereas 17/429 (4.0%) were detected by RT-PCR and 14/429 (3.3%) by virus isolation. The qRT-PCR assay showed significantly higher sensitivity than RT-PCR and virus isolation. Results from an epidemiological survey indicated that the positive rate of MRV in rectal swabs from sheep and goats tested in Shaanxi, Jiangsu, and Xinjiang were 9/80 (11.3%), 12/93 (12.9%) and 14/128 (10.9%), respectively. In goats and sheep, MRV prevalence was obviously associated with season and age, with a high positive rate of more than 8% during September to April and approximately 13% in small ruminant animals under two months of age. This is the first instance of MRV infection in sheep and goats in China, thus broadening our knowledge of MRV hosts. Consequently, primer optimization for qRT-PCR should not only prioritize amplification efficiency and specificity, but also sensitivity. This assay will contribute to more accurate and rapid MRV monitoring by epidemiological investigation, viral load, and vaccination efficacy.

Generation of whole-porcine neutralizing antibodies of an alphacoronavirus by single B cell antibody technology.

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that causes severe morbidity and mortality in piglets, resulting in substantial economic losses to the swine industry. While vaccination is currently the most effective preventive measure, existing vaccines fail to provide complete and reliable protection against PEDV infection. Consequently, there is a need to explore alternative or complementary strategies to address this issue. In this study, we utilized single B cell antibody technology to obtain a potent neutralizing antibody, C62, which specifically targets the receptor binding domain S1B of the PEDV-S1 protein. C62 exhibited potent neutralizing activity against PEDV and inhibited viral attachment to the cell surface in vitro. Furthermore, the effectiveness of C62 in mitigating PEDV infection was demonstrated in vivo, as evidenced by the delayed onset of diarrhea and reduced mortality rates observed in piglets following oral administration of C62. Our study provides an alternative approach for controlling PEDV infection. Meanwhile, C62 holds promise as a therapeutic biological agent to complement existing vaccines. More importantly, our study forms a solid foundation for the development of whole-porcine neutralizing antibodies against other swine coronaviruses, thus contributing to the overall improvement of swine health.

PLK3 facilitates replication of swine influenza virus by phosphorylating viral NP protein.

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.

Evaluation of the Mucosal Immunity Effect of Bovine Viral Diarrhea Virus Subunit Vaccine E2Fc and E2Ft.

Classified as a class B infectious disease by the World Organization for Animal Health (OIE), bovine viral diarrhea/mucosal disease is an acute, highly contagious disease caused by the bovine viral diarrhea virus (BVDV). Sporadic endemics of BVDV often lead to huge economic losses to the dairy and beef industries. To shed light on the prevention and control of BVDV, we developed two novel subunit vaccines by expressing bovine viral diarrhea virus E2 fusion recombinant proteins (E2Fc and E2Ft) through suspended HEK293 cells. We also evaluated the immune effects of the vaccines. The results showed that both subunit vaccines induced an intense mucosal immune response in calves. Mechanistically, E2Fc bonded to the Fc γ receptor (FcγRI) on antigen-presenting cells (APCs) and promoted IgA secretion, leading to a stronger T-cell immune response (Th1 type). The neutralizing antibody titer stimulated by the mucosal-immunized E2Fc subunit vaccine reached 1:64, which was higher than that of the E2Ft subunit vaccine and that of the intramuscular inactivated vaccine. The two novel subunit vaccines for mucosal immunity developed in this study, E2Fc and E2Ft, can be further used as new strategies to control BVDV by enhancing cellular and humoral immunity.

Antimicrobial susceptibility and multilocus sequence typing of Clostridium perfringens isolated from yaks in Qinghai-Tibet plateau, China.

Clostridium perfringens (C. perfringens) is an opportunistic pathogen that cause necrotic enteritis, food poisoning and even death in animals. In this study, we explored the prevalence, antibiotic resistance and genetic diversity of Clostridium perfringens isolated from yak in the Qinghai-Tibet plateau, China. A total of 744 yak fecal samples were collected and assessed for toxin genes, antimicrobial susceptibility and multilocus sequence typing (MLST). Results indicated that 144 out of 744 (19.35%) yak fecal samples were tested to be positive for C. perfringens, 75% (n = 108, 108/144) were C. perfringens type A, 17.36% (n = 25, 25/144) were C. perfringens type C, 2.78% (n = 4, 4/144) were C. perfringens type D, and 4.86% (n = 7, 7/144) were C. perfringens type F. In addition, 2.78% (n = 4, 4/144) of the isolates were positive for cpb2 toxin gene. Antimicrobial susceptibility testing revealed that 98.61% (142/144) of the isolates showed multiple-antibiotic resistance. According to MLST and phylogenetic tree, 144 yak-derived C. perfringens isolates had an average of 12.95 alleles and could be divided into 89 sequence types (STs) and clustered in 11 clonal complexes (CCs). The most of isolates belong to type A with a considerable genetic diversity, having Simpson index up to 0.9754. MLST and phylogenetic analysis showed that the isolates under the same clade came from multiple regions. Cross-transmission among isolates and interconnectedness were observed in the genetic evolution. According to the study, the most of the isolates exhibited broad-spectrum antibacterial resistance, diverse alleles, and multiple lethal toxin genes of C. perfringens.

Profiling and Functional Analysis of long non-coding RNAs in yak healthy and atretic follicles.

Yak is the livestock on which people live in plateau areas, but its fecundity is low. Follicular development plays a decisive role in yak reproductive performance. As an important regulatory factor, the expression of long non-coding RNA (lncRNAs) in yak follicular development and its regulatory mechanism remains unclear. To explore the differentially expressed lncRNAs between healthy and atretic follicular in yaks. We used RNA-seq to construct lncRNA, miRNA, and mRNA expression profiles in yak atretic and healthy follicles, and the RNA sequence results were identified by qPCR. In addition, the correlation of lncRNA and targeted mRNA was also analyzed by Starbase software. Moreover, lncRNA/miRNA/mRNA networks were constructed by Cytoscape software, and the network was verified by dual-luciferase analysis. A total of 682 novel lncRNAs, 259 bta-miRNAs, and 1704 mRNAs were identified as differentially expressed between healthy and atretic follicles. Among them, 135 mRNAs were positively correlated with lncRNA expression and 97 were negatively correlated, which may be involved in the yak follicular development. In addition, pathway enrichment analysis of differentially expressed lncRNA host genes by Kyoto Genome Encyclopedia (KEGG) showed that host genes were mainly involved in hormone secretion, granulosa cell apoptosis, and follicular development. In conclusion, we identified a series of novel lncRNAs, constructed the lncRNA ceRNA regulatory network, and provided comprehensive resources for exploring the role of lncRNAs in yak ovarian follicular development.

First detection and complete genome analysis of porcine circovirus-like virus P1 and porcine circovirus-2 in yak in China.

Porcine circovirus-like virus P1, like porcine circovirus type 2 (PCV2), is a potential pathogen of post-weaning multisystemic wasting syndrome in swine. Yaks are a valuable species and an iconic symbol of the Tibet Plateau which is the highest and largest plateau in the world. In this study, a total of 105 yak diarrheal samples, collected from 13 farms in Linzhi in the Tibet Plateau from January 2019 to December 2021, that were screened for P1 and PCV2 by polymerase chain reaction, 10.48% (n = 11) were positive for P1, 4.76% (n = 5) for PCV2, and 5.71% (n = 6) were positive for coinfection of P1 and PCV2. In addition, the whole genomes of eight P1 strains and eight PCV2 strains were sequenced. Alignment of deduced amino acid sequences of P1 ORF1 and PCV2 ORF2 gene revealed that ON012566 had one unique amino acid mutation at residues 137 (T to P). This mutation has important implication for the study of virus virulence, tissue tropism, and immune response. Phylogenetic analysis shows that the yak-origin P1 strains in this study with cattle-origin P1 reference strains were grouped into one cluster. The yak-origin PCV2 (ON012566) and a buffalo-origin PCV2 (KM116514) reference strain clustered in the same branch in the PCV2b regions. Meanwhile, the remaining PCV2 strains and buffalo-origin PCV2 reference strain (ON012565) clustered in the PCV2d regions. To summarize, to our knowledge, this is the first report on the molecular prevalence and genome characteristics of P1 and PCV2 in yaks in the world and will contribute to further study of the molecular epidemiology, source, and evolution of P1 and PCV2 strains.

Porcine circovirus-like virus P1 activates pancreatic secretion pathway by interacting with CHRM3 protein.

The porcine circovirus-like virus P1, a member of the circovirus family, causes post-weaning multisystemic wasting syndrome (PMWS) in weaned piglets with progressive wasting as the main clinical symptom. The pancreatic secretion pathway induces pancreatic acinar cells to secrete various digestive enzymes and as such is an important signaling pathway for the digestive system and somatic growth. This study examined the effects and mechanism of P1 virus infection on the pancreatic secretion pathway. The experiment was conducted by transfecting double-copy plasmid P1 into PK-15 and 3D4 cells and by infecting cells with the P1 virus. Samples were collected at various times after transfection or infection. The pathway's transcription and translation levels of CHRM3, Gq, PLC-ß2, PRKCA, Rab3D, RhoA, Rac1, and amyA proteins were detected by real-time PCR and Western blots; these analyses confirmed that the P1 virus infection could upregulate the expression level of key pancreatic secretion signaling molecules. Then, we confirmed that the VP1 protein of the P1 virus could interact with the pathway initiation protein CHRM3 using Co-IP, pull-downs, and confocal fluorescence microscopy. Finally, we demonstrated that the VP1 protein activates the pancreatic secretory pathway through the CHRM3 protein. In conclusion, this study demonstrated that the P1 virus can interact with the CHRM3 receptor protein to activate the pancreatic secretion pathway and promote the secretion of various digestive enzymes downstream of the pathway, thereby providing a basis for P1 virus pathogenesis.

Next-Generation Sequencing Reveals Four Novel Viruses Associated with Calf Diarrhea.

Calf diarrhea is one of the common diseases involved in the process of calf feeding. In this study, a sample of calf diarrhea that tested positive for bovine coronavirus and bovine astrovirus was subjected to high-throughput sequencing. The reassembly revealed the complete genomes of bovine norovirus, bovine astrovirus, bovine kobuvirus, and the S gene of bovine coronavirus. Phylogenetic analysis showed that the ORF2 region of bovine astrovirus had the lowest similarity with other strains and gathered in the Mamastrovirus unclassified genogroup, suggesting a new serotype/genotype could appear. Compared with the most closely related strain, there are six amino acid mutation sites in the S gene of bovine coronavirus, most of which are located in the S1 subunit region. The bovine norovirus identified in our study was BNoV-GIII 2, based on the VP1 sequences. The bovine kobuvirus is distributed in the Aichi virus B genus; the P1 gene shows as highly variable, while the 3D gene is highly conserved. These findings enriched our knowledge of the viruses in the role of calf diarrhea, and help to develop an effective strategy for disease prevention and control.

Metagenomic insights into the diversity of carbohydrate-degrading enzymes in the yak fecal microbial community.

BACKGROUND: Yaks are able to utilize the gastrointestinal microbiota to digest plant materials. Although the cellulolytic bacteria in the yak rumen have been reported, there is still limited information on the diversity of the major microorganisms and putative carbohydrate-metabolizing enzymes for the degradation of complex lignocellulosic biomass in its gut ecosystem. RESULTS: Here, this study aimed to decode biomass-degrading genes and genomes in the yak fecal microbiota using deep metagenome sequencing. A comprehensive catalog comprising 4.5 million microbial genes from the yak feces were established based on metagenomic assemblies from 92 Gb sequencing data. We identified a full spectrum of genes encoding carbohydrate-active enzymes, three-quarters of which were assigned to highly diversified enzyme families involved in the breakdown of complex dietary carbohydrates, including 120 families of glycoside hydrolases, 25 families of polysaccharide lyases, and 15 families of carbohydrate esterases. Inference of taxonomic assignments to the carbohydrate-degrading genes revealed the major microbial contributors were Bacteroidaceae, Ruminococcaceae, Rikenellaceae, Clostridiaceae, and Prevotellaceae. Furthermore, 68 prokaryotic genomes were reconstructed and the genes encoding glycoside hydrolases involved in plant-derived polysaccharide degradation were identified in these uncultured genomes, many of which were novel species with lignocellulolytic capability. CONCLUSIONS: Our findings shed light on a great diversity of carbohydrate-degrading enzymes in the yak gut microbial community and uncultured species, which provides a useful genetic resource for future studies on the discovery of novel enzymes for industrial applications.

Characterization of Metagenome-Assembled Genomes and Carbohydrate-Degrading Genes in the Gut Microbiota of Tibetan Pig.

Tibetan pig is an important domestic mammal, providing products of high nutritional value for millions of people living in the Qinghai-Tibet Plateau. The genomes of mammalian gut microbiota encode a large number of carbohydrate-active enzymes, which are essential for the digestion of complex polysaccharides through fermentation. However, the current understanding of microbial degradation of dietary carbohydrates in the Tibetan pig gut is limited. In this study, we produced approximately 145 gigabases of metagenomic sequence data for the fecal samples from 11 Tibetan pigs. De novo assembly and binning recovered 322 metagenome-assembled genomes taxonomically assigned to 11 bacterial phyla and two archaeal phyla. Of these genomes, 191 represented the uncultivated microbes derived from novel prokaryotic taxa. Twenty-three genomes were identified as metagenomic biomarkers that were significantly abundant in the gut ecosystem of Tibetan pigs compared to the other low-altitude relatives. Further, over 13,000 carbohydrate-degrading genes were identified, and these genes were more abundant in some of the genomes within the five principal phyla: Firmicutes, Bacteroidetes, Spirochaetota, Verrucomicrobiota, and Fibrobacterota. Particularly, three genomes representing the uncultivated Verrucomicrobiota encode the most abundant degradative enzymes in the fecal microbiota of Tibetan pigs. These findings should substantially increase the phylogenetic diversity of specific taxonomic clades in the microbial tree of life and provide an expanded repertoire of biomass-degrading genes for future application to microbial production of industrial enzymes.

Development of a DAS-ELISA for detection of H9N2 avian influenza virus.

H9N2 avian influenza virus is threatening animals and public health systems. Effective diagnosis is imperative to control the disease. Thus, we developed a panel of monoclonal antibodies (Mabs) against the H9N2 avian influenza virus (AIV) and implemented a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to detect the H9 viral antigen. Hybridomas 4D10 and 5G2 were screened to secrete immunoglobulin G (IgG) and IgA, respectively. Antibody 4D10 was used as the capture antibodies and HRP labeled 5G2 as the detector antibody. The specificity of the optimized DAS-ELISA was evaluated by using AIV subtypes H1, H3, H5, H9 and H10. Specimens containing AIV H9 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limit of the DAS-ELISA is 10-2.3 TCID50 (50% Tissue culture infective doses). Negative-positive threshold was 0.211 (OD630). In comparison with virus isolation the sensitivity and specificity of DAS-ELISA were found to be 98.9% and 98.1% respectively. Taken together, the newly developed Mab-based DAS-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H9 subtype AIV.

Investigation of antimicrobial resistance in Escherichia coli and enterococci isolated from Tibetan pigs.

OBJECTIVES: This study investigated the antimicrobial resistance of Escherichia coli and enterococci isolated from free-ranging Tibetan pigs in Tibet, China, and analyzed the influence of free-ranging husbandry on antimicrobial resistance. METHODS: A total of 232 fecal samples were collected from Tibetan pigs, and the disk diffusion method was used to examine their antimicrobial resistance. Broth microdilution and agar dilution methods were used to determine minimum inhibitory concentrations for antimicrobial agents for which disks were not commercially available. RESULTS: A total of 129 E. coli isolates and 84 Enterococcus isolates were recovered from the fecal samples. All E. coli isolates were susceptible to amoxicillin/clavulanic acid, and 40.4% were resistant to tetracycline. A small number of isolates were resistant to florfenicol (27.9%), ampicillin (27.9%), sulfamethoxazole/trimethoprim (19.4%), nalidixic acid (19.4%), streptomycin (16.2%) and ceftiofur (10.9%), and very low resistance rates to ciprofloxacin (7.8%), gentamicin (6.9%), and spectinomycin (2.3%) were observed in E. coli. All Enterococcus isolates, including E. faecium, E. faecalis, E. hirae, and E. mundtii, were susceptible to amoxicillin/clavulanic acid and vancomycin, but showed high frequencies of resistance to oxacillin (92.8%), clindamycin (82.1%), tetracycline (64.3%), and erythromycin (48.8%). Resistance rates to florfenicol (17.9%), penicillin (6.0%), ciprofloxacin (3.6%), levofloxacin (1.2%), and ampicillin (1.2%) were low. Only one high-level streptomycin resistant E. faecium isolate and one high-level gentamicin resistant E. faecium isolate were observed. Approximately 20% and 70% of E. coli and Enterococcus isolates, respectively, were defined as multidrug-resistant. CONCLUSIONS: In this study, E. coli and Enterococcus isolated from free-ranging Tibetan pigs showed relatively lower resistance rates than those in other areas of China, where more intensive farming practices are used. These results also revealed that free-range husbandry and absence of antibiotic use could decrease the occurrence of antimicrobial resistance to some extent.

Haematological and blood biochemical characteristics of Glyptosternum maculatum (Siluriformes: Sisoridae) in Xizang (Tibet).

This study investigated the haematological and blood biochemical characteristics of Glyptosternum maculatum. The haematological and biochemical parameters were measured in 30 adult fish collected from Nyingchi Reach of Yarlung Zangbo River in Tibet. The red blood cell count (RBC), haemoglobin concentration (Hb), haematocrit (Hct), erythrocyte osmotic fragility (maxEof and minEof), the erythrocyte sedimentation rate, mean cell volume (MCV), mean cellular haemoglobin content (MCH), and mean cell haemoglobin concentration (MCHC) were determined. Compared with other Siluriformes fishes, G. maculatum showed similar mean values for Hct, Hb, MCH, and MCHC and had slightly lower RBC and higher MCV. The biochemical parameters were assayed including alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, direct bilirubin, urea, creatinine, glucose, total cholesterol, and triglyceride. The result showed that the value of AST in G. maculatum was obviously higher than that in Rhamdia quelen as well as in Silurus merdionalis.