Natural hyperoside extracted from hawthorn exhibits antiviral activity against porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and death in piglets, resulting in significant economic losses for the pork industry. There is an urgent need for new treatment strategies. Here, we focused on optimizing the process of purifying natural hyperoside (nHYP) from hawthorn and evaluating its effectiveness against PEDV both in vitro and in vivo. Our findings demonstrated that nHYP with a purity >98% was successfully isolated from hawthorn with an extraction rate of 0.42 mg/g. Furthermore, nHYP exhibited strong inhibitory effects on PEDV replication in cells, with a selection index of 9.72. nHYP significantly reduced the viral load in the intestines of piglets and protected three of four piglets from death caused by PEDV infection. Mechanistically, nHYP could intervene in the interaction of PEDV N protein and p53. The findings implicate nHYP as having promising therapeutic potential for combating PEDV infections.

Licorice extract inhibits porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and even death in piglets, resulting in significant economic losses to the pig industry. Because of the ongoing mutation of PEDV, there might be variations between the vaccine strain and the prevailing strain, causing the vaccine to not offer full protection against different PEDV variant strains. Therefore, it is necessary to develop anti-PEDV drugs to compensate for vaccines. This study confirmed the anti-PEDV effect of licorice extract (Le) in vitro and in vivo. Le inhibited PEDV replication in a dose-dependent manner in vitro. By exploring the effect of Le on the life cycle of PEDV, we found that Le inhibited the attachment, internalization, and replication stages of the virus. In vivo, all five piglets in the PEDV-infected group died within 72 h. In comparison, the Le-treated group had a survival rate of 80 % at the same time, with significant relief of clinical symptoms, pathological damage, and viral loads in the jejunum and ileum. Our results suggested that Le can exert anti-PEDV effects in vitro and in vivo. Le is effective and inexpensive; therefore it has the potential to be developed as a new anti-PEDV drug.

A Mini-Review on the Common Antiviral Drug Targets of Coronavirus.

Coronaviruses in general are a zoonotic pathogen with significant cross-species transmission. They are widely distributed in nature and have recently become a major threat to global public health. Vaccines are the preferred strategy for the prevention of coronaviruses. However, the rapid rate of virus mutation, large number of prevalent strains, and lag in vaccine development contribute to the continuing frequent occurrence of coronavirus diseases. There is an urgent need for new antiviral strategies to address coronavirus infections effectively. Antiviral drugs are important in the prevention and control of viral diseases. Members of the genus coronavirus are highly similar in life-cycle processes such as viral invasion and replication. These, together with the high degree of similarity in the protein sequences and structures of viruses in the same genus, provide common targets for antiviral drug screening of coronaviruses and have led to important advances in recent years. In this review, we summarize the pathogenic mechanisms of coronavirus, common drugs targeting coronavirus entry into host cells, and common drug targets against coronaviruses based on biosynthesis and on viral assembly and release. We also describe the common targets of antiviral drugs against coronaviruses and the progress of antiviral drug research. Our aim is to provide a theoretical basis for the development of antiviral drugs and to accelerate the development and utilization of commonly used antiviral drugs in China.

A survey of fecal virome and bacterial community of the diarrhea-affected cattle in northeast China reveals novel disease-associated ecological risk factors.

Limited information on the virome and bacterial community hampers our ability to discern systemic ecological risk factors that cause cattle diarrhea, which has become a pressing issue in the control of disease. A total of 110 viruses, 1,011 bacterial genera, and 322 complete viral genomes were identified from 70 sequencing samples mixed with 1,120 fecal samples from 58 farms in northeast China. For the diarrheic samples, the identified virome and bacterial community varied in terms of composition, abundance, diversity, and geographic distribution in relation to different disease-associated ecological factors; the abundance of identified viruses and bacteria was significantly correlated with the host factors of clinical status, cattle type, and age, and with environmental factors such as aquaculture model and geographical location (P < 0.05); a significant interaction occurred between viruses and viruses, bacteria and bacteria, as well as between bacteria and viruses (P < 0.05). The abundance of SMB53, Butyrivibrio, Facklamia, Trichococcus, and Turicibacter was significantly correlated with the health status of cattle (P < 0.05). The proportion of BRV, BCoV, BKV, BToV, BoNoV, BoNeV, BoAstV, BEV, BoPV, and BVDV in 1,120 fecal samples varied from 1.61% to 12.05%. A series of significant correlations were observed between the prevalence of individual viruses and the disease-associated ecological factors. A genome-based phylogenetic analysis revealed high variability of 10 bovine enteric viruses. The bovine hungarovirus was initially identified in both dairy and beef cattle in China. This study elucidates the fecal virome and bacterial community signatures of cattle affected by diarrhea, and reveals novel disease-associated ecological risk factors, including cattle type, cattle age, aquaculture model, and geographical location.IMPORTANCEThe lack of data on the virome and bacterial community restricts our capability to recognize ecological risk factors for bovine diarrhea disease, thereby hindering our overall comprehension of the disease's cause. In this study, we found that, for the diarrheal samples, the identified virome and bacterial community varied in terms of composition, abundance, diversity, configuration, and geographic distribution in relation to different disease-associated ecological factors. A series of significant correlations were observed between the prevalence of individual viruses and the disease-associated ecological factors. Our study aims to uncover novel ecological risk factors of bovine diarrheal disease by examining the pathogenic microorganism-host-environment disease ecology, thereby providing a new perspective on the control of bovine diarrheal diseases.

Establishment and evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type canine distemper virus from vaccine strains.

This study sought to establish a real-time reverse transcription (RT)-PCR method to differentially detect canine distemper virus (CDV) wild-type and vaccine strains. To this end, a pair of CDV universal primers and two specific minor groove binder (MGB) probes, harboring a T/C substitution in the hemagglutinin (H) gene, were designed. Using a recombinant plasmid expressing the H gene of the CDV wild-type or vaccine strain as standards, a sensitive and specific multiplex real-time RT-PCR was established for quantitative and differential detection of CDV wild-type and vaccine strains. The limit of detection for this multiplex assay was 22.5 copies/µL and 2.98 copies/µL of viral RNA for wild-type and vaccine strains, respectively. Importantly, the wild-type and vaccine MGB probes specifically hybridized different genotypes of wild-type CDV circulating in China as well as globally administered vaccine viruses, respectively, with no cross-reactivity observed with non-CDV viruses. Moreover, this method was successfully applied for the quantitative detection of CDV RNA in tissue samples of experimentally infected breeding foxes, raccoon dogs, and minks. Additionally, the multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples as early as 3 days post-infection, 3 to 4 days prior to the onset of clinical signs in these CDV infection animals. Hence, the established multiplex real-time RT-PCR method is useful for differentiating wild-type CDV and vaccine strains in China, and for conducting canine distemper early diagnosis as well as dynamic mechanism of CDV replication studies in vivo.

DNA Vaccine Co-Expressing Hemagglutinin and IFN-γ Provides Partial Protection to Ferrets against Lethal Challenge with Canine Distemper Virus.

Canine distemper (CD), caused by canine distemper virus (CDV), is a highly contagious and lethal disease in domestic and wild carnivores. Although CDV live-attenuated vaccines have reduced the incidence of CD worldwide, low levels of protection are achieved in the presence of maternal antibodies in juvenile animals. Moreover, live-attenuated CDV vaccines may retain residual virulence in highly susceptible species and cause disease. Here, we generated several CDV DNA vaccine candidates based on the biscistronic vector (pIRES) co-expressing virus wild-type or codon-optimized hemagglutinin (H) and nucleocapsid (N) or ferret interferon (IFN)-γ, as a molecular adjuvant, respectively. Apparently, ferret (Mustela putorius furo)-specific codon optimization increased the expression of CDV H and N proteins. A ferret model of CDV was used to evaluate the protective immune response of the DNA vaccines. The results of the vaccinated ferrets showed that the DNA vaccine co-expressing the genes of codon-optimized H and ferret IFN-γ (poptiH-IRES-IFN) elicited the highest anti-CDV serum-neutralizing antibodies titer (1:14) and cytokine responses (upregulated TNF-α, IL-4, IL-2, and IFN-γ expression) after the third immunization. Following vaccination, the animals were challenged with a lethal CDV 5804Pe/H strain with a dose of 105.0 TCID50. Protective immune responses induced by the DNA vaccine alleviated clinical symptoms and pathological changes in CDV-infected ferrets. However, it cannot completely prevent virus replication and viremia in vivo as well as virus shedding due to the limited neutralizing antibody level, which eventually contributed to a survival rate of 75% (3/4) against CDV infection. Therefore, the improved strategies for the present DNA vaccines should be taken into consideration to develop more protective immunity, which includes increasing antigen expression or alternative delivery routes, such as gene gun injection.

The outer membrane protein of Fusobacterium necrophorum, 43K OMP, stimulates inflammatory cytokine production through nuclear factor kappa B activation.

OBJECTIVE: Fusobacterium necrophorum causes bovine hepatic abscess, foot rot, mastitis, and endometritis. The 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in infections by this bacterium, but the biological function and the pathogenesis of this protein are largely unknown. METHODS: In this study, we investigated the role of the 43 K OMP in bacterial infection of bovine mammary epithelial cells (MAC-T cells) by Tandem Mass Tag proteomic analysis. The RAW264.7 cells were incubated with recombinant 43 K OMP (12.5 µg/mL) for 2 h, 4 h, 6 h, and 12 h, and then the inflammatory related protein and inflammatory cytokine production were measured by Western blot analysis and ELISA, the mRNA expression levels of inflammatory cytokine were measured by Real-Time PCR. RESULTS: Proteomic analysis results demonstrated there were 224 differentially expressed proteins in the MAC-T cells stimulated with the 43 K OMP compared with control, and 118 proteins were upregulated and 106 proteins were downregulated. These differentially expressed proteins were mainly involved in NF-kappa B signaling, bacterial invasion of epithelial cells, cell adhesion, complement and coagulation cascades. The top six differentially expressed proteins were; MMP9, PLAU, STOM, PSMD13, PLAUR, and ITGAV, which were involved in a protein-protein interaction network. Furthermore, TLR/MyD88/NF-κB pathway related proteins and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) were assessed by Western blot analysis and ELISA. Results showed the 43 K OMP to enhance the expression of TLR4 protein at 2 h (P < 0.01) and the MyD88 protein at 4 h (P < 0.05) post-stimulation, and to decrease IκBα expression at 4 h, 6 h and 12 h (P < 0.05) post-infection, as well as induce phosphorylation at Ser536 (P < 0.01). Levels of IL-6, IL-1ß, and TNF-α in the supernatants of mouse macrophages were increased (P < 0.05), as were mRNA expression levels of IL-6, IL-1ß, and TNF-α (P < 0.05), while IL-4 mRNA expression was decreased (P < 0.05). CONCLUSIONS: Taken together, these results suggested the important role for 43 K OMP in F. necrophorum infection, promoting the production of pro-inflammatory cytokines (IL-6 and TNF-α) by activation of the TLR/MyD88/NF-κB pathway. These findings provided a theoretical basis for a better understanding of the pathogenesis of F. necrophorum infection.

Octyl gallate targeting the 3C-like protease exhibits highly efficient antiviral activity against swine enteric coronavirus PEDV.

Infection with porcine epidemic diarrhea virus (PEDV) causes severe watery diarrhea in newborn piglets, leading to substantial financial losses for the swine industry. In this study, we screened small molecule drugs targeting 3 C-like protease (3CLpro) by molecular docking, and further evaluated the antiviral activity of the screened drugs against PEDV. Results showed that octyl gallate (OG), a widely used food additive, exhibited strong binding affinity with the 3CLpro active sites of PEDV. Bio-layer interferometry and fluorescence resonance energy transfer revealed that OG directly interacts with PEDV 3CLpro (KD = 549 nM) and inhibits 3CLpro activity (IC50 = 22.15 µM). OG showed a strong inhibition of PEDV replication in vitro. Virus titers were decreased by 0.58 and 0.71 log10 TCID50/mL for the CV777 and HM2017 strains, respectively. In vivo, all piglets in the PEDV-infected group died at 48 h post-infection (hpi), while 75% of piglets in the OG treatment group showed significant relief from the clinical symptoms, pathological damage, and viral loads in the jejunum and ileum. Moreover, the western blotting results showed that OG also has strong antiviral activity against other swine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). Our findings revealed that OG could be developed as a novel antiviral drug against PEDV. The OG exhibited a potential broad-spectrum antiviral drug for control of other swine enteric coronaviruses.

A Catalog of over 5,000 Metagenome-Assembled Microbial Genomes from the Caprinae Gut Microbiota.

Most microbiome studies regarding the ruminant digestive tract have focused on the rumen microbiota, whereas only a few studies were performed on investigating the gut microbiota of ruminants, which limits our understanding of this important component. Herein, the gut microbiota of 30 Caprinae animals (sheep and goats) from six provinces in China was characterized using ultradeep (>100 Gbp per sample) metagenome shotgun sequencing. An inventory of Caprinae gut microbial species containing 5,046 metagenomic assembly genomes (MAGs) was constructed. Particularly, 2,530 of the genomes belonged to uncultured candidate species. These genomes largely expanded the genomic repository of the current microbes in the Caprinae gut. Several enzymes and biosynthetic gene clusters encoded by these Caprinae gut species were identified. In summary, our study extends the gut microbiota characteristics of Caprinae and provides a basis for future studies on animal production and animal health. IMPORTANCE We constructed a microbiota catalog containing 5,046 MAGs from Caprinae gut from six regions of China. Most of the MAGs do not overlap known databases and appear to be potentially new species. We also characterized the functional spectrum of these MAGs and analyzed the differences between different regions. Our study enriches the understanding of taxonomic, functional, and metabolic diversity of Caprinae gut microbiota. We are confident that the manuscript will be of utmost interest to a wide range of readers and be widely applied in future research.

Up-regulated 60S ribosomal protein L18 in PEDV N protein-induced S-phase arrested host cells promotes viral replication.

Coronavirus subverts the host cell cycle to create a favorable cellular environment that enhances viral replication in host cells. Previous studies have revealed that nucleocapsid (N) protein of the coronavirus porcine epidemic diarrhea virus (PEDV) interacts with p53 to induce cell cycle arrest in S-phase and promotes viral replication. However, the mechanism by which viral replication is increased in the PEDV N protein-induced S-phase arrested cells remains unknown. In the current study, the protein expression profiles of PEDV N protein-induced S-phase arrested Vero E6 cells and thymidine-induced S-phase arrested Vero E6 cells were characterized by tandem mass tag-labeled quantitative proteomic technology. The effect of differentially expressed proteins (DEPs) on PEDV replication was investigated. The results indicated that a total of 5709 proteins, including 20,560 peptides, were identified, of which 58 and 26 DEPs were identified in the PEDV N group and thymidine group, respectively (P < 0.05; ratio ≥ 1.2 or ≤ 0.8). The unique DEPs identified in the PEDV N group were mainly involved in DNA replication, transcription, and protein synthesis, of which 60S ribosomal protein L18 (RPL18) exhibited significantly up-regulated expression in the PEDV N protein-induced S-phase arrested Vero E6/IPEC-J2 cells and PEDV-infected IPEC-J2 cells (P < 0.05). Further studies revealed that the RPL18 protein could significantly enhance PEDV replication (P < 0.05). Our findings reveal a mechanism regarding increased viral replication when the PEDV N protein-induced host cells are in S-phase arrest. These data also provide evidence that PEDV maintains its own replication by utilizing protein synthesis-associated ribosomal proteins.

Isolation, identification, and pathogenicity of a goose astrovirus causing fatal gout in goslings.

Since November 2016, severe infectious diseases characterized by gout and kidney swelling and caused by goose astrovirus (GoAstV) have affected goslings in major goose-producing areas in China. In 2021, a similar serious infectious disease broke out in commercial goose farms in Heilongjiang Province, China. In this study, strain HLJ2021 was successfully isolated from goose embryos. Electron microscopy showed that the viral particles are spherical, with a diameter of about 28 nm. The complete genomic length of strain HLJ2021 is 7210 nt, and it encodes three viral proteins. A phylogenetic analysis showed that strain HLJ2021 belongs to GoAstV-2 (G2). Compared with the two original GoAstV strains, amino acid site 540Q of the strain HLJ2021 spike domain has a mutation that affects the protein structure. One potential recombination event occurred between strains HLJ2021 and AstV/HB01/Goose/0123/19, which led to the generation of recombinant strain AstV/HN03/Goose/0402/19. Strain HLJ2021 also showed strong pathogenicity in goslings. Goslings infected with GoAstV began to die at 48 h post-infection (hpi), with a mortality rate of 83.3% at 240 hpi. At autopsy, visceral urate deposits, severe renal hemorrhage and swelling, and urate in the ureter were observed in the dead goslings. These findings extend our understanding of the evolution of GoAstV, which causes gout. The isolated GoAstV strain HLJ2021 provides a potential resource for the development of biological products for the prevention of goose gout.

Goose Astrovirus in China: A Comprehensive Review.

Goose astroviruses (GoAstVs) are small non-enveloped viruses with a genome consisting of a single-stranded positive-sense RNA molecule. A novel GoAstV was identified in Shandong in 2016 and quickly spread to other provinces in China, causing gout in goslings, with a mortality rate of approximately 50%. GoAstV can also cause gout in chickens and ducks, indicating its ability to cross the species barrier. GoAstV has only been reported in China, where it has caused serious losses to the goose-breeding industry. However, in view of its cross-species transmission ability and pathogenicity in chickens and ducks, GoAstV should be a concern to poultry breeding globally. As an emerging virus, there are few research reports concerning GoAstV. This review summarizes the current state of knowledge about GoAstV, including the epidemiology, evolution analysis, detection methods, pathogenicity, pathogenesis, and potential for cross-species transmission. We also discuss future outlooks and provide recommendations. This review can serve as a valuable reference for further research on GoAstV.

Fusobacterium necrophorum Promotes Apoptosis and Inflammatory Cytokine Production Through the Activation of NF-κB and Death Receptor Signaling Pathways.

Fusobacterium necrophorum can cause liver abscess, foot rot in ruminants, and Lemire syndrome in humans, Also, its virulence factors can induce the apoptosis of macrophages and neutrophils. However, the detailed mechanism has not been fully clarified. This study investigated the mechanisms of apoptosis and inflammatory factor production in F. necrophorum-induced neutrophils and macrophages (RAW246.7). After infection of macrophages with F. necrophorum, 5-ethynyl-2'-deoxyuridine labeling assays indicated that F. necrophorum inhibited macrophage proliferation in a time- and dose-dependent manner. Hoechst staining and DNA ladder assays showed significant condensation of the nucleus and fragmentation of genomic DNA in F. necrophorum-infected macrophages, Annexin V (FITC) and propidium iodide (PI) assay confirmed the emergence of apoptosis in the macrophages and sheep neutrophils with F. necrophorum compared with the control. The group with significant apoptosis was subjected to RNA sequencing (RNA-Seq), and the sequencing results revealed 2581 up- and 2907 downregulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of the differentially expressed genes showed that F. necrophorum drove apoptosis and production of inflammatory factors by activating genes related to the Nuclear Factor-κB (NF-κB) and death receptor pathways. Meanwhile, quantitative reverse transcription PCR and Western blot validation results were consistent with the results of transcriptome sequencing analysis. In conclusion, F. necrophorum induced apoptosis and production of pro-inflammatory factors through the NF-κB and death receptor signaling pathway, providing a theoretical basis for further mechanistic studies on the prevention and control of F. necrophorum disease treatment.

Bovine Astrovirus-A Comprehensive Review.

Bovine astrovirus (BoAstV) is a small non-enveloped virus with a single-stranded positive-sense RNA. In 1978, BoAstV was first found in calf diarrhea fecal samples in the United Kingdom and since then it has been reported in many other countries. It has wide tissue tropism and can infect multiple organs, including the intestine, nerves and respiratory tract. Since BoAstV is prevalent in healthy as well as clinically infected bovines, and is mostly associated with co-infection with other viruses, the pathogenic nature of BoAstV is still unclear. At present, there are no stable passage cell lines available for the study of BoAstV and animal model experiments have not been described. In addition, it has been reported that BoAstV may have the possibility of cross-species transmission. This review summarizes the current state of knowledge about BoAstV, including the epidemiology, evolution analysis, detection methods, pathogenesis and potential cross species transmission, to provide reference for further research of BoAstV.

Advances in Bovine Coronavirus Epidemiology.

Bovine coronavirus (BCoV) is a causative agent of enteric and respiratory disease in cattle. BCoV has also been reported to cause a variety of animal diseases and is closely related to human coronaviruses, which has attracted extensive attention from both cattle farmers and researchers. However, there are few comprehensive epidemiological reviews, and key information regarding the effect of S-gene differences on tissue tendency and potential cross-species transmission remain unclear. In this review, we summarize BCoV epidemiology, including the transmission, infection-associated factors, co-infection, pathogenicity, genetic evolution, and potential cross-species transmission. Furthermore, the potential two-receptor binding motif system for BCoV entry and the association between BCoV and SARS-CoV-2 are also discussed in this review. Our aim is to provide valuable information for the prevention and treatment of BCoV infection throughout the world.

Interaction of 43K OMP of Fusobacterium necrophorum with fibronectin mediates adhesion to bovine epithelial cells.

Fusobacterium necrophorum, a Gram-negative anaerobe, is an important bovine pathogen that causes hepatic abscesses, foot rot, mastitis and endometritis. We have previously shown that the 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in bacterial infections; however, the molecular mechanisms by which this protein mediates adhesion remain unclear. In this study, we investigated the role of 43 K OMP in F. necrophorum adhesion to bovine epithelial cells using 43 K OMP-deficient mutants, and identified the protein that interacts with 43 K OMP by immunoprecipitation-mass spectrometry. Our results indicated that the native 43 K OMP and recombinant 43 K OMP could bind to the cell membrane of MAC-T or bovine endometrial epithelial cells (BEECs). When F. necrophorum was preincubated with antibodies against the recombinant 43 K OMP or bovine epithelial cells were preincubated with 43 K OMP, the adhesion of F. necrophorum to MAC-T or BEECs decreased significantly (P<0.01). We successfully constructed a 43 K OMP-deficient strain (A25Δ43 K OMP) and bacterial attachment to MAC-T or BEECs was significantly higher with the F. necrophorum A25 strain than with mutant strain A25Δ43 K OMP (P<0.01). The deficiency of 43 K OMP reduced the binding of F. necrophorum to bovine epithelial cells by 90.5 %-94.9 %. Among the 39 potential differential proteins, fibronectin, collagen and myosin were selected as the target proteins, and direct interaction between 43 K OMP of F. necrophorum and fibronectin was demonstrated. Taken together, these results suggest that 43 K OMP plays a key role in adhesion of F. necrophorum to bovine epithelial cells through its interaction with fibronectin. These findings provide a theoretical basis for the pathogenic mechanism of F. necrophorum.

Epidemiological survey and genetic diversity of bovine coronavirus in Northeast China.

In 2020, to trace the prevalence and evolution of bovine coronavirus (BCoV) in China, a total of 1383 samples (1016 fecal samples and 367 nasal swab samples) were collected from 1016 cattle exhibiting diarrhea symptoms on dairy farms and beef cattle farms in Heilongjiang Province, Northeast China. All samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) detection of the BCoV N gene, followed by an analysis of its epidemiology and genetic evolution. The results indicated that of the 1016 diarrhea-affected cattle, 15.45% (157/1016) were positive for BCoV, in which positive rates of the fecal and nasal swab samples were 12.20% (124/1016) and 21.53% (79/367), respectively. Of the 367 cattle whose nasal swab samples were collected, the BCoV positive rate of the corresponding fecal samples was 15.26% (56/367). BCoV infection was significantly associated with age, farming pattern, cattle type, farm latitude, sample type, and clinical symptom (p < 0.05). Of the 203 BCoV-positive samples, 20 spike (S) genes were successfully sequenced. The 20 identified BCoV strains shared nucleotide homologies of 97.7-100.0%, and their N-terminal domain of S1 subunit (S1-NTD: residues 15-298) differed genetically from the reference strains of South Korea and Europe. The 20 identified BCoV strains were clustered in the Asia-North America group (GII group) in the global strain-based phylogenetic tree and formed three clades in the Chinese strain-based phylogenetic tree. The HLJ/HH-10/2020 strain was clustered into the Europe group (GI group) in the S1-NTD-based phylogenetic tree, exhibiting N146/I, D148/G, and L154/F mutations that affect the S protein structure. Of the identified BCoV strains, one potential recombination event occurred between the HLJ/HH-20/2020 and HLJ/HH-10/2020 strains, which led to the generation of the recombinant BCV-AKS-01 strain. A selective pressure analysis on the S protein revealed one positively selected site (Asn509) among the 20 identified BCoV strains located inside the putative receptor binding domain (residues 326-540). These data provide a greater understanding of the epidemiology and evolution of BCoV in China.

Development of a Novel Double Antibody Sandwich ELISA for Quantitative Detection of Porcine Deltacoronavirus Antigen.

Porcine deltacoronavirus (PDCoV) can cause diarrhea and dehydration in newborn piglets. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-ELISA) for detection of PDCoV by using a specific monoclonal antibody against the PDCoV N protein and an anti-PDCoV rabbit polyclonal antibody. Using DAS-ELISA, the detection limit of recombinant PDCoV N protein and virus titer were approximately 0.5 ng/mL and 103.0 TCID50/mL, respectively. A total of 59 intestinal and 205 fecal samples were screened for the presence of PDCoV by using DAS-ELISA and reverse transcriptase real-time PCR (RT-qPCR). The coincidence rate of the DAS-ELISA and RT-qPCR was 89.8%. DAS-ELISA had a sensitivity of 80.8% and specificity of 95.6%. More importantly, the DAS-ELISA could detect the antigen of PDCoV inactivated virus, and the viral antigen concentrations remained unchanged in the inactivated virus. These results suggest that DAS-ELISA could be used for antigen detection of clinical samples and inactivated vaccines. It is a novel method for detecting PDCoV infections and evaluating the PDCoV vaccine.

Inhibitory Effect of Bovine Adipose-Derived Mesenchymal Stem Cells on Lipopolysaccharide Induced Inflammation of Endometrial Epithelial Cells in Dairy Cows.

Endometritis is a disease that affects reproductive health in dairy cows and causes serious economic damage to the dairy industry world-wide. Although in recent years, the application of mesenchymal stem cell (MSC) therapy for the treatment of inflammatory diseases has attracted much attention, there are few reports of the use of MSCs in dairy cows. In the present study, our objective was to explore the inhibitory effects of bovine adipose-derived mesenchymal stem cells (bAD-MSCs) on lipopolysaccharide (LPS) induced inflammation in bovine endometrial epithelial cells (bEECs) along with the potential underlying molecular mechanisms. We characterized isolated bAD-MSCs using cell surface marker staining and adipogenic/osteogenic differentiation, and analyzed them using immunofluorescence, flow cytometry (surface marker staining), and adipogenic and osteogenic differentiation. Furthermore, to understand the anti-inflammatory effects of bAD-MSCs on LPS induced bEEC inflammation, we used a bAD-MSC/bEEC co-culture system. The results showed that bAD-MSC treatments could significantly decrease LPS induced bEEC apoptosis and pro-inflammatory cytokine expression levels, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Furthermore, our results showed that bAD-MSC treatments could also significantly downregulate LPS induced p38, IkB-a, and JAK1 phosphorylation and Bax protein expression levels, which are closely related to inflammatory progress and cellular apoptosis in bEECs. Our findings demonstrate that bAD-MSCs play an inhibitory role in LPS induced bEEC inflammation and provide new insights for the clinical therapy of endometritis in dairy cows.

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.