Porcine Deltacoronavirus Infection Disrupts the Intestinal Mucosal Barrier and Inhibits Intestinal Stem Cell Differentiation to Goblet Cells via the Notch Signaling Pathway.

Goblet cells and their secreted mucus are important elements of the intestinal mucosal barrier, which allows host cells to resist invasion by intestinal pathogens. Porcine deltacoronavirus (PDCoV) is an emerging swine enteric virus that causes severe diarrhea in pigs and causes large economic losses to pork producers worldwide. To date, the molecular mechanisms by which PDCoV regulates the function and differentiation of goblet cells and disrupts the intestinal mucosal barrier remain to be determined. Here, we report that in newborn piglets, PDCoV infection disrupts the intestinal barrier: specifically, there is intestinal villus atrophy, crypt depth increases, and tight junctions are disrupted. There is also a significant reduction in the number of goblet cells and the expression of MUC-2. In vitro, using intestinal monolayer organoids, we found that PDCoV infection activates the Notch signaling pathway, resulting in upregulated expression of HES-1 and downregulated expression of ATOH-1 and thereby inhibiting the differentiation of intestinal stem cells into goblet cells. Our study shows that PDCoV infection activates the Notch signaling pathway to inhibit the differentiation of goblet cells and their mucus secretion, resulting in disruption of the intestinal mucosal barrier. IMPORTANCE The intestinal mucosal barrier, mainly secreted by the intestinal goblet cells, is a crucial first line of defense against pathogenic microorganisms. PDCoV regulates the function and differentiation of goblet cells, thereby disrupting the mucosal barrier; however, the mechanism by which PDCoV disrupts the barrier is not known. Here, we report that in vivo, PDCoV infection decreases villus length, increases crypt depth, and disrupts tight junctions. Moreover, PDCoV activates the Notch signaling pathway, inhibiting goblet cell differentiation and mucus secretion in vivo and in vitro. Thus, our results provide a novel insight into the mechanism underlying intestinal mucosal barrier dysfunction caused by coronavirus infection.

Preparation of monoclonal antibody against porcine saperovirus VP1 protein and identification of its antigenic epitope

As a member of the family Picornaviridae, porcine sapelovirus (PSV) is often infected with porcine epidemic diarrhea virus, teschovirus and so on. In recent years, PSV has been isolated from porcine in many provinces of China. It suggests that it is necessary to strengthen the research on PSV. In this study, according to the sequence of PSV HuN2 strain, VP1 gene was inserted into the pGEX-6 P-1 vector, and expressed the recombinant protein. BALB/c mice aged 6-8 weeks were immunized according to the standard procedure. After the third immunization, the mouse orbital blood was collected to identify the antibody level. The highly positive mouse spleen cells were selected for cell fusion. The positive hybridoma cells and two subclones were screened by IFA method, and then a PSV VP1 monoclonal antibody was obtained, named as 33-2 A. The results of IFA showed that PSV could be recognized by 33-2 A MAb, and specific green fluorescence appeared in the cytoplasm;The results of WB and IP showed that PSV infected porcine cell could specifically bind to 33-2 A, and there was a specific band at 32 ku. We also identified the B-cell antigen epitope of 33-2 A, it was at amino acids 40-46 of PSV VP1 protein, and the polypeptide sequence was 40PALTAAE46. The results showed that the monoclonal antibody can react with PSV VP1 protein. The epitope was analyzed with the PSV sequences uploaded in NCBI, 33-2 A antibody can react with most PSV strains and has a certain universal to PSV. This study laid a foundation for the study of the etiology and pathogenesis of PSV.

Evolution of Transmissible Gastroenteritis Virus (TGEV): A Codon Usage Perspective.

Transmissible gastroenteritis virus (TGEV) is a coronavirus associated with diarrhea and high mortality in piglets. To gain insight into the evolution and adaptation of TGEV, a comprehensive analysis of phylogeny and codon usage bias was performed. The phylogenetic analyses of maximum likelihood and Bayesian inference displayed two distinct genotypes: genotypes I and II, and genotype I was classified into subtypes Ia and Ib. The compositional properties revealed that the coding sequence contained a higher number of A/U nucleotides than G/C nucleotides, and that the synonymous codon third position was A/U-enriched. The principal component analysis based on the values of relative synonymous codon usage (RSCU) showed the genotype-specific codon usage patterns. The effective number of codons (ENC) indicated moderate codon usage bias in the TGEV genome. Dinucleotide analysis showed that CpA and UpG were over-represented and CpG was under-represented in the coding sequence of the TGEV genome. The analyses of Parity Rule 2 plot, ENC-plot, and neutrality plot displayed that natural selection was the dominant evolutionary driving force in shaping codon usage preference in genotypes Ia and II. In addition, natural selection played a major role, while mutation pressure had a minor role in driving the codon usage bias in genotype Ib. The codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index (SiD) analyses suggested that genotype I might be more adaptive to pigs than genotype II. Current findings contribute to understanding the evolution and adaptation of TGEV.