Effect Of PKV On PEDV Pathogenesis And Intestinal Pathology Of (Their) Co-infection

Porcine kobuvirus(PKV)is an enteric virus regularly found in piglets under four weeks old.It is more frequently detected in episodes of viral diarrhea,and tends to be accompanied by other viruses.Recent epidemiological studies have discovered that clinical samples with PKV infection are highly associated with Porcine Epidemic Diarrhoea Virus(PEDV)infection,but the mechanisms of their interaction have not been elucidated or reported.The relationship between PKV and other porcine diarrhoeal viruses is still unknown.In order to further investigate the impact of PKV infection on the pathogenic effect of PEDV and the associated immune responses,PKV polyclonal antibodies were prepared by prokaryotic expression of PKV VP1 protein and the localization of PKV in the small intestine of piglets was visualized by immunohistochemical methods.Piglets that were infected with PKV or PEDV alone and those infected with PEDV 48 hours post PKV infection served as infection models.Under different viral infection modes,the pathogenicity of infection,the morphology of small intestinal mucosa,the localization of virus in the small intestine of infected piglets and the change in viral loads,the number and localization of immune cells in the intestinal mucosa and the expression of IFN-βrelated factors in the small intestine were studied.The main research contents are as follows:1.Intestinal histopathological changes in piglets infected with PKV and PEDVThe piglets were artificially infected with PKV or PEDV,and the co-infection group was pre-infected with PKV for 48 hours before receiving PEDV inoculation.The small intestine tissues of piglets in each group were harvested to make paraffin sections for HE staining to observe the histopathological changes.The results showed that PKV infection did not cause obvious damage to the small intestine of piglets,and the tissue structure was intact,which was not significantly different from that of normal piglets.PEDV infection resulted in shortened and atrophic intestinal villi with damaged epithelial striated edge.Damage caused by co-infection was more severe than that caused by PEDV infection alone.2.Preparation of PKV polyclonal antibody and localization analysis of PKV in the intestine of pigletsThe PKV VP1 gene was amplified by RT-PCR and cloned into pET-42b vector to construct the recombinant plasmid pET42b-Nhis-PKV-VP1,which was transformed into BL21 competent cells.The recombinant protein was purified by nickel column affinity chromatography.The product was verified by SDS-PAGE and Western-blot analysis,and the results showed that the correct recombinant protein was obtained.The recombinant protein was pretreated to immunize rabbits.After immunization,the serum was collected for antibody purification.The titer of the antibody was detected by indirect ELISA,which indicated the titer of the prepared rabbit anti-pig PKV polyclonal antibody was 1:64000.The PKV polyclonal antibody prepared in this study and the PEDV antibody preserved in our laboratory were used as primary antibodies to locate PKV and PEDV in the small intestine of piglets by immunohistochemistry(IHC).IHC results showed that PKV was mainly localized in intestinal villus epithelial cells and lamina propria lymphocytes of piglets,and PEDV was mainly distributed in the intestinal villus epithelial cells.Compared with piglets infected with PEDV alone,those in the co-infection group showed increased number of PEDV positive cells in the small intestine.The results were consistent with the changes of PEDV viral load in the small intestine of piglets detected by Real-time fluorescence quantitative PCR.3.The effects of PKV and PEDV infection on intestinal immune cells in pigletsImmunohistochemical staining was used to localize and analyze the immune cells in the intestinal mucosa of piglets,and the positive rate and changes in the colonization sites were statistically analyzed.The results showed that compared with normal piglets,the numbers of CD3~+T lymphocytes,CD4~+T lymphocytes,and B lymphocytes in the small intestine of piglets in each infection group significantly decreased.The expression levels of CD8~+T lymphocytes in the small intestine of piglets were similarly not high in either control or infection groups,but their location differed.In normal piglets and PEDV groups,CD8~+T lymphocytes were mainly distributed in the intestinal villus epithelium,and a small amount were distributed in the lamina propria.However,in the PKV group and the PKV&PEDV co-infection group,it was mainly distributed in the lamina propria of the small intestine,and there was no positive signal in the intestinal villus epithelium.The above results indicated that PKV infection suppressed the immune function of the small intestinal mucosa in some piglets.4.Effect of PKV and PEDV infection on IFN-βand its related factors in small intestine of pigletsRIG-1,MDA5 and type Ⅰ interferon-regulated interferon-stimulated genes(ISGs)in IFN-βand RLRs pathways were detected in the infected piglet intestine via fluorescence quantitative PCR and Western-blot.The results showed that the expression of RIG-1,MDA5,IFN-β,MX1,MX2,OAS-1 and PKR in the small intestine of piglets in the PKV group and the PKV&PEDV group decreased to varying degrees at transcriptional level and protein level,which suggested that PKV infection inhibited the expression of IFN-βand antagonized the host antiviral response by inhibiting the activation of RLRs pathway.Through correlation analysis,it was found that IFN-βwas positively correlated with CD3~+T lymphocytes,CD4~+T lymphocytes and B lymphocytes in the small intestine of normal piglets and piglets in each infection group.The decreased expression of IFN-βmediated the lower number of intestinal mucosal immune cells in piglets,thus inhibiting the immune response of the host.In conclusion,PKV infection localized in the intestinal villous epithelial cells and lymphocytes of the lamina propria of piglets.Clinical symptoms such as diarrhea appeared 48 h after PEDV infection,and pre-infection with PKV could synergistically aggravate the clinical symptoms caused by PEDV,exacerbate the pathological damage caused to piglets’small intestine,and enhance the replication of PEDV in piglets’small intestine cells.PKV infection in piglets suppressed the immune function of small intestinal mucosa,inhibiting the expression of RLRs pathway and IFN-β,which mediated its immune evasion.