Study On The Superinfection Exclusion Between PDCoV And TGEV In Cultured Cells

Superinfection exclusion(SIE)means that a primary viral infection can prevent further viruses from infecting the same cells.Porcine transmissible gastroenteritis virus(TGEV)and porcine deltacoronavirus(PDCoV)are the main pathogens that cause piglet diarrhea and that can cause symptoms such as acute diarrhea,dehydration and vomiting in infected piglets.Among them,both of them are effective for infections under two weeks of age and have a high fatality rate for infected piglets,which seriously endangers the healthy development of pig industry in our country.In recent years,epidemiological investigations on pig farms across our country have shown that PDCoV and TGEV often exhibit mixed infections,and the positive rate of TGEV is gradually decreasing while the positive rate of PDCoV is gradually increasing.In addition,the infection of the two viruses has the following characteristics.First,both of them share the same tropism for swine intestinal epithelial cells.Second,there is no antigen cross-reaction between the two viruses.Third,both of them engage the same functional receptor,Porcine Aminopeptidase N,for infectious cellular entry.For this reason,we propose that thers is a phenomenon of superinfection exclusion between the two coronavirus.Although our country has many preventive measures against porcine enteric coronavirus,viral porcine diarrhea is still prevalent throughout the country,which makes it necessary to formulate new measures to reduce the impact of porcine enteric coronavirus on the pig industry.So the analysis of the relevant superinfection exclusion mechanism between TGEV and PDCoV,is of great significance for understanding the virus replication mechanism and construction of a new porcine enteric coronavirus prevention and control strategy.In this study,firstly,the time of establishment of rejection of two types of intestinal coronavirus super-infection is analyzed by fluorescence quantitative PCR and IFA.Secondly,the influence of different life cycles of the first virus on the establishment of exclusion is analyzed by drug inhibition and invasion experiments.Finally,by constructing an intestinal cell infection model of intestinal coronavirus,it lays a foundation for further research on the related mechanism of intestinal coronavirus on target cells.The specific research content is as follows:1.Developing a Taq Man-probe-based q PCR assay.Specific primers and probes were designed based on the TGEV-N,TGEV-ORF1a,PDCoV-M and PDCoV-ORF1a.Then constructing the specific recombinant plasmids for standard curves and improving the reaction situation to make E>90%and R~2>0.99.In addition,four detection systems were optimized through sensitivity,specificity and repeatability experiment,and finally establish the corresponding standard curve.2.Research on the superinfection exclusion.To characterize the timing,we utilized IFA and q PCR to analyze the infection in the cell.And the result show that both of the two coronavirus can establish the superinfection exclusion and majority of the first-infected cells establish superinfection exclusion by 4 h postinfection.3.Research on the mechanism of superinfection exclusion.To understand the role of viral life cycle in establishing superinfection exclusion,we altered infection through intrusion experiment and drug treatment.The result show that TGEV can not block the attachment of PDCoV to the cell suface and when TGEV replication is inhibited,PDCoV can infect the cell again.4.Construction of intestinal cell model.Through the method that IPEC-J2 cells were sub-cloned by finite continuous dilution,we obtain a cell pool highly susceptible to TGEV,PDCoV,PEDV and Se ACoV,which can be used as an excellent cell model for co-infection studies of different swine enteric CoVs.In summary,there is a superinfection exclusion phenomenon between TGEV and PDCoV,which can be established at 4 hpi.In addition,TGEV can not block the attachment of PDCoV to the cell surface,and PDCoV can infected the cell again when the TGEV replication was inhibited.