The Mechanism Of Porcine Epidemic Diarrhea Virus Spike Protein Affects Viral Trypsin Dependency

Porcine epidemic diarrhea virus(PEDV)is an important pathogen that causes acute intestinal infections in pigs.It is highly contagious and the main symptoms are diarrhea and vomiting.Since 2010,PEDV new variants have emerged worldwide,and its mortality rate to newborn piglets is extremely high,causing huge economic losses to the pig industry.Multiple mutations of PEDV lead to two genotypes:G1 and G2(classic strains and variant strains),while the antigenicity between G1 and G2 is changed.In recent years,the frequent outbreaks of PEDV in vaccinated pigs indicate that the current vaccines on the market have a low protection rate of the circulating strains.Therefore,the study of the PEDV infection mechanism and the novel vaccine products are urgent.Isolation and cultivation of PEDV is the research basis,which usually requires additional trypsin.Generally,PEDV is trypsin-dependent,which restricts the study of virus properties in vitro and vaccine production.In this study,PEDV strains YN200 and DR13 were taken as the research objects to explore the PEDV trypsin-dependent mechanism.The main research contents were as follows:1.The trypsin-dependent characteristics of PEDV strains YN200 and DR13The PEDV strains YN200 and DR13 are from G2 and G1 genotypes.By comparing the CPE,IFA,and multi-step growth curves of YN200 and DR13,we found that trypsin can promote YN200 infection,which represents rapid replication and syncytial lesions under the condition of trypsin;the proliferation rate of YN200 is slow without trypsin.However,DR13 can effectively infect cells with or without trypsin.Meanwhile,only the spike(S)protein of YN200 has the ability to induce cell-cell membrane fusion and can be cleaved into specific bands by trypsin,indicating that the S protein may affect the trypsin dependency of PEDV.2.S protein is the determinant of PEDV trypsin dependencyIn order to explore the role of S protein in the PEDV trypsin dependency,we used our laboratory's YN200 reverse genetic operating system platform to construct and obtained the PEDV recombinant virus r YN200-S_DR13,following with substitution of YN200 S gene by DR13 S gene.By characterizing virus trypsin dependency,the trypsin dependency of r YN200 was changed after replacing the S gene,which is r YN200-S_DR13is trypsin-independent,indicating that the S protein is the key factor of trypsin dependency.3.R892(S2'site)and R885 mutations do not affect YN200 trypsin dependencyThrough sequence analysis of the protease cleavage sites of PEDV S protein,we found that YN200 S protein exists arginine mutations in the predicted S2'892 site and the nearby 885 site.Since trypsin cleaves protein at arginine or lysine,we constructed and obtained mutant recombinant viruses r YN200-R885S,r YN200-R892G and r YN200-R885S,R892G.The results show that neither R892 nor R885 mutations affect the YN200 trypsin dependency.In addition,the cell infection ability and the size of plaque of r YN200-R885S,R892G is slightly weaker and smaller than that of the parent strain.The results of the virus competition assay show that R885 mutated recombinant viruses have a weak ability for competitive cell infection,indicating that R885 is a viral cell-adaptive mutation.4.The S2 subunit determines PEDV trypsin dependencyIn order to further explore the S protein domain of affecting virus trypsin dependency,we divided the S protein into S1 subunit,S2 subunit and NS2'(S2_720-892aa).The DR13 S gene fragment replaces the corresponding YN200 S gene to construct recombinant viruses r YN200-S1_DR13,r YN200-S2_DR13,and r YN200-NS2'_DR13.The results show that the virus trypsin dependency of r YN200-S2_DR13 is changed as a trypsin-independent strain,while r YN200-S1_DR13 and r YN200-NS2'_DR13 still need trypsin to infect cells,indicating that the S2 subunit determines virus trypsin dependency.Meanwhile,cell propagation of r YN200-NS2'_DR13 has significantly decreased,implied a vital role in of NS2'cell infection.The above results have suggested that the PEDV trypsin dependency is mainly controlled by the integral structure of the S2 subunit,rather than trypsin cleavage sites.In summary,this project explores the trypsin dependence of PEDV strains YN200and DR13,which confirmed that the mutation of the S2'cleavage site does not affect the trypsin dependency of YN200.Results also pointed out that the integral structure of the S2 subunit is the key to determining virus trypsin dependency,providing a new direction for PEDV isolation and vaccine research.