Construction And Application Of The Infectious Clone Of Porcine Epidemic Diarrhea Virus Strains

Porcine epidemic diarrhea virus（PEDV）is the causative agent of porcine epidemic diarrhea（PED）,a highly contagious disease characterized by enteritis and lethal watery diarrhea,dehydration,vomiting,weight loss and mental malaise which infects pigs of all ages but the morbidity and mortality rate in neonatal piglets is up to 80%-100%.Until 2010,a massive outbreak of PED that caused by a highly PEDV variant was occurred in China,resulting in high mortality and morbidity and significant ecomomic losses to swine industry.According to the phylogenetic tree of spike（S）gene,the the highly virulent PEDV variants are mainly from GII genogroup showed insertion,deletion and substitution mutations in S gene,and made the protection rate of the traditional vaccines based on PEDV CV777strains of GI genogroup lower.Therefore,isolate a PEDV strain of GII genogroup and continuous passage in vero cells are essential for studying pathogenicity,analysing molecular characterization and developing vaccine.To study the genetic factors and molecular pathogenesis,the reverse genetic manipulation platform was constructed.In this study,we describe a PEDV isolate strain of CH/HBXT/2018 which located in GIIa genogroup successfully obtained from small intestine contents in Hebei,China and inoculated into Vero cells.CH/HBXT/2018 strain can be stable passage in Vero cells and produce significant cytopathic effects（CPEs）,and the PEDV virus can cause highly morbidity and mortality in sucking piglets accompanied by vomiting,diarrhea,extreme emaciation and other typical clinical symptoms in animal experiments.The median pig diarrhea dose(PDD₅₀)was 8.625 PDD₅₀/3 mL at 7 Days of Postinfection（7 DPI）in newborn pigs indicated that the CH/HBXT/2018 belongs to highly pathogenic virulent strain.Swine industrys in China have been hit hard by PEDV strains,and our study has significant implications for developing effective vaccine against PEDV infection.In addition,the GIIa genotype of PEDV CH/HBXT/2018 strain and PEDV CH/HNPJ/2017 strain which located in GIIb genotype and previously preserved in our laboratory were used as parental epidemic strains of the construction of full-length genomes by in vitro ligation technology.Firstly,the genomes of the two strains were sequenced and synthesized as six contiguous subclones,and the 5′terminal of PEDV A fargment and the 3′terminal of PEDV F fargment contains a T7 start site and polyA structure,respectively.Then the cDNA clones of the two strains were successfully obtained by using restriction enzyme,T4 DNA ligase and the in vitro transcription,which establish a foundation for the virus recovery and the construction and application of reverse genetic platform.