Genetic Variation Of Porcine Epidemic Diarrhea Virus Prevailing In Henan Province During 2014-2015 And Preliminary Study Of Interaction Between ISG15 And PEDV

Porcine epidemic diarrhea(PED)is an intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV),the manifestations of which are diarrhea,vomiting and dehydration.Pigs of different ages are susceptible,especially the suckling piglets less than 2-3 weeks old.PED was first reported in Great Britain in 1971,and then was found in China in 1976.PED have outbroken again in China since 2010,and spread throughout the country,which caused enormous economic losses in the pork industry.Therefore,it is impotant to study the pathogens of this disease.ISG15 is a ubiquitin-like protein conjugated to a variety of proteins when cells are treated with interferon type I or lipopolysaccharide.High level expression of ISG15 can affect viral replication.Therefore,it's function in the innate cellular immunity cannot be ignored.But the relationship between innate immunity of ISG15 and PEDV remains unclear.In this study,we make a preliminary research on the role of ISG15 in PEDV replication useing the model of interferon-deficient monkey kidney cells(Vero).Based on the background above,we have done the following research: the variation of PEDV strains in Henan Province were monitored,at the same time,we established a stable cell line expressing ISG15 protein and explored the interaction between ISG15 and PEDV.It laid the foundation for further research of ISG15 antiviral mechanism and cellular innate immune escape of PEDV.The main contents contains the following three parts:1.The analysis of genetic Variation of PEDV Prevailing in Henan Province during 2014-2015To monitor and research the variation of PEDV in Henan Province,the S genes of 15 PEDV strains prevailing in Henan province during 2014-2015 were cloned and sequenced.Compared with Henan strains in 2011--2013 as well as domestic and foreign representative strains,the homology and genetic variation were analyzed and the N-glycosylation sites as well as the transmembrane helical regions were predicted.The results showed that 15 PEDV strains prevailing in Henan province had 97.0-99.8%(97.0-99.5%)nucleotide(deduced amino acid)identity with each other and 97.1-99.0%(97.2-98.8%),92.2%-99.0%(91.9%-99.0%),and 91.9%-99.0%(92.0%-92.0%)nucleotide(deduced amino acid)identity with members of Henan epidemic strains in 2011-2013,other reference strains and classical strains,respectively.Compared with classical strains,there were 15 bp insertion and 6bp deletion in the N-terminal of S1 region.Phylogenetic analysis showed that the current strains and classic strains were in different groups.According to the prediction of N-glycosylation sites and transmembrane helical regions,the PEDV strains prevailing in Henan province from 2014 to 2015 had mutation compared with classical strains,and partial mutation compared with the epidemic strains in 2010.It is imperative to develop an effective vaccine of the epidemic strain for preventing and controlling the disease.2.Establishment of stable Vero cell line expressing ISG15 proteins.To construct stable Vero cell line expressing ISG15 proteins,eukaryotic transposon system(Piggy Bac,PB)with ISG15 was transfected into Vero cells.The positive transfected cells were achieved by puromycin resistance screening and GFP detection.Cells were passaged and gene amplification was detected by means of real-time quantitative PCR and western-blotting.It showed that ISG15 was successfully expressed in the cell lines.It offered a basic research tool for further study of the function and mechanism of ISG15,which laied a foundation for the following studies.3.Researches on the interaction between ISG15 protein and PEDVTo probe into the mechanism of PEDV escaping the innate cellular immunity,the interaction between ISG15 protein and PEDV were studied.The Piggybac-ISG15-Vero cells and control cells were collected after infection with PEDV at different time and their S gene copies and PFU of PEDV were detected respectively.By drawing the growth kinetics of PEDV in different cell lines,we found that ISG15 protein promoted the replication of PEDV.In the meantime,compared with Piggybac-ISG15-Vero cells without infection,the expression of ISG15 protein in Piggybac-ISG15-Vero cells infected PEDV were up-regulated significantly.However,the exact mechanisms involved are still unknown and remain to be further researched.In conclusion,this study laid the foundation for the further researches.