Culture And Identification Of Porine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is a highly contacting transmitted disease that is characterized by acute enteritis,vomit,diarrhea and dehydration.The causative agent of PED is Porcine epidemic diarrhea virus(PEDV).Every time of the outbreak will cause serious economic loss because of the high lethality to newborn piglets.Since 2010 the incidence of PED increased significantly with larger scale in China.In May 2013,PED was first be reported in USA and spreaded rapidly in a short time. Qichen et al. successfully isolated two strains of PEDV.Our laboratory have been committed to the research of attenuated live vaccine of PEDV in recent years,and had made some achievements. We has obtained some different types of recombinant PEDVs,but has not tried the separation of PEDV field strain.In this study we attempts to isolate the PEDV-GJL wild strain using Vero cells,which will establish a foundation for the future vaccine developement and basic research releated to PEDV.The spike(S) protein plays an important role in determining PEDV host specificity,and the S gene which encodes it has high mutation rate.Therefore,the study on molecular epidemiology of S gene has more and more improtant significance.This study firstly did the amplification and sequence analysis of S gene of the field strains in samples,afterwards we tried to use Vero cells to isolate PEDV-GJL strain.During the test we employed QiChen’s culture program.By the phylogenetic analysis,we inferred the field strains in samples are in the same cladogenesis with some newly epidemic field strains in China.Then,we observed that the CPE caused by isolated strain in Vero cells is similar to DR13,and its proliferation dose not depend on the presence of trypsin,so ORF3 gene of PEDV was detected through RT-PCR to confirm the nature of the isolated strain.The result showed the sample contained mixed of PEDV-GJL and suspected attenuated DR13.However,the GJL strain was lost at the fourth generation.Plaque purification experiment was carried out to the second generation of virus,which again was confirmed to be the suspected attenuated DR13 strain.It could be inferred that the pigs in the farm from which the samples were collected had been immunized by attenuated DR13 vaccine. Qichen’s culture system may be more suitable for growth of this vaccine strain.This hypothesis still need future validation.Reverse genetic system of PEDV were also replicated successfully in this study, which provided powerful tool for rescue of recombinant PEDV in upcoming process.