Constuction Of Infectious CDNA Clones Of Porcine Epidemic Diarrhea Virus And The Effect Of S Gene Mutation On The Growth In Vitro And Pathogenicity Of Avirulent Strain

Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease of swine,caused by the porcine epidemic diarrhea virus(PEDV).This disease is characterized by acute enteritis,diarrhea,vomiting and dehydration.Clinically,all ages of pigs are shown to be susceptible to PEDV infection,with the mortality of 100%in the affected sucking piglets.In 2010,highly virulent PEDV strains with characteristic variations in S gene emerged and prevailed in China,causing the outbreaks of PED all over the country and huge economic losses to the pig industry.Therefore,the research on the pathogenesis of PEDV has important scientific significance for prevention and control of PED.In this study,the reverse genetic manipulation of PEDV isolates was constructed and the effect of S gene mutation on the growth and pathogenicity of PEDV was further analyzed,in order to provide necessary technical foundation for illuminating the molecular pathogenesis of PEDV.A PEDV strain was isolated from a pig farm in Beijing that clinical diarrhea occurred piglets and was designated CHM2013.This isolate was shown to be adaptative in Vero cells and could be passaged without trypsin.The primers were designed according to the PEDV sequences in GenBank and the complete genome of CHM2013 was amplified by RT-PCR and sequenced.The results showed that the complete genome was 27994 nt in length.Twenty-six representative strains of PEDV belonging to different clusters were chosen to draw the phylogenetic tree based on the whole genome nucleotide sequences among these strains and CHM2013.It was shown the isolate CHM2013 belonged to G1 group together with the classical PEDV strains CV777,SM98 and DR13,the whole genome homologue is 97.9%-99.9%;Compared to GII strains and recombinant strains,the whole genome homologue is 96.5%-96.8 and 97.0%-97.1%respectively.The primers were designed based on the complete genome sequence of CHM2013 and the highly virulent PEDV strain BJ-2011-C,and RT-PCR was performed,and appropriate enzyme site was induced by synonymous mutation.The full-length cDNA clones of CHM2013 and BJ-2011-C were constructed by using low copy plasmid pBeloBAC11 with the inserted cytomegalovirus promoter(CMV),hepatitis delta virus ribozyme and bovine growth hormone(BGH).The genetic marker at 1809 nt(T?C)of CHM2013 genome or 15932nt(G?A)of BJ-2011-C genome was created to distinguish the rescued viruses and their parental strains.Vero-CCL81 cells were transfected with the full-length cDNA clone plasmids,the cytopathic effects(CPE)were observed and the rescued viruses were identified.The results showed that the rescued viruses could cause the cytopathic effect(CPE)typical of PEDV as the parental viruses.The genetic marker site could be detected in the P3 virus through RT-PCR and sequencing,and the specificity positive signals of rescued viruses-infected cells could be seen by indirect immunofluorescence assay(IFA).The rescued clone viruses were named as rCHM2013 and rBJ-2011-C,respectively.These results showed that the full-length cDNA clones of both CHM2013 and BJ-2011-C were infectious.The growth feature of the rescued viruses in vitro was analyzed,and the pathogenicity of the rescued viruses was explored in sucking piglets.The multi-step growth curve indicated the growth dynamic of rescued viruses was similar to parental viruses in Vero-CCL81 cells.Animal trials indicated that rBJ-2011-C exhibited highly virulence for 2-day-old piglets with severe diarrhea and the mortality of 100%(4/4);while rCHM2013 could not cause clinical diarrhea in piglets,showing that CHM2013 is an avirulent strain.Compared with the classical strains,the S gene of highly virulent variant PEDV strains shared characteristic amino acid mutations.Five major variable regions including aa27-29 QST?SAN,aa57-59 MNS?GENQGVN<aa insert and mutant>,aa68-72 GTGIE?AGQHP,aa84-89 YIDSSQ?HIRGGH,and aal51-156 RDGKDI?SEHS<aa deletion and mutant>were chosen to construct five recombinat cDNA clones containing single mutation respectively based on the infectious clone of CHM2013.The results showed that R-C-GENQGVN and R-C-SEHS could be successfully recovered from the respective cDNA clones,but the other three could not.Then we constructed another recombinat clone plasmid carrying both mutant GENQGVN and SEHS.This infectious clone recombinat virus could also be recovered.In vitro growth dynamic showed that the three recombinant viruses had similar proliferation tendency to CHM2013.Animal trials revealed that the recombinant virus R-C-GENQGVN+SEHS had no pathogenicity for suckling piglets,and could not cause the clinical symptoms of diarrhea,suggesting that the characteristic insertion and deletion in S gene of highly virulent PEDV has no effect on the the growth and pathogenicity of the avirulent strain CHM2013.In conclusion,we successfully constructed the infectious clones of PEDV avirulent strain CHM2013 and highly virulent strain BJ-2011-C,and carried out a preliminary analysis regarding the effect of the characteristic insertion and deletion mutation in S gene of highly virulent PEDV strain on the growth and pathogenicity of PEDV.Our findings showed that the cDNA clones of both PEDV avirulent and highly virulent strains were infectious,and the cloned viruses could be rescued from the infectious clones.The characteristic mutations in the S gene of PEDV virulent strain had no effect on the replication and pathogenicity of the avirulent PEDV strain CHM2013.This work provides the foundation for the further investigation of molecular pathogenesis of PEDV.