Genotyping S1of Porcine Epidemic Diarrhea Virus In Zhejiang Province And The Development Of Indirect ELISA

【Objective】In this study, S1genefragment of porcine epidemic diarrhea virus (PEDV) from clinical samplesof diarrhea in piglets was sequenced for analysis of genetic variation and the epidemic situation of PEDV inZhejiang province. An indirect ELISA for detection of antibodies against PEDV was established using theprokaryotic expressed and purified S1of The recent PEDV isolate.【Methods】Afragment of963bp in length of porcine epidemic diarrhea virus S1gene was amplified by onestep RT-PCR and sequenced after cloned into pMD-18T. The sequence characteristics of S1gene fragment ofPEDV strains in Zhejiang province were analysis and the phylogenetic trees of nucleotide sequence anddeduced amino acid sequence were built to comparing with that from both other provinces of China andabroad for relationship of genetic evolution with molecμLar biology software. The epidemic regμLarity ofPED in Zhejiang province was analyzed based on the clinical data of PED. The963bp fragment of the5’endof PEDV S1gene was cloned into prokaryotic expression vector pET-28a, and the recombinant plasmid weretransformed into E.coli Rosetta. Recombinant S1protein were expressed by inducing with IPTG andimmunogenicity and stability of the recombinant protein was identified by Western blot. The expressed proteinwas purified by Ni chelate column affinity chromatography and then by electricity elution recovery forrowingSDS-PAGE electrophoresis. microtiter strips were coated with purified protein as antigen and an indirectELISA was established after optimization of reaction conditions.【ResμLts】The nucleotide sequence and deduced amino acid sequence of41PEDV strains from ZhejiangProvince in the past four years showed that both insertion and deletion mutation occurred in S1gene comparedwith previous isolates in China or the vaccine strain. The resμLts of the phylogenetic relationship analys isshowed that Zhejiang PEDV current epidemic strains had high sequence homology to Korea strains, while hadobvious variation to European strains and early isolates in China. The clinical data of PED in Zhejiang showedthat the total detection rate of69.30%with an upward trend year by year, up to73.24%and PEDV has becomethe main pathogens of diarrhea in piglets. The prokaryotic expression vector of pET28a-S was constructed andhigh purity target protein was obtained by especially established protein purification method. Afteroptimization the reaction conditions of the established ELISA for PEDV antibody were as follows: antigencoating at4℃overnight with the concentration of1ug/ml, serum diluted in1:50reacted with antigen for1h at37℃, mixture of5%skim milk and5%gelatin as blocking solution at37℃for4h， dilution of HRP labelingantibody at1:5000at37℃for30min, visualiztion of TMB substrate37℃for10min. The cutoff value of theoptical density at450nm (OD450) is0.282based on resμLts of30negative sera. The established indirectELISA method have no cross-reaction with antibodies for main pig pathogens such as PCV2. The variationcoefficients of repeated trials of Intra-assay and inter-assay were less than8%. the resμLts of ELISA clinicalapplication showed that the positive rate of antibody in sow group was81.7%but31.6%in piglets and general pig PEDV antibody positive rate was45.5%in Zhejiang Province. Serum antibody levels above0.4in sowherd with diarrhea piglets accounted for only26.67%, but up to63.33%in not diseased group【Conclusions】Therewas acertain genetic variation of PEDV current epidemic strain in Zhejiang Province，which resμLt in the high homology to Korea strains and obvious mutation compared with European andprevious isolates in China. The PEDV epidemic characteristics in Zhejiang province appeared the increaseinfection year by year recently and spread in whole province comprehensively and continuously. An indirectELISA for antibody detection based on PEDV-S protein as antigen was established with good specificity,sensitivity and reproducibility, and can be used for detection of serum antibody and epidemiological survey.On the incidence of the serum resμLts of the sow with diseased suckling piglets and not diseased ones, theproposed ELISA can be used for immune protection monitoring.