Isolation,Characterization,and Antigenicity Of A Porcine Epidemic Diarrhea Virus Strain CH/JX/01 From,Jiangxi,China In 2014

Since late 2010,porcine epidemic diarrhea virus（PEDV）,the causative agent of porcine epidemic diarrhea（PED）,has gained an increased attention in China as a result of several outbreaks of re-emerging PED,characterized by severe diarrhea,vomiting and dehydration along with high morbidity and mortalityamong neonatal piglets.Based on peers’and our previous research results,the variant PEDVs circulating in China have been undergoing dramatic alterations at nucleotide（nt）and amino acid（aa）level when compared to CV777-based vaccine strains of PEDV.In order to elucidate the differences between PEDV variant strains circulating in China and CV777 vaccine strains in the context of biological characteristics,phylogeny,and antigenicity,we performed the following experiments in this study.1.Isolation,propagation and identification of a Jiangxi field PEDV strain.To uncover the biological characteristics of currently circulating PEDV strains,12 intestinal samples were collected from diarrheal piglets in Jiujiang,Jiangxi 2014 for PEDV isolation.Of 12 samples,one,designated CH/JX/01,caused cytopathic effects（CPE）when passaged three times in Vero cell culture with DMEM medium supplement of 10μg/mL trypsin,and PEDV in cell culture supernatants was confirmed by RT-PCR.To date,CH/JX/01 strain has been stably passaged in Vero cell culture for over 60 passages,and the infectious titer of CH/JX/01reached2.7×10⁶ plaque forming units（PFU）/mL or 5.12 lg 50%tissue culture infectious dose(TCID₅₀),which was higher than that of CV777 since the infectious titer of CV777 was 5.86×10⁴PFU/mL or 4.6 log₁₀ TCID₅₀.But the plaque morphorlogy caused by CH/JX/01 strain is smaller than that caused by CV777.With the adaptation of CH/JX/01 in Vero cell culture,the viral titer of first three passages ranged from 6 to 7 lg genome equivalents per microliter,and the RNA titers of passage 3 to 20 could reach over 7lg genome equivalents per microliter when measured with qRT-PCR.2.A comparative analysis of the growth kinetics of PEDV Jiangxi strain,CH/JX/01 and CV777 vaccine strain:In this study,CH/JX/01 and CV777 growth kinetics in Vero cell culture were determined and compared.After six hours of inoculation,CH/JX/01 started causing CPE,characterized by single cells losing their demarcation and fusing peripheral infected cells to form syncytia containing multiple nuclei.The titer of CH/JX/01 in the supernatants could reach a peak at 27 hpi.However,in the case of CV777,the CPE was observed at 8 hpi,a two hr delay than that of CH/JX/01,and the virus titer started increasing and then reached the highest level at 31 hpi in the cell culture supernatants,and the△Ct value is smaller than CH/JX/01（11.34 to 15.30）.3.Sequencing and comparison analysis of PEDV Jiangxi strain CH/JX/01 and its derivatives:In this study,the complete genome sequences of CH/JX/01/P0（parental strain）,CH/JX/01/P5（passage 5）and CH/JX/01/P30（passage 30）were successfully cloned and sequenced.The full-length genome sequences of three PEDV Jiangxi strain with different passages were all 20,038 nt excluding ploy A tail.No deletion and insertion occurred in these three passages of virus.Like CV777,the genome organization of these three strains was5’UTR-ORF1a/1b-S-ORF3-E-M-N-3’UTR.The sequencing results indicated that the mutations among CH/JX/01 increased with passaging in Vero cell culture,and the mutations were mostly occurred in ORF1a/1b,S,E,and M gene.Ten,seven,one and one nt mutations in the ORF1a/1b,S,E,and M gene of CH/JX/01/P30 were observed which resulted in six,seven,one and one aa mutations in their corresponding ORFs,respectively,when compared with CH/JX/01/P0 strain.To better elucidate molecular basis of PEDV attenuation,this study compared the complete genome sequences of CH/JX/01-derived strains with PEDV virulent strains（virulent CV777 and virulent DR13）and attenuated strains（attenuated DR13 and attenuated CV777）.The results indicated that 24 and 49 nt deletions were observed at acidic domain and ORF3genes in the attenuated PEDV strains,respectively.Then,we predicted,compared and analyzed the RNA secondary and protein 3D structures in the Ac and ORF3 genes between attenuated and virulent PEDV strains.The results demonstrated that virulent DR13（JQ023161）and attenuated DR13（JQ023162）shared same RNA secondary structure in the Ac gene,while the RNA secondary structures among CV777（AF353511）,attenuated CV777（KT323979）and CH/JX/01/P0（KX058031）were different from one another.Compared with attenuated PEDV strains,the virulent PEDVs had two more internalβ-sheets,but they shared similar protein structure in Ac domain.The RNA secondary structures of the ORF3 gene of the attenuated strains of PEDV were similar in terms of topolgy,but different from virulent PEDV strains.Moreover,significant difference was also observed between virulent and attenuated PEDV strains in ORF3 gene regarding protein 3D structure.The virulent PEDVs had two moreα-helixes,two moreβ-sheets,and more random loops than attenuated PEDVs.Based on the phylogenetic trees generated on the complete genome,S,S1,S2,ORF3,and M gene of PEDV strains,all these three strains（CH/JX/01/P0,CH/JX/01/P5,and CH/JX/01/P30）determined in this study were classified to subgroup2a in G2,together with those isolated post-2010,including Chinese PEDVs,American and Korean PEDV strains circulating in recent years.However,the prototype CV777 strain was fallen into subgroup 1a in G1,excluding the phylogenetic tree based on ORF3 gene.Phylogenetic analysis based on complete genome sequence indicated that CH/JX/01 and three PEDV strains（CH/ZMDZY/11,CH/HNYF/14,and CH/HNQX-3/14）circulating in Henan,China in recent years belonged to one cluster.Based on the complete S gene,CH/JX/01 had the closest genetic relationship with CH/ZMDZY/11（KC196276）,whereas CH/JX/01 had a close relationship with CH/FJND-3/2011（JQ282909）in the S1 region.However,topologies of phylogenetic trees based on S2,E,and M gene were very much similar,clustering CH/JX/01 and American strains,Korean strains circulating in recent years and post-2010 Chinese PEDVs into one group in subgroup2a.Phylogenetic tree based on N gene showed that CH/JX/01 had the closest relationship with CH/JX-1/2013（KR095279）.In the ORF3 region,CH/JX/01 was more related to American（KF452323,KF650371,KJ399978,and KF272920）and Korean（KJ623926）strains,interestingly,CV777 fell into subgroup 2b based on phylogenetic analysis of ORF3 gene.Phylogenetic analysis based on N gene demonstrated that CH/JX/01had the closest relationship with CH/JX-1/2013（KF760557）isolated from Jiangxi,China,2013.In order to determine whether CH/JX/01 was originated from recombination events,recombination test was conducted using RDP 4.0 program.The results demonstrated that a strong recombination signal was detected by RDP,GENECOVN,Bootscan,MaxChi,Chimaera,Siscan,and 3Seq algorithms,when CH/JX/01/P0 served as query strain.CH/JX/01was originated from possible recombination events between CH/HNYF/14（KP890336）and JS-HZ2012（KC201147）.The ORF1b region of CH/JX/01 mightderive from CH/HNYF/14-like strains,whereas the structural protein and ORF3 genewere mostly likely from JS-HZ2012-like strains.4.Cross protection study of PEDV Jiangxi strain CH/JX/01:To reveal theantigenic relationships between CH/JX/01 and CV777 vaccine strain,two-way cross reactivity examinations were carried out by viral neutralization assay invitro.Rabbit anti-CV777 and CH/JX/01 hyperimmune antisera could fully neutralize homologous viruses,i.e.,CV777 and CH/JX/01,respectively.In contrast,the results from a in vitro cross neutralization assayindicated that rabbit anti-CV777 hyperimmune sera couldn’t neutralize PEDV Jiangxi strain CH/JX/01,and vice versa.