Investigation On The Prevalence Of Porcine Epidemic Diarrhea Virus Subtype G2b Variant Strains In Jiangsu Province And Development Of Attenuated Strains

Porcine epidemic diarrhea(PED)is an acute and highly contagious enteric disease caused by porcine epidemic diarrhea virus(PEDV).Since 1984,subtype G1a PEDV had been an important pathogen of piglets in China.From 2010 till now,the outbreaks of subtype G2b PEDV occurred and spread in many provinces in China.Compared with the classical subtype Gla strains,the emerging subtype G2b strains become more pathogenic and the subtype Gla CV777 vaccine could not offer effective protection.Thus,it's urgent to develop a subtype G2b vaccine.In this study,the prevalence of subtype G2b PEDV variant strains in Jiangsu Province between 2015 and 2017 was investigated,and an attenuated subtype G2b PEDV strain was developed by continuous passage on Vero cells,which laid the foundation for the further development of the attenuated vaccine.1 Investigation on the prevalence of subtype G2b PEDV variant strains in Jiangsu Province between 2015 and 2017In this study,176 clinical samples were collected from large-scale pig farms which have occurred diarrhea of piglet in Jiangsu Province between 2015 and 2017.19 of them were detected positive for PEDV by real-time quantitative PCR.PEDV was isolated and identified from these 19 positive samples and a G2b subtype strain was successfully isolated(named PED-JS-2016-05-4).We used PCR to amplify the PEDV spike(S)gene from these 19 positive samples.Phylogenetic analysis revealed that 17 of them were subtype G2b variant strains which had higher identity to the published subtype G2b epidemic strains and homology of S gene nucleotide was range from 98.5%to 99.7%.Nevertheless,when compared with the classic CV777 or other subtype Gla strains,homology of the S genes' nucleotide was only 93.6%to 94.2%.In the other 2 samples,genotype of PEDV was subtype Gla,demonstrated high homology with the USA OH851 strain,the nucleotide sequence homology was up to 99.5%.However,their nucleotide homology with the subtype G1a strains was only 95.6%to 96.8%.The study shows that the current prevalent PEDV strain in Jiangsu Province is subtype G2b variant strain.Compared with CV777,the neutralization epitopes in S protein of the subtype G2b variant strains which detected in this study had many amino acid differences,mutations were mainly concentrated on the COE epitope.There was only one mutation on the SS6 epitope and no mutation on the SS2 and 2C10 epitope.In addition,prediction of S protein potential glycosylation sites by CBS online software package found that,when compared with vaccine strain CV777,17 subtype G2b variant strains had 6 identical potential glycosylation site mutations,among which three mutations(N57V,N127I,T232I)destroyed N-glycosylated sites,however,another three mutations(S59N,I116T,T1193N)created three new N-glycosylated sites.Compared with CV777 strain,there was only one potential glycosylation site difference in two subtype G1b strains,that is,the 1193 amino acid produced a new potential glycosylation site from T mutation to N.2 Attenuation of the subtype G2b PEDV variant strain JSX2014In this study,the subtype G2b PEDV variant strain JSX2014 previously identified in our laboratory was selected for attenuated studies,in order to cultivate attenuated vaccine strain.The JSX2014 strain after continuous passage in Vero cell could produce typical CPE that was cell swelling,rounding,cell monolayer forming mesh-like and gradually disintegrating.JSX2014 strain could form plaques with different sizes on the cell,which was presumed to be related to the replication ability of different progeny virus.In order to screen out the strains with better replication ability,plaques with distinct morphological differences were selected during the passage.Then,plaque purification was carried out and the TCID50 was used to evaluate replication ability of different plaque clone strains.The results showed that the clone ability of the large plaque clone strains was significantly better than that of the small plaque clone strains.Therefore,large plaques were repeatedly picked for cloning and continue passed to 130 generations,named JSX2014/ATT.Three-day-old newborn piglets without sucking colostrum were selected for the pathogenicity test of JSX2014/ATT.Each piglet was injected intramuscularly with 2 x 107 TCID50 virus.Clinical symptoms such as vomiting and diarrhea were observed daily.Anal swabs as well as blood were collected daily for PEDV detection.Autopsies were made five days later.The small intestine and its contents were collected for PEDV detection.Also,histopathological examination of the intestine was performed.The results showed that there were no diarrhea-associated clinical symptoms in piglets,and there was no PEDV in the anal swab.However,PEDV was detected in the blood on the first day.The intestine tissue structure of piglets was intact,and the intestinal villi didn't shrink or fall off.The safety test indicated that the JSX2014/ATT strain had already been attenuated.The whole genome of the JSX2014/ATT strain was sequenced and then compared with the original JSX2014 strain.The S gene had 11 mutations.Both E and N gene had only one mutation.In addition to the two mutations in the M gene,there was a unique 5 amino acid insertion.Mutations of non-structural genes were mainly located on ORF1ab,which had 13 mutations.The T inserted to ORF3 at position 14 led to early termination of translation.In conclusion,the recent PEDV epidemic strains in Jiangsu province are mainly subtype G2b variant strains,and there is also a small number of subtype G1b strains.A subtype G2b PEDV strain was successfully attenuated by continuous passage combined with virus plaque purification technology,which laid the foundation for the further development of attenuated vaccines.