Analysis Of Genetic Variation Of Porcine Epidemic Diarrhea Virus And The Effects Of N-terminal Of S1 Protein On Viral Replication

Porcine epidemic diarrhea(PED)is caused by porcine epidemic diarrhea virus(PEDV).The main clinical signs are acute intestinal infections such as vomiting,diarrhea and dehydration.PED can occur in all ages of pigs and has a high mortality rate in piglets.In recent years,the genetic variation of PEDV has brought many problems to the prevention and control of PED.In this project,we analyzed the molecular epidemiology of PEDV and studied the newly emerged PEDV mutant strains,analyzed the genetic variation characteristics of PEDV,established a duplex TaqMan RT-qPCR method for differentiate and detection of PEDV classical strains and mutant strains,and studied the effects of the N-terminal region of the S1 protein on pathogenesis.Our discoveries are listed below:1.We assessed 129 clinical samples collected from July 2014 to July 2015,which were the intestinal tissues of piglets with severe diarrhea,from 17 cities in central China.Both the S1 subunit of spike(S)glycoprotein(S1,1-789 amino acids(aa))and the full-length ORF3gene of 21 representative field strains from 21 farms in 11 cities were sequenced and analysed.PEDV was detected by reverse transcription-polymerase chain reaction(RT-PCR).The S1and ORF3 sequences were aligned by the Clustal W method in DNAMAN 8 software.Phylogenetic trees were constructed by the neighbor-joining method using MEGA 6 software.The results showed that the prevalence of PEDV was 92.25%(119/129),with 94.03%(63/67)of pig farms harbouring the disease.According to the phylogenetic analysis of the S1 genes,our isolates all fell into group G2(variants)and showed a close relationship to isolates from Chinese(HN1303,CH/ZMDZY/11 and AJ1102),Korean(AD01),American(MN,IA1,IA2and 13-019349)sources,and these isolates differed genetically from other Chinese(LZC,CH/HNZZ/2011 and SD-M)and Korean(SM98)strains as well as Japanese(83-P5 and MK)strains.In addition,our isolates differed from attenuated vaccine strains,CV777(used in China)and DR13(used in Korea).According to our derived amino acid sequence analysis,we detected one novel variant PEDV,CH/HNLY,with 4-aa insertion/deletion(RSSS/T)at position 375 and 1-aa(D)deletion at position 430 compared to the CV777 attenuated strain.These mutations were located on the receptor binding domain.Our analyses on the ORF3genes analyses showed that the prevalent PEDV isolates were genetically variable,and the field strains differed genetically from the vaccine strains.These findings illustrated the existence of genetic diversity among geographically distinct PEDV strains.Our study has provided an impetus to conducting further research on the PEDV receptor binding domain and on the new and efficacious vaccines design.2.In the present study,a novel duplex TaqMan RT-qPCR was developed for detecting and differentiating PEDV strains in China.There was no cross-amplification between the two probes when using standard recombinant plasmids,and the specificity was further confirmed by using other seven non-PEDV swine pathogens.The minimum copies required for the detection of both classical and variant PEDV were 4.8×10~2 DNA copies/reaction.The repeatability of TaqMan RT-qPCR was evaluated using standard recombinant plasmids and gave coefficients of variation 0.19-4.93.In recent 5 years,79 clinical samples were collected from piglets with severe diarrhea in the Central China.Among these clinical samples,51 were confirmed as PEDV positive by conventional RT-PCR,whereas 63 variant PEDV,3co-infections and 1 classical PEDV were confirmed by this duplex TaqMan RT-qPCR,with viral loads of 10~2-10~8,10~2-10~3,and 10~4 copies/reaction,respectively.Therefore,the duplex TaqMan RT-qPCR could be a useful method for detecting and differentiating variant and classical PEDV strains.The results showed that variant PEDV was prevalent in clinical samples in central China.Moreover,in this study,co-infection by PEDV strains was detected for the first time and might help explain the emergence of the novel recombinant PEDV in recent years.3.Four types of PEDV variants with a large deletion in the S protein were detected together with the original US PEDV from pig fecal and oral fluid samples collected during April 2016 to April 2017 in the US.Two types of the variants are similar to those identified in Japan:one contains a 194-aa deletion as same as PEDV variant TTR-2/JPN/2014,and the other contains a 204-aa deletion as same as PEDV variant JKa-292/CS1de204.Two new S1NTD-del PEDV variants were found:one contains a 201-aa deletion located at residues30-230 and the other contains a 202-aa deletion located at residues 24-225 aa of the S protein.This is the first report on coinfection of S1 NTD-del PEDV variants and the original US PEDV in US pigs,indicating that PEDV continues to evolve in pigs and might be responsible for disease pattern changes.4.Porcine epidemic diarrhea virus(PEDV)variants having a large deletion in the N-terminal domain of the S1 subunit of spike(S)protein were designated as S1 NTD-del PEDVs.They replicate well in experimentally infected pigs.However,on farms they often co-infect pigs with the PEDV containing an intact S protein(S-intact PEDV).We aimed to characterize viral replication and pathogenesis in neonatal gnotobiotic pigs infected simultaneously with the two types of PEDV using two recombinant PEDVs:icPC22A and its S1 NTD-del form icPC22A-S1?197.Additionally,viral replication was compared in Vero and IPEC-DQ cells at the presence of bovine mucin(BM),porcine gastric mucin(PGM),swine bile and bile acids during inoculation.In the pigs coinfected with icPC22A and icPC22A-S1?197,icPC22A replicated to a higher peak titer than its infection of pigs without the presence of icPC22A-S1?197.The severity of diarrhea and intestinal atrophy were similar between icPC22A and the coinfection groups,but were significantly higher than icPC22A-S1?197 group.In Vero and IPEC-DQ cells,certain concentrations of BM,PGM,bile and bile acids increased significantly the infectivity of icPC22A but had no or negative effects on icPC22A-S1?197.These results indicated that the replication of the S-intact PEDV was enhanced during coinfection in piglets.This observation may be explained partially by the fact that mucin,bile and bile acids in gastrointestinal tract had facilitating effects on the infection of S-intact PEDV,but no/inhibition effects on S1 NTD-del PEDV.