Molecular Characterization And Genetic Evolution Analysis Of Porcine Epidemic Diarrhea Virus

ObjectiveTo understand the current epidemic diversity of porcine epidemic diarrhea virus(Porcine epidemic diarrhea virus,PEDV)in China,and to explore the genetic evolution and recombination of PEDV,lay a foundation for PEDV prevention control and vaccine development.Methods1.Identification of common viruses in samples: RNA was extracted from small intestine of diarrhea pigs collected from Liaoning,Jiangsu and Jiangxi provinces,and c DNA was synthesized by reverse transcription of RNA.This was used as a template to detect PEDV,porcine transmissible gastroenteritis virus(Transmissible gastroenteritis virus,TGEV),porcine deltacoronavirus(Porcine deltacoronavirus,PDCo V),porcine acute diarrhea syndrome coronavirus(Swine acute diarrhea syndrome coronavirus(SADS-Co V)and classical swine fever virus(Classical swine fever virus,CSFV).2.Virus isolation and identification:The PEDV positive samples were isolated from Vero cell and identified by indirect immunofluorescence(Indirect Immunofluorescence Assay,IFA).The S1 gene of PEDV epidemic strains were amplified by reverse transcription polymerase chain reaction(Reverase transcript polymerase chain reaction,RT-PCR)and sequenced3.Whole genome amplification of PEDV epidemic strains:(1)Primer design : According to the results of S1 gene sequencing,ten whole genome sequences of PEDV with high Blast similarity in NCBI were selected as references.After alignment,conservative region sequences were selected for whole genome primer design.(2)The genome of PEDV epidemic strains were amplified by the method of gene segment overlap.RT-PCR was used to amplify the genome of PEDV epidemic strains.Every two adjacent sequence fragments had 100-200 bp overlap region to ensure the sequence accuracy.(3)The 5’ UTR and 3’ UTR regions of PEDV genome were amplified by c DNA terminal rapid cloning(rapid-amplification of c DNA ends,RACE).(4)Whole genome splicing The sequencing results were spliced according to the sequencing peak map to check whether the peak corresponds to the bases.The sequencing results of 26 gene fragments were assembled to obtain the whole genome sequence.Use ORFfinder in NCBI to predict the location of its open reading frames.4.Genetic evolution analysis of PEDV epidemic strains(1)Construction of phylogenetic tree and homology analysis Phylogenetic tree construction and homology analysis of the epidemic strains determined in this study and sixty-seven domestic and foreign reference strains were used to construct the phylogenetic trees;The Phylo Suite v1.2.2 software was used to multiple sequence alignment of sixty-nine sequences by MAFFT method.The IQTree program followed 1000 repeated bootstraps.The phylogenetic trees were constructed by maximum likelihood(ML)method.The bat coronavirus of Bt Co V/512/2005(Gen Bank:DQ648858)was used as outgroup reference.The homology of whole genome and structural gene from epidemic strains and representative strains that CV777(GIa),attenuated CV777(GIb),AH2012(GIIa),AJ1102(GIIb)and OH851(GIIc)were analyzed by Meg Align in DNAstar.(2)Recombination analysis Collected all PEDV whole genome sequences from Gen Bank database.After screening,artificial mutants,repeated copy sequences matched with sampling position and time,cell passage sequences,vaccine strains and low quality sequences were excluded.Four hundred and ninety PEDV whole genome sequences were obtained for recombination analysis.Seven algorithms of RDP,GENECONV,Boot Scan,Si Scan,Max Chi,3Seq and Chimaera in the recombination detection software RDP v5.0 were used to detect recombination events.The default setting and threshold pair 0.01 were used to identify potential recombinant.If four algorithms had recombination signals at least and P values were less than 1 × 10-6.The recombination event was considered to be reliable;Sim Plot was used to further verify sequence similarity and potential recombination events;IQTree program and maximum likelihood(ML)were used to construct phylogenetic trees of non-recombination regions and recombination regions for further analysis.(3)Selection stress analysis Two hundred and three Chinese PEDV sequences were selected from four hundred and ninety PEDV sequences for selective stress analysis.Four pairs of site model(M0 vs M3,Mla vs M2 a,M7 vs M8 and M8 a vs M8)in Easy Code ML were used to analyze the difference by LRTs method.In order to further screen the results,FUBAR,MEME,FEL and SLAC methods of online website Datamonkey were used to predict the positive selection sites of PEDV structural gene sequences(FEL and MEME P < 0.05,SLAC P < 0.1,FUBAR P > 0.9).Results1.Identification of common viruses in samples: Virus identification of tissues from pigs with diarrhea in three provinces,the result showed that the samples from Liaoning and Jiangsu were positive for PEDV.TGEV、PDCo V、SADS-Co V and CSFV were negative.2.Virus isolation and identification: The virus was isolated from the positive material of Jiangsu province,and the isolated virus was determined to be PEDV by IFA detection.The PEDV S1 genes were successfully amplified.3.Whole genome amplification of PEDV epidemic strain: The whole genome sequences of CN/Jiangsu002/2022 and CN/Liaoning25/2018 were obtained by sequence amplification,sequencing and splicing.The full length of CN/Jiangsu002/2022 was 28038 nt.The full length of CN/Liaoning25/2018 was 27961 nt.The genomes were typical PEDV structure.4.Genetic evolution analysis of PEDV epidemic strain(1)Phylogenetic tree construction and homology analysis CN/Jiangsu002/2022 strain,the phylogenetic tree of whole genome and S gene showed that its genotype was GIIa type,which was far from the classical vaccine strain of GI type.The phylogenetic trees of E gene,M gene and N gene all showed that its genotype was GII type.Through homology analysis with five representative strains: CV777(GIa),attenuated CV777(GIb),AH2012(GIIa),AJ1102(GIIb)and OH851(GIIc),the homology of the whole genome and S gene of CN/Jiangsu002/2022 strain was the highest with that of GIIa AH2012 strain and there were 8 unique amino acid mutation sites on S protein.E,M and N genes had the highest homology with GIIc type OH851 strain,in which M protein had one amino acid mutation site and N protein had two amino acid mutation sites.The phylogenetic tree of the whole genome of CN/Liaoning25/2018 strain showed that its genotype was GIb type,which was far from GII variant strain;The phylogenetic trees of E gene,M gene and N gene all showed that its genotype was GI;The phylogenetic tree of S gene showed that its genotype was GIIa type,which was far from GI classical vaccine strain.The results of homology analysis showed that the whole genome,E gene,M gene and N gene had the highest homology with GIb attenuated CV777 strain,while S gene had the highest homology with GIIa AH2012 strain.(2)Recombination analysis The recombinant CN/Jiangsu002/2022 strain was a new type of recombinant strain formed by Spanish ES/1508 Zaragoza Tauste/2014(GIIc)and Chinese CH/HM/2017(GIIb).The recombinant region was ORF1b-S1,located at 17723-21556 nt;The recombinant CN/Liaoning25/2018 strain was produced by CH/SQ2014/2014(GIb)and CH/GDZQ/2014(GIIa),and the recombinant region was S,located at 20597-24758 nt.(3)Selection stress analysis There were positive selection sites in S,E,M and N genes of two hundred and three epidemic strains of PEDV in China.The amino acids at position 312,357,361,367,524,636,888 of S protein.The 42 th amino acid of M protein and the 412 th amino acid of N protein were in positive selection effect.However,no positive selection site was detected in E protein.Conclusions1.Virus detection was successfully carried out on diarrhea samples,and samples from Jiangsu and Liaoning were positive for PEDV.2.The CN/Jiangsu002/2022 strain was successfully isolated.3.The CN/Jiangsu002/2022 and CN/Liaoning25/2018 whole genome sequences were successfully obtained.4.CN/Jiangsu002/2022 strain was successfully identified as a variant of GIIa.It was the natural recombinant strain produced by the Spanish ES/1508 Zaragoza Tauste/2014 strain of GIIc type and the Chinese CH/HM/2017 strain of GIIb type.There were many amino acid mutation sites on its S protein,and most of them were located in S1 domain.CN/Laoning25/2018 strain was a recombinant strain of GIb,which was produced by the CH/SQ2014/2014 strain of GIb type and the CH/GDZQ/2014 strain of GIIa type.5.Positive selection sites for structural genes of PEDV epidemic strains in China were analyzed,indicating that S gene was more representative at the genomic level,and the evolution of S gene was highly correlated with S1 domain.