Genome Analysis Of Porcine Epidemic Diarrhea Virus In Yunnan Province

Porcine epidemic diarrhea virus(PEDV)is α Members of the coronavirus genus can cause severe diarrhea symptoms in piglets,causing huge economic losses to the pig industry.In order to understand the epidemic variation of PEDV strains in Yunnan Province,this study designed primers for piecewise amplification of PEDV complete gene sequences.Three PEDV strains(YN2021,YNLP 2022,and YNBS 2022)and one commercially available attenuated vaccine strain(Attenuated AJ1102-R)that were previously isolated and preserved were subjected to RT-PCR segmented amplification,sequencing,and sequence splination.The spliced gene sequences were compared with representative PEDV strains at home and abroad(classic strains CV777,DR13,Attenuated CV777,Attenuated DR13,mutated strain AJ1102,etc.),and analyzed from the aspects of gene homology,genetic evolution,amino acid sites derived from key genes,and strain recombination.The results were as follows:1.33 pairs of PEDV whole gene primers were independently designed for segmented amplification,and specific amplification,molecular cloning,sequencing,assembly and splicing were performed by RT-PCR method.The full gene sequences of three PEDV Yunnan endemic strains(YN2021,YNLP 2022,and YNBS 2022)and one commercially available Attenuated AJ1102-R were obtained.The full length of YN2021,YNLP 2022,YNBS 2022 and Attenuated AJ1102-R strains are 27953 bp,28036bp,28031 bp and28042bp,respectively.2.The bioinformatics software Megalign was used to analyze the homology of 4PEDV complete gene sequences and 58 reference strains: The nucleotide homology of YN2021,YNLP 2022 and YNBS 2022 from Yunnan was 96.7 ～ 99.3%,and the nucleotide homology of YN2021,YNLP 2022 and YNBS 2022 was 96.6% ～ 97.6% with the classical strain CV777.The nucleotide homology with mutant AJ1102 was 97.0%-98.9%.MEGA 11 software was used to construct an NJ phylogenetic tree based on the full gene sequence.It was found that the YN2021 strain was in the same evolutionary branch as the Attencv777 strain,belonging to the GI-b gene subtype.YNLP 2022 and YNBS 2022 strains formed a large branch with the representative strains of ZJCZ4 and Atten AJ1102-R variants currently prevalent in China,belonging to the GII-b gene subtype.3.Megalign software was used for comparative analysis of amino acid sequences derived from S,ORF3,M and N genes of PEDV endemic strain in Yunnan Province sequenced in this study.Amino acid homology analysis based on S gene deduction showed that YN2021,YNLP 2022 and YNBS 2022 had 96.7%,93.9% and 93.6% amino acid homology with classical strain CV777,and 93.6%,99.1% and 98.9% amino acid homology with mutant strain AJ1102.The amino acid sequence alignment of S protein showed that compared with the classical strain,the mutant strain had 1,5 and 1 amino acid insertion(56G,59QGVNS63 and 146N/D)at the 56 g,59QGVNS63 and 146N/D)at the 56 th position,and L(leucine)and N(methionine)substitution at the 58 th position.YN2021 strain was consistent with classic virus representative strain CV777 in the above areas,while YNLP 2022 and YNBS 2022 were consistent with mutant strain AJ1102 in the above areas,which further indicated that YN2021 belonged to classic epidemic strain,and YNLP2022 and YNBS 2022 belonged to mutant strain.In the amino acid sequence derived from ORF3 gene,YN2021 strain had a large number of amino acid deletion at positions 82-113,115-135,139-144 and 150-169,and lacked TM4 transmembrane domain,which was consistent with the deletion characteristics of classical attenuated vaccine strains and cellular adaptive strains.In amino acid sequences derived from M and N genes,3 PEDV endemic strains in Yunnan had different amino acid mutations from 58 reference strains.In addition,compared with the variant strain AJ1102,the fully sequenced Attenuated AJ1102-R was used in the present study with five amino acid insertions(1280AIVDV1284)and two amino acid deletions(228HL229)in the M protein sequence compared with the variant attenuated strain AJ1102.4.According to the analysis of 7 algorithms of RDP4 software and Sim Plot,among the strains YN2021,YNLP 2022 and YNBS 2022,YNLP 2022 is a recombinant of PEDV-7C subtype GII-b strain and YN1 subtype GII-a strain as the primary parent.The recombination region is nt4994-7605;YNBS 2022 strain is a recombinant of GII-b subtype strain 17GXZC-1ORF3 c and GII-a subtype strain PEDV-CHZ as the secondary parent,and the recombinant region is nt16399-22326,indicating that the virus gene recombination may increase the difficulty of prevention and control.In this study,the complete genome sequence of PEDV endemic strain in Yunnan Province was taken as the entry point,and the molecular characteristics,genetic evolution and recombination among the complete genome sequences of PEDV endemic strain in Yunnan Province and related vaccine strains were analyzed and analyzed by bioinformatics method,providing strong theoretical support for the safe production of first-line pigs.