猪流行性腹泻病毒广西变异株全基因组序列测定与分子遗传进化分析

Complete Genome Sequencing and Molecular Genetic Evolution Analysis of Guangxi Mutant of Porcine Epidemic Diarrhea Virus

为了解近年广西地区猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)基因组特征和分子遗传多样性,本研究设计11对特异性引物,对中国广西南宁猪腹泻病例中检测到的1株PEDV GXNN进行了全基因组测定,同时进行相似性比对、遗传进化、基因变异及S基因重组分析。结果显示,广西变异株GXNN与其他PEDV分离株具有相似的基因组特征,基因组全长28035 bp,与不同参考株核苷酸相似性为96.4%~98.7%,S、ORF3、M和N基因核苷酸相似性分别为93.7%~98.9%、90.9%~99.4%、97.4%~99.7%和95.6%~99.2%;氨基酸相似性分别为92.9%~99.5%、91.3%~99.1%、97.4%~99.1%和96.4%~99.5%。GXNN与近年国内大多数分离株亲缘关系较近,处于同一分支,为GII-b亚型,而与经典疫苗毒株、国内早期流行株、国外流行株及广西CH-GX-2015-750A分离株亲缘关系都较远,为不同亚型。与目前常用5个疫苗毒株相比,S基因变异较大,在118、844和905位插入氨基酸Q,在COE(Core neutralizing epitope)区和主要抗原表位区发生4个独特的氨基酸位点突变,其他区域发生14个位点突变;ORF3、M和N基因分别在126位、4D4区和PN-D4区发生126 T/A、199 A/V和103 T/A突变。重组分析发现在S基因的高变区826~3142 nt存在1个潜在的重组区域。本研究成功获得1株PEDV全基因组序列,对其进行遗传变异分析,为PEDV分子流行病学研究和新型疫苗研发提供参考依据。

In order to understand the genomic characteristics and molecular genetic diversity of porcine epidemic diarrhea virus(PEDV)in Guangxi in recent years,11 pairs of specific primers were designed to detect the whole genome of PEDV GXNN isolated from porcine diarrhea in Nanning,Guangxi,China,and similarity comparison,genetic evolution,gene variation and S gene recombination were also analyzed.The results showed that full length of the GXNN strain was 28035 bp,had similar genomic characteristics with other PEDV isolates,about 96.4%~98.7%nucleotide similarity with different reference strains,and the nucleotide similarity of S,ORF3,M and N genes was 93.7%~98.9%,90.9%~99.4%,97.4%~99.7%and 95.6%~99.2%;the amino acid similarity of them was 92.9%~99.5%,91.3%~99.1%,97.4%~99.1%and 96.4%~99.5%.GXNN is closely related to most domestic isolates in recent years.Phylogenetic tree showed that GXNN closely related to most strains isolated in China recent years,belonged to GII-b subtype.However,it was low relatedness to classic vaccine strains,domestic early epidemic strains,foreign epidemic strains and Guangxi CH-GX-2015-750 A,they belong to different subtypes.Compared with the 5 vaccine strains,the S gene of GXNN stain has a large variation,by inserting amino acid Q at positions 118844 and 905 sizes,four unique amino acid mutations in the core neutralizing epitope(COE)region and the main epitope region,and 14 mutations in other regions.126 T/A,199 A/V and 103 T/A site mutations of ORF3,M and N genes were happened at position 126,4 D4 region and PN-D4 region,respectively.Recombination analysis revealed that there was a potential recombination region in the hypervariable region of S gene at 826~3142 nt.This study successfully obtained the complete genome sequence of a PEDV strain,and analyzed its genetic variation and provided a reference for PEDV molecular epidemiology research and new vaccine development.