猪非典型瘟病毒广西流行株的鉴定与遗传进化分析  

Identification and phylogenetic analysis of atypical porcine pestivirus strains prevalent in Guangxi

作　　者：谢守玉 魏园园 谢颖 黄张玲[1] 周明旭 颜健华[3] 梁凤 李军[1] 熊毅[1] XIE Shou-yu;WEI Yuan-yuan;XIE Ying;HUANG Zhang-ling;ZHOU Ming-xu;YAN Jian-hua;LIANG Feng;LI Jun;XIONG Yi(Guangxi Animal Disease Prevention and Control Center,Nanning 530001,China;Guangxi Vocational University of Agriculture,Nanning 530007,China;Guangxi University,Nanning 530005,China)

机构地区：[1]广西动物疫病预防控制中心,广西南宁530001 [2]广西农业职业技术大学,广西南宁530007 [3]广西大学,广西南宁530005

出　　处：《中国预防兽医学报》2024年第4期418-425,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31660713)。

摘　　要：为了解猪非典型瘟病毒(APPV)广西流行株的遗传进化特点,本研究从广西3个地区共采集23份患先天性震颤(CT)症状的新生仔猪脑、肝脏、脾脏、肾脏等组织样品,采用荧光定量RT-PCR (RT-q PCR)检测APPV,经RT-PCR分段扩增APPV阳性样品的全基因组序列,结果显示,检出18份APPV阳性样品,RT-PCR分段扩增并经测序拼接后共获得4条APPV广西流行株全基因组序列,结合Gen Bank登录的共有9条APPV广西流行株全基因组序列,采用BLAST及Bio Edit软件分析APPV广西流行株全基因组序列的相似性;利用Mega 7.0软件绘制ORF、N^(pro)、E^(rns)及E2基因的系统发育树;通过Bepipred 2.0软件预测APPV囊膜糖蛋白E^(rns)、E2蛋白的线性抗原表位;利用Net NGlyc 1.0在线软件预测APPV E^(rns)、E2蛋白N-糖基化位点;利用RDP5和Sim Plot软件分析APPV广西流行株全基因组的重组性。相似性结果显示,9条APPV广西流行株全基因组序列与Gen Bank中APPV流行株全基因组序列的相似性最高为89.9%~99.8%,且这些流行株分别来源于河北、湖北、湖南、四川、广西及广东等地;APPV广西流行株之间及与国内外APPV参考株全基因组序列的相似性分别为79.4%~99.6%及77.1%~98.3%,表明APPV广西流行株基因组进化来源广泛且复杂,并且APPV广西流行株之间的差异较大。遗传进化分析显示,ORF系统发育树分为3个基因型,APPV广西流行株GX01-2019属于I-2基因亚型,GX02-2018株属于I-3基因亚型,GX02-2019株属于Ⅱ基因型,其余则属于I-1基因亚型,N^(pro)、E^(rns)、E2基因的系统发育趋势与ORF基本一致。表明APPV广西流行株以I基因型为主,且无时间地域分布特点,并具有生物遗传多样性特征;抗原表位和N-糖基化位点预测结果显示,E^(rns)蛋白主要含aa7~aa14、aa24~aa36、aa39~aa66等7个抗原表位,E2蛋白主要含aa5~aa9、aa16~aa25、aa35~aa111等5个抗原表位;APPV广西流行株E^(rns)蛋白N-糖基化位点分别为^(12)NSTG^(15)、^(43)NIn order to understand genetic feature of atypical porcine pestivirus(APPV)strains prevalent in Guangxi,a total of 23 samples including brain,liver,spleen and kidney were collected from newborn piglets with congenial tremors(CT)symptoms from three regions of Guangxi.The APPV was detected by fluorescence quantitation PCR(RT-qPCR)and the whole genome sequence of APPV positive samples was segmentally amplified by RT-PCR.The results showed 18 positive samples were detected(positive rate 78.26%)and 4 whole genome sequences of APPV were obtained after sequencing and splicing.A total of 9 APPV whole genome sequences of endemic strains in Guangxi including 4 strains that identified in this study were further analyzed.The similarity of these APPV epidemic strains was analyzed by BLAST and BioEdit,the linear antigen epitopes and N-glycosylation sites of E^(rns)and E2 glycoprotein were predicted by Bepipred 2.0 software and NetNGlyc 1.0 online software;phylogenetic and recombination analysis with Mega 7.0 and RDP5 and SimPlot software was conducted.The results showed that the highest similarity of APPV Guangxi strains and GenBank strains was 89.9%-99.8%,which identified from Hebei,Hubei,Hunan,Sichuan,Guangxi and Guangdong provinces.Also,the Guangxi strains inter-and reference strains shared genome similarity with 79.4%-99.6%and 77.1%-98.3%respectively,suggested that genome evolution of the APPV was diversity and the differences among the APPV strains in Guangxi are relatively large.Phylogenetic analysis based on nucleotide sequences of ORF showed that there were three genotype clusters in the trees,the GX01-2019 strain and GX02-2018 strain belonged to I-2,and I-3 sub genotype,respectively.The GX02-2019 strain belonged toⅡgenotype,and the others belonged to I-1 sub genotype.Besides,the genetic trend of N^(pro),E^(rns)and E2 was similar with ORF suggesting that APPV of Guangxi strains were mainly genotype I and without association to spatiotemporal dynamics,and genetic was diversity.Epitope prediction of E^(rns)and E2 prote

关 键 词：猪非典型瘟病毒 广西流行株 遗传进化 

分 类 号：S852.65[农业科学—基础兽医学]