浙江省猪流行性腹泻病毒S1基因克隆与序列分析
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摘要
本研究旨在分析浙江省猪流行性腹泻病毒(PEDV)的遗传变异情况,利用实时荧光定量RT-PCR方法对2015-2016年浙江省内收集的58份猪腹泻样品进行检测,设计2对特异性引物对16份来自浙江不同地区PEDV阳性样品的S1基因进行RT-PCR扩增、克隆及序列测定,并应用生物信息学软件对16株PEDV浙江毒株的S1基因进行分析。结果显示,48份样品为PEDV阳性。16个毒株之间S1基因片段核苷酸和氨基酸同源性分别为93.1%~99.8%和92.4%~99.7%,与疫苗株CV777的核苷酸同源性为92.3%~95.7%,氨基酸同源性为90.7%~95.7%。与疫苗株CV777相比,15个毒株在S1基因区域存在着15个核苷酸插入和6个核苷酸缺失。系统进化分析表明,大部分毒株与国内外流行的基因Ⅱ型PEDV毒株亲缘关系较近,15个毒株与2011-2016年中国流行的基因Ⅱ型PEDV毒株核苷酸和氨基酸同源性均在96.6%以上;与早期分离的CV777株、LZC株亲缘关系较远,核苷酸和氨基酸同源性均在93.4%以下;1个毒株(ZJ16NB6)与国内外流行的S-INDEL样毒株较近,核苷酸和氨基酸的同源性较高,均在98.4%~99.5%之间。本研究结果表明,2015-2016年浙江省仔猪腹泻主要是由PEDV感染引起的,浙江省流行的PEDV同时存在着基因Ⅱ型和S-INDEL样毒株,但以基因Ⅱ型毒株为主。
To analyze the genetic variation of porcine epidemic diarrhea virus(PEDV),the piglet diarrhea samples from Zhejiang province during 2015 to 2016 were detected by Real-time RTPCR.Two pairs of primers were designed and the S1 gene of 16 PEDV positive samples from different regions were amplified by RT-PCR,cloned and sequenced.The sequence of S1 gene was analyzed by biological information software.The results showed that 48 samples were positive for PEDV.Homologous analysis showed that the S1 gene of 16 PEDV strains shared 93.1% to99.8% nucleotide homology and 92.4%to 99.7%amino acid homology.The S1 gene of 16 PEDV strains shared 92.3%to 95.7% nucleotide homology and 90.7%to 95.7% amino acid homology with vaccine strain CV777.Compared to vaccine strain CV777,15 nucleotides were inserted and 6 nucleotides were absent in the S1 gene of 15 epidemic strains.The phylogenetic tree based on sequences of S1 gene suggested that 15 PEDV strains had a close relationship with the genotypeⅡPEDV,nucleotide and amino acid homologous were all more than 96.6%.Compared with the early separation of CV777 and LZC strains,nucleotide and amino acid homologous were all less than93.4%.However,strain ZJ16 NB6 had a close relationship with the S-INDEL-like strain,nucleotide and amino acid homologous were all 98.4% to 99.5%.The results indicated that PEDV infection was the main pathogen of piglets diarrhea in Zhejiang province.The genotypeⅡand S-INDEL-like PEDV strain existed in Zhejiang province,but mainly prevailing the genotypeⅡ PEDV strains.
To analyze the genetic variation of porcine epidemic diarrhea virus(PEDV),the piglet diarrhea samples from Zhejiang province during 2015 to 2016 were detected by Real-time RTPCR.Two pairs of primers were designed and the S1 gene of 16 PEDV positive samples from different regions were amplified by RT-PCR,cloned and sequenced.The sequence of S1 gene was analyzed by biological information software.The results showed that 48 samples were positive for PEDV.Homologous analysis showed that the S1 gene of 16 PEDV strains shared 93.1% to99.8% nucleotide homology and 92.4%to 99.7%amino acid homology.The S1 gene of 16 PEDV strains shared 92.3%to 95.7% nucleotide homology and 90.7%to 95.7% amino acid homology with vaccine strain CV777.Compared to vaccine strain CV777,15 nucleotides were inserted and 6 nucleotides were absent in the S1 gene of 15 epidemic strains.The phylogenetic tree based on sequences of S1 gene suggested that 15 PEDV strains had a close relationship with the genotypeⅡPEDV,nucleotide and amino acid homologous were all more than 96.6%.Compared with the early separation of CV777 and LZC strains,nucleotide and amino acid homologous were all less than93.4%.However,strain ZJ16 NB6 had a close relationship with the S-INDEL-like strain,nucleotide and amino acid homologous were all 98.4% to 99.5%.The results indicated that PEDV infection was the main pathogen of piglets diarrhea in Zhejiang province.The genotypeⅡand S-INDEL-like PEDV strain existed in Zhejiang province,but mainly prevailing the genotypeⅡ PEDV strains.
引文
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[17]杜晓莉,王一成,吴润,等.2010-2013年浙江省猪流行性腹泻病毒临床检测及PEDV-S基因型分析[J].浙江农业学报,2014,26(3):581-587.DU X L,WANG Y C,WU R,et al.Genotyping and clinical detection of porcine epidemic diarrhea virus during 2010-2013in Zhejiang province[J].Acta Agriculture Zhejiangensis,2014,26(3):581-587.(in Chinese)
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[19]郑逢梅,霍金耀,赵军,等.2010-2012年华中地区猪流行性腹泻病毒分子特征和遗传进化分析[J].病毒学报,2013,29(2):197-205.ZHENG F M,HUO J Y,ZHAO J,et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central China during 2010-2012outbreaks[J].Chinese Journal of Virology,2013,29(2):197-205.(in Chinese)
[20]张志,程小娜,樊雅婷,等.猪流行性腹泻病毒变异株的鉴定和分子特征分析[J].中国畜牧兽医,2015,42(7):1654-1660.ZHANG Z,CHENG X N,FAN Y T,et al.Identification and molecular characteristics analysis of porcine epidemic diarrhea virus variants[J].China Animal Husbandry&Veterinary Medicine,2015,42(7):1654-1660.(in Chinese)
[21]毛黎红,张双翔,周碧君,等.6株猪流行性腹泻病毒贵州株S基因序列分析[J].中国畜牧兽医,2017,44(6):1621-1629.MAO L H,ZHANG S X,ZHOU B J,et al.Sequence analysis of Sgene of 6strains of porcine epidemic diarrhea virus from Guizhou[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1621-1629.(in Chinese)
[22]刘孝珍,陈建飞,时洪艳,等.2011年猪流行性腹泻病毒的遗传变异分析[J].中国预防兽医学报,2012,34(3):180-183.LIU X Z,CHEN J F,SHI H Y,et al.Genetic variation analysis of porcine epidemic diarrhea virus isolated in 2011[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(3):180-183.(in Chinese)
[2]PENSAERT M B,DEBOUCK P.A new coronaviruslike particles associated with diarrhea in swine[J].Archives of Virology,1978,58(3):243-247.
[3]CHEN Q,LI Q,STASKO J,et al.Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013disease outbreak among swine in the United States[J].Journal of Clinical Microbiology,2014,52(1):234-243.
[4]SONG D,PARK B.Porcine epidemic diarrhoea virus:A comprehensive review of molecular epidemiology,diagnosis,and vaccines[J].Virus Genes,2012,44(2):167-175.
[5]SUN M,MA J,WANG Y,et al.Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia[J].Journal of Clinical Microbiology,2015,53(5):1484-1492.
[6]KOCHERHANS R,BRIDGEN A,ACKERMANN M,et al.Completion of the porcine epidemic diarrhea coronavirus(PEDV)genome sequence[J].Virus Genes,2001,23(2):137-144.
[7]PARK S J,KIM H K,SONG D S.Complete genome sequences of a Korean virulent porcine epidemic diarrhea virus and its attenuated counterpart[J].Journal of Virology,2012,86(10):5964.
[8]YEO S G,HERNANDEZ M,KRELL P J,et al.Cloning and sequence analysis of the spike gene of porcine epidemic diarrhea virus Chinuj99[J].Virus Genes,2003,26(3):239-246.
[9]CHANG S H,BAE J L,KANG T J,et al.Identification of the epitope region capable of inducing neutralizing antibody against the porcine epidemic diarrhea virus[J].Molecules and Cells,2002,14(2):295-299.
[10]SUN D,FENG L,SHI H,et al.Identification of two novel B epitopes on porcine epidemic diarrhea virus spike protein[J].Veterinary Microbiology,2008,131(1-2):73-81.
[11]LI B X,GE J W,LI Y J.Porcine aminopeptidase N is a functional receptor for the PEDV coronavirus[J].Virology,2007,365(1):166-172.
[12]陈慧娟,吴志明,闫若潜,等.2012年河南猪流行性腹泻病毒分离株S1基因遗传变异分析[J].中国畜牧兽医,2014,41(3):14-18.CHEN H J,WU Z M,YAN R Q,et al.Genetic variation analysis of S1genes of porcine epidemic diarrhea virus isolated from Henan in 2012[J].China Animal Husbandry&Veterinary Medicine,2014,41(3):14-18.(in Chinese)
[13]SU Y F,LIU Y C,CHEN Y M,et al.Detection and phylogenetic analysis of porcine epidemic diarrhea virus in central China based on the ORF3gene and the S1gene[J].Virology Journal,2016,13(1):192-200.
[14]罗六龙,黄冬妮,黄良宗,等.广东省猪流行性腹泻病毒S1基因的克隆与序列分析[J].中国兽医杂志,2016,52(3):19-21.LUO L L,HUANG D N,HUANG L Z,et al.Cloning and sequence analysis of Sl genes of porcine epidemic diarrhea virus in Guangdong province[J].Chinese Journal of Veterinary Medicine,2016,52(3):19-21.(in Chinese)
[15]WANG E Y,GUO D H,LI C Q,et al.Molecular characterization of ORF3and S1genes of porcine epidemic diarrhea virus non S-INDEL strains in seven regions of China,2015[J].PLoS One,2016,11(8):e0160561.
[16]TIAN P F,JIN Y L,XING G,et al.Evidence of recombinant strains of porcine epidemic diarrhea virus,united states,2013[J].Emerging Infectious Diseases,2014,20(10):1735-1738.
[17]杜晓莉,王一成,吴润,等.2010-2013年浙江省猪流行性腹泻病毒临床检测及PEDV-S基因型分析[J].浙江农业学报,2014,26(3):581-587.DU X L,WANG Y C,WU R,et al.Genotyping and clinical detection of porcine epidemic diarrhea virus during 2010-2013in Zhejiang province[J].Acta Agriculture Zhejiangensis,2014,26(3):581-587.(in Chinese)
[18]吴旺霞,王一成,吴润,等.2011-2016年浙江省猪流行性腹泻病毒S基因与ORF3基因遗传进化分析[J].浙江农业学报,2017,29(3):380-388.WU W X,WANG Y C,WU R,et al.Genetic analysis of Sand ORF3gene in porcine epidemic diarrhea virus during 2011-2016in Zhejiang province[J].Acta Agriculture Zhejiangensis,2017,29(3):380-388.(in Chinese)
[19]郑逢梅,霍金耀,赵军,等.2010-2012年华中地区猪流行性腹泻病毒分子特征和遗传进化分析[J].病毒学报,2013,29(2):197-205.ZHENG F M,HUO J Y,ZHAO J,et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central China during 2010-2012outbreaks[J].Chinese Journal of Virology,2013,29(2):197-205.(in Chinese)
[20]张志,程小娜,樊雅婷,等.猪流行性腹泻病毒变异株的鉴定和分子特征分析[J].中国畜牧兽医,2015,42(7):1654-1660.ZHANG Z,CHENG X N,FAN Y T,et al.Identification and molecular characteristics analysis of porcine epidemic diarrhea virus variants[J].China Animal Husbandry&Veterinary Medicine,2015,42(7):1654-1660.(in Chinese)
[21]毛黎红,张双翔,周碧君,等.6株猪流行性腹泻病毒贵州株S基因序列分析[J].中国畜牧兽医,2017,44(6):1621-1629.MAO L H,ZHANG S X,ZHOU B J,et al.Sequence analysis of Sgene of 6strains of porcine epidemic diarrhea virus from Guizhou[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1621-1629.(in Chinese)
[22]刘孝珍,陈建飞,时洪艳,等.2011年猪流行性腹泻病毒的遗传变异分析[J].中国预防兽医学报,2012,34(3):180-183.LIU X Z,CHEN J F,SHI H Y,et al.Genetic variation analysis of porcine epidemic diarrhea virus isolated in 2011[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(3):180-183.(in Chinese)