PRRSV广东株XH-GD的分离及其NSP2、ORF3、ORF5的序列分析
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摘要
将采集的广东某猪场疑似猪高热症病料处理后接种于Marc-145细胞,出现细胞聚集、固缩、脱落等特异性细胞病变。根据GenBank中猪繁殖与呼吸综合征病毒(PRRSV)的基因序列设计并合成针对NSP2、ORF3和ORF5基因的引物,扩增目的基因并测序分析。结果表明:分离毒株XH-GD株PRRRSV属于美洲型,且NSP2基因发生30个氨基酸的不连续缺失;XH-GD的NSP2、ORF3和ORF5基因与HUB1、NX06、BJsy06、VR-2332、HB-1(sh)2002、HB-2(sh)2002、CH-la和RespPRRS MLV等毒株等位基因的核苷酸同源性分别为83.3%-98.9%、88.6%-99.2%、88.1%-99.2%,推导的氨基酸同源性分别为76.8%-98.3%、83.7%-98.8%、88.1%-99.2%;而与LV的等位基因核苷酸同源性分别为52.9%、65.0%和64.3%,推导的氨基酸同源性分别为31.3%、57.5%和58.9%。基因系统发育树分析表明,XH-GD与NX06、BJsy06株的亲缘关系很近,与HUB1、NX06、BJsy06、VR-2332、HB-1(sh)2002、HB-2(sh)2002、CH-la和RespPRRS MLV亲缘关系较近,与LV毒株处于不同的分支。
The samples collected from Guangdong province were inoculated to Marc-145 cell,the PRRSV specific cytopathogenic effect(CPE) was observed and the isolates named XH-GD obtained.After reverse transcription polymerase chain reaction,the amplified NSP2、ORF3 and ORF5 fragments were analyzed by sequence comparison.The results demonstrated that XH-GD belonged to the North American genotype and its NSP2 had 30 amino acids deletion.The NSP2,ORF3 and ORF5 of XH-GD shared 83.3%-98.9%,88.6%- 99.2% and 88.1%-99.2% of nucleotide similarity,and 76.8%-98.3%,83.7%-98.8% and 88.1%-99.2% of deduced amino acid similarity,with that of HUB1,NX06,BJsy06,VR-2332,HB-1(sh)2002,HB-2(sh)2002,CH-la and RespPRRS MLV respectively;and shared only 52.9%,65.0% and 64.3% of nucleotide similarity,and 31.3%,57.5% and 58.9% of deduced amino acid similarity with that of LV respectively.The phylogenetic tree revealed XH-GD had closer relationship with HUB1,NX06,BJsy06,VR-2332,HB-1(sh)2002,HB-2(sh)2002,CH-la and RespPRRS MLV strains than that of LV strain.
The samples collected from Guangdong province were inoculated to Marc-145 cell,the PRRSV specific cytopathogenic effect(CPE) was observed and the isolates named XH-GD obtained.After reverse transcription polymerase chain reaction,the amplified NSP2、ORF3 and ORF5 fragments were analyzed by sequence comparison.The results demonstrated that XH-GD belonged to the North American genotype and its NSP2 had 30 amino acids deletion.The NSP2,ORF3 and ORF5 of XH-GD shared 83.3%-98.9%,88.6%- 99.2% and 88.1%-99.2% of nucleotide similarity,and 76.8%-98.3%,83.7%-98.8% and 88.1%-99.2% of deduced amino acid similarity,with that of HUB1,NX06,BJsy06,VR-2332,HB-1(sh)2002,HB-2(sh)2002,CH-la and RespPRRS MLV respectively;and shared only 52.9%,65.0% and 64.3% of nucleotide similarity,and 31.3%,57.5% and 58.9% of deduced amino acid similarity with that of LV respectively.The phylogenetic tree revealed XH-GD had closer relationship with HUB1,NX06,BJsy06,VR-2332,HB-1(sh)2002,HB-2(sh)2002,CH-la and RespPRRS MLV strains than that of LV strain.
引文
[1]Wensvoort G,Terpstra C,Pol J M A,et al.Mystery swinedisease in the Netherlands:The isolation of Lelystad virus[J].Vet Microbiol,1991,13:121-130.
[2]Snijder E J,Meulenberg J M.The molecular biology ofarteriviruses[J].J Gen Virol,1998,271:4684-4691.
[3]童光志,周艳君,都晓芳,等.高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流行病学分析[J].中国预防兽医学报,2007,29(5):323-327.
[4]赵红妮,许信刚,童德文,等.表达PRRSVGP3蛋白减毒鼠伤寒沙门氏菌疫苗株的构建[J].西北农业学报,2011,20(10):6-11.
[5]Jiang Y,Xiao S,Fang L,et al.DNA vaccines co-expressingGP5 and M proteins of porcine reproductive and respiratorysyndrome virus(PRRSV)display enhanced immunogenicity[J].Vaccine,2006,24:2869-2879.
[6]Roques E,Girard A,St-Louis M C,et al.Immunogenic andprotective properties of GP5 and M structural proteins ofporcine reproductive and respiratory syndrome virus expressedfrom replicating but nondisseminating adenovectors[J].Vet Res,2013,44(1):17.
[7]Cao Z X,Jiao P R,Huang Y M,et al.Genetic diversityanalysis of porcine reproductive and respiratory syndrome virusisolated in south china from 2007 to 2009 based on ORF5gene[J].Acta Veterinaria Hungarica,2012,60(1):157-164.
[8]Stadejek T,Stankevicius A,Murtaugh M P,et al.Molecularevolution of PRRSV in Europe:Current state of play[J].VetMicrobiol,2013,165(1-2):21-28.
[9]Liu J K,Wei C H,Yang X Y,et al.Genetic diversity andevolutionary characterization of Chinese porcine reproductiveand respiratory syndrome viruses based on NSP2 and ORF5[J].Arch Virol,2013.DOI:10.1007/s00705-013-1669-8.
[10]Li Z,Leng X,Qi Q,et al.Complete genome sequence of ahighly pathogenic porcine reproductive and respiratory syndromevirus NM1 strain from northern China[J].J Virol,2012,86(24):13863-13864.
[11]王文华,秦宏阳,曹宗喜,等.猪繁殖与呼吸综合征病毒广州株的分离鉴定与全基因序列分析[J].黑龙江畜牧兽医,2011(11):101-103.
[12]曹宗喜,潘全会,张原江,等.猪繁殖与呼吸综合征病毒各结构蛋白的融合表达[J].中国预防兽医学报,2009,31(2):85-88.
[13]Suárez P,Dlaz-Guerra M,Prieto C,et al.Open reading frame5 of porcine reproductive and respiratory syndrome virus as acause of virus-induced apoptosis[J].J Virol,1996,70:2876-2882.
[14]Tian K,Yu X,Zhao T,et al.Emergence of fatal PRRSVvariants:unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmark[J].Plos one,2007,2(6):526.
[15]Tong G Z,Zhou Y J,Hao X F,et al.Highly pathogenic porcinereproductive and respiratory syndrome,China[J].Emerg Infect Dis,2007,13(9):1434-1435.
[2]Snijder E J,Meulenberg J M.The molecular biology ofarteriviruses[J].J Gen Virol,1998,271:4684-4691.
[3]童光志,周艳君,都晓芳,等.高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流行病学分析[J].中国预防兽医学报,2007,29(5):323-327.
[4]赵红妮,许信刚,童德文,等.表达PRRSVGP3蛋白减毒鼠伤寒沙门氏菌疫苗株的构建[J].西北农业学报,2011,20(10):6-11.
[5]Jiang Y,Xiao S,Fang L,et al.DNA vaccines co-expressingGP5 and M proteins of porcine reproductive and respiratorysyndrome virus(PRRSV)display enhanced immunogenicity[J].Vaccine,2006,24:2869-2879.
[6]Roques E,Girard A,St-Louis M C,et al.Immunogenic andprotective properties of GP5 and M structural proteins ofporcine reproductive and respiratory syndrome virus expressedfrom replicating but nondisseminating adenovectors[J].Vet Res,2013,44(1):17.
[7]Cao Z X,Jiao P R,Huang Y M,et al.Genetic diversityanalysis of porcine reproductive and respiratory syndrome virusisolated in south china from 2007 to 2009 based on ORF5gene[J].Acta Veterinaria Hungarica,2012,60(1):157-164.
[8]Stadejek T,Stankevicius A,Murtaugh M P,et al.Molecularevolution of PRRSV in Europe:Current state of play[J].VetMicrobiol,2013,165(1-2):21-28.
[9]Liu J K,Wei C H,Yang X Y,et al.Genetic diversity andevolutionary characterization of Chinese porcine reproductiveand respiratory syndrome viruses based on NSP2 and ORF5[J].Arch Virol,2013.DOI:10.1007/s00705-013-1669-8.
[10]Li Z,Leng X,Qi Q,et al.Complete genome sequence of ahighly pathogenic porcine reproductive and respiratory syndromevirus NM1 strain from northern China[J].J Virol,2012,86(24):13863-13864.
[11]王文华,秦宏阳,曹宗喜,等.猪繁殖与呼吸综合征病毒广州株的分离鉴定与全基因序列分析[J].黑龙江畜牧兽医,2011(11):101-103.
[12]曹宗喜,潘全会,张原江,等.猪繁殖与呼吸综合征病毒各结构蛋白的融合表达[J].中国预防兽医学报,2009,31(2):85-88.
[13]Suárez P,Dlaz-Guerra M,Prieto C,et al.Open reading frame5 of porcine reproductive and respiratory syndrome virus as acause of virus-induced apoptosis[J].J Virol,1996,70:2876-2882.
[14]Tian K,Yu X,Zhao T,et al.Emergence of fatal PRRSVvariants:unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmark[J].Plos one,2007,2(6):526.
[15]Tong G Z,Zhou Y J,Hao X F,et al.Highly pathogenic porcinereproductive and respiratory syndrome,China[J].Emerg Infect Dis,2007,13(9):1434-1435.