2013年-2014年江西地区PRRSV分离株NSP2及ORF5基因的遗传变异分析
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摘要
为研究江西地区猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因的变异情况及NSP2基因的结构特征,采用RT-PCR方法扩增了12份江西地区猪场疑似患PRRS的猪肺脏样品中的ORF5全序列和NSP2部分序列,应用DNAStar和Mega 6.0等软件对所得序列进行同源性比对及遗传变异分析。12株PRRSV ORF5核苷酸同源性为83.7%~99.8%,氨基酸同源性为82.1%~99.5%;与参考毒株JXA1、VR-2332和LV的核苷酸同源性分别为84.9%~99.7%、85.2%~91.0%和62.4%~64.8%。对阳性病料进行了NSP2基因部分序列的扩增,测序结果显示12株PRRSV均属于美洲型毒株,12株PRRSV的NSP2部分序列均存在30个氨基酸的不连续缺失,与高致病性PRRSV有相同的缺失特征。12株PRRSV的ORF5遗传进化树分析显示,10株与高致病性PRRSV处在同一进化分支,进一步说明高致病性PRRSV已成为江西地区的优势流行毒株。
To study the genetic varations of ORF5 and NSP2genes of porcine reproductive and respiratory syndrome virus(PRRSV)isolates in Jiangxi province from 2013 to 2014,the complete ORF5 gene and subsequence of NSP2 gene of PRRSV were amplified by RT-PCR from lung samples of swine in Jiangxi.Sequence comparison and genetic variation analysis were conducted by DNAStar and Mega 6.0.The results showed that the homologies of ORF5 genes among the 12 PRRSV isolates were 83.7% to 99.8% and 82.1% to 99.5%in nucleotide and amino acid sequences,respectively.Compared to the reference strains JXA1,VR-2332 and LV,the homologies were 84.9% to 99.7%,85.2% to 91.0% and 62.4% to 64.8%,respectively.Nsp2 sequences were amplified from positive samples and compared with the other published sequences,it indicated that 12 PRRSV isolates from Jiangxi belonged to Northern American genotype,12 isolates had30amino acids discontinuous deletions in NSP2 protein which showed a typical structure as previously reported for highly pathogenic PRRSV.The phylogenetic tree of ORF5 of 12isolates indicated that 10 PRRSV isolates were in the same branch with the highly pathogenic PRRSV,whichshowed that the highly pathogenic PRRSV had become the dominant PRRSV epidemic strain in Jiangxi.
To study the genetic varations of ORF5 and NSP2genes of porcine reproductive and respiratory syndrome virus(PRRSV)isolates in Jiangxi province from 2013 to 2014,the complete ORF5 gene and subsequence of NSP2 gene of PRRSV were amplified by RT-PCR from lung samples of swine in Jiangxi.Sequence comparison and genetic variation analysis were conducted by DNAStar and Mega 6.0.The results showed that the homologies of ORF5 genes among the 12 PRRSV isolates were 83.7% to 99.8% and 82.1% to 99.5%in nucleotide and amino acid sequences,respectively.Compared to the reference strains JXA1,VR-2332 and LV,the homologies were 84.9% to 99.7%,85.2% to 91.0% and 62.4% to 64.8%,respectively.Nsp2 sequences were amplified from positive samples and compared with the other published sequences,it indicated that 12 PRRSV isolates from Jiangxi belonged to Northern American genotype,12 isolates had30amino acids discontinuous deletions in NSP2 protein which showed a typical structure as previously reported for highly pathogenic PRRSV.The phylogenetic tree of ORF5 of 12isolates indicated that 10 PRRSV isolates were in the same branch with the highly pathogenic PRRSV,whichshowed that the highly pathogenic PRRSV had become the dominant PRRSV epidemic strain in Jiangxi.
引文
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[12]时建立,李俊,徐绍建,等.两株高致病性PRRSV ORF6和ORF7基因克隆及序列分析[J].中国畜牧兽医,2008,35(1):39-42.
[13]Fang Y,Christopher-Henning J,Brown E,et al.Development of genetic markers in the non-structural protein 2region of a US type 1porcine reproductive and respiratory syndrome virus:Implications for future recombinant marker vaccine development[J].J Gen Virol,2008,89(12):3086-3096.
[14]冷雪,李真光,王凤雪,等.高致病性猪繁殖与呼吸综合征病毒ORF5基因的变异分析[J].吉林农业大学学报,2012,34(4):443-448.
[2]曹宗喜,潘全会,林哲敏,等.PRRSV广东株XH-GD的分离及其NSP2、ORF3、ORF5的序列分析[J].广东农业科学,2013,40(12):159-163.
[3]郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国畜禽传染病,1996,12(2):1-4.
[4]贾怀杰,陈国华,房永祥,等.猪繁殖与呼吸综合征病毒JX0708株的分离鉴定及其Nsp2基因的遗传变异分析[J].中国畜牧兽医,2010,37(10):58-62.
[5]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.
[6]Jiang Y,Xiao S,Fang L,et al.DNA vaccines co-expressing GP5and M proteins of porcine reproductive and respiratory syndrome virus(PRRSV)display enhanced immunogenicity[J].Vaccine,2006,24(1):2869-2879.
[7]Roques E,Girard A,St-Louis M C,et al.Immunogenic and protective properties of GP5and M structural proteins of porcine reproductive and respiratory syndrome virus expressed from replicating but nondisseminating adenovectors[J].Vet Res,2013,44(1):17.
[8]Cao Z X,Jiao P R,Huang Y M,et al.Genetic diversity analysis of porcine reproductive and respiratory syndrome virus isolated in South China from 2007to2009based on ORF5gene[J].Acta Veterinaria Hungarica,2012,60(1):157-164.
[9]Liu J K,Wei C H,Yang X Y,et al.Genetic diversity and evolutionary characterization of Chinese porcine reproductive and respiratory syndrome viruses based on NSP2and ORF5[J].Arch Virol,2013,158(3):1811-1816.
[10]Li Z,Leng X,Qi Q,et al.Complete genome sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus NM1strain from Northern China[J].Virol,2012,86(24):13863-13864.
[11]Nielsen H S,Oleksiewicz M B,Forsberg R,et al.Reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations[J].Gen Virol,2001,82(6):1263-1272.
[12]时建立,李俊,徐绍建,等.两株高致病性PRRSV ORF6和ORF7基因克隆及序列分析[J].中国畜牧兽医,2008,35(1):39-42.
[13]Fang Y,Christopher-Henning J,Brown E,et al.Development of genetic markers in the non-structural protein 2region of a US type 1porcine reproductive and respiratory syndrome virus:Implications for future recombinant marker vaccine development[J].J Gen Virol,2008,89(12):3086-3096.
[14]冷雪,李真光,王凤雪,等.高致病性猪繁殖与呼吸综合征病毒ORF5基因的变异分析[J].吉林农业大学学报,2012,34(4):443-448.