猪圆环病毒3型全基因克隆及遗传演化分析
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摘要
辽宁地区某猪场发现疑似断奶仔猪多系统综合征(PMWS),经临床症状、病理变化和PCR检测,确诊为猪圆环病毒3型(PCV3)感染;根据Gen Bank中收录的PCV3基因序列,设计2对特异性引物,从患有PMWS的仔猪内脏组织中扩增PCV3全基因序列,克隆到p MD18-T载体中,经测序、拼接获得PCV3全长c DNA序列,利用DNAStar和DNAman生物软件对全基因组进行遗传变异分析。结果显示:PCV3毒株基因组全长2 000 bp,命名为CN/Liaoning-2017 (简称LN株)(GenBank登录号:MH177453. 1),基因组有3个开放阅读框(ORF),其中2个ORF编码的蛋白与圆环病毒的复制相关蛋白(Rep)和衣壳蛋白(Cap)同源。Cap基因大小为645 bp,编码214个氨基酸。PCV3国内株可分为3大分支(3a簇、3b簇与3c簇),LN株处于3a簇分支中,与湖北株(KY354039. 1)同源性最高,达到99. 7%; LN株与PCV3a簇代表株(PCV3-USMN2016,PCV3-US-MO2015)同源性在98. 8%～99. 2%之间,与PCV3b簇代表株(PCV3-US-SD2016)的同源性为98. 8%,与PCV3c簇代表株(PCV3-China-GD-2016)同源性为98. 7%。Cap蛋白氨基酸位点分析显示,LN株在第68位发生突变,与其他代表株都不相同。以上试验结果表明,成功从患有PMWS的仔猪体内克隆了PCV3全基因,并完成了序列分析。LN株是辽宁省首次发现的PCV3毒株,这些数据将为研究PCV3的遗传变异和流行病学特征提供分子生物学依据。
A pig farm in Liaoning Province was suspected of incidence of multi-system syndrome( PMWS) in weaned piglets. Clinical symptoms,pathological changes and PCR detection showed that porcine circovirus type 3 caaused the incidence. According to the PCV3 gene sequence contained in Gen Bank,two pairs of specific primers were designed. The whole PCV3 gene sequence was amplified from the visceral tissues of the piglets with PMWS,was cloned into the p MD18-T vector,sequenced and spliced to obtain a PCV3 full-length c DNA sequence. Genetic variation analysis was performed of the whole genomes using the biologic software of DNAStar and DNAMAN biologic software. The results showed that the PCV3 strain was isolated and the genome was 2 000 bp in length,named CN/Liaoning-2017( LN strain)( Gen Bank accession number: MH177453). The genome had three open reading frames( ORF),two of which encoded proteins that were homologous to the replication-associated protein( Rep) and the capsid protein( Cap) of the circovirus. The encoded structural protein Cap was645 bp in length and it encoded 214 amino acids. The PCV3 domestic strain could be divided into three major branches( the 3a cluster,3b cluster and 3c cluster). The LN strain was in the 3a cluster branch, and possessed the highest homology with the Hubei strain( KY354039. 1),up to 99. 7%; with the PCV3 a cluster representative strains,PCV3-US-MN2016 and PCV3-US-MO2015,between98. 8% and 99. 2%; with the PCV3 b cluster representative strain,PCV3-US-SD2016,at 98. 8%,and and the PCV3 c cluster representative strain,PCV3-China-GD-2016 at 98. 7%. The amino acid site analysis of the Cap protein showed that the LN strain was mutated at position68,which was different from the other representative strains. The LN strain was the first PCV3 strain found in Liaoning Province. The above results indicated that the PCV3 gene was successfully cloned from piglets with PMWS and sequence analysis was completed. These data might provide molecular biological basis for research on the genetic variation and epidemiological characteristics of the novel virus,PCV3.
A pig farm in Liaoning Province was suspected of incidence of multi-system syndrome( PMWS) in weaned piglets. Clinical symptoms,pathological changes and PCR detection showed that porcine circovirus type 3 caaused the incidence. According to the PCV3 gene sequence contained in Gen Bank,two pairs of specific primers were designed. The whole PCV3 gene sequence was amplified from the visceral tissues of the piglets with PMWS,was cloned into the p MD18-T vector,sequenced and spliced to obtain a PCV3 full-length c DNA sequence. Genetic variation analysis was performed of the whole genomes using the biologic software of DNAStar and DNAMAN biologic software. The results showed that the PCV3 strain was isolated and the genome was 2 000 bp in length,named CN/Liaoning-2017( LN strain)( Gen Bank accession number: MH177453). The genome had three open reading frames( ORF),two of which encoded proteins that were homologous to the replication-associated protein( Rep) and the capsid protein( Cap) of the circovirus. The encoded structural protein Cap was645 bp in length and it encoded 214 amino acids. The PCV3 domestic strain could be divided into three major branches( the 3a cluster,3b cluster and 3c cluster). The LN strain was in the 3a cluster branch, and possessed the highest homology with the Hubei strain( KY354039. 1),up to 99. 7%; with the PCV3 a cluster representative strains,PCV3-US-MN2016 and PCV3-US-MO2015,between98. 8% and 99. 2%; with the PCV3 b cluster representative strain,PCV3-US-SD2016,at 98. 8%,and and the PCV3 c cluster representative strain,PCV3-China-GD-2016 at 98. 7%. The amino acid site analysis of the Cap protein showed that the LN strain was mutated at position68,which was different from the other representative strains. The LN strain was the first PCV3 strain found in Liaoning Province. The above results indicated that the PCV3 gene was successfully cloned from piglets with PMWS and sequence analysis was completed. These data might provide molecular biological basis for research on the genetic variation and epidemiological characteristics of the novel virus,PCV3.
引文
[1]张永宁,梅琳,张舟,等.猪圆环病毒3型研究进展[J].东北农业大学学报,2017,48(9):89-96.
[2]Sun J,Wei L,Lu Z,et al.Retrospective study of porcine circovirus3 infection in China[J].Transbound Emerg Dis,2018,65(3):607-613.
[3]刘远佳,余伟权,李刚,等.新型圆环病毒-PCV3研究进展及展望(2017)[J].猪业科学,2017,34(5):105-106.
[4]Li X,Qiao M,Sun M,et al.A duplex Real-Time PCR assay for the simultaneous detection of porcine circovirus 2 and circovirus 3[J].Virol Sin,2018,33(2):181-186.
[5]湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084.
[6]谢永兴,黄立平.猪圆环病毒3型Cap蛋白表达及单克隆抗体的制备与鉴定[C]//中国免疫学会.第十二届全国免疫学学术大会摘要汇编.中国天津:2017:450.
[7]靳玉芬.检测猪圆环病毒2型DNA的套式PCR方法的建立及应用[J].安徽农业科学,2007(7):1950-1951.
[8]韩勇,李文刚,项朝荣,等.猪伪狂犬病病毒的分离和鉴定[J].动物医学进展,2007(3):43-45.
[9]宋勤叶,马兴树,陈春花.猪断奶后多系统衰竭综合征PCR检测方法的建立[C].京津冀畜牧兽医科技创新交流会暨新思想、新观点、新方法论坛.河北保定,2008:467-471.
[10]徐朋丽,张鸿鑫,张宇,等.猪圆环病毒3型PCR检测方法的建立及应用[J].中国预防兽医学报,2017,39(9):727-730.
[11]庄金秋,梅建国,王艳,等.猪圆环病毒3型的病原学特征及检测技术研究进展[J].中国动物检疫,2018,35(2):59-61,101.
[12]Kedkovid R,Woonwong Y,Arunorat J,et al.Porcine circovirus type 3(PCV3)shedding in sow colostrum[J].Vet Microbiol,2018,220:12-17.
[13]Kedkovid R,Woonwong Y,Arunorat J,et al.Porcine circovirus type 3(PCV3)infection in grower pigs from a Thai farm suffering from porcine respiratory disease complex(PRDC)[J].Vet Microbiol,2018,215:71-76.
[14]Zhang L,Luo Y,Liang L,et al.Phylogenetic analysis of porcine circovirus type 3 and porcine circovirus type 2 in China detected by duplex nanoparticle-assisted PCR[J].Infect Genet Evol,2018,60:1-6.
[15]张忠国.近年来PCV3在国内外的研究进展[J].畜牧兽医科技信息,2017(12):17-18.
[2]Sun J,Wei L,Lu Z,et al.Retrospective study of porcine circovirus3 infection in China[J].Transbound Emerg Dis,2018,65(3):607-613.
[3]刘远佳,余伟权,李刚,等.新型圆环病毒-PCV3研究进展及展望(2017)[J].猪业科学,2017,34(5):105-106.
[4]Li X,Qiao M,Sun M,et al.A duplex Real-Time PCR assay for the simultaneous detection of porcine circovirus 2 and circovirus 3[J].Virol Sin,2018,33(2):181-186.
[5]湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084.
[6]谢永兴,黄立平.猪圆环病毒3型Cap蛋白表达及单克隆抗体的制备与鉴定[C]//中国免疫学会.第十二届全国免疫学学术大会摘要汇编.中国天津:2017:450.
[7]靳玉芬.检测猪圆环病毒2型DNA的套式PCR方法的建立及应用[J].安徽农业科学,2007(7):1950-1951.
[8]韩勇,李文刚,项朝荣,等.猪伪狂犬病病毒的分离和鉴定[J].动物医学进展,2007(3):43-45.
[9]宋勤叶,马兴树,陈春花.猪断奶后多系统衰竭综合征PCR检测方法的建立[C].京津冀畜牧兽医科技创新交流会暨新思想、新观点、新方法论坛.河北保定,2008:467-471.
[10]徐朋丽,张鸿鑫,张宇,等.猪圆环病毒3型PCR检测方法的建立及应用[J].中国预防兽医学报,2017,39(9):727-730.
[11]庄金秋,梅建国,王艳,等.猪圆环病毒3型的病原学特征及检测技术研究进展[J].中国动物检疫,2018,35(2):59-61,101.
[12]Kedkovid R,Woonwong Y,Arunorat J,et al.Porcine circovirus type 3(PCV3)shedding in sow colostrum[J].Vet Microbiol,2018,220:12-17.
[13]Kedkovid R,Woonwong Y,Arunorat J,et al.Porcine circovirus type 3(PCV3)infection in grower pigs from a Thai farm suffering from porcine respiratory disease complex(PRDC)[J].Vet Microbiol,2018,215:71-76.
[14]Zhang L,Luo Y,Liang L,et al.Phylogenetic analysis of porcine circovirus type 3 and porcine circovirus type 2 in China detected by duplex nanoparticle-assisted PCR[J].Infect Genet Evol,2018,60:1-6.
[15]张忠国.近年来PCV3在国内外的研究进展[J].畜牧兽医科技信息,2017(12):17-18.