广西地区猪圆环病毒2型和猪圆环病毒3型基因型监测及遗传进化分析
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摘要
为了明确猪圆环病毒2型(Porcine circovirus type 2,PCV-2)和猪圆环病毒3型(Porcine circovirus type 3,PCV-3)在广西地区的分子流行病学情况,试验通过PCR方法对2017年来自广西不同地区的80份猪肺脏样品进行PCV-2和PCV-3核酸检测,并对PCV-2和PCV-3的阳性样品进行ORF2基因的扩增和测序,同时利用生物信息学软件DNAStar和MEGA 6.06对PCV-2和PCV-3的ORF2基因进行同源性分析和构建遗传进化树。结果表明:2017年广西地区PCV-2和PCV-3的总感染率分别为43.8%(35/80)和33.8%(27/80),PCV-2和PCV-3的共感染率为16.3%(13/80)。试验获得的19个PCV-2 ORF2基因序列之间的核苷酸同源性为93.6%～99.9%,对应氨基酸同源性为93.1%～100%;19个PCV-2 ORF2基因序列与国内外参考毒株ORF2基因序列的核苷酸同源性为82.8%～99.9%,对应氨基酸同源性为83.3%～99.6%。试验获得的2个PCV-3 ORF2基因序列之间的核苷酸同源性为99.8%,对应氨基酸同源性为100%;2个PCV-3 ORF2基因序列与国内外参考毒株ORF2基因序列的核苷酸同源性为97.7%～99.8%,对应氨基酸同源性为96.2%～100%。获得的19个PCV-2 ORF-2基因序列中,12个为PCV-2b亚型,7个为PCV-2d亚型;获得的2个PCV-3 ORF2基因序列为PCV-3a亚型,与丹麦和中国湖南分离得到的PCV-3亲缘关系较近。说明PCV-2和PCV-3在广西地区猪群中感染率很高,PCV-2感染主要以PCV-2b亚型为主,PCV-3之间高度保守,分布无地域性规律。
To understand the molecular epidemiology of PCV-2 and PCV-3 in Guangxi, a total of 80 lung samples from Guangxi in 2017 were detected for PCV-2 and PCV-3 by using PCR method. The positive samples of PCV-2 and PCV-3 were amplified and sequenced for ORF2 gene. Homology analysis and genetic tree construction of ORF2 genes of PCV-2 and PCV-3 were conducted using bioinformatics software DNAStar and MEGA 6.06.The result showed that the total infection rate of PCV-2 and PCV-3 were 43.8%(35/80) and 33.8%(27/80) respectively, co-infection rate of PCV-2 and PCV-3 was 16.3%(13/80). The nucleotide homology was 93.6%-99.9%, and the corresponding amino acid homology were 93.1%-100%, respectively. The nucleotide homology of ORF2 gene sequences of 19 ORF-2 strains between domestic and foreign reference strains B were 82.8%-99.9%, and the homology of corresponding amino acids were 83.3%-99.6%.The nucleotide homology between the ORF2 gene sequences of the two PCV-3 strains was 99.8%, and the homology of the corresponding amino acids was 100%.The nucleotide homology between ORF2 gene sequence of two PCV-3 strains and ORF2 gene sequence of foreign reference strains were 97.7%-99.8%, and the homology of corresponding amino acids were 96.2%-100%.Of the 19 PCV-2 ORF-2 gene sequences obtained, 12 were PCV-2 b subtypes and 7 were PCV-2 d subtypes. The two PCV-3 ORF-2 gene sequences obtained were PCV-3 a subtypes, which were closely related to PCV-3 isolated from Denmark and Hunan, China. It indicated that PCV-2 and PCV-3 had a high infection rate in pigs farms in Guangxi. PCV-2 infection was mainly caused by PCV-2 b subtype, and PCV-3 strains were highly conserved and distributed without regional rules.
To understand the molecular epidemiology of PCV-2 and PCV-3 in Guangxi, a total of 80 lung samples from Guangxi in 2017 were detected for PCV-2 and PCV-3 by using PCR method. The positive samples of PCV-2 and PCV-3 were amplified and sequenced for ORF2 gene. Homology analysis and genetic tree construction of ORF2 genes of PCV-2 and PCV-3 were conducted using bioinformatics software DNAStar and MEGA 6.06.The result showed that the total infection rate of PCV-2 and PCV-3 were 43.8%(35/80) and 33.8%(27/80) respectively, co-infection rate of PCV-2 and PCV-3 was 16.3%(13/80). The nucleotide homology was 93.6%-99.9%, and the corresponding amino acid homology were 93.1%-100%, respectively. The nucleotide homology of ORF2 gene sequences of 19 ORF-2 strains between domestic and foreign reference strains B were 82.8%-99.9%, and the homology of corresponding amino acids were 83.3%-99.6%.The nucleotide homology between the ORF2 gene sequences of the two PCV-3 strains was 99.8%, and the homology of the corresponding amino acids was 100%.The nucleotide homology between ORF2 gene sequence of two PCV-3 strains and ORF2 gene sequence of foreign reference strains were 97.7%-99.8%, and the homology of corresponding amino acids were 96.2%-100%.Of the 19 PCV-2 ORF-2 gene sequences obtained, 12 were PCV-2 b subtypes and 7 were PCV-2 d subtypes. The two PCV-3 ORF-2 gene sequences obtained were PCV-3 a subtypes, which were closely related to PCV-3 isolated from Denmark and Hunan, China. It indicated that PCV-2 and PCV-3 had a high infection rate in pigs farms in Guangxi. PCV-2 infection was mainly caused by PCV-2 b subtype, and PCV-3 strains were highly conserved and distributed without regional rules.
引文
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[17] POGRANICHNYY R M, YOON K J, HARMS P A, et al. Characterization of immune response of young pigs to Porcine circovirus type 2 infection [J]. Viral Immunol, 2000, 13(2): 143-153.
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[19] 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.
[20] SEGALES J, OLVERA A, GRAU-ROMA L, et al. PCV-2 genotype definition and nomenclature [J]. Vet Rec, 2008, 162(26): 867-868.
[21] 王苗苗,王婷婷,朱建国.华东地区猪圆环病毒2型基因型鉴别及ORF遗传变异分析[J].畜牧与兽医,2018,50(7):92-97.
[22] SUN J, WEI L, LU Z, et al. Retrospective study of Porcine circovirus 3 infection in China [J].Transbound Emerg Dis, 2018, 65(3): 607-613.
[23] SARAIVA G L, VIDIGAL P M P, FIETTO J L R, et al. Evolutionary analysis of Porcine circovirus 3 (PCV3) indicates an ancient origin for its current strains and a worldwide dispersion [J]. Virus Genes, 2018, 54(3): 376-384.
[2] SAHA D, LEFEBVRE D J, DUCATELLE R, et al. Outcome of experimental Porcine circovirus type 1 infections in mid-gestational porcine foetuses [J]. BMC Vet Res, 2011, 7:64.
[3] BAEKBO P, KRISTENSEN C S, LARSEN L E. Porcine circovirus diseases: a review of PMWS [J]. Transbound Emerg Dis, 2012, 59(Suppl 1):60-67.
[4] OPRIESSNIG T, MENG X J, HALBUR P G. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies [J]. J Vet Diagn Invest, 2007, 19(6): 591-615.
[5] PALINSKI R, PINEYRO P, SHANG P, et al. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure [J]. J Virol, 2017, 91(1): e01879-16.
[6] CHEN G H, MAI K J, ZHOU L, et al. Detection and genome sequencing of Porcine circovirus 3 in neonatal pigs with congenital tremors in South China [J]. Transbound Emerg Dis, 2017, 64(6): 1650-1654.
[7] 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.
[8] OLVERA A, CORTEY M, SEGALéS J. Molecular evolution of Porcine circovirus type 2 genomes: phylogeny and clonality [J]. Virology, 2007, 357(2): 175-185.
[9] XIAO C T, HALBUR P G, OPRIESSNIG T. Global molecular genetic analysis of Porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d [J]. J Gen Virol, 2015, 96(Pt7): 1830-1841.
[10] KWON T, LEE D U, YOO S J, et al. Genotypic diversity of Porcine circovirus type 2 (PCV2) and genotype shift to PCV2d in Korean pig population [J]. Virus Res, 2017, 228:24-29.
[11] DUPONT K, NIELSEN E O, BAEKBO P, et al. Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time [J]. Vet Microbiol, 2008, 128(1/2): 56-64.
[12] DAVIES B, WANG X, DVORAK C M, et al. Diagnostic phylogenetics reveals a new Porcine circovirus 2 cluster [J]. Virus Res, 2016, 217:32-37.
[13] KU X, CHEN F, LI P, et al. Identification and genetic characterization of Porcine circovirus type 3 in China [J]. Transbound Emerg Dis, 2017, 64(3): 703-708.
[14] KWON T, YOO S J, PARK C K, et al. Prevalence of novel Porcine circovirus 3 in Korean pig populations [J]. Vet Microbiol, 2017, 207:178-180.
[15] TOCHETTO C, LIMA D A, VARELA A P M, et al. Full-genome dequence of Porcine circovirus type 3 recovered from serum of sows with stillbirths in Brazil [J]. Transbound Emerg Dis, 2018, 65(1): 5-9.
[16] FRANZO G, LEGNARDI M, HJULSAGER C K, et al. Full-genome sequencing of porcine circovirus 3 field strains from denmark, italy and spain demonstrates a high within-europe genetic heterogeneity [J]. Transbound Emerg Dis, 2018, 65(3): 602-606.
[17] POGRANICHNYY R M, YOON K J, HARMS P A, et al. Characterization of immune response of young pigs to Porcine circovirus type 2 infection [J]. Viral Immunol, 2000, 13(2): 143-153.
[18] JIANG C G, WANG G, TU Y B, et al. Genetic analysis of Porcine circovirus type 2 in China [J]. Arch Virol, 2017, 162(9): 2715-2726.
[19] 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.
[20] SEGALES J, OLVERA A, GRAU-ROMA L, et al. PCV-2 genotype definition and nomenclature [J]. Vet Rec, 2008, 162(26): 867-868.
[21] 王苗苗,王婷婷,朱建国.华东地区猪圆环病毒2型基因型鉴别及ORF遗传变异分析[J].畜牧与兽医,2018,50(7):92-97.
[22] SUN J, WEI L, LU Z, et al. Retrospective study of Porcine circovirus 3 infection in China [J].Transbound Emerg Dis, 2018, 65(3): 607-613.
[23] SARAIVA G L, VIDIGAL P M P, FIETTO J L R, et al. Evolutionary analysis of Porcine circovirus 3 (PCV3) indicates an ancient origin for its current strains and a worldwide dispersion [J]. Virus Genes, 2018, 54(3): 376-384.