猪细环病毒k2型福建株ORF2 基因克隆

doi:10.11924/j.issn.1000-6850.casb15060043

摘要/Abstract

摘要： 为丰富福建省猪细环病毒的分子流行病学数据，本研究根据猪细环病毒1a 型、1b 型和k2 型特异性检测引物对临床顽固性腹泻仔猪病料组织进行PCR检测，结果从1 例病料中检测到猪细环病毒k2 型阳性。并通过设计猪细环病毒k2 型ORF2 基因特异性引物对阳性病料扩增后进行克隆测序，获得猪细环病毒k2 型ORF2 基因完整编码区序列。分析发现所克隆的猪细环病毒k2 型福建株ORF2 基因全长为203 bp，编码有67 个氨基酸。本实验株ORF2 基因和猪细环病毒k2 型代表株德国家猪分离株2p 株（GenBank 登录号AY823991）核苷酸同源性高达97.5%；和猪细环病毒1a 型（Sd_TTV31 株）和1b 型（1p 株）代表株核苷酸同源性均低于50.0%，分别为43.9%和47.3%。从遗传进化关系上看，本实验株ORF2 基因和GenBank中猪细环病毒k2 型处于同一遗传进化分支，而猪细环病毒1a 型和猪细环病毒1b型均处于其他遗传分支。本研究首次在福建猪群中检测到猪细环病毒k2型感染。

关键词: 玉米, 玉米, 株型, 密度, 生理性状, 产量, 品质

Abstract: To supplement molecular epidemiology data of porcine Torque teno sus virus in Fujian Province, specific primers targeting the porcine Torque teno sus virus genotype 1a, 1b and k2 were used to test the samples from piglets with refractory diarrhea syndrome. One sample was tested to be positive with porcine Torque teno sus virus k2, then the ORF2 gene fragments were cloned and sequenced. The results showed that the ORF2 gene of the porcine Torque teno sus virus k2 isolated in Fujian was 203 bp in length, coding 67 amino acids. Comparing with other porcine Torque teno sus virus strains, the cloned ORF2 gene sequence of Fujian isolate in this study shared 97.5% homologous sequence with the porcine Torque teno sus virus k2 isolate 2p strain; and shared 43.9% and 47.3% homologous sequence with porcine Torque teno sus virus 1a (Sd_TTV31 strain) and 1b (1p strain), respectively. According to the phylogenetic tree based on the ORF2 gene, the results demonstrated that the porcine Torque teno sus virus k2 isolated in Fujian shared the same branch with porcine Torque teno sus virus k2, while porcine Torque teno sus virus 1a and porcine Torque teno sus virus 1b strains occupied individual phylogenetic branch. All the results showed that the porcine Torque teno sus virus k2 was spreading in Fujian Province.

黄秋宇,陈鹏强,胡崇伟,陈秋勇,林群群,修金生. 猪细环病毒k2型福建株ORF2 基因克隆[J]. 中国农学通报, 2015, 31(35): 18-22.

修金生. Clone of the ORF2 Gene of Porcine Torque teno sus virus k2 in Fujian[J]. Chinese Agricultural Science Bulletin, 2015, 31(35): 18-22.

参考文献

[1]http://ictvonline.org/virusTaxonomy.asp
[2]http://ictvonline.org/proposals/2007.075a-xxV.v4.Anelloviridae
[3]Biagini P, Gallian P, Attoui H, et al. Genetic analysis of full-length genomes and subgenomic sequences of TT virus-like mini virus human isolates [J]. J Gen Virol, 2001, 82(2): 379-383.
[4]Okamoto H, Takahashi M, Nishizawa T, et al. Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias [J]. J Gen Virol, 2002, 83(6): 1291-1297.
[5]Okamoto H, Nishizawa T, Tawara A, et al. Species-specific TT viruses in humans and nonhuman primates and their phylogenetic relatedness [J]. Virology, 2000, 277(2): 368-378.
[6]Zhu C, Shan T, Cui L, et al. Molecular detection and sequence analysis of feline Torque teno virus (TTV) in China [J]. Virus Res, 2011, 156(1-2):13-16.
[7]Burián Z, Szabó H, Székely G, et al. Detection and follow-up of torque teno midi virus ("small anelloviruses") in nasopharyngeal aspirates and three other human body fluids in children [J]. Arch Virol, 2011, 156(9):1537-1541
[8]Zhai SL, Long JX, Wei WK, et al. High prevalence of torque teno sus virus in China and genetic diversity of the 5' non-coding region [J]. Arch Virol, 2013, 158(7):1567-1573.
[9]Biagini, P. Create genus Kappatorquevirus in the family Anelloviridae [J]. ICTV, 2010.005.a-dV.
[10]Biagini, P. Create genus Kappatorquevirus in the family Anelloviridae [J]. ICTV, 2010.004.a-dV.
[11]Lan D, Hua X, Cui L, et al. Sequence analysis of a Torque teno canis virus isolated in China [J]. Virus Res, 2011, 160(1-2): 98-101.
[12]Okamoto H, Nishizawa T, Takahashi M, et al. Genomic and evolutionary characterization of TT virus (TTV) in tupaias and comparison with species-specific TTVs in humans and non-human primates [J]. J Gen Virol, 2001, 82(9): 2041-2050
[13]胡崇伟,陈如敬,陈秋勇,等．猪细环病毒1b型ORF3基因克隆及序列分析[J] ．中国畜牧兽医,2014,41(8)：83-87．
[14]刘建波．猪细环病毒与猪圆环病毒 2型分子生物学特性研究[M]．中国农业科学院博士论文,2014．
[15]王礞礞．猪源TTV：分子流行病学,全长序列分析及感染性分子克隆的构建[M]．中国农业科学院博士论文,2014．
[16]Xie Z, Deng X, Xie L, et al. Full-Genome Sequence of Chicken Anemia Virus Strain GXC060821, Isolated from a Guangxi Sanhuang Chicken [J]. Genome Announc, 2014,2(1): pii: e00040-14.
[17]候军委．猪源TTV转录本的鉴定及荧光定量PCR检测方法的建立[M]．中国农业科学院硕士论文,2011．
[18]赵玲,徐志文,朱玲,等．猪输血性传播病毒2型全基因组的克隆与序列分析[J]．中国兽医科学,2011,41(06)：582-587．
[19]刘建波,刘长明．猪细环病毒流行病学与遗传变异研究进展[J]．动物医学进展,2012,33(3)：89-93．
[20]刘建波．猪输血传播病毒流行病学调查与Cap蛋白表达及感染性克隆构建[M]．中国农业科学院硕士论文,2011．
[21]Huang Y, Ni Y, Dryman B, et al. Multiple infection of porcine Torque teno virus in a single pig and characterization of the full-length genomic sequences of four U.S. prototype PTTV strains: implication for genotyping of PTTV [J]. Virology, 2010, 396(2): 289-297.
[22]孙泉云,沈素芳,汤赛冬,等．Torque Teno 病毒感染的研究进展 [J]．动物医学进展,2009,30(5)：90-93．
[23]Niel C, Diniz-Mendes L, Devalle S. Rolling-circle amplification of Torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup [J]. J Gen Virol, 2005, 86: 1343-1347.
[24]Aramouni M, Segalés J, Cortey M, et al. Age-related tissue distribution of swine Torque teno sus virus 1 and 2 [J]. Vet Microbiol, 2010, 146(3-4): 350-353.
[25]Martínez-Guinó L, Kekarainen T, Segalés J. Evidence of Torque teno virus (TTV) vertical transmission in swine [J]. Theriogenology, 2009, 71(9): 1390-1395.
[26]翟少伦,龙进学,岳城,等．中国部分地区猪细环病毒1型和2型的分子检测[J] ．微生物和感染,2010,5(2)：84-88．
[27]韦建兴,梁兆强,欧阳康,等．猪细环病毒的检测及与多种病原混合感染的调查分析[J] ．中国畜牧兽医,2013,40(10)：197-202．
[28]伍志伟,王红宁,杨鑫,等．中国西南地区猪群中TTV分子流行病学调查[J] ．中国兽医学报,2011,31(6)：779-782．
[29]Cortey M, Macera L, Segalés J, et al. Genetic variability and phylogeny of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) based on complete genomes[J]. Vet Microbiol, 2011, 148(2-4):125-31.
[30]McKeown N, Fenaux M, Halbur P, et al. Molecular characterization of porcine TT virus, an orphan virus, in pigs from six different countries [J]. Vet Microbiol, 2004, 104(1-2): 113-117.
[31]Cadar D, Kiss T, ádám D, et al. Phylogeny, spatio-temporal phylodynamics and evolutionary scenario of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) in wild boars: fast dispersal and high genetic diversity [J]. Vet Microbiol, 2013, 166(1-2): 200-213.
[32]王礞礞,周艳君,陈宗艳,等．我国猪群中TTV的鉴定及其分子流行病学分析[J]．中国预防兽医学报,2009,31(10)：751-755．
[33]陈轶雯．广东TTSVs流行病学调查与TTSV2 ORF1抗原性分析[M]．南京农业大学硕士论文,2013．
[34]彭宜红,彭进,胡烈谱,等．一种新的TTV基因群全基因序列测定及基因结构分析[J]．中华微生物学和免疫学杂志,2003,23(4)：307-310．
[35]刘畅,井申荣．人类输血传播病毒基因型别及流行率的研究进展[J]．中国生物制品学杂志,2012,25(5)：649-652．
[36]邹力．猪细环病毒(TTSuV) 1型和2型PCR检测方法的建立及应用[M]．四川农业大学硕士论文,2012．
[37]李坤,钞安军,王子馨,等．猪细环病毒两种基因型双重 PCR 检测方法的建立[J]．中国预防兽医学报,2013,35(3)：214-217．
[38]Pozzuto T, Mueller B, Meehan B, et al. In utero transmission of porcine torque teno viruses [J]. Vet Microbiol, 2009, 137(3-4): 375-379.
[39]Martinez L, Kekarainen T, Sibila M, et al. Torque teno virus (TTV) is highly prevalent in the European wild boar (Sus scrofa) [J]. Vet Microbiol, 2006, 118(3-4): 223-229.
[40]Kekarainen T, Segalés J. Torque teno virus infection in the pig and its potential role as a model of human infection [J]. Vet J, 2009, 180(2): 163-168.
[41]Huang Y, Ni Y, Dryman B, et al. Multiple infection of porcine Torque teno virus in a single pig and characterization of the full-length genomic sequences of four U.S. prototype PTTV strains: implication for genotyping of PTTV [J]. Virology, 2010, 396(2): 289-297.
[42]Li K, Wang L, Wu Y, et al. Molecular detection and genomic characterization of Torque teno sus virus 1 and 2 from domestic pigs in central China [J]. Virus Genes, 2013, 46(3): 479-486.
[43]Liu J, Guo L, Zhang L, et al. Three new emerging subgroups of Torque teno sus viruses (TTSuVs) and co-infection of TTSuVs with porcine circovirus type 2 in China [J]. Virol J, 2013, 10:189.
[44]Okamoto H. TT viruses in animals [J]. Curr Top Microbiol Immunol, 2009, 331:35-52.
[45]Biagini, P. Classification of TTV and related viruses (anelloviruses) [J]. Curr Top Microbiol Immunol, 2009, 331: 21-33.
[46]Martinez L, Kekarainen T, Sibila M, et al. Torque teno virus (TTV) is highly prevalent in the European wild boar (Sus scrofa) [J]. Vet Microbiol, 2006, 118(3-4): 223-229.
[47]Mei M, Zhu L, Xu Z, et al. Molecular investigation of Torque teno sus virus in geographically distinct porcine breeding herds of Sichuan, China [J]. Virol J, 2013, 10:161.
[48]Niel C, Diniz-Mendes L, Devalle S. Rolling-circle amplification of Torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup [J]. J Gen Virol, 2005, 86: 1343-1347.
[49]Aramouni M, Segalés J, Sibila M, et al. Torque teno sus virus 1 and 2 viral loads in postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) affected pigs [J]. Vet Microbiol, 2011, 153(3-4): 377-381.
[50]Nieto D, Aramouni M, Grau-Roma L, et al. Dynamics of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) DNA loads in serum of healthy and postweaning multisystemic wasting syndrome (PMWS) affected pigs [J]. Vet Microbiol, 2011, 152(3-4): 284-190.
[51]刘建波．猪输血传播病毒流行病学调查与Cap蛋白表达及感染性克隆构建[M]．中国农业科学院硕士论文,2011．
[52]赵玲．猪输血性传播病毒1型和2型PCR检测方法的建立及其全基因组克隆与序列分析[M]．四川农业大学硕士论文,2011．
[53]Wang M, Zhou Y, Zhang Q, et al. Complete Genome Sequences of Highly Divergent Torque Teno Virus Type II from Swine Herds [J]. J Virol, 2012, 86 (22): 12465.
[54]Lee S, Sunyoung S, Jung H, et al. Quantitative detection of porcine Torque teno virus in Porcine circovirus-2-negative and Porcine circovirus-associated disease-affected pigs [J]. J Vet Diagn Invest, 2010, 22(2): 261-264.
[55]Huang Y, Harrall K, Dryman B, et al. Serological profile of torque teno sus virus species 1 (TTSuV1) in pigs and antigenic relationships between two TTSuV1 genotypes (1a and 1b), between two species (TTSuV1 and -2), and between porcine and human anelloviruses [J]. J Virol, 2012, 86(19):10628-10639.
[56]Lang C, S?llner H, Barz A, et al. Investigation of the prevalence of swine torque teno virus in Austria [J]. Berl Munch Tierarztl Wochenschr, 2011, 124(3-4):142-147.
[57]De Arruda Leme R, Ribeiro J, Alfieri A, et al. Simultaneous infection with distinct strains of torque teno sus virus (TTSuV) in healthy slaughter-age pigs [J]. Vet Res Commun, 2013, 37(2): 183-186.
[58]胡崇伟,陈如敬,陈秋勇,等．猪细环病毒1b型ORF3基因克隆及序列分析[J] ．中国畜牧兽医,2014,41(8)：83-87．
[59]王俊,单晶晶,孟凡伟,等．猪细环病毒1型real time PCR检测方法的建立[J]．中国兽医科学,2014,44(09)：927-932．
[60]单晶晶,王俊,孟凡伟,等．猪细环病毒2型real time PCR检测方法的建立[J]．中国兽医科学,2014,44(02)：146-151．
[61]向海洋,聂福平,杨俊,等．猪细环病毒LAMP检测方法的建立[J]．中国预防兽医学报,2013,35(9)：747-751．
[62]杨春华,周延,孙思扬,等．猪细环病毒PCR-DHPLC检测技术的建立及应用[J]．畜牧兽医学报,2012,43(9)：1422-14