虎源副流感病毒5型的分离鉴定及全基因序列分析
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摘要
本研究旨在了解虎源副流感病毒5型(parainfluenza virus 5,PIV5)的遗传变异情况,阐明其基因组与遗传进化等特征。从广东省某动物园疑似虎PIV5感染的病料处理后接种Vero细胞并通过一系列的鉴定,将分离的毒株命名为HMZ。根据GenBank中PIV5基因序列,设计包含全长的12对特异性引物,并扩增其全基因组。将扩增产物克隆到pMD-18T载体上,对鉴定为阳性的重组质粒送检测序,并对基因全序列进行同源性分析及遗传进化分析。结果显示,HMZ与各病毒株全基因序列同源性为97.2%～99.8%。其中与1168-1 (KC237064.1)、ZJQ-221 (KX100034.1)以及Mammalian rubulavirus 5 (KY685075.1)亲缘性最高,为99.8%;而与D277的亲缘性最低,为97.2%。全基因系统进化树分析结果表明,HMZ与ZJQ-221、Mammalian rubulavirus 5属于同一分支。
The purpose of this study is to understand the genetic variation of parainfluenza virus5(PIV5) and to elucidate its of genome and genetic evolution charactertion.Vero cells were inoculated with sample suspect infected PIV5 of a tiger from a zoo in Guangdong Province.The isolated strain was named as HMZ after a series of identification.According to the sequence of PIV5 gene in Gen Bank,Twelve overlapping fragments covering the whole viral genome were designed and the full-genome were amplified.The amplified products were cloned into p MD-18 T vector,then sequenced the positive recombinant plasmids.The whole gene sequence was analyzed using homology analysis and genetic evolution analysis.The results showed that HMZ was homologous with 97.2%—99.8% of all virus strains.Alignment analysis indicated that HMZ had the most proximate consensus(99.8%)to 1168-1(KC237064.1),ZJQ-221(KX100034.1)and Mammalian rubulavirus 5(KY685075.1),while the least consensus(97.2%) to D277(KC237065.1).Phylogenetic tree analysis revealed that HMZ strain,ZJQ-221 strain and Mammalian rubulavirus 5 belonged to the same branch.
The purpose of this study is to understand the genetic variation of parainfluenza virus5(PIV5) and to elucidate its of genome and genetic evolution charactertion.Vero cells were inoculated with sample suspect infected PIV5 of a tiger from a zoo in Guangdong Province.The isolated strain was named as HMZ after a series of identification.According to the sequence of PIV5 gene in Gen Bank,Twelve overlapping fragments covering the whole viral genome were designed and the full-genome were amplified.The amplified products were cloned into p MD-18 T vector,then sequenced the positive recombinant plasmids.The whole gene sequence was analyzed using homology analysis and genetic evolution analysis.The results showed that HMZ was homologous with 97.2%—99.8% of all virus strains.Alignment analysis indicated that HMZ had the most proximate consensus(99.8%)to 1168-1(KC237064.1),ZJQ-221(KX100034.1)and Mammalian rubulavirus 5(KY685075.1),while the least consensus(97.2%) to D277(KC237065.1).Phylogenetic tree analysis revealed that HMZ strain,ZJQ-221 strain and Mammalian rubulavirus 5 belonged to the same branch.
引文
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[8]卢昕,倪淑媛,李煜生.两种单负链RNA呼吸道病毒---流感病毒和呼吸道合胞病毒感染对黏蛋白1表达调控差异的研究[J].病毒学报,2012,28(06):639-644.LU Xin,NI Shu-yuan,LI Yu-sheng.Differences of the regulation on the expression of mucin 1 induced by two single-strand RNA viruses,respiratory syncytial virus and influenza virus[J].Chinese Journal of Virology,2012,28(06):639-644.(in Chinese)
[9]BAGLA V P,MCGAW L J,ELOFF J N.The antiviral activity of six South African plants traditionally used against infections in ethnoveterinary medicine[J].Veterinary Microbiology,2012,155(2-4):198-206.
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[14]齐宾宾,宋彩玲,陈小微,等.犬副流感病毒分子克隆的构建及全基因组序列分析[J].动物医学进展,2015,36(10):10-15.QI Bin-bin,SONG Cai-ling,CHEN Xiao-wei,et al.Construction and genome sequence analysis of molecular clone of canine parainfluenza virus[J].Progress In Veterinary Medicine,2015,36(10):10-15.(in Chinese)
[15]BINN L N,EDDY G A,LAZAR E R,et al.Viruses recovered from laboratory dogs with respiratory disease[J].Proceedings of the Society for Experimental Biology and Medicine,1967,126(1):140-145.
[16]李长磊.犬副流感病毒的分离鉴定及结构蛋白基因的克隆与序列分析[D].长春:吉林农业大学,2013.LI Chang-lei.Isolation and identification of canine parainfluenza virus and cloning and sequence analysis of it’s structural protein genes[D].Changchun:Jilin Agricultural University,2013.(in Chinese)
[17]ERLES K,DUBOVI E J,BROOKS H W,et al.Longitudinal study of viruses associated with canine infectious respiratory disease[J].Journal of Clinical Microbiology,2004,42(10):4524-4529.
[18]姚月琴.猪细小病毒的PCR检测、分离鉴定及其单克隆抗体的研制[D].扬州:扬州大学,2006.YAO Yue-qin.Porcine Parvovirus:PCR detection,isolation,identification,and its monoclonal antibody development[D].Yangzhou:Yangzhou University,2006.(in Chinese)
[19]殷震,刘景华.动物病毒学[M].2版.北京:科学出版社,1997:336-340.YIN Zhen,LIU Jing-hua.Animal Virology[M].2nd ed.Beijing:Science Press,1997:336-340.(in Chinese)
[20]任桂杰,王志玉.副粘病毒包膜糖蛋白分子生物学研究进展[J].病毒学报,2005(01):72-75.REN Gui-jie,WANG Zhi-yu.Advances in molecular biology of glycoprotein from paramyxovirus[J].Chinese Journal of Virology,2005(01):72-75.(in Chinese)
[21]夏咸柱,高玉伟,扈荣良,等.通过病毒分离鉴定和基因检测首次发现虎流感[J].中国兽医学报,2003(02):107-110.XIA Xian-zhu,GAO Yu-wei,HU Rong-liang,et al.Virus isolation,identification and gene detection of flu isolate from tiger for the first time[J].Chinese Journal of Veterinary Science,2003(02):107-110.(in Chinese)
[22]孙贺廷,夏咸柱,高玉伟,等.虎血清中犬副流感病毒的抗体流行病学调查研究[J].畜牧与兽医,2004(09):4-6.SUN He-ting,XIA Xian-zhu,GAO Yu-wei,et al.Seroepidemiological investigation of canine parainfluenza virus in tiger[J].Acta Veterinaria et Zootechnica Sinica,2004(09):4-6.(in Chinese)
[23]翟俊琼,周霞,邹舒展,等.虎源、小熊猫源副流感病毒5型的鉴定及F基因遗传进化分析[J].中国兽医学报,2018,38(04):700-703.ZHAI Jun-qiong,ZHOU Xia,ZOU Shu-zhan,et al.I-dentification of the parainfluenza virus type 5from tiger source,red panda source and analysis of F gene genetic evolution in genus[J].Chinese Journal of Veterinary Science,2018,38(04):700-703.(in Chinese)
[24]YUAN P,LESER G P,DEMELER B,et al.Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase(HN)protein[J].Journal of Virology,2008,378(2):282-291.
[25]周天赐,毛立,李文良,等.山羊副流感病毒3型HN蛋白单克隆抗体的制备与鉴定[J]中国兽医科学,2016,46(04):436-441.ZHOU Tian-ci,MAO Li,LI Weng-liang,et al.Preparation and characterization of monoclonal antibodies specific for hemagglutinin neuraminidase proteins of caprine parainfluenza virus type 3[J].Chinese Veterinary Science,2016,46(04):436-441.(in Chinese)
[26]袁小远,杨金兴,张玉霞,等.NDV和APMV-4的F和HN蛋白糖基化位点的差异分析比较[J].家禽科学,2017(12):8-10.YUAN Xiao-yuan,YANG Jin-xing,ZHANG Yu-xia,et al.Comparison of glycosylation sites of F and HNproteins between NDV and APMV-4[J].Poultry Science,2017(12):8-10.(in Chinese)
[27]LEE Y N,LEE C.Complete genome sequence of a novel porcine parainfluenza virus 5 isolate in Korea[J].Archives of Virology,2013,158(8):1765-1772.
[28]LIU C,LI X,ZHANG J,et al.Isolation and genomic characterization of a canine parainfluenza virus type 5 strain in China[J].Archives of Virology,2017,162(8):2337-2344.
[2]高菽蔓,靳红亮,张守峰,等.犬副流感病毒CC-14株全基因组测序及分析[J].中国预防兽医学报,2015,37(10):802-804.GAO Shu-man,JIN Hong-liang,ZHANG Shou-feng,et al.The complete genome sequencing and analysis of canine parainfluenza virus strain CC-14[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(10):802-804.(in Chinese)
[3]MITCHELL S A,ZWIJNENBERG R J,HUANG J,et al.Duration of serological response to canine parvovirus-type 2,canine distemper virus,canine adenovirus type 1 and canine parainfluenza virus in client-owned dogs in Australia[J].Australian Veterinary Journal,2012,90(12):468-473.
[4]焦库华,岑皓,张小荣,等.犬副流感病毒单克隆抗体的研制及其特性鉴定[J].中国预防兽医学报,2009,31(12):985-987.JIAO Ku-hua,CEN Hao,ZHANG Xiao-rong,et al.Production and characterization of the monoclonal antibodies against canine parainfluenza virus[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(12):985-987.(in Chinese)
[5]王凤雪,程世鹏,温永俊.犬副流感病毒反转录-环介导等温扩增检测方法的建立[J].中国兽医科学,2016,46(04):486-490.WANG Feng-xue,CHENG Shi-peng,WEN Yong-jun.Development of loop-mediated isothermal amplification(LAMP)method for detection of canine parainfluenza virus[J].Chinese Veterinary Science,2016,46(04):486-490.(in Chinese)
[6]孙明,刘巧荣,秦亚嫚,等.犬副流感病毒的分离鉴定及部分基因序列分析[J].中国比较医学杂志,2016,26(02):77-82.SUN Ming,LIU Qiao-rong,QIN Ya-man,et al.Isolation and identification of canine parainfluenza virus and partial gene sequence analysis of the isolate[J].Chinese Journal of Comparative Medicine,2016,26(02):77-82.(in Chinese)
[7]岑皓.犬副流感病毒的分离及单克隆抗体的制备[D].扬州:扬州大学,2007.CEN Hao.Isolation of canine parainfluenza virus(CPIV)and preparation of monocloneal antibodies against CPIV[D].Yangzhou:Yangzhou University,2007.(in Chinese)
[8]卢昕,倪淑媛,李煜生.两种单负链RNA呼吸道病毒---流感病毒和呼吸道合胞病毒感染对黏蛋白1表达调控差异的研究[J].病毒学报,2012,28(06):639-644.LU Xin,NI Shu-yuan,LI Yu-sheng.Differences of the regulation on the expression of mucin 1 induced by two single-strand RNA viruses,respiratory syncytial virus and influenza virus[J].Chinese Journal of Virology,2012,28(06):639-644.(in Chinese)
[9]BAGLA V P,MCGAW L J,ELOFF J N.The antiviral activity of six South African plants traditionally used against infections in ethnoveterinary medicine[J].Veterinary Microbiology,2012,155(2-4):198-206.
[10]SOUTHERN J A,YOUNG D F,HEANEY F,et al.Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics[J].Jour nal of General Virology,1991,72(Pt7):1551-1557.
[11]中国农业科学院哈尔滨兽医研究所.兽医微生物学[M].北京:中国农业出版社,1998:414-415.Harbin Veterinary Research Institute.Veterinary Microbiology[M].China Agriculture Press,1998:414-415.(in Chinese)
[12]LAMB R A,KOLAKOFSKY D.Paramyxoviridae:the viruses and their replication.In Fields Virology[M].4th ed.Philadelphia,PA:Lippincott-Raven,2001:1305-1340.
[13]师新川.牛副流感病毒3型NM09株反向遗传系统的建立[D].北京:中国农业科学院研究生院,2012.SHI Xin-chuan.Reverse genetics system of bovine parainfluenza virus 3 NM09 strain[D].Beijing:Graduate School of Chinese Academy of Agricultural Sciences,2012.(in Chinese)
[14]齐宾宾,宋彩玲,陈小微,等.犬副流感病毒分子克隆的构建及全基因组序列分析[J].动物医学进展,2015,36(10):10-15.QI Bin-bin,SONG Cai-ling,CHEN Xiao-wei,et al.Construction and genome sequence analysis of molecular clone of canine parainfluenza virus[J].Progress In Veterinary Medicine,2015,36(10):10-15.(in Chinese)
[15]BINN L N,EDDY G A,LAZAR E R,et al.Viruses recovered from laboratory dogs with respiratory disease[J].Proceedings of the Society for Experimental Biology and Medicine,1967,126(1):140-145.
[16]李长磊.犬副流感病毒的分离鉴定及结构蛋白基因的克隆与序列分析[D].长春:吉林农业大学,2013.LI Chang-lei.Isolation and identification of canine parainfluenza virus and cloning and sequence analysis of it’s structural protein genes[D].Changchun:Jilin Agricultural University,2013.(in Chinese)
[17]ERLES K,DUBOVI E J,BROOKS H W,et al.Longitudinal study of viruses associated with canine infectious respiratory disease[J].Journal of Clinical Microbiology,2004,42(10):4524-4529.
[18]姚月琴.猪细小病毒的PCR检测、分离鉴定及其单克隆抗体的研制[D].扬州:扬州大学,2006.YAO Yue-qin.Porcine Parvovirus:PCR detection,isolation,identification,and its monoclonal antibody development[D].Yangzhou:Yangzhou University,2006.(in Chinese)
[19]殷震,刘景华.动物病毒学[M].2版.北京:科学出版社,1997:336-340.YIN Zhen,LIU Jing-hua.Animal Virology[M].2nd ed.Beijing:Science Press,1997:336-340.(in Chinese)
[20]任桂杰,王志玉.副粘病毒包膜糖蛋白分子生物学研究进展[J].病毒学报,2005(01):72-75.REN Gui-jie,WANG Zhi-yu.Advances in molecular biology of glycoprotein from paramyxovirus[J].Chinese Journal of Virology,2005(01):72-75.(in Chinese)
[21]夏咸柱,高玉伟,扈荣良,等.通过病毒分离鉴定和基因检测首次发现虎流感[J].中国兽医学报,2003(02):107-110.XIA Xian-zhu,GAO Yu-wei,HU Rong-liang,et al.Virus isolation,identification and gene detection of flu isolate from tiger for the first time[J].Chinese Journal of Veterinary Science,2003(02):107-110.(in Chinese)
[22]孙贺廷,夏咸柱,高玉伟,等.虎血清中犬副流感病毒的抗体流行病学调查研究[J].畜牧与兽医,2004(09):4-6.SUN He-ting,XIA Xian-zhu,GAO Yu-wei,et al.Seroepidemiological investigation of canine parainfluenza virus in tiger[J].Acta Veterinaria et Zootechnica Sinica,2004(09):4-6.(in Chinese)
[23]翟俊琼,周霞,邹舒展,等.虎源、小熊猫源副流感病毒5型的鉴定及F基因遗传进化分析[J].中国兽医学报,2018,38(04):700-703.ZHAI Jun-qiong,ZHOU Xia,ZOU Shu-zhan,et al.I-dentification of the parainfluenza virus type 5from tiger source,red panda source and analysis of F gene genetic evolution in genus[J].Chinese Journal of Veterinary Science,2018,38(04):700-703.(in Chinese)
[24]YUAN P,LESER G P,DEMELER B,et al.Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase(HN)protein[J].Journal of Virology,2008,378(2):282-291.
[25]周天赐,毛立,李文良,等.山羊副流感病毒3型HN蛋白单克隆抗体的制备与鉴定[J]中国兽医科学,2016,46(04):436-441.ZHOU Tian-ci,MAO Li,LI Weng-liang,et al.Preparation and characterization of monoclonal antibodies specific for hemagglutinin neuraminidase proteins of caprine parainfluenza virus type 3[J].Chinese Veterinary Science,2016,46(04):436-441.(in Chinese)
[26]袁小远,杨金兴,张玉霞,等.NDV和APMV-4的F和HN蛋白糖基化位点的差异分析比较[J].家禽科学,2017(12):8-10.YUAN Xiao-yuan,YANG Jin-xing,ZHANG Yu-xia,et al.Comparison of glycosylation sites of F and HNproteins between NDV and APMV-4[J].Poultry Science,2017(12):8-10.(in Chinese)
[27]LEE Y N,LEE C.Complete genome sequence of a novel porcine parainfluenza virus 5 isolate in Korea[J].Archives of Virology,2013,158(8):1765-1772.
[28]LIU C,LI X,ZHANG J,et al.Isolation and genomic characterization of a canine parainfluenza virus type 5 strain in China[J].Archives of Virology,2017,162(8):2337-2344.