猪源副黏病毒JL-1株全基因组克隆及遗传变异分析
摘要
近几年,在我国吉林、上海、浙江、安徽及福建等省市在发病猪体内相继分离出副黏病毒病,经研究初步表明分离株为新城疫病毒。新城疫病毒(NDV)的感染宿主主要是鸡,但经过世界范围内的几次大流行后,其宿主范围已经明显扩大。迄今已知能自然或人工感染的鸟类超过250余种,而且可能有更多的易感宿主还没有被发现。但1997年以后鹅新城疫病毒病的出现并持续流行,则打破了不引起水禽发病的这一规则。目前,研究表明新城疫病毒已跨越种间屏障感染哺乳动物(猪)。这种现象揭示,新城疫病毒的感染和致病宿主范围正在不断扩大,给新城疫的防控带来新的挑战。因此对猪源副黏病毒的进化关系分析及寻找来源是非常必要的。
本研究从发病猪体内分离出一株病毒,电镜观察初步鉴定为副黏病毒,分子鉴定确定该病毒为新城疫病毒变异株,暂命名为JL-1株。该病毒可在SPF鸡胚尿囊腔和猪肾细胞中增殖,并能导致鸡胚发生病变。为进一步鉴定及明确该病毒的起源及所处的分类地位,本研究采用RT-PCR和RACE法对该株病毒含有6个蛋白及末端序列的全基因组进行了克隆和测序。对F、HN蛋白氨基酸序列疏水性和抗原表位预测及对比,并将该病毒的结构和非结构蛋白与同属的其它病毒进行了同源性比较和进化关系分析,对猪源副黏病毒的来源进行了推测,明确了猪源副黏病毒JL-1株的病毒起源分类地位。本研究为阐明导致新城疫病毒感染宿主特性改变的原因,决定NDV宿主特异性改变的分子机理奠定了基础。
Avian Paramyxovirus consisted of nine serotypes, APMV 1-9, but APMV-1 only included Newcastle disease virus. There existed tremendous virulence diversity among different hosts in which chicken was the most sensitive, pigeon and parrot also could be infected and falled ill. Waterfowls such as geese or ducks constantly were not considered to be infected by NDV. For the past few years, uninterruptly expanding of the host infection spectrum about NDV, chicken, pigeon, parrot and some rare birds could be infected, as well as waterfowls. The outbreak of goose-host paramyxovirus disease broken traditionary theory which said waterfowls could not be infected by NDV. However, since 1999, researchers isolated paramyxovirus from swine. After studying the virus is the variation of NDV strains that are infected the mammals (swine).
In this study, our lab isolated NDV from illed swines in 2005, named JL-1. Electron microscopy revealed the morphology of a paramyxovirus. Hemagglutination and hemagglutination inhibition tests confirmed the JL-1 and APMV-1 have the relationship between the serum. Through MDT, EID50, ICPI, IVPI tests determind that JL-1 is avirulent. After molecular identification confirm the porcine paramyxovirus strain JL-1 is the Newcastle disease virus variant. Cloning and sequence analysis of the F protein found that the encoding F protein cleavage site amino acid composition for the 112-G-R-Q-G-R-L-117, which is characteristics of avirulent, that confirm the JL-1 is avirulent in molecular level again, LaSota, and homology of 99.5%, belong to F typeⅡgene. To confirmed virus origin taxonomic status and the source of the porcine paramyxovirus strain JL-1. A total of ten overlapping cDNA clones, covering the entire genome of JL-1, were obtained by either target gene walking RT-PCR or 5’- and 3’-RACE. The genomes of nine NDV strains including Aus/32, Beaudette C, Herts/33, LaSota, Ulster, HB92 V4, SF02, ZJ1 and NA-1 were compared to the nucleotide sequence of the gene of strain JL-1. JL-1 was isolated from swine; NA-1, SF02 and ZJ1 were all isolated from geese, others from chicken. The results showed that NP nucleotide homology of 85.37% - 99.32%; P nucleotide homology of 82.66% - 99.92%; M nucleotide homology of 84.84% - 99.82%; F nucleotide homology 84.60 % - 99.52; HN nucleotide homology of 81.66% - 100%; L nucleotide homology of 86.20% - 99.83%; 5’nucleotide homology of 60.2% - 99.1%; 3’nucleotide homology of 85.5% - 100%. The results showed that JL-1 strains was closely related to the LaSota, Beaudette C strain, with higher nucleotide homology, but low nucleotide homology with geese NDV。
Sequence compiled from these clones showed that the genome of strain JL-1 was 15,021 nt long (GenBank accession # EU546165). Analysis of sequence shown that JL-1 had changed in 1590-1798 nt, 1821-1894 nt, 2298-2418 nt, 3091-3263 nt, 4418-4670 nt, 15010-15090 nt. That confirmed infect swine is the Newcastle disease virus variant first. Phylogeny of JL-1 showed that it is the same evolutionary branch with LaSota, Beaudette C, and Clone 30. It revealed NDV has infected mammal (swine). This study provide a theoretical basis for the origin and prevalence characteristics of porcine paramyxovirus, and had founded basic for the reasons for change that Characteristics of infected host and the molecular mechanism change of NDV specificity host.
本研究从发病猪体内分离出一株病毒,电镜观察初步鉴定为副黏病毒,分子鉴定确定该病毒为新城疫病毒变异株,暂命名为JL-1株。该病毒可在SPF鸡胚尿囊腔和猪肾细胞中增殖,并能导致鸡胚发生病变。为进一步鉴定及明确该病毒的起源及所处的分类地位,本研究采用RT-PCR和RACE法对该株病毒含有6个蛋白及末端序列的全基因组进行了克隆和测序。对F、HN蛋白氨基酸序列疏水性和抗原表位预测及对比,并将该病毒的结构和非结构蛋白与同属的其它病毒进行了同源性比较和进化关系分析,对猪源副黏病毒的来源进行了推测,明确了猪源副黏病毒JL-1株的病毒起源分类地位。本研究为阐明导致新城疫病毒感染宿主特性改变的原因,决定NDV宿主特异性改变的分子机理奠定了基础。
Avian Paramyxovirus consisted of nine serotypes, APMV 1-9, but APMV-1 only included Newcastle disease virus. There existed tremendous virulence diversity among different hosts in which chicken was the most sensitive, pigeon and parrot also could be infected and falled ill. Waterfowls such as geese or ducks constantly were not considered to be infected by NDV. For the past few years, uninterruptly expanding of the host infection spectrum about NDV, chicken, pigeon, parrot and some rare birds could be infected, as well as waterfowls. The outbreak of goose-host paramyxovirus disease broken traditionary theory which said waterfowls could not be infected by NDV. However, since 1999, researchers isolated paramyxovirus from swine. After studying the virus is the variation of NDV strains that are infected the mammals (swine).
In this study, our lab isolated NDV from illed swines in 2005, named JL-1. Electron microscopy revealed the morphology of a paramyxovirus. Hemagglutination and hemagglutination inhibition tests confirmed the JL-1 and APMV-1 have the relationship between the serum. Through MDT, EID50, ICPI, IVPI tests determind that JL-1 is avirulent. After molecular identification confirm the porcine paramyxovirus strain JL-1 is the Newcastle disease virus variant. Cloning and sequence analysis of the F protein found that the encoding F protein cleavage site amino acid composition for the 112-G-R-Q-G-R-L-117, which is characteristics of avirulent, that confirm the JL-1 is avirulent in molecular level again, LaSota, and homology of 99.5%, belong to F typeⅡgene. To confirmed virus origin taxonomic status and the source of the porcine paramyxovirus strain JL-1. A total of ten overlapping cDNA clones, covering the entire genome of JL-1, were obtained by either target gene walking RT-PCR or 5’- and 3’-RACE. The genomes of nine NDV strains including Aus/32, Beaudette C, Herts/33, LaSota, Ulster, HB92 V4, SF02, ZJ1 and NA-1 were compared to the nucleotide sequence of the gene of strain JL-1. JL-1 was isolated from swine; NA-1, SF02 and ZJ1 were all isolated from geese, others from chicken. The results showed that NP nucleotide homology of 85.37% - 99.32%; P nucleotide homology of 82.66% - 99.92%; M nucleotide homology of 84.84% - 99.82%; F nucleotide homology 84.60 % - 99.52; HN nucleotide homology of 81.66% - 100%; L nucleotide homology of 86.20% - 99.83%; 5’nucleotide homology of 60.2% - 99.1%; 3’nucleotide homology of 85.5% - 100%. The results showed that JL-1 strains was closely related to the LaSota, Beaudette C strain, with higher nucleotide homology, but low nucleotide homology with geese NDV。
Sequence compiled from these clones showed that the genome of strain JL-1 was 15,021 nt long (GenBank accession # EU546165). Analysis of sequence shown that JL-1 had changed in 1590-1798 nt, 1821-1894 nt, 2298-2418 nt, 3091-3263 nt, 4418-4670 nt, 15010-15090 nt. That confirmed infect swine is the Newcastle disease virus variant first. Phylogeny of JL-1 showed that it is the same evolutionary branch with LaSota, Beaudette C, and Clone 30. It revealed NDV has infected mammal (swine). This study provide a theoretical basis for the origin and prevalence characteristics of porcine paramyxovirus, and had founded basic for the reasons for change that Characteristics of infected host and the molecular mechanism change of NDV specificity host.
引文
[1] Alexander D J, Wilson G W C, Russell P H, et al. Newcastle disease out breaks in fowl in Great Britain during 1984[J]. Veterinary Record, 1985, 117, 429-434.
[2] Alexander D J. Newcastle disease and other paramyxoviridae infections [M]. In: Calnek , B W (Ed) , Diseases of Poultry, 10th Ed. Mosby-Wolfe, London,1997 : 541-569.
[3] Kirkland P D. Virulent Newcastle disease virus in Australia: in through the‘back door’[J]. Austral Vet J, 2000, 78 : 331-333.
[4] West bury H. Newcastle disease virus: an evolving pathogen [J]. Avian Pathology, 2001, 30 : 5-11.
[5]丁壮,王承宇,向华,等.鹅副粘病毒分离株生物学特性的研究[A].中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C].南京, 2002, 127-129.
[6]赵晓春,孙刚,张加勇,等.黑龙江省鹅副粘病毒的分离与鉴定[J].黑龙江省畜牧兽医, 2002, 8 : 24-25.
[7]万洪全,吴艳涛,刘秀梵,等. 4株鹅源新城疫病毒融合蛋白基因的克隆及序列分析[J].微生物学报, 2002, 42(2) : 208-213.
[8] Scott J. Goebel, Jill Taylor, Bradd C. Barr, et al. Isolation of Avian Paramyxovirus 1 from a Patient with a Lethal Case of Pneumonia Journal of virology, Nov. 2007, p. 12709-12714.
[9]王永坤,严维巍,周继宏,等.鸡副粘病毒病防制的探讨[J].中国禽业导刊,2000,17(11) : 4-5.
[10]毕玉海,李志杰,吴昊,等.猪源副粘病毒的分离鉴定及其生物学特性分析[A].中国畜牧兽医学会动物传染病学分会第12次学术研讨会论文集[C].井冈山: 2007,440-443.
[11]程龙飞,柯美峰,郑腾,等.猪源新城疫病毒的分离鉴定[J].中国兽医科技,2004,34(12) : 66-67.
[12]刘文博,万洪全,吴艳涛,等.鸡新城疫标准强毒感染鹅实验[J].中国家禽,2001,23(19) : 10-11.
[13]吴洪俊,刘文斌,彭永刚,等.新城疫的流行特点[J],中国兽医,2004,40(1) : 34-37.
[14] Alexander D J, Arp H G. Newcastle disease and other paramyxoviorus infection [A]. Disease of Poultry 9th Edition [M]. Lowa State University Press, USA, 1991. 496-519.
[15] Morrison TG. Structure and function of a paramyxovirus fusion protein. Biochim Biophys Acta. 2003 Jul 11; 1614(1) : 73-84.
[16] Panda A, Huang Z, Elankumaran S, et al. Role of fusion protein cleavage site in the virulence of Newcastle disease virus. Microb Pathog. 2004 Jan; 36(1) : 1-10.
[17] Aldous E W, A lexander D J. Detection and differentiation of Newcastle disease virus (avian paramyoviorus type 1) [J]. A vian Patho l, 2001, 30: 117-128.
[18] Alexander D J. N ewcastle disease and other avian paramyovioruses [J]. Rev Sci Tech Off Int Ep iz, 2000, 19 : 443-462.
[19] Meulemans, Gvanden, Berg T P, Decaesstecke, et al. Evolution of pigeon Newcastle disease virus strains [J]. Avian Pathol, 2002, 31 (5) : 515-519.
[20] Peeters B P, De Leeuw O S, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence [J]. J Vir, 1999, 73 (6) : 5001-5009.
[21] Panda A, Elankumaran S, Krishnamurt hy S, et al. Loss of Nlinked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus [J]. J Virol, 2004, 78 (10) : 4965-4975.
[22] Alexander D J, Wilson G W C, Russell P H, et al. Newcastle disease out breaks in fowl in Great Britain during 1984 [J]. Veterinary Record, 1985, 117, 429-434.
[23] West bury H. Newcastle disease virus: an evolving pathogen [J]. Avian Pathology, 2001, 30 : 5-11.
[24] Collins M S, Franklin S, Strong I, et al. Antigenic and phylogenetic studies on a variation Newcastle disease virus using antifusion protein monoclonal antibodies and partial sequencing of the fusion protein gene [J]. Avain Pathology, 1998, 27 : 90-96.
[25] Shengqing Y, Kishida N, Ito H, et al. Generation of velogenic Newcastle disease viruses from a nonpat hogenic waterfowl isolate by passaging in chickens [J]. Virology, 2002, 301 (2) : 206-211.
[26] De Leeuw O S, Koch G, Hartog L, et al. Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem regionand globular head of the haemagglutinin-neuraminidase protein [J]. Gen Virol, 2005, 86 (6) : 1759-1769.
[27]金扩世,金宁一,袁书智,等.猪副粘病毒的分离[J].中国兽医学报,2001,( 4) : 323.
[28]吴祖立,高诚,叶陈梁,等.猪副粘病毒感染的初步诊断和病毒分离[J].上海交通大学学报:农业科学版,2002,20(1) : 67-68.
[29]洪健,周雪平. ICTV第八次报告的最新病毒分类系统中[J].国病毒学,2006,21(1) : 84-96.
[30]殷震,刘景华,主编.动物病毒学第二版[M],北京:科学出版社, 1997,736-737.
[31]毕玉海,丛彦龙,李志杰,等.猪源副粘病毒的分子鉴定[J].中国兽医学报,2008,28(4) : 343-349.
[32]胡祥壁.家畜传染病[M]. 7版.北京:农业出版社,1988 :794-798.
[33]费恩阁.动物传染病学[M].长春:吉林科技出版社,1995 :378-383.
[34] GW贝兰.人畜共患的病毒性疾病[M].徐启丰,译.北京:人民军医出版社,1985 : 371-376.
[35]徐明,丁壮,毕玉海,等.鹅副粘病毒NA-1株全基因组的克隆及特性分析[J].中国兽医学报,2006,26(6) : 610-613.
[36]王学理,武迎红,丁壮.鹅源禽副粘病毒NA-1株生物学特性的研究[J].吉林农业大学学报,2006,28(1) : 73-97.
[37] Hoffman, G., Neumannn, G., Hobom, G. et al.“Ambisense”Appronch for the generation of Influenza A virus rRNA and mRNA synthesis from one trmplate[J]. Virology. 2001, 267: 310-317.
[38] Bermingham, A. & Collins, P.L. The M2-2 protein of human respirotary syncytial virus is a regulatory factor involved in balance between RNA replication and transcription.[J] Proceeding of the National Academy of Science, 1999, 96: 11259-11264.
[39] Curran, J., Marq, J.B. & kolakofsky, D. An N-terminal domain of the sendai paramyxovirus P protein acs as a chaperone for the NP protein during the nascent chain assembly step of genome replication[J]. Journal of Virology. 1995, 69: 849-855.
[40]李文良,卞汝霖,冯太兰,等.鹅类新城疫病原研究[J].畜牧与兽医, 1999, 31(2): 1-3.
[41] Peeters BP, Gruijthuijsen YK, de Leeuw OS, Gielkens AL (2000) Genome replication of Newcastle disease virus: involvement of the rule-of six. Arch Virol 145:1829–1845
[42]覃德鑫,邓绍基.鸡新城疫Ⅰ系苗防治新生仔猪流行性腹泻疗效观察[J].广西农业科学,1995(2) :86-871.
[43]曾仕康,刘晓颖,黄大明,等.猪血、猪脾白细胞的培养及干扰素的诱生[J].中国生化药物杂志,1996 ,17 (3) : 116-118.
[2] Alexander D J. Newcastle disease and other paramyxoviridae infections [M]. In: Calnek , B W (Ed) , Diseases of Poultry, 10th Ed. Mosby-Wolfe, London,1997 : 541-569.
[3] Kirkland P D. Virulent Newcastle disease virus in Australia: in through the‘back door’[J]. Austral Vet J, 2000, 78 : 331-333.
[4] West bury H. Newcastle disease virus: an evolving pathogen [J]. Avian Pathology, 2001, 30 : 5-11.
[5]丁壮,王承宇,向华,等.鹅副粘病毒分离株生物学特性的研究[A].中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集[C].南京, 2002, 127-129.
[6]赵晓春,孙刚,张加勇,等.黑龙江省鹅副粘病毒的分离与鉴定[J].黑龙江省畜牧兽医, 2002, 8 : 24-25.
[7]万洪全,吴艳涛,刘秀梵,等. 4株鹅源新城疫病毒融合蛋白基因的克隆及序列分析[J].微生物学报, 2002, 42(2) : 208-213.
[8] Scott J. Goebel, Jill Taylor, Bradd C. Barr, et al. Isolation of Avian Paramyxovirus 1 from a Patient with a Lethal Case of Pneumonia Journal of virology, Nov. 2007, p. 12709-12714.
[9]王永坤,严维巍,周继宏,等.鸡副粘病毒病防制的探讨[J].中国禽业导刊,2000,17(11) : 4-5.
[10]毕玉海,李志杰,吴昊,等.猪源副粘病毒的分离鉴定及其生物学特性分析[A].中国畜牧兽医学会动物传染病学分会第12次学术研讨会论文集[C].井冈山: 2007,440-443.
[11]程龙飞,柯美峰,郑腾,等.猪源新城疫病毒的分离鉴定[J].中国兽医科技,2004,34(12) : 66-67.
[12]刘文博,万洪全,吴艳涛,等.鸡新城疫标准强毒感染鹅实验[J].中国家禽,2001,23(19) : 10-11.
[13]吴洪俊,刘文斌,彭永刚,等.新城疫的流行特点[J],中国兽医,2004,40(1) : 34-37.
[14] Alexander D J, Arp H G. Newcastle disease and other paramyxoviorus infection [A]. Disease of Poultry 9th Edition [M]. Lowa State University Press, USA, 1991. 496-519.
[15] Morrison TG. Structure and function of a paramyxovirus fusion protein. Biochim Biophys Acta. 2003 Jul 11; 1614(1) : 73-84.
[16] Panda A, Huang Z, Elankumaran S, et al. Role of fusion protein cleavage site in the virulence of Newcastle disease virus. Microb Pathog. 2004 Jan; 36(1) : 1-10.
[17] Aldous E W, A lexander D J. Detection and differentiation of Newcastle disease virus (avian paramyoviorus type 1) [J]. A vian Patho l, 2001, 30: 117-128.
[18] Alexander D J. N ewcastle disease and other avian paramyovioruses [J]. Rev Sci Tech Off Int Ep iz, 2000, 19 : 443-462.
[19] Meulemans, Gvanden, Berg T P, Decaesstecke, et al. Evolution of pigeon Newcastle disease virus strains [J]. Avian Pathol, 2002, 31 (5) : 515-519.
[20] Peeters B P, De Leeuw O S, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence [J]. J Vir, 1999, 73 (6) : 5001-5009.
[21] Panda A, Elankumaran S, Krishnamurt hy S, et al. Loss of Nlinked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus [J]. J Virol, 2004, 78 (10) : 4965-4975.
[22] Alexander D J, Wilson G W C, Russell P H, et al. Newcastle disease out breaks in fowl in Great Britain during 1984 [J]. Veterinary Record, 1985, 117, 429-434.
[23] West bury H. Newcastle disease virus: an evolving pathogen [J]. Avian Pathology, 2001, 30 : 5-11.
[24] Collins M S, Franklin S, Strong I, et al. Antigenic and phylogenetic studies on a variation Newcastle disease virus using antifusion protein monoclonal antibodies and partial sequencing of the fusion protein gene [J]. Avain Pathology, 1998, 27 : 90-96.
[25] Shengqing Y, Kishida N, Ito H, et al. Generation of velogenic Newcastle disease viruses from a nonpat hogenic waterfowl isolate by passaging in chickens [J]. Virology, 2002, 301 (2) : 206-211.
[26] De Leeuw O S, Koch G, Hartog L, et al. Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem regionand globular head of the haemagglutinin-neuraminidase protein [J]. Gen Virol, 2005, 86 (6) : 1759-1769.
[27]金扩世,金宁一,袁书智,等.猪副粘病毒的分离[J].中国兽医学报,2001,( 4) : 323.
[28]吴祖立,高诚,叶陈梁,等.猪副粘病毒感染的初步诊断和病毒分离[J].上海交通大学学报:农业科学版,2002,20(1) : 67-68.
[29]洪健,周雪平. ICTV第八次报告的最新病毒分类系统中[J].国病毒学,2006,21(1) : 84-96.
[30]殷震,刘景华,主编.动物病毒学第二版[M],北京:科学出版社, 1997,736-737.
[31]毕玉海,丛彦龙,李志杰,等.猪源副粘病毒的分子鉴定[J].中国兽医学报,2008,28(4) : 343-349.
[32]胡祥壁.家畜传染病[M]. 7版.北京:农业出版社,1988 :794-798.
[33]费恩阁.动物传染病学[M].长春:吉林科技出版社,1995 :378-383.
[34] GW贝兰.人畜共患的病毒性疾病[M].徐启丰,译.北京:人民军医出版社,1985 : 371-376.
[35]徐明,丁壮,毕玉海,等.鹅副粘病毒NA-1株全基因组的克隆及特性分析[J].中国兽医学报,2006,26(6) : 610-613.
[36]王学理,武迎红,丁壮.鹅源禽副粘病毒NA-1株生物学特性的研究[J].吉林农业大学学报,2006,28(1) : 73-97.
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