羊口疮病毒OV-HLJ05株及其VEGF毒力因子的遗传特征
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摘要
羊口疮在我国呈区域流行态势,各地分离株基因型混杂不清,严重制约该病的区域性防控。本研究针对黑龙江大庆地区某小型私营羊场出现的疑似羊口疮病例,采集发病的波尔山羊痂皮病料,通过病毒的分离、电镜形态学观察、本体动物回归试验、血清学检测和分子生物学鉴定,获得一株羊口疮病毒(Orf virus,ORFV),命名为OVHLJ05株。进而,病毒分子遗传学研究发现,OV-HLJ05在其主要的囊膜蛋白编码基因F1L上,与近年来国内众多分离株相差不大;同源性达95%～98.5%。而在其毒力因子编码基因VEGF方面,OV-HLJ05与9个国内外代表毒株相比,则出现了较大分歧。OV-HLJ05株与OV-SA00(USA)、OV-GO(China)和OV-SJ1(China)等3个毒株亲缘关系密切;同源性分别是93.2%、91.9%和87.2%。而与NZ2(New Zealand)、IA82(USA)、B029(Germany)、D1701(Germany)OV-YX(China)和OV-NA-1-11(China)距离较远。但是,VEGF分子同源建模及其功能性结构域VHD分析表明,各毒株毒力因子仅在Loop3处差别明显,Loop3多态性是否决定ORFV毒力的多样性有待于进一步比较研究。本研究结果说明ORFV不同基因型在我国南北分布的复杂性,这为羊口疮分子流行病学和羊口疮的区域防控研究提供了参考。
The orf possessing territorial spread of tendency in China,and the mixed genotypes of isolates in different regions,severely restricted the regional prevention and control of the disease. In view of suspected cases of infection by the orf virus(ORFV)from an outbreak in Daqing(Heilongjiang Province,China),clinical samples were collected from infected Boer goats. After isolation of the ORFV,morphologic observations,animal regression test,serologic detection and molecular-biologic identification revealed it to be the main pathogen responsible for the epidemic,and it was named the"OV-HLJ05 isolate". Molecular genetic studies showed that OV-HLJ05 was not significantly different from many isolates in China in recent years with regard to its main envelope protein-coding gene F1 L. Homology reached 95%～98.5%. However,in terms of its virulence factor encoding gene, VEGF, OV-HLJ05 showed greater divergence compared with nine representative strains in China and overseas. OV-HLJ05 was closely related to OV-SA00(USA),OV-GO(China)and OV-SJ1(China),and homology was 93.2%,91.9% and 87.2%,respectively. OV-HLJ05 was not closely related to NZ2(New Zealand),IA82(USA),B029(Germany),D1701(Germany)OV-YX(China) or OV-NA-1-11(China). Homologous modeling of VEGF molecules and VHD analyses of the functional structural domains of VEGF showed that the virulence factors of each strain were significantly different only at Loop3. Whether polymorphisms of Loop3 determined the diversity of ORFV virulence merits further comparative studies. The present study indicated the complexity of the distribution of different ORFV genotypes in north and south China. In this way,we provided a reference for the molecular epidemiology of the ORFV as well as the regional prevention and control of ORFV in goats.
The orf possessing territorial spread of tendency in China,and the mixed genotypes of isolates in different regions,severely restricted the regional prevention and control of the disease. In view of suspected cases of infection by the orf virus(ORFV)from an outbreak in Daqing(Heilongjiang Province,China),clinical samples were collected from infected Boer goats. After isolation of the ORFV,morphologic observations,animal regression test,serologic detection and molecular-biologic identification revealed it to be the main pathogen responsible for the epidemic,and it was named the"OV-HLJ05 isolate". Molecular genetic studies showed that OV-HLJ05 was not significantly different from many isolates in China in recent years with regard to its main envelope protein-coding gene F1 L. Homology reached 95%～98.5%. However,in terms of its virulence factor encoding gene, VEGF, OV-HLJ05 showed greater divergence compared with nine representative strains in China and overseas. OV-HLJ05 was closely related to OV-SA00(USA),OV-GO(China)and OV-SJ1(China),and homology was 93.2%,91.9% and 87.2%,respectively. OV-HLJ05 was not closely related to NZ2(New Zealand),IA82(USA),B029(Germany),D1701(Germany)OV-YX(China) or OV-NA-1-11(China). Homologous modeling of VEGF molecules and VHD analyses of the functional structural domains of VEGF showed that the virulence factors of each strain were significantly different only at Loop3. Whether polymorphisms of Loop3 determined the diversity of ORFV virulence merits further comparative studies. The present study indicated the complexity of the distribution of different ORFV genotypes in north and south China. In this way,we provided a reference for the molecular epidemiology of the ORFV as well as the regional prevention and control of ORFV in goats.
引文
[1]Hosamani M,Scagliarini A,Bhanuprakash V,McInnes C J,Singh R K.Orf:an update on current research and future perspectives[J].Expert Rev Anti Infect Ther,2009,7(7):879-893.
[2]McElroy M C,Bassett H F.The development of oral lesions in lambs naturally infected with orf virus[J].Vet J2007,174(3):663-664.
[3]Eisa M,Tagel Deen M H,Omer E A.An outbreak of contagious ecthyma in sheep and goats in the Sudan[J].Bulletin of Animal Health and Production in Africa,1985,33,169-170.
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[7]Zhao K,Song D,He W,Lu H,Zhang B,Li C,Chen K,Gao F.Identification and phylogenetic analysis of an Orf virus isolated from an outbreak in sheep in the Jilin province of China[J].Vet Microbiol,2010,142(3-4):408-415.
[8]葛士坤,张凯照,鲁荣,刘健新,宁章勇.2006-2016年中国羊口疮病毒的遗传演化分析[J].中国畜牧兽医,2018,45(2):439-447.
[9]Mercer A A,Haig D M.Parapoxviruses[M].In:Granoff,A.,Webster,R.G.(Eds.),Encyclopedia of Virology,second ed.Academic Press,1999,1140-1146.
[10]Haig D M,McInnes C J.Immunity and counter-immunity during infection with the parapoxvirus orf virus[J].Virus Res,2002,88(1-2):3-16.
[11]Haig D M,Mercer A A.Ovine diseases[J].Orf Vet Res,1998,29(3-4):311-326.
[12]López-Cedeno Angella,Canedo Gustavo,Knopfel Nicole,Colmenero Isabel,Pérez-Pastrana Esperanza,Torrelo Antonio.Erythema multiforme after orf virus infection[J/OL].Pediatr Dermatol,2018,35(4):e237-e238.
[13]Wittek R,Kuenzle C C,Wyler R.High C+G content in parapoxvirus DNA[J].J Gen Virol,1979,43(1):231-234.
[14]McInnes C J,Wood A R,Nettleton P E,Gilray J A.Genomic comparison of an avirulent strain of Orf virus with that of a virulent wild type isolate reveals that the Orf virus G2L gene is non-essential for replication[J].Virus Genes,2001,22(2):141-150.
[15]Scagliarini A,Ciulli S,Battilani M,Jacoboni I,Montesi F,Casadio R,Prosperi S.Characterisation of immunodominant protein encoded by the F1L gene of orf virus strains isolated in Italy[J].Arch Virol,2002,147(10):1989-1995.
[16]Sullivan J T,Mercer A A,Fleming S B,Robinson A J.Identification and characterization of an Orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K[J].Virology,1994,202(2):968-973.
[17]Gallina L,Scagliarini A,Ciulli S,Prosperi S.Cloning and expression of the Orf virus F1L gene:possible use as a subunit vaccine[J].Vet Res Commu,2004,28(1):291-293.
[18]Scagliarini A,Gallina L,DalPozzo F,Battilani M,Ciulli S,Prosperi S.Heparin bindin gactivity of orf virus F1L protein[J].Virus Res,2004,105(1):107-112.
[19]Savory L J,Stacker S A,Fleming S B,Niven B E,Mercer A A.Viral vascular endothelial growth factor plays a critical role in orf virus infection[J].J Virol,2000,74(22):10699-10706.
[20]Lyttle D J,Fraser K M,Fleming S B,Mercer A A,Robinson A J.Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus[J].J Virol,1994,68(1):84-92.
[21]Wise L M,Veikkola T,Mercer A A,Savory L J,Fleming S B,Caesar C,Vitali A,Makinen T,Alitalo K,Stacker S A.Vascular endothelial growth factor(VEGF)-like protein from orf virus NZ2 binds to VEG-FR2 and neuropilin-1[J].Proc Natl Acad Sci USA,1999,96(6):3071-3076.
[22]Mercer A A,Wise L M,Scagliarini A,McInnes C J,Büttner M,Rziha H J,McCaughan C A,Fleming S B,Ueda N,Nettleton P F.Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved,functionally relevant structure[J].J Gen Virol,2002,83(Pt11):2845-2855.
[23]Rziha H J,Henkel M,Cottone R,Bauer B,Auge U,G?tz F,Pfaff E,R?ttgen M,Dehio C,Büttner M.Generation of recombinant parapoxviruses:non-essential genes suitable for insertion and expression of foreign genes[J].J Biotech,2000,83(1-2):137-145.
[24]Savory L J,Stacker S A,Fleming S B,Niven B E,Mercer A A.Viral vascular endothelial growth factor plays a critical role in Orf virus infection[J].J Virol,2000,74(22):10699-10706.
[25]Rziha H J,Henkel M,Cottone R,Meyer M,Dehio C,Büttner M.Parapoxviruses:potential alternative vectors for directing the immune response in permissive and nonpermissive hosts[J].J Biotechnol,1999,73(2-3):235-242.
[26]Bordoli L,Kiefer F,Arnold K,Benkert P,Battey J,Schwede T.Protein structure homology modeling using SWISS-MODEL workspace[J].Nat Protoc,2009,4(1):1-13.
[27]Brozzo M S,Bjeli?S,Kisko K,Schleier T,Lepp?nen V M,Alitalo K,Winkler F K,Ballmer-Hofer K.Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization[J].Blood,2012,119(7):1781-1788.
[28]Pieren M,Prota AE,Ruch C,Kostrewa D,Wagner A,Biedermann K,Winkler F K,Ballmer-Hofer K.Crystal structure of the Orf virus NZ2 variant of vascular endothelial growth factor-E.Implications for receptor specificity[J].J Biol Chem,2006,281(28):19578-19587.
[29]Chi X,Zeng X,Li W,Hao W,Li M,Huang X,Huang Y,Daniel L R,Luo S,Wang S.Genome analysis of orf virus isolates from goats in the Fujian Province of southern China[J].Front Microbiol,2015,6:1135.
[30]Li W,Ning Z,Hao W,Song D,Gao F,Zhao K,Liao X,Li M,Rock D L,Luo S.Isolation and phylogenetic analysis of orf virus from the sheep herd outbreak in northeast China[J].BMC Vet Res,2012,8:229.
[2]McElroy M C,Bassett H F.The development of oral lesions in lambs naturally infected with orf virus[J].Vet J2007,174(3):663-664.
[3]Eisa M,Tagel Deen M H,Omer E A.An outbreak of contagious ecthyma in sheep and goats in the Sudan[J].Bulletin of Animal Health and Production in Africa,1985,33,169-170.
[4]Hawkins C D,Ellis T M,Davies M K,Peet R L,Parkinson J.An unusual outbreak of contagious ovine ecthyma[J].Aust Vet J,1991,68(6):210-211.
[5]Chan K W,Lin J W,Lee S H,Liao C J,Tsai M C,Hsu W L,Wong M L,Shih H C.Identification and phylogenetic analysis of orf virus from goats in Taiwan[J].Virus genes,2007,35(3):705-712.
[6]GU S,SHI X,SHI Z,WANG Z,ZHANG M.Identification and phylogenetic analysis of an Orf virus isolated from an outbreak in boer goat in Shanxi Province[J].Agricultural Sciences in China,2011,10(6):946-953.
[7]Zhao K,Song D,He W,Lu H,Zhang B,Li C,Chen K,Gao F.Identification and phylogenetic analysis of an Orf virus isolated from an outbreak in sheep in the Jilin province of China[J].Vet Microbiol,2010,142(3-4):408-415.
[8]葛士坤,张凯照,鲁荣,刘健新,宁章勇.2006-2016年中国羊口疮病毒的遗传演化分析[J].中国畜牧兽医,2018,45(2):439-447.
[9]Mercer A A,Haig D M.Parapoxviruses[M].In:Granoff,A.,Webster,R.G.(Eds.),Encyclopedia of Virology,second ed.Academic Press,1999,1140-1146.
[10]Haig D M,McInnes C J.Immunity and counter-immunity during infection with the parapoxvirus orf virus[J].Virus Res,2002,88(1-2):3-16.
[11]Haig D M,Mercer A A.Ovine diseases[J].Orf Vet Res,1998,29(3-4):311-326.
[12]López-Cedeno Angella,Canedo Gustavo,Knopfel Nicole,Colmenero Isabel,Pérez-Pastrana Esperanza,Torrelo Antonio.Erythema multiforme after orf virus infection[J/OL].Pediatr Dermatol,2018,35(4):e237-e238.
[13]Wittek R,Kuenzle C C,Wyler R.High C+G content in parapoxvirus DNA[J].J Gen Virol,1979,43(1):231-234.
[14]McInnes C J,Wood A R,Nettleton P E,Gilray J A.Genomic comparison of an avirulent strain of Orf virus with that of a virulent wild type isolate reveals that the Orf virus G2L gene is non-essential for replication[J].Virus Genes,2001,22(2):141-150.
[15]Scagliarini A,Ciulli S,Battilani M,Jacoboni I,Montesi F,Casadio R,Prosperi S.Characterisation of immunodominant protein encoded by the F1L gene of orf virus strains isolated in Italy[J].Arch Virol,2002,147(10):1989-1995.
[16]Sullivan J T,Mercer A A,Fleming S B,Robinson A J.Identification and characterization of an Orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K[J].Virology,1994,202(2):968-973.
[17]Gallina L,Scagliarini A,Ciulli S,Prosperi S.Cloning and expression of the Orf virus F1L gene:possible use as a subunit vaccine[J].Vet Res Commu,2004,28(1):291-293.
[18]Scagliarini A,Gallina L,DalPozzo F,Battilani M,Ciulli S,Prosperi S.Heparin bindin gactivity of orf virus F1L protein[J].Virus Res,2004,105(1):107-112.
[19]Savory L J,Stacker S A,Fleming S B,Niven B E,Mercer A A.Viral vascular endothelial growth factor plays a critical role in orf virus infection[J].J Virol,2000,74(22):10699-10706.
[20]Lyttle D J,Fraser K M,Fleming S B,Mercer A A,Robinson A J.Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus[J].J Virol,1994,68(1):84-92.
[21]Wise L M,Veikkola T,Mercer A A,Savory L J,Fleming S B,Caesar C,Vitali A,Makinen T,Alitalo K,Stacker S A.Vascular endothelial growth factor(VEGF)-like protein from orf virus NZ2 binds to VEG-FR2 and neuropilin-1[J].Proc Natl Acad Sci USA,1999,96(6):3071-3076.
[22]Mercer A A,Wise L M,Scagliarini A,McInnes C J,Büttner M,Rziha H J,McCaughan C A,Fleming S B,Ueda N,Nettleton P F.Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved,functionally relevant structure[J].J Gen Virol,2002,83(Pt11):2845-2855.
[23]Rziha H J,Henkel M,Cottone R,Bauer B,Auge U,G?tz F,Pfaff E,R?ttgen M,Dehio C,Büttner M.Generation of recombinant parapoxviruses:non-essential genes suitable for insertion and expression of foreign genes[J].J Biotech,2000,83(1-2):137-145.
[24]Savory L J,Stacker S A,Fleming S B,Niven B E,Mercer A A.Viral vascular endothelial growth factor plays a critical role in Orf virus infection[J].J Virol,2000,74(22):10699-10706.
[25]Rziha H J,Henkel M,Cottone R,Meyer M,Dehio C,Büttner M.Parapoxviruses:potential alternative vectors for directing the immune response in permissive and nonpermissive hosts[J].J Biotechnol,1999,73(2-3):235-242.
[26]Bordoli L,Kiefer F,Arnold K,Benkert P,Battey J,Schwede T.Protein structure homology modeling using SWISS-MODEL workspace[J].Nat Protoc,2009,4(1):1-13.
[27]Brozzo M S,Bjeli?S,Kisko K,Schleier T,Lepp?nen V M,Alitalo K,Winkler F K,Ballmer-Hofer K.Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization[J].Blood,2012,119(7):1781-1788.
[28]Pieren M,Prota AE,Ruch C,Kostrewa D,Wagner A,Biedermann K,Winkler F K,Ballmer-Hofer K.Crystal structure of the Orf virus NZ2 variant of vascular endothelial growth factor-E.Implications for receptor specificity[J].J Biol Chem,2006,281(28):19578-19587.
[29]Chi X,Zeng X,Li W,Hao W,Li M,Huang X,Huang Y,Daniel L R,Luo S,Wang S.Genome analysis of orf virus isolates from goats in the Fujian Province of southern China[J].Front Microbiol,2015,6:1135.
[30]Li W,Ning Z,Hao W,Song D,Gao F,Zhao K,Liao X,Li M,Rock D L,Luo S.Isolation and phylogenetic analysis of orf virus from the sheep herd outbreak in northeast China[J].BMC Vet Res,2012,8:229.
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