山东省某地鹦鹉喙羽病病毒的全基因组序列及对SPF鸡胚的致病性探究
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摘要
2017年4月,山东省某鹦鹉场发生疑似喙羽病感染,鹦鹉出现异形喙和羽毛脱落,甚至急性猝死。目的:了解该地区鹦鹉喙羽病病毒(Psittacine beak and feather disease,PBFD)的流行现状、分子生物学特征和遗传变异规律,为PBFDV的防控提供参考依据。方法:收集怀疑有喙羽病病毒(PBFDV)感染的非洲灰鹦鹉的新鲜肝脏组织和羽毛样本,根据PBFDV全基因组序列设计特异性引物,从患病灰鹦鹉肝脏和羽毛组织中扩增PBFDV的全基因组序列,克隆到p MD19-T载体中,经测序获得PBFDV全长c DNA序列,利用DNAstar软件对全基因组进行遗传变异分析。由于目前尚未发现适合PBFDV繁殖的培养细胞或胚胎,我们尝试使用患病鹦鹉的羽毛研磨液注入7日龄SPF鸡胚的卵黄膜中。结果:分离到3株PBFDV (Gen Bank登录号:MH279594,MH180298和MH190788),全基因组序列测序后,发现它们彼此具有93. 3%—99. 9%的相似性,并且它们也与Gen Bank中的一些参考序列具有86. 1%—98. 8%的相似性。卵黄囊注射病毒液后胚胎发育变缓,48—72 h内鸡胚死亡,全身出血。接着我们收集鸡胚的肝组织的研磨液在鸡胚上传代至第5代,均能使鸡胚死亡。提取来自每一代鸡胚肝组织的DNA都可以通过PCR扩增出PBFDV的目的条带。结论:以上实验结果表明,成功从患病鹦鹉体内克隆出PBFDV的全基因,并完成了序列分析,同时PBFDV接种鸡胚卵黄膜后成功复制。我们针对在该地区新出现的病毒从分子方面进行相关研究,分析其遗传变异特征,以研究该病毒与其他地区喙羽病病毒之间的亲缘关系,这些数据为研究该病毒的遗传变异和流行病学提供分子生物学依据。
In April 2017,a few parrots in a farm had abnormally shaped beaks and feathers,and even experienced acute sudden death in Shandong Province. This study was conducted to understand the prevalence,molecular biological characteristics and genetic variation of psittacine beak and feather disease( PBFD) in a parrot farm of Shandong Province,and provide reference for disease prevention and control. We collected fresh liver tissue and feather samples of African grey parrots suspected of PBFDV infection, and designed a pair of specific primers based on the PBFDV genome-wide sequence to detect PBFDV in the feathers and liver of the diseased parrots.The whole genome sequence of PBFDV was amplified,cloned into p MD19-T vector,and the fulllength c DNA sequence of PBFDV was obtained by sequencing and then analyzed by DNAstar software. No cultured cells or embryos suitable for PBFDV reproduction were found. We injected the yolk membrane of 7-day-old SPF chicken embryos with the feather slurry of the diseased parrot.Three strains of PBFDVs were isolated( Gen Bank accession numbers: MH279594, MH180298 and MH190788). The whole genome sequence of the three strains shared 93. 3%-99. 9% similarity and they also shared 86. 1%-98. 8% similarity with other reference sequences in Gen Bank.After the injection of virus solution,the embryonic development of chicken was slowed and embryos died after 48-72 h,due to systemic hemorrhage. We then collected the grinding fluid of the liver tissue of the chicken embryo and passed it on to the fifth generation,after which all embryos died. The target band of PBFDV was amplified by PCR in DNA extracted from liver tissue of each generation of chicken embryo. Our experimental results showed that the complete gene of PBFDV was successfully cloned from the diseased parrot and sequence analysis was completed. At the same time,PBFDV was successfully replicated in chicken embryos. We conducted a molecular study on this emerging virus and analyzed its genetic variation to study the genetic relationship between the virus and other areas of the feather disease virus. These data provide a molecular biology basis for studying the genetic variation of the virus and epidemiology.
In April 2017,a few parrots in a farm had abnormally shaped beaks and feathers,and even experienced acute sudden death in Shandong Province. This study was conducted to understand the prevalence,molecular biological characteristics and genetic variation of psittacine beak and feather disease( PBFD) in a parrot farm of Shandong Province,and provide reference for disease prevention and control. We collected fresh liver tissue and feather samples of African grey parrots suspected of PBFDV infection, and designed a pair of specific primers based on the PBFDV genome-wide sequence to detect PBFDV in the feathers and liver of the diseased parrots.The whole genome sequence of PBFDV was amplified,cloned into p MD19-T vector,and the fulllength c DNA sequence of PBFDV was obtained by sequencing and then analyzed by DNAstar software. No cultured cells or embryos suitable for PBFDV reproduction were found. We injected the yolk membrane of 7-day-old SPF chicken embryos with the feather slurry of the diseased parrot.Three strains of PBFDVs were isolated( Gen Bank accession numbers: MH279594, MH180298 and MH190788). The whole genome sequence of the three strains shared 93. 3%-99. 9% similarity and they also shared 86. 1%-98. 8% similarity with other reference sequences in Gen Bank.After the injection of virus solution,the embryonic development of chicken was slowed and embryos died after 48-72 h,due to systemic hemorrhage. We then collected the grinding fluid of the liver tissue of the chicken embryo and passed it on to the fifth generation,after which all embryos died. The target band of PBFDV was amplified by PCR in DNA extracted from liver tissue of each generation of chicken embryo. Our experimental results showed that the complete gene of PBFDV was successfully cloned from the diseased parrot and sequence analysis was completed. At the same time,PBFDV was successfully replicated in chicken embryos. We conducted a molecular study on this emerging virus and analyzed its genetic variation to study the genetic relationship between the virus and other areas of the feather disease virus. These data provide a molecular biology basis for studying the genetic variation of the virus and epidemiology.
引文
[1] FOGELL D J,MARTIN R O,GROOMBRIDGE J J.Beak and feather disease virus in wild and captive parrots:an analysis of geographic and taxonomic distribution and methodological trends[J].Archives of Virology,2016,161(8):2059-2074.
[2] JULIAN L,LORENZO A,CHENUET J P,et al.Evidence of multiple introductions of beak and feather disease virus into the Pacific islands of Nouvelle-Calédonie(New Caledonia)[J].Journal of General Virology,2012,93(11):2466-2472.
[3] RAIDAL S R,CROSS G M.The haemagglutination spectrum of psittacine beak and feather disease virus[J].Avian Pathology,1994,23(4):621-630.
[4] STEWART M E,BONNE N,SHEARER P,et al.Baculovirus expression of beak and feather disease virus(BFDV)capsid protein capable of self-assembly and haemagglutination[J].Journal of Virological Methods,2007,141(2):181-187.
[5] BASSAMI M R,YPELAAR I,BERRYMAN D,et al.Genetic diversity of beak and feather disease virus detected in psittacine species in Australia[J].Virology,2001,279(2):392-400.
[6] VARSANI A,REGNARD G L,BRAGG R,et al.Global genetic diversity and geographical and host-species distribution of beak and feather disease virus isolates[J].Journal of General Virology,2011,92(4):752-767.
[7] RITCHIE B W,NIAGRO F D,LUKERT P D,et al.Characterization of a new virus from cockatoos with psittacine beak and feather disease[J].Virology,1989,171(1):83-88.
[8] CROWTHER R A,BERRIMAN J A,CURRAN W L,et al.Comparison of the structures of three circoviruses:Chicken anemia virus,Porcine circovirus type 2,and Beak and feather disease virus[J].Journal of Virology,2003,77(24):13036-13041.
[9] YILMAZ A,KALETA E F.Disinfectant tests at 20 and 10 degrees C to determine the virucidal activity against circoviruses[J].Deutsche Tierarztliche Wochenschrift,2004,111(6):248-251.
[10] PASS D A,PERRY R A.The pathology of psittacine beak and feather disease[J].Australian Veterinary Journal,1984,61(3):69-74.
[11] STENZEL T,PESTKA D,CHOSZCZ D.The prevalence and genetic characterization of Chlamydia psittaci from domestic and feral pigeons in Poland and the correlation between infection rate and incidence of pigeon circovirus[J].Poultry Science,2014,93(12):3009-3016.
[12] RITCHIE B W,NIAGRO F D,LATIMER K S,et al.Ultrastructural,protein composition,and antigenic comparison of psittacine beak and feather disease virus purified from four genera of psittacine birds[J].Journal of Wildlife Diseases,1990,26(2):196-203.
[13] MCORIST S,BLACK D G,PASS D A,et al.Beak and feather dystrophy in wild sulphur-crested cockatoos(Cacatua galerita)[J].Journal of Wildlife Diseases,1984,20(2):120-124.
[14] GUO J,TIAN J,TAN X,et al.Pathological observations of an experimental infection of geese with goose circovirus[J].Avian Pathology,2011,40(1):55-61.
[15] RITCHIE B W,NIAGRO F D,LATIMER K S,et al.Routes and prevalence of shedding of psittacine beak and feather disease virus[J].American Journal of Veterinary Research,1991,52(11):1804-1809.
[16] RAHAUS M,DESLOGES N,PROBST S,et al.Detection of beak and feather disease virus DNA in embryonated eggs of psittacine birds[J].Veterinarni Medicina,2008,53(1):53-58.
[17] DAVIDSON I,ARTZI N,SHKODA I,et al.The contribution of feathers in the spread of chicken anemia virus[J].Virus Research,2008,132(1/2):152-159.
[18] DUVENAGE L,HITZEROTH I I,MEYERS A E,et al.Expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides[J].Journal of Virological Methods,2013,191(1):55-62.
[19] PATTERSON E I,SWARBRICK C M D,ROMAN N,et al.Differential expression of two isolates of beak and feather disease virus capsid protein in Escherichia coli[J].Journal of Virological Methods,2013,189(1):118-124.
[20] BERT E,TOMASSONE L,PECCATI C,et al.Detection of beak and feather disease virus(BFDV)and avian polyomavirus(APV)DNA in psittacine birds in Italy[J].Journal of Veterinary Medicine,Series B Banner,2005,52(2):64-68.
[21] YANG S Q,WANG L Y,SUN S H.Natural infection with Avian hepatitis E virus and Marek's disease virus in brown layer chickens in China[J].Avian Diseases,2016,60(3):698-704.
[22] HEATH L,MARTIN D P,WARBURTON L,et al.Evidence of unique genotypes of beak and feather disease virus in southern Africa[J].Journal of Virology,2004,78(17):9277-9284.
[23] HEATH L,WILLIAMSON A L,RYBICKI E P.The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus[J].Journal of Virology,2006,80(14):7219-7225.
[24] BONNE N,SHEARER P,SHARP M,et al.Assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease[J].Journal of General Virology,2009,90(3):640-647.
[25] JOHNE R,RAUE R,GRUND C,et al.Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests[J].Avian Pathology,2004,33(3):328-336.
[26] KHALESI B.Studies of beak and feather disease virus infection[D].Murdoch University,2007.
[27] GERLACH H,LEIPOLD R.Feather loss syndrome in cockatoos[J].DTW Deutsche Tierrztliche Wochenschrift,1986,93(1):24-26.
[28] RAIDAL S R,SABINE M,CROSS G M.Laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition[J].Australian Veterinary Journal,1993,70(4):133-137.
[2] JULIAN L,LORENZO A,CHENUET J P,et al.Evidence of multiple introductions of beak and feather disease virus into the Pacific islands of Nouvelle-Calédonie(New Caledonia)[J].Journal of General Virology,2012,93(11):2466-2472.
[3] RAIDAL S R,CROSS G M.The haemagglutination spectrum of psittacine beak and feather disease virus[J].Avian Pathology,1994,23(4):621-630.
[4] STEWART M E,BONNE N,SHEARER P,et al.Baculovirus expression of beak and feather disease virus(BFDV)capsid protein capable of self-assembly and haemagglutination[J].Journal of Virological Methods,2007,141(2):181-187.
[5] BASSAMI M R,YPELAAR I,BERRYMAN D,et al.Genetic diversity of beak and feather disease virus detected in psittacine species in Australia[J].Virology,2001,279(2):392-400.
[6] VARSANI A,REGNARD G L,BRAGG R,et al.Global genetic diversity and geographical and host-species distribution of beak and feather disease virus isolates[J].Journal of General Virology,2011,92(4):752-767.
[7] RITCHIE B W,NIAGRO F D,LUKERT P D,et al.Characterization of a new virus from cockatoos with psittacine beak and feather disease[J].Virology,1989,171(1):83-88.
[8] CROWTHER R A,BERRIMAN J A,CURRAN W L,et al.Comparison of the structures of three circoviruses:Chicken anemia virus,Porcine circovirus type 2,and Beak and feather disease virus[J].Journal of Virology,2003,77(24):13036-13041.
[9] YILMAZ A,KALETA E F.Disinfectant tests at 20 and 10 degrees C to determine the virucidal activity against circoviruses[J].Deutsche Tierarztliche Wochenschrift,2004,111(6):248-251.
[10] PASS D A,PERRY R A.The pathology of psittacine beak and feather disease[J].Australian Veterinary Journal,1984,61(3):69-74.
[11] STENZEL T,PESTKA D,CHOSZCZ D.The prevalence and genetic characterization of Chlamydia psittaci from domestic and feral pigeons in Poland and the correlation between infection rate and incidence of pigeon circovirus[J].Poultry Science,2014,93(12):3009-3016.
[12] RITCHIE B W,NIAGRO F D,LATIMER K S,et al.Ultrastructural,protein composition,and antigenic comparison of psittacine beak and feather disease virus purified from four genera of psittacine birds[J].Journal of Wildlife Diseases,1990,26(2):196-203.
[13] MCORIST S,BLACK D G,PASS D A,et al.Beak and feather dystrophy in wild sulphur-crested cockatoos(Cacatua galerita)[J].Journal of Wildlife Diseases,1984,20(2):120-124.
[14] GUO J,TIAN J,TAN X,et al.Pathological observations of an experimental infection of geese with goose circovirus[J].Avian Pathology,2011,40(1):55-61.
[15] RITCHIE B W,NIAGRO F D,LATIMER K S,et al.Routes and prevalence of shedding of psittacine beak and feather disease virus[J].American Journal of Veterinary Research,1991,52(11):1804-1809.
[16] RAHAUS M,DESLOGES N,PROBST S,et al.Detection of beak and feather disease virus DNA in embryonated eggs of psittacine birds[J].Veterinarni Medicina,2008,53(1):53-58.
[17] DAVIDSON I,ARTZI N,SHKODA I,et al.The contribution of feathers in the spread of chicken anemia virus[J].Virus Research,2008,132(1/2):152-159.
[18] DUVENAGE L,HITZEROTH I I,MEYERS A E,et al.Expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides[J].Journal of Virological Methods,2013,191(1):55-62.
[19] PATTERSON E I,SWARBRICK C M D,ROMAN N,et al.Differential expression of two isolates of beak and feather disease virus capsid protein in Escherichia coli[J].Journal of Virological Methods,2013,189(1):118-124.
[20] BERT E,TOMASSONE L,PECCATI C,et al.Detection of beak and feather disease virus(BFDV)and avian polyomavirus(APV)DNA in psittacine birds in Italy[J].Journal of Veterinary Medicine,Series B Banner,2005,52(2):64-68.
[21] YANG S Q,WANG L Y,SUN S H.Natural infection with Avian hepatitis E virus and Marek's disease virus in brown layer chickens in China[J].Avian Diseases,2016,60(3):698-704.
[22] HEATH L,MARTIN D P,WARBURTON L,et al.Evidence of unique genotypes of beak and feather disease virus in southern Africa[J].Journal of Virology,2004,78(17):9277-9284.
[23] HEATH L,WILLIAMSON A L,RYBICKI E P.The capsid protein of beak and feather disease virus binds to the viral DNA and is responsible for transporting the replication-associated protein into the nucleus[J].Journal of Virology,2006,80(14):7219-7225.
[24] BONNE N,SHEARER P,SHARP M,et al.Assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease[J].Journal of General Virology,2009,90(3):640-647.
[25] JOHNE R,RAUE R,GRUND C,et al.Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests[J].Avian Pathology,2004,33(3):328-336.
[26] KHALESI B.Studies of beak and feather disease virus infection[D].Murdoch University,2007.
[27] GERLACH H,LEIPOLD R.Feather loss syndrome in cockatoos[J].DTW Deutsche Tierrztliche Wochenschrift,1986,93(1):24-26.
[28] RAIDAL S R,SABINE M,CROSS G M.Laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition[J].Australian Veterinary Journal,1993,70(4):133-137.