鸭副粘病毒分离鉴定、F基因序列分析及生物学特性研究
摘要
鸭副粘病毒病(Duck Paramyxo Virus Disease)是由鸭副粘病毒(Duck Paramyxo Virus)引起的一种病毒性急性传染病。该病以气管环出血,肺脏弥漫性出血,脾脏肿大为主要特征。鸭副粘病毒属于副粘病毒科(Paramyxoviridae),副粘病毒亚科,腮腺炎病毒属(Rubulavirus)。长期以来,禽1型副粘病毒不感染水禽,水禽即使带毒也不发病。但近年来,该病的流行又呈现出新的特点:高抗体禽群发生该病,禽1型副粘病毒对水禽的致病力逐渐增强,水禽(鹅、鸭、企鹅等)不再仅是禽1型副粘病毒的宿主和储存库,已成为禽1型副粘病毒的易感禽类。其中鹅、鸭的易感性最高,且不同日龄的鹅、鸭均易感,日龄越小,发病率和死亡率越高,半个月内的雏鸭、雏鹅发病率和死亡率最高。近年来,该病在我国呈上升趋势,给我国养鸭业乃至整个养禽业带来巨大的经济损失。
2006-2008年在山东地区部分养鸭场出现鸭子大量死亡现象,本实验室从来自潍坊、滨州、肥城、东营、泰安、济南的鸭病料和鸭胚中分离得到六株鸭副粘病毒。应用RT-PCR技术对这六株副粘病毒的F基因扩增后进行序列测定,并与传统疫苗株、标准强毒株F48E9和已发表的24株鸭副粘病毒进行同源性比较、进化树分析,旨在从分子生物学水平阐明山东地区鸭副粘病毒分离株之间的亲缘关系以及与其它地区流行株之间的亲缘关系,为该病的预防和控制提供理论依据。
本课题分二部分进行研究。
第一部分:一株经鸭胚传递的鸭副粘病毒的分离鉴定及生物学特性研究2008年6月份山东省某种鸭场260日龄的种鸭出现产蛋下降,但未出现死亡,该种鸭群所产种蛋连续3批在孵化至20~25胚龄时出现约10%的死胚,出壳后雏鸭弱雏较多,部分雏鸭出壳后即出现神经症状,表现为头颈扭转、角弓反张、斜颈、左右摇摆、站立不稳、瘫痪等,剖检可见脑膜出血、肺脏出血、十二指肠黏膜弥漫性出血。为确定发病原因,我们对死亡鸭胚、1日龄的病死鸭及出现神经症状的雏鸭进行了病原的分离、鉴定,确定为副粘病毒,命名为SDFCH株,对该分离株的生物学特性进行了研究。毒力测定结果表明该病毒鸡肧平均致死亡时间(MDT)为56.6 h;1日龄SPF鸡脑内接种分离病毒致病指数(ICPI)为1.78;6周龄SPF鸡静脉接种致病指数(IVPI)为2.59。攻毒雏鸭生长缓慢,精神沉郁,剖检可见气管弥漫性出血、肺脏出血、腺胃乳头出血、胰脏出血、十二指肠黏膜弥漫性出血,个别雏鸭脑膜出血。
第二部分:鸭副粘病毒山东株F基因克隆与序列分析
对2006-2008年从山东省不同地区规模化鸭场采集的疑似鸭副粘病毒病症状的病料进行了鸭副粘病毒的分离鉴定及F基因克隆和序列分析,结果表明F基因的核苷酸序列较稳定,不同地区6株鸭副粘病毒毒株的F基因全基因组序列均由1662bp组成,彼此间核苷酸序列同源性达95.3%～99.8%,亲缘关系密切;与Genbank已发表的鸭副粘病毒参考毒株的同源性介于87.4%~97.8%;与Genbank已发表的鹅副粘病毒参考毒株的同源性介于95.7~98.4%;与传统的疫苗株Lasota株的同源性在87.7%~88.3%之间;与F48E9的同源性在90.3%~91.0%之间。系统进化树分析表明这6株毒株与江苏、黑龙江、广东的鹅副粘病毒毒株和鸭副粘病毒毒株亲缘性较近,属于同一分支。本研究对所测定的6株鸭副粘病毒毒株F基因所对应的氨基酸序列分析表明:6株毒株之间的氨基酸同源性为95.3%~98.0%;与Genbank已发表的鸭源副粘病毒的氨基酸同源性为87.4%~97.5%;与鹅源副粘病毒的氨基酸同源性为96.4%~98.4%。6株毒株与鹅源副粘病毒的亲缘性较近,因此推测鸭副粘病毒是由鹅源副粘病毒进化而来。
Duck Paramyxo virus disease is regarded as one of the most devaetating diseases of poultry in the world, caused by Duck Paramyxo Virus. It is included as an Office International des Epizooties(OIE)list A disease.In the past two decades,an intensive vaccination program against Duck Paramyxo virus disease had been practiced in both large-poultry operations and village poultry farming.This disease breaks out infrequently in some vaccinated flocks.
Six Duck Paramyxo Viruses were isolated from shandong province. The Fusion (F) gene of these isolate strains were amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique.The F genes were sequenced after the products of amplification being cloned.So that it can give great help for the study of the prevalence and control. This study included two parts.
Part I: Biological Identification of Paramyxovirus Isolated from Ducklings and Duck-embryos
A strain virus isolated from duckling and duck-embryos was identified as paramyxovirus by HA , HI and neutralization of virus. The virus was named SDFCH strain. The MDT,ICPI and IVPI for SDFCH strain were 56.6h,1.78 and 2.59.LD50 of duck-embryos is 10-8.5. The results indicated that SDFCH isolate was virulent strain.Ducklings and duck-embryos which were challenged by paramyxovirus SDFCH strain had visible pathological change. The fusion (F) gene of SDFCH strain was amplified by the RT-PCR using three pairs of primers designed according to the published gene sequences . The F gene of SDFCH strain exhibited 96.4%~98.0% nucleotide and 96.4%~98.0% amino acid homology to those of paramyxoviruses isolated from ducks and gooses.But only 84.4% nucleotide and 87.9% amino acid homology to Lasota strain. The results indicated that SDFCH strain was genetically closer to paramyxoviruses isolated from ducks and gooses than to Lasota strain
Part II: Cloning and sequencing of complete F genome of Duck Paramyxo virus Shandong strains.
Six paramyxovirus isolates were obtained from lungs, livers and brains of ducks.The The result of RT-PCR showed that isolatesSDFCH,SDTA,SDDY,SDBZH,SDJN,SDWF were velogenic strains of NDV.Then the F fusion gene of those isolates were sequenced.A phylogenetic tree based on published sequences of NDV reference strains were constructed and showed that isolates belong to the genotypeⅥNDV.
2006-2008年在山东地区部分养鸭场出现鸭子大量死亡现象,本实验室从来自潍坊、滨州、肥城、东营、泰安、济南的鸭病料和鸭胚中分离得到六株鸭副粘病毒。应用RT-PCR技术对这六株副粘病毒的F基因扩增后进行序列测定,并与传统疫苗株、标准强毒株F48E9和已发表的24株鸭副粘病毒进行同源性比较、进化树分析,旨在从分子生物学水平阐明山东地区鸭副粘病毒分离株之间的亲缘关系以及与其它地区流行株之间的亲缘关系,为该病的预防和控制提供理论依据。
本课题分二部分进行研究。
第一部分:一株经鸭胚传递的鸭副粘病毒的分离鉴定及生物学特性研究2008年6月份山东省某种鸭场260日龄的种鸭出现产蛋下降,但未出现死亡,该种鸭群所产种蛋连续3批在孵化至20~25胚龄时出现约10%的死胚,出壳后雏鸭弱雏较多,部分雏鸭出壳后即出现神经症状,表现为头颈扭转、角弓反张、斜颈、左右摇摆、站立不稳、瘫痪等,剖检可见脑膜出血、肺脏出血、十二指肠黏膜弥漫性出血。为确定发病原因,我们对死亡鸭胚、1日龄的病死鸭及出现神经症状的雏鸭进行了病原的分离、鉴定,确定为副粘病毒,命名为SDFCH株,对该分离株的生物学特性进行了研究。毒力测定结果表明该病毒鸡肧平均致死亡时间(MDT)为56.6 h;1日龄SPF鸡脑内接种分离病毒致病指数(ICPI)为1.78;6周龄SPF鸡静脉接种致病指数(IVPI)为2.59。攻毒雏鸭生长缓慢,精神沉郁,剖检可见气管弥漫性出血、肺脏出血、腺胃乳头出血、胰脏出血、十二指肠黏膜弥漫性出血,个别雏鸭脑膜出血。
第二部分:鸭副粘病毒山东株F基因克隆与序列分析
对2006-2008年从山东省不同地区规模化鸭场采集的疑似鸭副粘病毒病症状的病料进行了鸭副粘病毒的分离鉴定及F基因克隆和序列分析,结果表明F基因的核苷酸序列较稳定,不同地区6株鸭副粘病毒毒株的F基因全基因组序列均由1662bp组成,彼此间核苷酸序列同源性达95.3%～99.8%,亲缘关系密切;与Genbank已发表的鸭副粘病毒参考毒株的同源性介于87.4%~97.8%;与Genbank已发表的鹅副粘病毒参考毒株的同源性介于95.7~98.4%;与传统的疫苗株Lasota株的同源性在87.7%~88.3%之间;与F48E9的同源性在90.3%~91.0%之间。系统进化树分析表明这6株毒株与江苏、黑龙江、广东的鹅副粘病毒毒株和鸭副粘病毒毒株亲缘性较近,属于同一分支。本研究对所测定的6株鸭副粘病毒毒株F基因所对应的氨基酸序列分析表明:6株毒株之间的氨基酸同源性为95.3%~98.0%;与Genbank已发表的鸭源副粘病毒的氨基酸同源性为87.4%~97.5%;与鹅源副粘病毒的氨基酸同源性为96.4%~98.4%。6株毒株与鹅源副粘病毒的亲缘性较近,因此推测鸭副粘病毒是由鹅源副粘病毒进化而来。
Duck Paramyxo virus disease is regarded as one of the most devaetating diseases of poultry in the world, caused by Duck Paramyxo Virus. It is included as an Office International des Epizooties(OIE)list A disease.In the past two decades,an intensive vaccination program against Duck Paramyxo virus disease had been practiced in both large-poultry operations and village poultry farming.This disease breaks out infrequently in some vaccinated flocks.
Six Duck Paramyxo Viruses were isolated from shandong province. The Fusion (F) gene of these isolate strains were amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique.The F genes were sequenced after the products of amplification being cloned.So that it can give great help for the study of the prevalence and control. This study included two parts.
Part I: Biological Identification of Paramyxovirus Isolated from Ducklings and Duck-embryos
A strain virus isolated from duckling and duck-embryos was identified as paramyxovirus by HA , HI and neutralization of virus. The virus was named SDFCH strain. The MDT,ICPI and IVPI for SDFCH strain were 56.6h,1.78 and 2.59.LD50 of duck-embryos is 10-8.5. The results indicated that SDFCH isolate was virulent strain.Ducklings and duck-embryos which were challenged by paramyxovirus SDFCH strain had visible pathological change. The fusion (F) gene of SDFCH strain was amplified by the RT-PCR using three pairs of primers designed according to the published gene sequences . The F gene of SDFCH strain exhibited 96.4%~98.0% nucleotide and 96.4%~98.0% amino acid homology to those of paramyxoviruses isolated from ducks and gooses.But only 84.4% nucleotide and 87.9% amino acid homology to Lasota strain. The results indicated that SDFCH strain was genetically closer to paramyxoviruses isolated from ducks and gooses than to Lasota strain
Part II: Cloning and sequencing of complete F genome of Duck Paramyxo virus Shandong strains.
Six paramyxovirus isolates were obtained from lungs, livers and brains of ducks.The The result of RT-PCR showed that isolatesSDFCH,SDTA,SDDY,SDBZH,SDJN,SDWF were velogenic strains of NDV.Then the F fusion gene of those isolates were sequenced.A phylogenetic tree based on published sequences of NDV reference strains were constructed and showed that isolates belong to the genotypeⅥNDV.
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