一株H5N1亚型禽流感病毒的生物学特性及其感染性克隆的研究
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摘要
本论文对１株H5N1亚型的禽流感病毒中国大陆分离株进行了分子遗传衍化、生物学特性的研究并以该病毒为基础建立了反向遗传操作系统。
首先,对此株病毒进行全基因组序列的测定、并与1996—2004年的东亚不同地区H5N1亚型HPAI代表株基因组的8个基因序列比较进行了分子遗传衍化的分析，结果表明此毒株各基因(除NA基因外)的核苷酸序列的同源性及分子进化关系与中国广东1996、1997年分离到的GGD/1/96、GGD/3/97两个H5N1亚型禽流感毒株亲缘关系较近，而与香港1997年禽流感事件的H5N1亚型代表株CHK/220/97﹑HK/483/97﹑HK/486/97的亲缘关系相对较远。因此，可以推测本实验毒株DSH/8/01很可能是由中国华南地区的早期的H5N1亚型禽流感毒株变异衍化而来，为一株GGD/1/96-like的H5N1亚型禽流感的病毒。
其次，通过此毒株对鸡的致病性试验发现：其EID50为10-8.33/0.1ml，感染鸡的IVPI为3.00、鸡的鼻腔感染平均死亡时间为3.82天、鸡的静脉接种的平均死亡时间为1.00天，故此毒株为高致病力禽流感病毒毒株。此株病毒经鼻腔接种感染后，病毒在鸡的不同组织器官中的病毒含量有所不同，在肺组织中的病毒含量最高高达6.98㏒10EID50/0.1ml，其它依次为心肌、法氏囊、肾脏、脑组织、脾脏、肝脏，而仅有个别鸡可从小肠分离到病毒，胰腺中分离不到病毒。由此可见，此株高致病力禽流感病毒对鸡具有广泛的组织嗜性。
第三,通过此毒株对小鼠的致病性试验发现：此毒株不仅对鸡具有高致病力，而且其可感染小鼠，并表现一定的致病性。此毒株在大剂量攻毒（50微升106、105EID50）时，小鼠表现出明显的临床症状(皮毛卷曲,弓背团缩,嗜睡等)、体重显著下降，甚至引起小鼠死亡，故对小鼠有较高的致病性。小剂量50微升104～101EID50攻毒时，小鼠体重变化基本上不明显。另外，此毒株MLD50为104.16EID50，故其对小白鼠表现为中等致病力。从此毒株感染小鼠的组织嗜性试验结果来看，经鼻腔接种感染小鼠后，禽流感病毒在肺脏中一直保持较高的含量；在脑中病毒的含量次之，并随时间的增加有所增高；在肾脏﹑脾脏﹑肝脏、心脏、血液可分离到病毒但含量并不高；小肠、胰腺几乎分离不到病毒。因此。此株H5N1亚型禽流感病毒经鼻腔感染小鼠后，病毒可通过呼吸系统侵入其他大部分组织器官，并可突破血脑屏障侵入大脑组织，最终引起小鼠死亡。这些结果表明对禽类有高致病力的H5N1亚型禽流感病毒，经哺乳动物体内复制增殖后，对哺乳动物的致病性有明显的差异。同时，也证明H5N1禽流感病毒可以直接对哺乳动物发病，甚至死亡。因此，可推测本实验毒株DSH/8/01具有潜在的感染人的可能性。
最后,应用8质粒反向遗传操作系统救获出了具有感染性的重组病毒粒子，为进一步对此毒株进行反向遗传操作奠定了实验基础。
总之,本研究对此株H5N1亚型高致病力禽流感病毒的生物学特性(包括分子生物学特性)进行了系统,并建立了此毒株的反向遗传操作系统,为今后进一步研究我国H5N1亚型禽流感病毒的致病机制、病毒的结构与功能、筛选新型疫苗毒株奠定实验和理论基础，同时也为我国H5N1亚型高致病力禽流感的遗传与衍化的研究提供了实验数据。
An H5N1 avian influenza virus (AIV) isolated from China was studied its molecular evolution and biological characteristics and established an eight-plasmid reverse genetics system for this virus in this paper.
Firstly, this virus named as DSH/8/01(H5N1) was sequenced and molecular evolutionarily analyzed, which compared with fifteen H5N1 AIV isolated from different regions in East Asia during the period of 1996 to 2004.DNA sequence analysis of these viruses genes showed that this virus excluded from NA gene in this study was closely related with GGD/3/97and GGD/1/96,which isolated from gooses in Guangdong, China in 1996 and 1997.But, it was comparatively far related with CHK/220/97,HK/483/97 and HK/486/97,which isolated from chickens and human in HongKong during the 1997 outbreak of AIV. Therefore, I suppose that DSH/8/01 in this study possibly came from early H5N1 AIV in Southern China, which is a GGD/1/96-like virus.
Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. Fifty percent of Egg Infective Doses (EID50), Intravenous Pathogenecity index (IVPI) to chicken of this virus, Mean Death Time to chicken of intranasal and intravenous inoculated virus were 10-8.33/0.1ml, 3.00,3.82days and 1.00 day respectively. This virus titer of chicken’s lung was most high, which was 6.98(log10EID50/0.1ml). The replicated virus also was isolated from the heart muscle, bursa of Fabricius, kidney, brain, spleen, liver in chickens, whereas only a few chickens’ intestine can isolated this virus and chickens’ pancreas cannot. Accordingly, this virus is a H5N1 Highly Pathogenic Avian Influenza Virus.
This virus intranasal inoculated BALB/c mice experiment showed that it had had evident pathogenicity to mice. The majority of mice infected intranasally with 106 to 105EID50 of this virus showed clinical signs, more significant weight loss, even led to death. Whenas, mice infected intranasally with 104 to 101EID50 of this virus had no significant weight loss. 50% Lethal Doses to BALB/c mice (MLD50) of this virus was 104.16EID50.The highest virus titers in the mice’s tissue and organs were lung and brain, but This virus titer in the brain was higher on day 6 than on day 4.The replicated virus also was recovered from kidney, spleen, liver, the heart muscle and blood, whereas intestine and pancreas nearly can not isolated this virus. Thus, pathogenicity of this virus to chickens and mice was obviously
different, and these test also proved that the virulence mechanism responsible for the lethality of influenza virus in birds also operates in mammalian host.
I established an eight-plasmid reverse genetics system for this virus, and succeeded to rescue infectious AIV in order to research its structure and function and its pathogenicity mechanism for the future.
In conclusion, the research is a good base for development of new-type AIV vaccine, the further study for the molecular evolution, structure, and function and pathogenicity mechanism of H5N1 AIV.
首先,对此株病毒进行全基因组序列的测定、并与1996—2004年的东亚不同地区H5N1亚型HPAI代表株基因组的8个基因序列比较进行了分子遗传衍化的分析，结果表明此毒株各基因(除NA基因外)的核苷酸序列的同源性及分子进化关系与中国广东1996、1997年分离到的GGD/1/96、GGD/3/97两个H5N1亚型禽流感毒株亲缘关系较近，而与香港1997年禽流感事件的H5N1亚型代表株CHK/220/97﹑HK/483/97﹑HK/486/97的亲缘关系相对较远。因此，可以推测本实验毒株DSH/8/01很可能是由中国华南地区的早期的H5N1亚型禽流感毒株变异衍化而来，为一株GGD/1/96-like的H5N1亚型禽流感的病毒。
其次，通过此毒株对鸡的致病性试验发现：其EID50为10-8.33/0.1ml，感染鸡的IVPI为3.00、鸡的鼻腔感染平均死亡时间为3.82天、鸡的静脉接种的平均死亡时间为1.00天，故此毒株为高致病力禽流感病毒毒株。此株病毒经鼻腔接种感染后，病毒在鸡的不同组织器官中的病毒含量有所不同，在肺组织中的病毒含量最高高达6.98㏒10EID50/0.1ml，其它依次为心肌、法氏囊、肾脏、脑组织、脾脏、肝脏，而仅有个别鸡可从小肠分离到病毒，胰腺中分离不到病毒。由此可见，此株高致病力禽流感病毒对鸡具有广泛的组织嗜性。
第三,通过此毒株对小鼠的致病性试验发现：此毒株不仅对鸡具有高致病力，而且其可感染小鼠，并表现一定的致病性。此毒株在大剂量攻毒（50微升106、105EID50）时，小鼠表现出明显的临床症状(皮毛卷曲,弓背团缩,嗜睡等)、体重显著下降，甚至引起小鼠死亡，故对小鼠有较高的致病性。小剂量50微升104～101EID50攻毒时，小鼠体重变化基本上不明显。另外，此毒株MLD50为104.16EID50，故其对小白鼠表现为中等致病力。从此毒株感染小鼠的组织嗜性试验结果来看，经鼻腔接种感染小鼠后，禽流感病毒在肺脏中一直保持较高的含量；在脑中病毒的含量次之，并随时间的增加有所增高；在肾脏﹑脾脏﹑肝脏、心脏、血液可分离到病毒但含量并不高；小肠、胰腺几乎分离不到病毒。因此。此株H5N1亚型禽流感病毒经鼻腔感染小鼠后，病毒可通过呼吸系统侵入其他大部分组织器官，并可突破血脑屏障侵入大脑组织，最终引起小鼠死亡。这些结果表明对禽类有高致病力的H5N1亚型禽流感病毒，经哺乳动物体内复制增殖后，对哺乳动物的致病性有明显的差异。同时，也证明H5N1禽流感病毒可以直接对哺乳动物发病，甚至死亡。因此，可推测本实验毒株DSH/8/01具有潜在的感染人的可能性。
最后,应用8质粒反向遗传操作系统救获出了具有感染性的重组病毒粒子，为进一步对此毒株进行反向遗传操作奠定了实验基础。
总之,本研究对此株H5N1亚型高致病力禽流感病毒的生物学特性(包括分子生物学特性)进行了系统,并建立了此毒株的反向遗传操作系统,为今后进一步研究我国H5N1亚型禽流感病毒的致病机制、病毒的结构与功能、筛选新型疫苗毒株奠定实验和理论基础，同时也为我国H5N1亚型高致病力禽流感的遗传与衍化的研究提供了实验数据。
An H5N1 avian influenza virus (AIV) isolated from China was studied its molecular evolution and biological characteristics and established an eight-plasmid reverse genetics system for this virus in this paper.
Firstly, this virus named as DSH/8/01(H5N1) was sequenced and molecular evolutionarily analyzed, which compared with fifteen H5N1 AIV isolated from different regions in East Asia during the period of 1996 to 2004.DNA sequence analysis of these viruses genes showed that this virus excluded from NA gene in this study was closely related with GGD/3/97and GGD/1/96,which isolated from gooses in Guangdong, China in 1996 and 1997.But, it was comparatively far related with CHK/220/97,HK/483/97 and HK/486/97,which isolated from chickens and human in HongKong during the 1997 outbreak of AIV. Therefore, I suppose that DSH/8/01 in this study possibly came from early H5N1 AIV in Southern China, which is a GGD/1/96-like virus.
Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. Fifty percent of Egg Infective Doses (EID50), Intravenous Pathogenecity index (IVPI) to chicken of this virus, Mean Death Time to chicken of intranasal and intravenous inoculated virus were 10-8.33/0.1ml, 3.00,3.82days and 1.00 day respectively. This virus titer of chicken’s lung was most high, which was 6.98(log10EID50/0.1ml). The replicated virus also was isolated from the heart muscle, bursa of Fabricius, kidney, brain, spleen, liver in chickens, whereas only a few chickens’ intestine can isolated this virus and chickens’ pancreas cannot. Accordingly, this virus is a H5N1 Highly Pathogenic Avian Influenza Virus.
This virus intranasal inoculated BALB/c mice experiment showed that it had had evident pathogenicity to mice. The majority of mice infected intranasally with 106 to 105EID50 of this virus showed clinical signs, more significant weight loss, even led to death. Whenas, mice infected intranasally with 104 to 101EID50 of this virus had no significant weight loss. 50% Lethal Doses to BALB/c mice (MLD50) of this virus was 104.16EID50.The highest virus titers in the mice’s tissue and organs were lung and brain, but This virus titer in the brain was higher on day 6 than on day 4.The replicated virus also was recovered from kidney, spleen, liver, the heart muscle and blood, whereas intestine and pancreas nearly can not isolated this virus. Thus, pathogenicity of this virus to chickens and mice was obviously
different, and these test also proved that the virulence mechanism responsible for the lethality of influenza virus in birds also operates in mammalian host.
I established an eight-plasmid reverse genetics system for this virus, and succeeded to rescue infectious AIV in order to research its structure and function and its pathogenicity mechanism for the future.
In conclusion, the research is a good base for development of new-type AIV vaccine, the further study for the molecular evolution, structure, and function and pathogenicity mechanism of H5N1 AIV.
引文
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Matrosovich MN, Matrosovich TY, et al. Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A. 2004 Mar 30; 101(13): 4620-4.
Ha Y; Stevens D.J; et al. X-ray structure of hemagglutinin of a potential H3 avain progenitor of the 1968 HongKong pandemic influenza virus. Virology 2003, 209-218.
Watsuki-Horimoto K, Kanazawa R, et al. The index influenza A virus subtype H5N1 isolated from a human in 1997 differs in its receptor-binding properties from a virulent avian influenza virus. J Gen Virol. 2004 Apr; 85(Pt 4): 1001-1005.
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Suzuki Y., Ito T., et al. Sialic acid species as a determinant of the host range of influenza viruses. J. Virol .2000,74, 11825–11831.
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