A型流感病毒冷适应减毒株拯救体系的建立
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摘要
流感(Influenza)是由流行性感冒病毒(Influenza virus)感染引起的急性呼吸道传染病。流感病毒属于正粘病毒科,可感染人和多种动物。其中A型流感病毒对人类威胁最大,曾引起人类历史上4次流感大流行。A型流感病毒抗原性易发生变异,抗原的漂移和转换给流感的防治带来了较大困难。接种疫苗仍然是预防和控制流感的主要手段。目前使用的疫苗包括全病毒疫苗、裂解疫苗和减毒活疫苗。减毒活疫苗可通过鼻腔接种诱导局部的黏膜免疫应答和系统免疫应答,是流感疫苗发展的新方向。反向遗传学的快速发展,为开发流感病毒疫苗提供了新手段,1999年以后Neumann等、Hoffmann等分别建立了完全以质粒为基础的流感病毒反向遗传操作系统,在开发新型流感疫苗、快速制备疫苗候选株等方面显示了良好应用前景。本研究选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60 (H2N2)(MDV-A)流感病毒株作为骨架病毒,运用8质粒系统拯救出冷适应减毒的A型人流感病毒,建立A型流感病毒冷适应拯救体系,并运用于06～07 A型流感病毒推荐疫苗组分株的拯救,为流感病毒新型疫苗的开发奠定了基础。
1.MDV-A 6个内部骨架基因双向转录/表达重组质粒的构建与验证
设计一对引物,以pcDNA3.1(+)质粒为模板扩增多聚A(polyA)信号序列,取代双向转录/表达载体pHW2000上牛生长激素(BGH)的多聚腺苷酸信号序列,构建双向转录/表达载体pAD3000。
人工合成的MDV-A株6个内部基因片段,克隆至双向转录/表达载体pAD3000,构建6个polⅠ-polⅡ系统的转录/表达质粒,经测序验证6个内部基因片段均在设计位点引入突变,期望6个质粒通过同一个载体,转染后利用细胞中的polⅠ和polⅡRNA聚合酶,实现MDV-A 6个内部基因vRNA和mRNA的转录和表达。
用MDV-A任意一个内部基因重组质粒与A/PR/8/34的基因组合,即7+1组合形式共转染COS-1细胞,37℃或33℃、5%CO2培养48h后将转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价。实验结果表明,6种组合均能产生有血凝性的流感病毒,说明双向转录/表达载体pAD3000能正常工作,含有MDV-A 6个内部基因片段的质粒,在同一载体上实现了vRNA和mRNA的转录和表达,并能装配成病毒,为下一步拯救MDV-A冷适应骨架病毒毒株打下了基础。
2冷适应减毒重配A型流感病毒rMDV-A的拯救及部分生物学特性的鉴定
用MDV-A 6个内部基因质粒与A/PR/8/34的表面HA和NA基因质粒组合,共转染COS-1细胞,33℃、5%CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价,得到有血凝性的重组A型流感病毒rMDV-A,电镜形态观察rMDV-A,与野生型流感病毒相似。rMDV-A在鸡胚中传至4代后,收集尿囊液,RT-PCR扩增6个内部基因片段,测序,序列分析与MDV-A 6个内部基因片段序列一致,说明rMDV-A能在鸡胚中稳定增殖。rMDV-A在不加胰酶的情况下感染MDCK细胞,能引起细胞变圆、脱落等病变。收集PR8和rMDV-A病毒在33℃、37℃、38℃和39℃四个温度培养的鸡胚尿囊液,测定两种病毒在MDCK细胞上空斑形成单位(PFU),结果表明rMDV-A效价随着温度的升高有明显下降趋势。到39℃时,病毒效价急剧下降,可见rMDV-A对温度敏感,较低温度(如33℃)更适于rMDV-A病毒的繁殖。rMDV-A的拯救和鉴定,说明成功建立了A型流感病毒冷适应减毒株拯救体系,为开发A型流感病毒减毒疫苗奠定了基础。
3.06～07 A型流感病毒冷适应候选疫苗株的拯救
RT-PCR扩增06～07 A型流感病毒推荐疫苗组分株A/New Caledonia/20/99(H1N1)和A/Wisconsin/67/2005(H3N2)的HA和NA基因,连接pGEM-T载体并测序,得到序列正确的HA和NA基因,并克隆至双向转录/表达载体pAD3000上,分别与MDV-A 6个内部基因共转染COS-1细胞,33℃、5%CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价,得到有血凝性的重组A型流感病毒rMDV-A-H1,rMDV-A-H3,拯救出06～07 A型流感病毒冷适应候选疫苗株,为06～07流感病毒减毒三价疫苗的开发与生产奠定基础。
本研究运用8质粒拯救系统建立了A型流感病毒冷适应拯救体系,为流感病毒新型疫苗的开发提供了新的手段。
Influenza is a highly contagious respiratory disease caused by Influenza virus. Influenza virus, a member of the family Orthomyxoviridae, can infect humans and many kinds of animals. Influenza A virus that had caused fourth pandemic in 20th century is a mass threaten to humans. The fragmented genome of the influenza A virus is more vulnerable to genetic variations which leads antigenic shift and drift. This phenomenon makes it’s more difficult to prevent and control this disease. Compare to other measures, vaccination remains the most effective approach to prevent this disease. Currently, influenza vaccines used in humans contains“inactivated”vaccines、split vaccine and live attenuated vaccines. Live attenuated vaccines, an intranasal vaccine, can induce both local mucosa immune response and system immune response. Due to their safety and efficacy, the live attenuated vaccines are seen as viable alternatives to the“inactivated vaccines”. Reverse genetic systems entirely based on cloned cDNAs of influenza virus established by Neumann, Hoffmann, et al. since 1999 is being employed to specially design“recombinant”A and B viruses, and it has opened the way to develop live attenuated vaccines. In our research , cold-adapted、temperature sensitive and attenuated influenza A virus A/Ann Arbor/6/60 (H2N2) is used as a master donor virus(MDV-A) to generate an attenuated recombinants influenza A virus. This system is also used to rescue the strains for the manufacture of seasonal influenza vaccine for 2006/2007.
1. Construction of six transcription/expression plasmids contained six inner gene of MDV-A
PolyA signal sequence was amplified from plasmid pcDNA3.1(+)by PCR to replace the bovine growth hormone polyadenylation signals of pHW2000, the reconstructed vector named as pAD3000.
Six inner fragments contained five mutations of MDV-A were artificial synthesized. This six cDNAs were separately cloned into pAD3000 to construct six transcription/expression plasmids for the purpose of synthesizing of vRNAs and mRNAs by polⅠand polⅡfrom one template after being transfected into eukaryotic cells such as COS-1. All plasmids were sequenced to ensure it’s the same with the consensus MDV-A sequence except the five mutations.
The COS-1 cell was cotransfected with eight plasmids which contained one plasmid of one fragment from MDV-A and seven plasmids had other seven fragments from PR8. The transfected cells were incubated at 35℃or 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs . The HA titer were determined. A series 7+1 reassortant virus was rescued. It showed that all the six plasmids contained MDV-A genes were shown to be functionally expressed. With this progress, a cold-adapted、temperature sensitive and attenuated strain would be rescued next.
2. Generation of cold-adapted attenuated reassortant influenza virus rMDV-A by reverse genetic and identification some of its biological characterizations
Six plasmids contained MDV-A genes were transfected into COS-1 cells in combination with plasmids incorporating the HA and NA genes of A/PR/8/34. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The reassortant rMDV-A virus was determined by HA assay. The rescued viruses were indistinguishable from the wild-type in morphology. Recovered rMDV-A viruses was passed in eggs for four generations, and then its six inner segments were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. MDCK cells were infected with rMDV-A virus without trypsin. Temperature sensitivity of rMDV-A was determined by plaque assay on MDCK cells. The results showed that the inner gene of the fourth generation reassortant virus was the same with the consensus MDV-A sequence. Recovered rMDV-A viruses could replicate in eggs stably. The MDCK cells infected with rMDV-A became rounding, shedding and formed plaque. Plaque assay showed that rMDV-A have significant reduction in plaque at 39℃compared with 33℃. Recovered rMDV-A viruses was sensitive to temperature, lower temperature such as 33℃was more suitable for its replication. This reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.
3. Rescue of the seasonal influenza vaccine composition strains for the season 2006/2007 by cold-adapted attenuated reverse genetic system
The HA and NA gene of A/New Caledonia/20/99(H1N1)、A/Wisconsin/67/2005(H3N2) were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. The correct genes were then be cloned to pAD3000. This two plasmids contained HA and NA genes were transfected into COS-1 cells in combination with six plasmid incorporating the inner genes of MDV-A respectively. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The recovered virus named rMDV-A-H1 and rMDV-A-H3.Generation of this two ressortant virus is the base of the development of trivalent vaccine for 2006/2007.
Overall, this reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.
1.MDV-A 6个内部骨架基因双向转录/表达重组质粒的构建与验证
设计一对引物,以pcDNA3.1(+)质粒为模板扩增多聚A(polyA)信号序列,取代双向转录/表达载体pHW2000上牛生长激素(BGH)的多聚腺苷酸信号序列,构建双向转录/表达载体pAD3000。
人工合成的MDV-A株6个内部基因片段,克隆至双向转录/表达载体pAD3000,构建6个polⅠ-polⅡ系统的转录/表达质粒,经测序验证6个内部基因片段均在设计位点引入突变,期望6个质粒通过同一个载体,转染后利用细胞中的polⅠ和polⅡRNA聚合酶,实现MDV-A 6个内部基因vRNA和mRNA的转录和表达。
用MDV-A任意一个内部基因重组质粒与A/PR/8/34的基因组合,即7+1组合形式共转染COS-1细胞,37℃或33℃、5%CO2培养48h后将转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价。实验结果表明,6种组合均能产生有血凝性的流感病毒,说明双向转录/表达载体pAD3000能正常工作,含有MDV-A 6个内部基因片段的质粒,在同一载体上实现了vRNA和mRNA的转录和表达,并能装配成病毒,为下一步拯救MDV-A冷适应骨架病毒毒株打下了基础。
2冷适应减毒重配A型流感病毒rMDV-A的拯救及部分生物学特性的鉴定
用MDV-A 6个内部基因质粒与A/PR/8/34的表面HA和NA基因质粒组合,共转染COS-1细胞,33℃、5%CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价,得到有血凝性的重组A型流感病毒rMDV-A,电镜形态观察rMDV-A,与野生型流感病毒相似。rMDV-A在鸡胚中传至4代后,收集尿囊液,RT-PCR扩增6个内部基因片段,测序,序列分析与MDV-A 6个内部基因片段序列一致,说明rMDV-A能在鸡胚中稳定增殖。rMDV-A在不加胰酶的情况下感染MDCK细胞,能引起细胞变圆、脱落等病变。收集PR8和rMDV-A病毒在33℃、37℃、38℃和39℃四个温度培养的鸡胚尿囊液,测定两种病毒在MDCK细胞上空斑形成单位(PFU),结果表明rMDV-A效价随着温度的升高有明显下降趋势。到39℃时,病毒效价急剧下降,可见rMDV-A对温度敏感,较低温度(如33℃)更适于rMDV-A病毒的繁殖。rMDV-A的拯救和鉴定,说明成功建立了A型流感病毒冷适应减毒株拯救体系,为开发A型流感病毒减毒疫苗奠定了基础。
3.06～07 A型流感病毒冷适应候选疫苗株的拯救
RT-PCR扩增06～07 A型流感病毒推荐疫苗组分株A/New Caledonia/20/99(H1N1)和A/Wisconsin/67/2005(H3N2)的HA和NA基因,连接pGEM-T载体并测序,得到序列正确的HA和NA基因,并克隆至双向转录/表达载体pAD3000上,分别与MDV-A 6个内部基因共转染COS-1细胞,33℃、5%CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价,得到有血凝性的重组A型流感病毒rMDV-A-H1,rMDV-A-H3,拯救出06～07 A型流感病毒冷适应候选疫苗株,为06～07流感病毒减毒三价疫苗的开发与生产奠定基础。
本研究运用8质粒拯救系统建立了A型流感病毒冷适应拯救体系,为流感病毒新型疫苗的开发提供了新的手段。
Influenza is a highly contagious respiratory disease caused by Influenza virus. Influenza virus, a member of the family Orthomyxoviridae, can infect humans and many kinds of animals. Influenza A virus that had caused fourth pandemic in 20th century is a mass threaten to humans. The fragmented genome of the influenza A virus is more vulnerable to genetic variations which leads antigenic shift and drift. This phenomenon makes it’s more difficult to prevent and control this disease. Compare to other measures, vaccination remains the most effective approach to prevent this disease. Currently, influenza vaccines used in humans contains“inactivated”vaccines、split vaccine and live attenuated vaccines. Live attenuated vaccines, an intranasal vaccine, can induce both local mucosa immune response and system immune response. Due to their safety and efficacy, the live attenuated vaccines are seen as viable alternatives to the“inactivated vaccines”. Reverse genetic systems entirely based on cloned cDNAs of influenza virus established by Neumann, Hoffmann, et al. since 1999 is being employed to specially design“recombinant”A and B viruses, and it has opened the way to develop live attenuated vaccines. In our research , cold-adapted、temperature sensitive and attenuated influenza A virus A/Ann Arbor/6/60 (H2N2) is used as a master donor virus(MDV-A) to generate an attenuated recombinants influenza A virus. This system is also used to rescue the strains for the manufacture of seasonal influenza vaccine for 2006/2007.
1. Construction of six transcription/expression plasmids contained six inner gene of MDV-A
PolyA signal sequence was amplified from plasmid pcDNA3.1(+)by PCR to replace the bovine growth hormone polyadenylation signals of pHW2000, the reconstructed vector named as pAD3000.
Six inner fragments contained five mutations of MDV-A were artificial synthesized. This six cDNAs were separately cloned into pAD3000 to construct six transcription/expression plasmids for the purpose of synthesizing of vRNAs and mRNAs by polⅠand polⅡfrom one template after being transfected into eukaryotic cells such as COS-1. All plasmids were sequenced to ensure it’s the same with the consensus MDV-A sequence except the five mutations.
The COS-1 cell was cotransfected with eight plasmids which contained one plasmid of one fragment from MDV-A and seven plasmids had other seven fragments from PR8. The transfected cells were incubated at 35℃or 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs . The HA titer were determined. A series 7+1 reassortant virus was rescued. It showed that all the six plasmids contained MDV-A genes were shown to be functionally expressed. With this progress, a cold-adapted、temperature sensitive and attenuated strain would be rescued next.
2. Generation of cold-adapted attenuated reassortant influenza virus rMDV-A by reverse genetic and identification some of its biological characterizations
Six plasmids contained MDV-A genes were transfected into COS-1 cells in combination with plasmids incorporating the HA and NA genes of A/PR/8/34. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The reassortant rMDV-A virus was determined by HA assay. The rescued viruses were indistinguishable from the wild-type in morphology. Recovered rMDV-A viruses was passed in eggs for four generations, and then its six inner segments were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. MDCK cells were infected with rMDV-A virus without trypsin. Temperature sensitivity of rMDV-A was determined by plaque assay on MDCK cells. The results showed that the inner gene of the fourth generation reassortant virus was the same with the consensus MDV-A sequence. Recovered rMDV-A viruses could replicate in eggs stably. The MDCK cells infected with rMDV-A became rounding, shedding and formed plaque. Plaque assay showed that rMDV-A have significant reduction in plaque at 39℃compared with 33℃. Recovered rMDV-A viruses was sensitive to temperature, lower temperature such as 33℃was more suitable for its replication. This reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.
3. Rescue of the seasonal influenza vaccine composition strains for the season 2006/2007 by cold-adapted attenuated reverse genetic system
The HA and NA gene of A/New Caledonia/20/99(H1N1)、A/Wisconsin/67/2005(H3N2) were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. The correct genes were then be cloned to pAD3000. This two plasmids contained HA and NA genes were transfected into COS-1 cells in combination with six plasmid incorporating the inner genes of MDV-A respectively. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The recovered virus named rMDV-A-H1 and rMDV-A-H3.Generation of this two ressortant virus is the base of the development of trivalent vaccine for 2006/2007.
Overall, this reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.
引文
[1] Radecke F, Billeter MA. Reverse genetics meets the nonsegmented negative-strand RNA viruses[J]. Med Virol, 1997, 7:49~63.
[2] Nagai Y, Kato A. Paramyxovirus reverse genetics is coming of ag[eJ]. Microbiol Immunol, 1999, 43(7): 615~24.
[3] 范宝昌,杨佩英. RNA 病毒的反向遗传学操作[J].军事医学科学院院刊,2001,25(3):223～27.
[4] 甘孟侯主编. 禽流感[M],第二版,北京:北京农业大学出版社,2002,15～79.
[5] Webster, RG,Bean WJ, Gorman OT, et al. Evolution and ecology of influenza A viruses[J]. Microbiol Rev,1992,56:152~79.
[6] Cox NJ, Subbarao K. Global epidemiology of influenza: past and present. Annu Rev Med[J], 2000,51:407~21.
[7] Alexander DJ. A review of avian influenza in different bird species[J]. Vet Microbiol, 2000,74:3~13.
[8] Chen W, Calvol PA, Malide D, et al. A novel influenza A virus mitochondrial protein that induces cell death[J]. Nature Medicine, 2001,7(12):1306~12.
[9] Zambon M C. Epidemiology and pathogenesis of influenza[J]. Antimicrobial Chemotherapy, 1999, 44:3~9.
[10] Earn DJ, Dushoff J, Levin SA, Ecology and evoluation of the flu[J]. Trends in Ecology & Evolution, 2002, 17(7):334~40.
[11] Neumann G, Hughes MT, Kawaoka Y. Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1[J]. EMBO J, 2000,19: 6751~58.
[12] Leahy MB, Pritlove DC, Poon LL, et al. Mutagenic analysis of the 5’ arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity[J]. Virol, 2001,75(1):134~42.
[13] Portela A, Zurcher T, Nieto A, et al. Replication of orthomyxoviruses[J]. Adv Virus Res, 1999,54:319~48.
[14] Julkunen I, Sareneva T, Pirhonen J, et al. Molecular pathogenesis of influenza A virus infection and virus- induced regulation of cytokine gene expression[J]. Cytokine Growth Factor Rev,2001,12:171~80.
[15] Briedis DJ, Conti G, Munn EA, et al. Migration of influenza virus-specific polypeptides from cytoplasm to nucleus of infected cells[J]. Virology,1981,111(1):154~64.
[16] Choppin PW, Scheid A. The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses[J]. Rev Infect Dis,1980,2(1):40~61.
[17] Kawakami K. Activation of influenza virus-associated RNA polymerase by Cap-1 structure (m7GpppNm) [J]. Biochem,1985,97:655.
[18] Plotch SJ. A unique cap (m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription[J]. Cell,1981,23:84.
[19] Herz C, Stavnezer E, Krug R, et al. Influenza virus, an RNA virus, synthesis its messenger RNA in thenucleus of infected cells[J]. Cell,1981,26:391~400.
[20] Jackson DA, Caton AJ, McCready SJ, et al. Influenza virus RNA is synthesized at fixed sites in the nucleus[J]. Natrue, 1982,296:366~68.
[21] Krug RM. Transcription and replication of influenza virus.In:Genetics of Influenza Viruse[sJ]. Edited by Palese P. New York: Springer Verlag.1983.
[22] Lamb RA, Choppin PW. The gene structure and replication of influenza virus[J]. Annu Rev Biochem.1983.52:467~506.
[23] Smith GL, Hay AJ. Replication of the influenza virus genome[J]. Virology, 1982,118(1):96~108.
[24] Hay AJ, Lomniczi B, Bellamy AR, et al. Transcription of the influenza virus genome[J]. Virology, 1977,83(2):337~355.
[25] Krug RM, Soeiro R. Studies on the intranuclear localization of influenza virus-specific proteins[J]. Virology, 1975,64(2):378~87.
[26] Compans RW, Meier-Ewert H, Palese P. Assembly of lipid-containing viruses[J]. J Supramol Struct, 1974,2(2-4):496~511.
[27] Neumann G, Kawaoka Y. Synthesis of influenza virus: new impetus from an old enzyme, RNA polymerase I[J]. Virus Res, 2002,82:153~58.
[28] Taniguchi T, Palmieri M,Weissmann C. QB DNA containing hybrid plasmids giving rise to QB phage formation in the bacterial host[J]. Nature, 1978, 247:223~8.
[29] Racaniello VR, Baltimore, D. Cloned poliovirus complementary DNA is infectious in mammalian cells[J]. Science, 1981, 214:916~9.
[30] Schnell MJ, MebatsionT, Conzelman KK. Infectious rabies virus from cloned cDNA[J]. EMBO J,1994,13:4195~203.
[31] Garcin D, Pelet T, Calain P,et al. A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus[J]. EMBO Journal , 1995,14: 6087~94.
[32] Lawson ND, Stillman EA, Whitt, M A, et al. Recombinant vesicular stomatitis viruses from DNA[J]. Proc Natl Acad Sci USA, 1995,92: 4477~81.
[33] Radecke F, Spielhofer P, Schneider H, et al. Rescue of Measles viruses from cloned DNA[J]. EMBO J, 1995, 14:5773~84.
[34] Baron, M D, Barrett T. Rescue of rinderpest virus from cloned cDNA[J]. J Virol, 1997, 71: 1265~71.
[35] Durbin AP, Hall SL, Siew JW, et al. Recovery of infectious human parainfluenza virus type 3 from cDNA[J]. Virology, 1997, 235(2): 323~32.
[36] Hoffman MA, Banerjee AK. An infectious clone of human parainfluenza virus type 3[J]. Virol, 1997, 71:4272~7.
[37] Bridgen A, Elliott RM. Rescue of a segment negative strand RNA virus entirely from cloned complimentary DNAs[J]. Proc Natl Acad Sci USA, 1996, 92(1): 15400~4.
[38] Honda A, Ueda K, Nagata, K, et al. Identification of the RNA polymerase-binding site on genome RNA of influenza virus[J]. J Biochem(Tokyo),1987, 102: 1241~49.
[39] Szewczyk B, Laver WG, Summers DF. Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase[J]. Proc Natl Acad Sci USA, 1988, 85: 7907~11.
[40] Parvin JD, Palese P Honda A, et al. Promoter analysis of influenza virus RNA polymerase[J]. Virol, 1989,63: 5142~52.
[41] Honda A, Mukaigawa J, Yokoiyama A, et al. Purification and molecular structure of RNA polymerase from influenza virus A/PR8[J]. J Biochem(Tokyo),1990,107: 624~28.
[42] Huang TS, Palese P, Krystal M. Determination of influenza virus proteins required for genome replication[J]. Virol,1990,64:5669~73.
[43] de la Luna S, Martin J, Portela A, et al. Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses[J]. J Gen Virol, 1993,74:535~9.
[44] Kimura N, Nishida M, Nagata K, et al. Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes[J]. J Gen Virol, 1992,73:1321~28.
[45] Mena I, de la Luna S, Albo C, et al. Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system[J]. J Gen Virol, 1994,75:2109~14.
[46] Perales B, Sanz-Ezquerro JJ, Gastamiza P, et al. The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis[J]. J Virol, 2000,74:1307~12.
[47] Luytjes W, Krystal M, Enami M, et al. Amplification, expression, and packaging of foreign gene by influenza virus[J]. Cell, 1989,59:1107~13.
[48] Enami M, Luytjes W, Krystal M, et al. Introduction of site-specific mutations into the genome of influenza virus[J]. Proc Natl Acad Sci USA, 1990, 87: 3802~5.
[49] Neumann G, Zobel A, Hobom G. RNA polymerase I-mediated expression of influenza viral RNA molecules[J]. Virology, 1994,202: 477~9.
[50] Yamanaka K, Ogasawara N, Yoshikawa H, et al. In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA[J]. Proc Natl Acad Sci USA, 1991, 88: 5369~73.
[51] Li X, Palese P. Mutational analysis of the promoter required for influenza virus virion RNA synthesis[J]. J Virol, 1992, 66:4331~8.
[52] Piccone ME, Fernandez-Sesma A, Palese P. Mutational analysis of the influenza virus vRNA promoter[J]. Virus Res, 1993, 28: 99~112.
[53] Fodor E, Pritlove DC, Brownlee GG. Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus[J]. J Virol, 1995, 69: 4012~9.
[54] Grassauer A, Egorov AY, Ferko B, et al. A host restriction-based selection system for influenza haemagglutinin transfectant viruses[J]. J Gen Virol, 1998,79:1405~9.
[55] Jin H, Subbarao K, Bagai S, et al. Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity[J]. J Virol, 1996,70:1406~14.
[56] Lin YP, Wharton SA, Martin J, et al. Adaptation of egg-grown and transfectant influenza viruses for growth in mammalian cells: selection of hemagglutinin mutants with elevated pH of membrane fusion[J]. Virology, 1997, 233: 402~10.
[57] Castrucci MR, Kawaoka Y. Reverse genetics system for generation of an influenza A virus mutant containing a deletion of the carboxyl-terminal residue of M2 protein[J]. J Virol, 1995,69: 2725~8.
[58] Yasuda J, Bucher DJ, Ishihama A. Growth control of influenza A virus by M1 protein: analysis oftransfectant viruses carrying the chimeric M gene[J]. J Virol, 1994,68:8141~6.
[59] Neumann G, Watanabe T, Ito H, et al. Generation of influenza A viruses entirely from cloned cDNAs[J]. Proc Natl Acad Sci USA, 1999, 96, 9345~50.
[60] Fodor E, Devenish L, Engelhardt OG, et al. Rescue of influenza A virus from recombinant DNA[J]. J Virol,1999,73:9679~82.
[61] Hoffmann E, Neumann G, Hobom G, et al. 'Ambisense' approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template[J]. Virology, 2000,267, 310~317.
[62] Hoffmann E, Neumann G, Kawaoka Y, et al. A DNA transfection system for generation of influenza A virus from eight plasmids[J]. Proc Natl Acad Sci USA,2000,97:6108~6113.
[63] Hoffmann E, Webster RG. Unidirectional RNA polymerase I–polymerase II transcription system for the generation of influenza A virus from eight plasmids[J]. J Gen Virol, 2000, 81:2843~7.
[64] Hoffmann E, Zhou N, Webster RG. Eight-plasmid rescue system for influenza A virus[J]. International Congress Series, 2001, 1219:1007~13.
[65] Hoffmann E, Krauss S, Perez D. Eight-plasmid system for rapid generation of influenza virus vaccines[J]. Vaccine, 2002,20:3165~70.
[66] Hoffmann E, Mahmood K, Yang CF, et al. Rescue of influenza B virus from eight plasmids[J]. Proc Natl Acad Sci USA, 2002, 99(17):11411~6.
[67] Neumann G, Ken Fujii, Yoichiro, et al. An improved reverse genetics system for influenza virus generation and its implications for vaccine production[J]. PANS,2005,102(46):16825～16829.
[68] Webster RG. A molecular whodunit[J]. Science, 2001, 293(5536):1773~5.
[69] Basler CF, Reid AH, Dybing JK, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant virus bearing the 1918 NS genes[J]. Proc Natl Acad Sci USA,2001,98(5):2746~51.
[70] Gao P, Watanabe S, Ito T, et al. Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong[J]. J Virol, 1999,73:3184~9.
[71] Lu X, Tumpey TM, Morken T, et al. A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans[J]. J Virol, 1999,73:5903~11.
[72] Hatta M, Gao P, Halfmann P, et al. Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses[J]. Science,2001,293:1840~1842.
[73] Hoffmann E, Stech J, Leneva I, et al. Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1[J]? J Virol, 2000, 74(14):6309~15.
[74] Watanabe T, Watanabe S, Ito H, et al. Influenza a virus can undergo multiple cycles of replication without M2 ion channel activity[J]. J Virol, 2001,75: 5656~62.
[75] Hui EK, Barman S, Yang TY, et al. Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs[J]. J Virol, 2003, 77(12):7078~92.
[76] O'Neill RE, Talon J, Palese P. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins[J]. EMBO J,1998,17, 288~96.
[77] Castrucci MR, Bilsel P, Kawaoka Y. Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase[J]. J Virology, 1992, 66:4647~53.
[78] Castrucci MR, Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus[J]. J Virol, 1993, 67:759~64.
[79] Luo G, Chung J, Palese P. Alterations of the stalk of the influenza virus neuraminidase : deletions and insertions[J]. Virus Res, 1993, 29:141~53.
[80] Li S, Schulman J, Itamura S, Palese P. Glycosylation of neuraminidase determines the neurovirulence of influenza A/WSN/33 virus[J]. J Virol, 1993, 67:6667~73.
[81] Fodor E, Poon LL, Mikulasova A. Transcription of influenza A virus genes[J]. International Congress Series, 2001, 1219:427~34.
[82] Duhaut SD, Dimmock NJ. Defective influenza A virus generated entirely from plasmids: its RNA is expressed in infected mouse lung and modulates disease[J]. J Virological Methods, 2003, 108:75~82.
[83] Garcia-Sastre A, Palese P. Influenza virus vectors[J]. Biologicals, 1995, 23:171~8.
[84] Muster T, Guinea R, Trkola A, et al. Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS[J]. J Virol, 1994,68(6):4031~4.
[85] Muster T, Ferko B, Klima A, et al. Mucosal model of immunization against human immunodeficiency virus type 1 with a chimeric influenza virus[J]. J Virol, 1995, 69(11):6678~86.
[86] Ferko B, Stasakova J, Sereinig S, et al. Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice[J]. J Virol,2001,75(19):8899~908.
[87] Castrucci MR, Hou S, Doherty PC, et al. Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes[J]. J Virol, 1994, 68(6):3486~90.
[88] Miyahira Y, Garcia-Sastre A, Rodriguez D, et al. Recombinant viruses expressing a human malaria antigen can elicit potentially protective immune CD8+ responses in mic[eJ]. Proc Natl Acad Sci USA, 1998, 95(7):3954~9.
[89] Mena I, Vivo A, Perez E, et al. Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids[J]. J Virol,1996,70(8):5016~24.
[90] Gomez-Puertas P, Mena I, Castillo M, et al. Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins[J]. J Gen Virol, 1999,80:1635~45.
[91] Neumann G, Watanabe T, Kawaoka Y. Plasmid-driven formation of influenza virus-like particles[J]. J Virol, 2000,74(1):547~51.
[92] Gomez-Puertas P, Mena I, Castillo M, et al. Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins[J]. J Gen Virol, 1999, 80:1635~45.
[93] Maassab HF, Bryant ML. The development of live attenuated cold-adapted influenza virus vaccine for humans[J]. Rev Med Virol, 1999,9:237~44.
[94] Belshe RB, Mendelman PM, Treanor J, et al. The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children[J]. New England J Med,1998,338:1405~12.
[95] Jin H, Lu B, Zhou H, et al. Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60[J]. Virology 306(2003): 18～24.
[96] Talon J, Salvatore M, O'Neill RE, et al. Influenza A and B viruses expressing altered NS1 proteins: Avaccine approach[J]. Proc Natl Acad Sci USA,2000,97:4309~14.
[97] Subbarao K, Chen H, Swayne D, et al. Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics [ J ] . Virology,2003,305:192~200.
[98] Li S, Jerome L, Schulman, et al. Influenza A virus transfectants with chimeric hemagglutinins containing epitopes from different subtypes[J]. J Virol,1992,66(1):399~404.
[99] Neumann G, Kawaoka Y. Reverse genetics of influenza virus[J]. Virology, 2001:243~50.
[100] Murphy,Coelingh.Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines[J]. Viral Immunol.2002.15:295~323
[101] Hoffmann E, Stech J, Guan Y,et al. Universal primer set for the full-length amplification of all influenza A viruses[J]. Arch Virol ,2001,146:2275-2289.
[102] 陈稚峰,张立国,董捷等. 应用反向遗传学技术在哺乳动物细胞中产生甲型流感病毒[J]. 病毒学报. 2002.18(3):193～197
[103] 卢建红,龙进学,邵卫星等. 用反向遗传操作技术产生致弱的 H5 亚型重组流感病毒[J].微生物学报. 2005.45(1):53～57
[104] 卢建红,邵卫星,刘玉良等. 用 8 质粒病毒拯救系统产生 H9N2/WSN 重组 A 型流行性感冒病毒[J].病毒学报. 2005.21(1):48～53
[105] 龙进学,吴艳涛,张小荣等.H5N1 亚型禽流感病毒拯救体系的建立[J].微生物学报.2006.46(1):55～59
[106] Neumann G, Hobom G. Mutational analysis of influenza virus promoter elements in viro[J]. J Gen Virol, 1995, 76:1709~17.
[107] Zhongying Chen, Amy Aspelund, George Kemble, Hong Jin. Genetic mapping of the cold-adapted phenotype of B/Ann Arbor/1/66,the master donor virus for live attenuated influenza vaccines (FluMistR) [J].Virology 345 (2006) :416 ~ 423
[108] Linda Widjaja, Natalia Ilyushina , Robert G. Webster, Richard J. Webby .Molecular changes associated with adaptation of human influenza A virus in embryonated chicken eggs[J].Virology.2006.350(1):137~45
[109] 殷 震,刘景华.动物病毒学[M].第二版.北京:科学出版社,1997.
[110] Sambrook J, Fritsch EF, Maniatis T 著,金冬雁译,黎孟枫校.分子克隆实验指南(现代生物技术译丛)[M],第二版,北京:科学出版社,2002,1～911.
[111] Clade H,Francoise M,James P. Immunogenicity and protective efficacy of influenza vaccination[J].Virus Research,2004,103:133-138.
[112] Reid A A,Fanning T G,Hultin J V.Origin and evolution of the 1918“Spinish”influenza virus hemagglutinin gene[J].Proc Natl Sci,1999,96:1651～1656.
[113] Ferguson N M,Galvani A P,Bush R M.Ecological and immunological determinants of influenza evolution[J].Nature,2003,422:428～433.
[114] Takada A,Kuboki N,Okazaki K. Avirulent avian influenza virus as a vaccine strain aginast a potential human pandemic[J].J Virol,1999,73(10):8303～8307.
[115] Subbarao K, Katz JM. Influenza vaccines generated by reverse genetics[J]. Curr Top Microbiol Immunol ,2004 ,283 :31～34.
[116] Simonsen, L., M. J. Clarke, G. D. Williamson, D. F. Stroup, N. H. Arden, and L. B. Schonberger.1997. The impact of influenza epidemics on mortality: introducing a severity index[J]. Am. J. Public Health 87:1944–1950.
[117] Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox, L. J.Anderson, and K. Fukuda. 2003. Mortality associated with influenza and respiratory syncytial virus in the United States[J]. JAMA 289:179–186.
[118] N.Bardiya.J.H.Bae Influenza vaccines: recent advancrs in production technologies[J]. Appl Microbiol Biotechnol (2005) 67: 299～305.
[2] Nagai Y, Kato A. Paramyxovirus reverse genetics is coming of ag[eJ]. Microbiol Immunol, 1999, 43(7): 615~24.
[3] 范宝昌,杨佩英. RNA 病毒的反向遗传学操作[J].军事医学科学院院刊,2001,25(3):223～27.
[4] 甘孟侯主编. 禽流感[M],第二版,北京:北京农业大学出版社,2002,15～79.
[5] Webster, RG,Bean WJ, Gorman OT, et al. Evolution and ecology of influenza A viruses[J]. Microbiol Rev,1992,56:152~79.
[6] Cox NJ, Subbarao K. Global epidemiology of influenza: past and present. Annu Rev Med[J], 2000,51:407~21.
[7] Alexander DJ. A review of avian influenza in different bird species[J]. Vet Microbiol, 2000,74:3~13.
[8] Chen W, Calvol PA, Malide D, et al. A novel influenza A virus mitochondrial protein that induces cell death[J]. Nature Medicine, 2001,7(12):1306~12.
[9] Zambon M C. Epidemiology and pathogenesis of influenza[J]. Antimicrobial Chemotherapy, 1999, 44:3~9.
[10] Earn DJ, Dushoff J, Levin SA, Ecology and evoluation of the flu[J]. Trends in Ecology & Evolution, 2002, 17(7):334~40.
[11] Neumann G, Hughes MT, Kawaoka Y. Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1[J]. EMBO J, 2000,19: 6751~58.
[12] Leahy MB, Pritlove DC, Poon LL, et al. Mutagenic analysis of the 5’ arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity[J]. Virol, 2001,75(1):134~42.
[13] Portela A, Zurcher T, Nieto A, et al. Replication of orthomyxoviruses[J]. Adv Virus Res, 1999,54:319~48.
[14] Julkunen I, Sareneva T, Pirhonen J, et al. Molecular pathogenesis of influenza A virus infection and virus- induced regulation of cytokine gene expression[J]. Cytokine Growth Factor Rev,2001,12:171~80.
[15] Briedis DJ, Conti G, Munn EA, et al. Migration of influenza virus-specific polypeptides from cytoplasm to nucleus of infected cells[J]. Virology,1981,111(1):154~64.
[16] Choppin PW, Scheid A. The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses[J]. Rev Infect Dis,1980,2(1):40~61.
[17] Kawakami K. Activation of influenza virus-associated RNA polymerase by Cap-1 structure (m7GpppNm) [J]. Biochem,1985,97:655.
[18] Plotch SJ. A unique cap (m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription[J]. Cell,1981,23:84.
[19] Herz C, Stavnezer E, Krug R, et al. Influenza virus, an RNA virus, synthesis its messenger RNA in thenucleus of infected cells[J]. Cell,1981,26:391~400.
[20] Jackson DA, Caton AJ, McCready SJ, et al. Influenza virus RNA is synthesized at fixed sites in the nucleus[J]. Natrue, 1982,296:366~68.
[21] Krug RM. Transcription and replication of influenza virus.In:Genetics of Influenza Viruse[sJ]. Edited by Palese P. New York: Springer Verlag.1983.
[22] Lamb RA, Choppin PW. The gene structure and replication of influenza virus[J]. Annu Rev Biochem.1983.52:467~506.
[23] Smith GL, Hay AJ. Replication of the influenza virus genome[J]. Virology, 1982,118(1):96~108.
[24] Hay AJ, Lomniczi B, Bellamy AR, et al. Transcription of the influenza virus genome[J]. Virology, 1977,83(2):337~355.
[25] Krug RM, Soeiro R. Studies on the intranuclear localization of influenza virus-specific proteins[J]. Virology, 1975,64(2):378~87.
[26] Compans RW, Meier-Ewert H, Palese P. Assembly of lipid-containing viruses[J]. J Supramol Struct, 1974,2(2-4):496~511.
[27] Neumann G, Kawaoka Y. Synthesis of influenza virus: new impetus from an old enzyme, RNA polymerase I[J]. Virus Res, 2002,82:153~58.
[28] Taniguchi T, Palmieri M,Weissmann C. QB DNA containing hybrid plasmids giving rise to QB phage formation in the bacterial host[J]. Nature, 1978, 247:223~8.
[29] Racaniello VR, Baltimore, D. Cloned poliovirus complementary DNA is infectious in mammalian cells[J]. Science, 1981, 214:916~9.
[30] Schnell MJ, MebatsionT, Conzelman KK. Infectious rabies virus from cloned cDNA[J]. EMBO J,1994,13:4195~203.
[31] Garcin D, Pelet T, Calain P,et al. A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus[J]. EMBO Journal , 1995,14: 6087~94.
[32] Lawson ND, Stillman EA, Whitt, M A, et al. Recombinant vesicular stomatitis viruses from DNA[J]. Proc Natl Acad Sci USA, 1995,92: 4477~81.
[33] Radecke F, Spielhofer P, Schneider H, et al. Rescue of Measles viruses from cloned DNA[J]. EMBO J, 1995, 14:5773~84.
[34] Baron, M D, Barrett T. Rescue of rinderpest virus from cloned cDNA[J]. J Virol, 1997, 71: 1265~71.
[35] Durbin AP, Hall SL, Siew JW, et al. Recovery of infectious human parainfluenza virus type 3 from cDNA[J]. Virology, 1997, 235(2): 323~32.
[36] Hoffman MA, Banerjee AK. An infectious clone of human parainfluenza virus type 3[J]. Virol, 1997, 71:4272~7.
[37] Bridgen A, Elliott RM. Rescue of a segment negative strand RNA virus entirely from cloned complimentary DNAs[J]. Proc Natl Acad Sci USA, 1996, 92(1): 15400~4.
[38] Honda A, Ueda K, Nagata, K, et al. Identification of the RNA polymerase-binding site on genome RNA of influenza virus[J]. J Biochem(Tokyo),1987, 102: 1241~49.
[39] Szewczyk B, Laver WG, Summers DF. Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase[J]. Proc Natl Acad Sci USA, 1988, 85: 7907~11.
[40] Parvin JD, Palese P Honda A, et al. Promoter analysis of influenza virus RNA polymerase[J]. Virol, 1989,63: 5142~52.
[41] Honda A, Mukaigawa J, Yokoiyama A, et al. Purification and molecular structure of RNA polymerase from influenza virus A/PR8[J]. J Biochem(Tokyo),1990,107: 624~28.
[42] Huang TS, Palese P, Krystal M. Determination of influenza virus proteins required for genome replication[J]. Virol,1990,64:5669~73.
[43] de la Luna S, Martin J, Portela A, et al. Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses[J]. J Gen Virol, 1993,74:535~9.
[44] Kimura N, Nishida M, Nagata K, et al. Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes[J]. J Gen Virol, 1992,73:1321~28.
[45] Mena I, de la Luna S, Albo C, et al. Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system[J]. J Gen Virol, 1994,75:2109~14.
[46] Perales B, Sanz-Ezquerro JJ, Gastamiza P, et al. The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis[J]. J Virol, 2000,74:1307~12.
[47] Luytjes W, Krystal M, Enami M, et al. Amplification, expression, and packaging of foreign gene by influenza virus[J]. Cell, 1989,59:1107~13.
[48] Enami M, Luytjes W, Krystal M, et al. Introduction of site-specific mutations into the genome of influenza virus[J]. Proc Natl Acad Sci USA, 1990, 87: 3802~5.
[49] Neumann G, Zobel A, Hobom G. RNA polymerase I-mediated expression of influenza viral RNA molecules[J]. Virology, 1994,202: 477~9.
[50] Yamanaka K, Ogasawara N, Yoshikawa H, et al. In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA[J]. Proc Natl Acad Sci USA, 1991, 88: 5369~73.
[51] Li X, Palese P. Mutational analysis of the promoter required for influenza virus virion RNA synthesis[J]. J Virol, 1992, 66:4331~8.
[52] Piccone ME, Fernandez-Sesma A, Palese P. Mutational analysis of the influenza virus vRNA promoter[J]. Virus Res, 1993, 28: 99~112.
[53] Fodor E, Pritlove DC, Brownlee GG. Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus[J]. J Virol, 1995, 69: 4012~9.
[54] Grassauer A, Egorov AY, Ferko B, et al. A host restriction-based selection system for influenza haemagglutinin transfectant viruses[J]. J Gen Virol, 1998,79:1405~9.
[55] Jin H, Subbarao K, Bagai S, et al. Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity[J]. J Virol, 1996,70:1406~14.
[56] Lin YP, Wharton SA, Martin J, et al. Adaptation of egg-grown and transfectant influenza viruses for growth in mammalian cells: selection of hemagglutinin mutants with elevated pH of membrane fusion[J]. Virology, 1997, 233: 402~10.
[57] Castrucci MR, Kawaoka Y. Reverse genetics system for generation of an influenza A virus mutant containing a deletion of the carboxyl-terminal residue of M2 protein[J]. J Virol, 1995,69: 2725~8.
[58] Yasuda J, Bucher DJ, Ishihama A. Growth control of influenza A virus by M1 protein: analysis oftransfectant viruses carrying the chimeric M gene[J]. J Virol, 1994,68:8141~6.
[59] Neumann G, Watanabe T, Ito H, et al. Generation of influenza A viruses entirely from cloned cDNAs[J]. Proc Natl Acad Sci USA, 1999, 96, 9345~50.
[60] Fodor E, Devenish L, Engelhardt OG, et al. Rescue of influenza A virus from recombinant DNA[J]. J Virol,1999,73:9679~82.
[61] Hoffmann E, Neumann G, Hobom G, et al. 'Ambisense' approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template[J]. Virology, 2000,267, 310~317.
[62] Hoffmann E, Neumann G, Kawaoka Y, et al. A DNA transfection system for generation of influenza A virus from eight plasmids[J]. Proc Natl Acad Sci USA,2000,97:6108~6113.
[63] Hoffmann E, Webster RG. Unidirectional RNA polymerase I–polymerase II transcription system for the generation of influenza A virus from eight plasmids[J]. J Gen Virol, 2000, 81:2843~7.
[64] Hoffmann E, Zhou N, Webster RG. Eight-plasmid rescue system for influenza A virus[J]. International Congress Series, 2001, 1219:1007~13.
[65] Hoffmann E, Krauss S, Perez D. Eight-plasmid system for rapid generation of influenza virus vaccines[J]. Vaccine, 2002,20:3165~70.
[66] Hoffmann E, Mahmood K, Yang CF, et al. Rescue of influenza B virus from eight plasmids[J]. Proc Natl Acad Sci USA, 2002, 99(17):11411~6.
[67] Neumann G, Ken Fujii, Yoichiro, et al. An improved reverse genetics system for influenza virus generation and its implications for vaccine production[J]. PANS,2005,102(46):16825～16829.
[68] Webster RG. A molecular whodunit[J]. Science, 2001, 293(5536):1773~5.
[69] Basler CF, Reid AH, Dybing JK, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant virus bearing the 1918 NS genes[J]. Proc Natl Acad Sci USA,2001,98(5):2746~51.
[70] Gao P, Watanabe S, Ito T, et al. Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong[J]. J Virol, 1999,73:3184~9.
[71] Lu X, Tumpey TM, Morken T, et al. A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans[J]. J Virol, 1999,73:5903~11.
[72] Hatta M, Gao P, Halfmann P, et al. Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses[J]. Science,2001,293:1840~1842.
[73] Hoffmann E, Stech J, Leneva I, et al. Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1[J]? J Virol, 2000, 74(14):6309~15.
[74] Watanabe T, Watanabe S, Ito H, et al. Influenza a virus can undergo multiple cycles of replication without M2 ion channel activity[J]. J Virol, 2001,75: 5656~62.
[75] Hui EK, Barman S, Yang TY, et al. Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs[J]. J Virol, 2003, 77(12):7078~92.
[76] O'Neill RE, Talon J, Palese P. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins[J]. EMBO J,1998,17, 288~96.
[77] Castrucci MR, Bilsel P, Kawaoka Y. Attenuation of influenza A virus by insertion of a foreign epitope into the neuraminidase[J]. J Virology, 1992, 66:4647~53.
[78] Castrucci MR, Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus[J]. J Virol, 1993, 67:759~64.
[79] Luo G, Chung J, Palese P. Alterations of the stalk of the influenza virus neuraminidase : deletions and insertions[J]. Virus Res, 1993, 29:141~53.
[80] Li S, Schulman J, Itamura S, Palese P. Glycosylation of neuraminidase determines the neurovirulence of influenza A/WSN/33 virus[J]. J Virol, 1993, 67:6667~73.
[81] Fodor E, Poon LL, Mikulasova A. Transcription of influenza A virus genes[J]. International Congress Series, 2001, 1219:427~34.
[82] Duhaut SD, Dimmock NJ. Defective influenza A virus generated entirely from plasmids: its RNA is expressed in infected mouse lung and modulates disease[J]. J Virological Methods, 2003, 108:75~82.
[83] Garcia-Sastre A, Palese P. Influenza virus vectors[J]. Biologicals, 1995, 23:171~8.
[84] Muster T, Guinea R, Trkola A, et al. Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS[J]. J Virol, 1994,68(6):4031~4.
[85] Muster T, Ferko B, Klima A, et al. Mucosal model of immunization against human immunodeficiency virus type 1 with a chimeric influenza virus[J]. J Virol, 1995, 69(11):6678~86.
[86] Ferko B, Stasakova J, Sereinig S, et al. Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice[J]. J Virol,2001,75(19):8899~908.
[87] Castrucci MR, Hou S, Doherty PC, et al. Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes[J]. J Virol, 1994, 68(6):3486~90.
[88] Miyahira Y, Garcia-Sastre A, Rodriguez D, et al. Recombinant viruses expressing a human malaria antigen can elicit potentially protective immune CD8+ responses in mic[eJ]. Proc Natl Acad Sci USA, 1998, 95(7):3954~9.
[89] Mena I, Vivo A, Perez E, et al. Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids[J]. J Virol,1996,70(8):5016~24.
[90] Gomez-Puertas P, Mena I, Castillo M, et al. Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins[J]. J Gen Virol, 1999,80:1635~45.
[91] Neumann G, Watanabe T, Kawaoka Y. Plasmid-driven formation of influenza virus-like particles[J]. J Virol, 2000,74(1):547~51.
[92] Gomez-Puertas P, Mena I, Castillo M, et al. Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins[J]. J Gen Virol, 1999, 80:1635~45.
[93] Maassab HF, Bryant ML. The development of live attenuated cold-adapted influenza virus vaccine for humans[J]. Rev Med Virol, 1999,9:237~44.
[94] Belshe RB, Mendelman PM, Treanor J, et al. The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children[J]. New England J Med,1998,338:1405~12.
[95] Jin H, Lu B, Zhou H, et al. Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60[J]. Virology 306(2003): 18～24.
[96] Talon J, Salvatore M, O'Neill RE, et al. Influenza A and B viruses expressing altered NS1 proteins: Avaccine approach[J]. Proc Natl Acad Sci USA,2000,97:4309~14.
[97] Subbarao K, Chen H, Swayne D, et al. Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics [ J ] . Virology,2003,305:192~200.
[98] Li S, Jerome L, Schulman, et al. Influenza A virus transfectants with chimeric hemagglutinins containing epitopes from different subtypes[J]. J Virol,1992,66(1):399~404.
[99] Neumann G, Kawaoka Y. Reverse genetics of influenza virus[J]. Virology, 2001:243~50.
[100] Murphy,Coelingh.Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines[J]. Viral Immunol.2002.15:295~323
[101] Hoffmann E, Stech J, Guan Y,et al. Universal primer set for the full-length amplification of all influenza A viruses[J]. Arch Virol ,2001,146:2275-2289.
[102] 陈稚峰,张立国,董捷等. 应用反向遗传学技术在哺乳动物细胞中产生甲型流感病毒[J]. 病毒学报. 2002.18(3):193～197
[103] 卢建红,龙进学,邵卫星等. 用反向遗传操作技术产生致弱的 H5 亚型重组流感病毒[J].微生物学报. 2005.45(1):53～57
[104] 卢建红,邵卫星,刘玉良等. 用 8 质粒病毒拯救系统产生 H9N2/WSN 重组 A 型流行性感冒病毒[J].病毒学报. 2005.21(1):48～53
[105] 龙进学,吴艳涛,张小荣等.H5N1 亚型禽流感病毒拯救体系的建立[J].微生物学报.2006.46(1):55～59
[106] Neumann G, Hobom G. Mutational analysis of influenza virus promoter elements in viro[J]. J Gen Virol, 1995, 76:1709~17.
[107] Zhongying Chen, Amy Aspelund, George Kemble, Hong Jin. Genetic mapping of the cold-adapted phenotype of B/Ann Arbor/1/66,the master donor virus for live attenuated influenza vaccines (FluMistR) [J].Virology 345 (2006) :416 ~ 423
[108] Linda Widjaja, Natalia Ilyushina , Robert G. Webster, Richard J. Webby .Molecular changes associated with adaptation of human influenza A virus in embryonated chicken eggs[J].Virology.2006.350(1):137~45
[109] 殷 震,刘景华.动物病毒学[M].第二版.北京:科学出版社,1997.
[110] Sambrook J, Fritsch EF, Maniatis T 著,金冬雁译,黎孟枫校.分子克隆实验指南(现代生物技术译丛)[M],第二版,北京:科学出版社,2002,1～911.
[111] Clade H,Francoise M,James P. Immunogenicity and protective efficacy of influenza vaccination[J].Virus Research,2004,103:133-138.
[112] Reid A A,Fanning T G,Hultin J V.Origin and evolution of the 1918“Spinish”influenza virus hemagglutinin gene[J].Proc Natl Sci,1999,96:1651～1656.
[113] Ferguson N M,Galvani A P,Bush R M.Ecological and immunological determinants of influenza evolution[J].Nature,2003,422:428～433.
[114] Takada A,Kuboki N,Okazaki K. Avirulent avian influenza virus as a vaccine strain aginast a potential human pandemic[J].J Virol,1999,73(10):8303～8307.
[115] Subbarao K, Katz JM. Influenza vaccines generated by reverse genetics[J]. Curr Top Microbiol Immunol ,2004 ,283 :31～34.
[116] Simonsen, L., M. J. Clarke, G. D. Williamson, D. F. Stroup, N. H. Arden, and L. B. Schonberger.1997. The impact of influenza epidemics on mortality: introducing a severity index[J]. Am. J. Public Health 87:1944–1950.
[117] Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox, L. J.Anderson, and K. Fukuda. 2003. Mortality associated with influenza and respiratory syncytial virus in the United States[J]. JAMA 289:179–186.
[118] N.Bardiya.J.H.Bae Influenza vaccines: recent advancrs in production technologies[J]. Appl Microbiol Biotechnol (2005) 67: 299～305.