表达和共表达H9亚型禽流感病毒血凝素基因和鸡Ⅱ型干扰素基因的重组鸡痘病毒
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摘要
H9亚型禽流感病毒（AIV）近年在我国部分地区广泛流行，给养禽业造成了严
重的经济损失。油乳剂全病毒灭活苗虽对该亚型禽流感的控制发挥了重要作用，但
由于其存在干扰免疫监测及成本较高等缺点，使用受到了限制。为了研制新型禽流
感疫苗以克服上述缺点，我们对H9亚型AIV中国分离株的血凝素（HA）基因和
鸡Ⅱ型干扰素基因（IFN-Ⅱ）进行了克隆、测序和分析，构建了表达HA基因及共
表达HA基因和鸡IFN-Ⅱ基因的两种重组鸡痘病毒（rFPV-HA、rFPV-HA-IFN-Ⅱ），
并测定了它们的免疫效力。
1 H9亚型AIV中国分离株HA基因cDNA的克隆及序列初步分析
以酚-SDS法提取H9N2亚型AIV中国分离株A/Chicken/China/F/1998（简称F
株）的基因组RNA，以此为模板，采用cDNA合成法及反转录聚合酶链式反应
（RT-PCR），扩增到长度分别为1.1Kb和0.8Kb的两段HA基因片段。测序结果表
明，这两个片段覆盖了HA基因的完整阅读框架，且在它们的重叠区有单一的PstⅠ
酶切位点。利用这一酶切位点，将这两个片段连接成插入在质粒pUC18的HindⅢ
和SalⅠ位点之间的全长HA基因，得重组质粒pUCHA。测定pUCHA位于HindⅢ
和SalⅠ位点之间外源片段的核苷酸序列。
本研究获得的HA基因片段共有1708个核苷酸，包含了HA完整的阅读区的
1680个核苷酸，推测其编码560个氨基酸，其中前18个氨基酸为信号肽序列，其
余542个氨基酸组成的前体蛋白在第320位氨基酸处被切割为HA1和HA2多肽。
F株HA裂解位点的氨基酸序列为RSSR↓GLF，不含连续的碱性氨基酸，具有低
致病性AIV HA裂解位点的特征。F株与H9亚型AIV原型毒株
A/Turkey/Wisconsin/1/66（H9N2）的HA基因的同源性较低，氨基酸和核苷酸的同源
性分别为84.1％和82.9％。
n 扬州大学博士学位论文
以 HAI CDNA的 55－1004 n酸为基元，建立 1992－1999年间 HgNZ亚型 AIV
中国及绷边国家和地区分离株的遗传发生树。结果表明，近年来在这一地区流行
的HgNZ亚型AJV招可敞为三憎系，同一谱系内，毒株间HA艄酸的同源
性在95％以上。F株与髓地区的部分俯株及94年北京分离株的亲龈系较近，
形成谱系卜 香港地区的另一些分离株和巴基斯坦分离株形成谱系2，它们与某些
南美分离株的亲缘关系很近；与谱系1的亲缘关系也较近，它们之间HA核昔酸的
同源性在 gi％R右；韩国分离株形成的谱系 3，该谱系与谱系 1、二的亲缘关系很
远，它们之间HA核昔酸的同源性只有83％左右。
2 表达HA辜因的直组鸡痘病霉的构巨
以 Hnd皿及 Sal消化PUCHA，得到 1．7Kb的 HA基因片段，将n向插人
FPV插入载体 11 75的 Hnd Ill和 Sal位点之间，使之处于痘苗病商启动子 P7．5的
下游，得到转移删 11 75HA。按常规的B眠脓染方法将其转染至已感染 FPV中
国疫酣282E4（Wt郴）的鸡胚成纤测胞（CEF），通过筛选蓝色的病毒腑
获得并纯化重组鸡痘病毒uV－HA。以PCR ＄＄1；ill实，dq，v－HA基因组中插有
HA基因：以间接兔疫荧光肌实，fPV－HA感染的C扭釉了HA。
3 表达鸡IFN－H羹因的匣组鸡痘病羹的钩 来达产物的生物活性
根据已发表的鸡 WN0基因序列，设计一对5！物，以 RTPCR方法从经 Con A
刺激的鸡脾脏细胞中扩增出全长的鸡IFN二基因的cDNA序列。将扩增的基因片
段定向插人质粒 AF09的 Sal和 Hnd IH位点之间，使其位于痘苗病毒早晚期启动
子PEI的下游。
根据启动子 P肌的序列设计上游引物，鸡 IFNl基因的 cDNA 3 M列设计．
下游5！物，以PCR法扩增包含痘苗病毒早晚期启动子PM在内的鸡IFN刁的基因
片段。经一系列步骤，将扩增片段定向插人 FPV JWA载体 11 75中，得到转移载体
1175IFN，用以转染已感染tFPV的CEF，并按上述重组病毒的构建和纯化方法，
获得插谰 rw－n基因的靴重赈毒 ev－r：w－11。在 rrnv－lrx－11中，rw－11
基因处于PDe的转录调控下。
以细胞病鲫制W侧－IFNI感染的CEF越了具有抗水泡性口炎病
毒（VSV）活性的鸡IFN刁。fPV－mI（M．O．H．m）感染CEF 72小时后，培
养上清中 IFN刁的表达量为 1280 U／ITl。动物硼表明，接种 rFPV－IFN刁 天或
14天后，其表达的 IFNJ可显著减轻载体鸡痘病毒对 1日龄 SPF tills｛＄重增长的
程坚：表达和共表达Hg亚型肖流感病毒HA基因和鸡！FN坝基因的重组鸡痘病毒 ＊
抑制作用。
4A表达HA墓因和鸡HN－11基因的直组鸡扈清羹的构建
经一系列步骤，将IFN－11的基因及其上游的启动子PM插人含全长HAcDNA
的质粒pUCm中，得重组质粒PHAIFN。在PHAJFN中，HA基因和IFN刁基因
的3’糊邻。将串联在一起的HA基因和IFNJ基因（包含上游启动子Py）片
段切下，将其定向插人 FPV e载体 11 75中，得到转移狲 11 75HAIFN。在
11 75HAff：N中，HA基因和 IFN。11基因分别处于启动子 P7．5和 PWh的转录控制下，
且转录方向正好相对。在IFN－11基因3’端有一痘苗病毒转录终止子，可避免这两
个基?
H9 subtype avian influenza virus (MV) is widely distzibuted and has resulted in considerable economic loss to poultry industiy in China in recent years. In order to protect poultry against this low pathogenic subtype of API, inactivated vaccines in oil emulsion have been developed and proved to be effective in reducing severity of disease and spread of MV in field situation. However, the use of inactivated vaccines was restricted because of the disadvantages they inherited, including higher cost and interference of routine surveillance by serological test Here, we describe the construction of recombinant fowlpox viruses, rFP V-HA and rFPV-HA-IFN- II, expressing hemagglutinin (HA) from subtype H9N2 of API and coexpressmg the HA and chicken type II interferon(IFN- II) respectively, and their protective efficacies against homologous challenge in chickens for the development of better vaccines.
1. Cloning and analyzing the HA cDNA
The genomic RNA of H9N2 subtype AIV AlChickenlCbina/F11998 (F strain for abbreviation), was extracted by phenol-SDS. When the purified genomic RNA was used as template for cDNA s~thesis and RT-PCR with two pairs of primers, two overlapping partial segments covering the complete HA cDNA, having the length of 1.1Kb and 0.8Kb nucleotides respectively, were obtained. Sequencs of the two eDNA segments showed that there was a unique Pst I site in the overlapping region of these two segments. By use of the unique Pst I site, we ligated these two segments into the complete HA cDNA, which was cloned in the recombinant plasmid pUCHA.
vi ~H )~f#眫{~
The cloned HA gene was 1708 nucleotides long with a single open reading frame of 1680 nucleotides by sequence analysis. The open reading frame encoded a polypeptide of 560 amino acids, which included the signal peptide of the initial 18 amino acids at the N-terminal and the remaining segment of 542 amino acids. The latter was cleaved into two peptides, HAl and HA2, at the 320 amino acid site. The amino acids ofF strain at the cleavage site were RSSR ~ CILF, revealing the typical cleavage site features of low pathogenic AIV Compared with the H9 prototype AJV strain, A]Turkey/Wisconsin/1/66, the HA gene ofF strain shared 82.90/a sequence homology with that of it.
According to the phylogenetic tree established for the H9N2 AJV isolates in China and neighboring countries and regions from 1992 to 1999, these isolates formed three lineages. F strain, the isolate from Beijing in 1994 and some 9 isolates from Hong Kong in recent years formed the first phylogenetic lineage, which showed significant phylogenetic difference with another lineage consisting of Korea isolates. The third lineage, also formed by isolates from Hong Kong, bad close phylogenetic relationship with the first lineage.
2. Construction of the rFPV expressing HA (rFP V-HA)
For the construction of rFPV-HA, the HA gene, removed from pUCI-IA as Sal I
-Hind III fragment, was directionally inserted into the plasmid 1175, resulting in the transferring vector 11 75HA, in which the HA gene was under the transcriptional control of the vaccinia promoter P7.5. Then the transferring vector 11 75HA was used to transfect the chicken embryo fibroblast cells (CEF) pre-infected with wild type FPV (wt-FPV). The recombinant viruses were obtained and purified by blue plaque selection. The presence of HA gene in the genome of the purified recombinant virus was assayed by PCR and the expression of HA in the rFPV-HA-infected CEF was detected by indirect immunofluorescence.
3. Cloning and expressing the chicken IFN- II gene
A pair of primers were designed on the published data to amplify chicken WN- II gene with the Con A-stimulated chicken spleen cells. The cloned WN- II gene was then directionally inserted into the Sal I-Hind LII site of the plasmid pAFO9, resulting in the recombinant plasmid AWN, in which the IFN- II gene was under the control of the
H9 *5~~ HA ~ I N-ll~ Iffl~1~ vii
vaccinia promoter PE/L.
For the amplification
重的经济损失。油乳剂全病毒灭活苗虽对该亚型禽流感的控制发挥了重要作用，但
由于其存在干扰免疫监测及成本较高等缺点，使用受到了限制。为了研制新型禽流
感疫苗以克服上述缺点，我们对H9亚型AIV中国分离株的血凝素（HA）基因和
鸡Ⅱ型干扰素基因（IFN-Ⅱ）进行了克隆、测序和分析，构建了表达HA基因及共
表达HA基因和鸡IFN-Ⅱ基因的两种重组鸡痘病毒（rFPV-HA、rFPV-HA-IFN-Ⅱ），
并测定了它们的免疫效力。
1 H9亚型AIV中国分离株HA基因cDNA的克隆及序列初步分析
以酚-SDS法提取H9N2亚型AIV中国分离株A/Chicken/China/F/1998（简称F
株）的基因组RNA，以此为模板，采用cDNA合成法及反转录聚合酶链式反应
（RT-PCR），扩增到长度分别为1.1Kb和0.8Kb的两段HA基因片段。测序结果表
明，这两个片段覆盖了HA基因的完整阅读框架，且在它们的重叠区有单一的PstⅠ
酶切位点。利用这一酶切位点，将这两个片段连接成插入在质粒pUC18的HindⅢ
和SalⅠ位点之间的全长HA基因，得重组质粒pUCHA。测定pUCHA位于HindⅢ
和SalⅠ位点之间外源片段的核苷酸序列。
本研究获得的HA基因片段共有1708个核苷酸，包含了HA完整的阅读区的
1680个核苷酸，推测其编码560个氨基酸，其中前18个氨基酸为信号肽序列，其
余542个氨基酸组成的前体蛋白在第320位氨基酸处被切割为HA1和HA2多肽。
F株HA裂解位点的氨基酸序列为RSSR↓GLF，不含连续的碱性氨基酸，具有低
致病性AIV HA裂解位点的特征。F株与H9亚型AIV原型毒株
A/Turkey/Wisconsin/1/66（H9N2）的HA基因的同源性较低，氨基酸和核苷酸的同源
性分别为84.1％和82.9％。
n 扬州大学博士学位论文
以 HAI CDNA的 55－1004 n酸为基元，建立 1992－1999年间 HgNZ亚型 AIV
中国及绷边国家和地区分离株的遗传发生树。结果表明，近年来在这一地区流行
的HgNZ亚型AJV招可敞为三憎系，同一谱系内，毒株间HA艄酸的同源
性在95％以上。F株与髓地区的部分俯株及94年北京分离株的亲龈系较近，
形成谱系卜 香港地区的另一些分离株和巴基斯坦分离株形成谱系2，它们与某些
南美分离株的亲缘关系很近；与谱系1的亲缘关系也较近，它们之间HA核昔酸的
同源性在 gi％R右；韩国分离株形成的谱系 3，该谱系与谱系 1、二的亲缘关系很
远，它们之间HA核昔酸的同源性只有83％左右。
2 表达HA辜因的直组鸡痘病霉的构巨
以 Hnd皿及 Sal消化PUCHA，得到 1．7Kb的 HA基因片段，将n向插人
FPV插入载体 11 75的 Hnd Ill和 Sal位点之间，使之处于痘苗病商启动子 P7．5的
下游，得到转移删 11 75HA。按常规的B眠脓染方法将其转染至已感染 FPV中
国疫酣282E4（Wt郴）的鸡胚成纤测胞（CEF），通过筛选蓝色的病毒腑
获得并纯化重组鸡痘病毒uV－HA。以PCR ＄＄1；ill实，dq，v－HA基因组中插有
HA基因：以间接兔疫荧光肌实，fPV－HA感染的C扭釉了HA。
3 表达鸡IFN－H羹因的匣组鸡痘病羹的钩 来达产物的生物活性
根据已发表的鸡 WN0基因序列，设计一对5！物，以 RTPCR方法从经 Con A
刺激的鸡脾脏细胞中扩增出全长的鸡IFN二基因的cDNA序列。将扩增的基因片
段定向插人质粒 AF09的 Sal和 Hnd IH位点之间，使其位于痘苗病毒早晚期启动
子PEI的下游。
根据启动子 P肌的序列设计上游引物，鸡 IFNl基因的 cDNA 3 M列设计．
下游5！物，以PCR法扩增包含痘苗病毒早晚期启动子PM在内的鸡IFN刁的基因
片段。经一系列步骤，将扩增片段定向插人 FPV JWA载体 11 75中，得到转移载体
1175IFN，用以转染已感染tFPV的CEF，并按上述重组病毒的构建和纯化方法，
获得插谰 rw－n基因的靴重赈毒 ev－r：w－11。在 rrnv－lrx－11中，rw－11
基因处于PDe的转录调控下。
以细胞病鲫制W侧－IFNI感染的CEF越了具有抗水泡性口炎病
毒（VSV）活性的鸡IFN刁。fPV－mI（M．O．H．m）感染CEF 72小时后，培
养上清中 IFN刁的表达量为 1280 U／ITl。动物硼表明，接种 rFPV－IFN刁 天或
14天后，其表达的 IFNJ可显著减轻载体鸡痘病毒对 1日龄 SPF tills｛＄重增长的
程坚：表达和共表达Hg亚型肖流感病毒HA基因和鸡！FN坝基因的重组鸡痘病毒 ＊
抑制作用。
4A表达HA墓因和鸡HN－11基因的直组鸡扈清羹的构建
经一系列步骤，将IFN－11的基因及其上游的启动子PM插人含全长HAcDNA
的质粒pUCm中，得重组质粒PHAIFN。在PHAJFN中，HA基因和IFN刁基因
的3’糊邻。将串联在一起的HA基因和IFNJ基因（包含上游启动子Py）片
段切下，将其定向插人 FPV e载体 11 75中，得到转移狲 11 75HAIFN。在
11 75HAff：N中，HA基因和 IFN。11基因分别处于启动子 P7．5和 PWh的转录控制下，
且转录方向正好相对。在IFN－11基因3’端有一痘苗病毒转录终止子，可避免这两
个基?
H9 subtype avian influenza virus (MV) is widely distzibuted and has resulted in considerable economic loss to poultry industiy in China in recent years. In order to protect poultry against this low pathogenic subtype of API, inactivated vaccines in oil emulsion have been developed and proved to be effective in reducing severity of disease and spread of MV in field situation. However, the use of inactivated vaccines was restricted because of the disadvantages they inherited, including higher cost and interference of routine surveillance by serological test Here, we describe the construction of recombinant fowlpox viruses, rFP V-HA and rFPV-HA-IFN- II, expressing hemagglutinin (HA) from subtype H9N2 of API and coexpressmg the HA and chicken type II interferon(IFN- II) respectively, and their protective efficacies against homologous challenge in chickens for the development of better vaccines.
1. Cloning and analyzing the HA cDNA
The genomic RNA of H9N2 subtype AIV AlChickenlCbina/F11998 (F strain for abbreviation), was extracted by phenol-SDS. When the purified genomic RNA was used as template for cDNA s~thesis and RT-PCR with two pairs of primers, two overlapping partial segments covering the complete HA cDNA, having the length of 1.1Kb and 0.8Kb nucleotides respectively, were obtained. Sequencs of the two eDNA segments showed that there was a unique Pst I site in the overlapping region of these two segments. By use of the unique Pst I site, we ligated these two segments into the complete HA cDNA, which was cloned in the recombinant plasmid pUCHA.
vi ~H )~f#眫{~
The cloned HA gene was 1708 nucleotides long with a single open reading frame of 1680 nucleotides by sequence analysis. The open reading frame encoded a polypeptide of 560 amino acids, which included the signal peptide of the initial 18 amino acids at the N-terminal and the remaining segment of 542 amino acids. The latter was cleaved into two peptides, HAl and HA2, at the 320 amino acid site. The amino acids ofF strain at the cleavage site were RSSR ~ CILF, revealing the typical cleavage site features of low pathogenic AIV Compared with the H9 prototype AJV strain, A]Turkey/Wisconsin/1/66, the HA gene ofF strain shared 82.90/a sequence homology with that of it.
According to the phylogenetic tree established for the H9N2 AJV isolates in China and neighboring countries and regions from 1992 to 1999, these isolates formed three lineages. F strain, the isolate from Beijing in 1994 and some 9 isolates from Hong Kong in recent years formed the first phylogenetic lineage, which showed significant phylogenetic difference with another lineage consisting of Korea isolates. The third lineage, also formed by isolates from Hong Kong, bad close phylogenetic relationship with the first lineage.
2. Construction of the rFPV expressing HA (rFP V-HA)
For the construction of rFPV-HA, the HA gene, removed from pUCI-IA as Sal I
-Hind III fragment, was directionally inserted into the plasmid 1175, resulting in the transferring vector 11 75HA, in which the HA gene was under the transcriptional control of the vaccinia promoter P7.5. Then the transferring vector 11 75HA was used to transfect the chicken embryo fibroblast cells (CEF) pre-infected with wild type FPV (wt-FPV). The recombinant viruses were obtained and purified by blue plaque selection. The presence of HA gene in the genome of the purified recombinant virus was assayed by PCR and the expression of HA in the rFPV-HA-infected CEF was detected by indirect immunofluorescence.
3. Cloning and expressing the chicken IFN- II gene
A pair of primers were designed on the published data to amplify chicken WN- II gene with the Con A-stimulated chicken spleen cells. The cloned WN- II gene was then directionally inserted into the Sal I-Hind LII site of the plasmid pAFO9, resulting in the recombinant plasmid AWN, in which the IFN- II gene was under the control of the
H9 *5~~ HA ~ I N-ll~ Iffl~1~ vii
vaccinia promoter PE/L.
For the amplification
引文
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20. Philpoot M, Easterday BC, Hinshaw VS. 1989. Neutralizing epitopes of the H5 Heamagglutinin from a virulent avian influenza virus and their relationship to pathogenicity. J Virol: 63:3453-8
21. Roges GN, Paulson JC. 1983. Receptor determinants of human and animal influenza virus isolates: difference in receptor cpecificity of the H3 hemaglutinin based on species of origin. Virology 127:361-373.
22. Toshihiro Ito and Yoshihiro Kawaoka. 2000. Host range barrier of influenza A viruses. Vet Microbiology, 74: 71-75.
23. Huang R T C, Rott R, Klenk H D. 1981. Influenza viruses cause hemolysis and fusion of cells. Virology, 110:243-247
24. White, J. M. 1994. Fusion of influenza virus in endosomes: role of hemagglutinin. In "Cellular Receptors for Animal Viruses"(E. Wimmer, ed.). Cold Spring Harbor Laboratory Press. Cold Sping Harbor, NY, 281-301.
25. Chistian F., Britta S. D., Andreas H. et al. 1998. Acylation of the Influenza Hemagglutinin Modulates Fusion Activity Virology, 248: 284-294.
26. Alexander DJ. 2000. A review of avian influenza in different bird species. Vet Microbio: 74: 3-13.
27. Wood GW, McCauley JW, Bashiruddin JB, et, al. 1993. Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtype. Arch Virol, 130: 209-217.
28. Senne D A. 1996. Survey of the hemagglutinin(HA) cleavage site sqience of H5 and H7 avian influenza: amino acid sequence at the HA cleavage site as a marker of
pathogenicity potential. Avian disease, 40:425-437．
29． Vey. M, Olich M, Adler S, et al. 1992． Haemagglutinin activation of pathogenic avian influenza viruses of subtype H7requires the recognition motif R-X-R/K-R.Virology 188,408-413
30． Horimoto T, Kawaoka Y . 1997． Biological effects of introducing additional basic amino acid residues into hemaglutinin in cleavage site of a virulent avian influenza virus. Virus Res, 50(1) : 35-40．
31． Khatchikian E, Orlich M, Rott.R. 1989． Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into hemagglutinin gene of an avian influenza virus. Nature, 340: 156-157．
32． Perdue. M L, Garcia M, Senne. D, et al. 1997． Virulence-associated sequence duplication at the haemagglutinin cleavage site of avian influenza viruses. Virus Res. 49:173-186．
33． Perdue. M L, Suarze D L. 2000． Structural features of the avian influenza virus haemagglutinin that influence virulence. Vet Micro,74:77-86
34． Perdue. M L, Latimer. J W, Crawford J M, et al. 1995． A novel carbohydrate addition site on the hemagglutinin protein of a highly pathogenic H7 subtype avian influenza virus. Virology, 213: 276-281
35． Ruarze D L, Garcia M, Latimer J, et al. 1998． Comparison of highly virulent H5N1 influenza viruses isolated from human and chickens from Hong Kong. J Virol. 72: 6678-6688．
36． Matrosovich M, Zhou N, Kawaoka Y, et, al. 1999． The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J .Virol, 73: 1146-1145．
37． Kawaoka Y and Webster RG. 1985． Evolution of the A/chicken/ Pennsylvania/83(H5N3) influenza virus. Virology, 146:130-137．
38． Reinhard U and Scholtissek C. 1988． Comparison of the nucleoprotein gene of a chicken and a mink influenza A H10 vitus. Arch Virol, 103:139-145．
39． Scholtissek C, Burger H, Kistner O, et, al. 1985． The nucleoprotein as possible factor in determining host specificity of influenza H3n2 virus. Virology, 147:287-294．
40． Snyder MH, Buckler-white AJ, London EL, et, al. 1987． The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus
polymerase genes each specify attenuation of avian-human influenza A/pintail/79 reassortant virus for monkeys. J. Virol, 61:2887-2863．
41． Ruigrok, RW., Calder LJ., Wharton SA. 1989． Electron microscopy of the influenza virus submembranal structure. Virology, 173:311-316
42． Dale B, Brown R, Miller J., et, al. 1986． nucletide and deduced amino acid sequence of the influenza neuraminidase genes of two equine serotype. Virol, 155:460-468．
43． Blok J., Air GM., Laver WG., et, al. 1982． Study on the size ,chemical composition and partial seqyence of the neuraminidase(NA) from type A influenza virus show that the N-terminal region of the NA is not processed and serves to anchor the Nain the viral membrane. Virol, 119:109-121．
44． Choppin PW. 1975． The structure of influenza virus. In: the influenza virus and influenza. Edited by Kilbourne ED. New York, Academic Press, 15-47．
45． Blok J and Air GM. 1982． Variation in the membrane-insertion and "stalk" sequences in eight subtype of influenza type A virus neuraminidase. Biochemistry, 21:4001-4007．
46． Varghes JN. 1983． Structure of the influenza virus glycoprotein antigen neuraminidase at 2． 9A resolution. Nature, 303:35-37．
47． Colman PM. 1983． Structure of the catalytic and antigen site in influenza virus neuraminidase. Nature, 303:41-43．
48． Lentz MR. 1987． Site-directed mutation of the active site of the influenza neuraminidase and implication for the catalytic mechanism. Biochemistry, 26:5351-5360．
49． Webster RG. 1987． Antigenic structure and variation in influenza virus N9 neuraminidase. J Virol,61:2910-2915．
50． 殷震，刘景华，动物病毒学．1997． 科学出版社, 671-703．
51． Fujiyoshi Y, Kume NP, Sakata K., et al. 1994． Fine structure of influenza A virus observed by electron cryo-microscope. EMBO J, 13:318-326．
52． Zhirnov OP. 1990． Solubilization of Matrix protein M1/M from virions occurs at different pH for orthomyxo and paramyxoviruses. Virology,1 76:274-279．
53． Bui M, Whittaker G, Helenius A. 1996． Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins. J Virol, 70:8391-8401．
54． Garcia-Sastre A, Egorov A, Matassov D, et,al. 1998． Influenza A virus lacking the
NS1 gene replicates in interferon-deficient systems. Virology, 252(2) :324-330．
55． O'Neill RE., Talon J., Palese P. 1998． The influenza virus NEP(NS2 protein) mediates the nuclear transport of viral ribonucleoproteins. EMBO J, 17:288-296．
56． Suarez DL and Schultz-Cherry S. 2000． Immunology of avian influenza virus: a review. Development and comparative Immunology, 24:269-283．
57． Johansson BE, Bucher DJ, Kilbourne ED. 1989． Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virol, 63: 1239-46．
58． Rott R. Becht H, Orlich M. 1974． The significance of the influenza virus neuraminidase in immunitity. J Gen Virol, 22:35-41．
59． McNulty MS, Allan GM, Adair BM. 1986． Efficacy of avian influenza neraminidase-specific vaccines in chickens. Avian Pathol, 15:107-15
60． Chen Z, Matsuo K, Asanuma H, Takahashi H, et, al. 1999． Enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin-and neuraminidase-expressing DNAs. Vaccine, 17:653-9．
61． Slepushkin VA, Katz JM, Black PA, et, al. 1995． Protection of mice against influenza Avirus challenge by vaccination with baculovirus-expressed M2 protein. Vaccine, 13:1399-402
62． Zebedee SL and Lamb RA. 1988． Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol, 62: 2762-72．
63． Frace AM, Klimov AI, Rowe T, et, al. 1999． Modified M2 proteins produce heterotypic immunity against influenza A virus. Vaccine, 17:2237-44
64． Beard CW. 1970． Avian influenza antibody detection by immunodiffussion. Avian Dis, 14:337-41．
65． Higgins DA, Shortridge KF, Ng PLK. 1987． Bile immunoglobulin of duck.Ⅱ. Antibody response in influenza A virus infections. Immunology, 62:499-504．
66． Magor KE, Warr GW, Bando Y, et, al. 1998． Secretory immune system of duck. Identification and expression of the gene encoding IgA and IgM heavy chains. Eur J Immunol, 28:1063-8．
67． Gelb JJ, Nix WA, Gellman SD. 1998． Infectious bronchitis virus antibodies in tears and their relationship to immunity. Avian Dis, 42:364-74．
68． Jayawardane GWL, Spradbrow PB. 1995． Mucosal immunity in chickens vaccinated with the V4 strain of Newcastle Disease virus. Vet Microbiol, 46:69-77．
69． Takada A, Kida H. 1996． Protective immune response of chickens against Newcastle Disease,induced by the intranasal vaccination with inactivated virus. Vet Microbiol, 50: 17-25．
70． Russell PH and Ezeifeka GO. 1995． The Hitchner B1 strain of Newcastle Disease virus induces high levels of IgA, IgG, IgM in newlyhatched chicks. Vaccine, 13:61-66
71． Holt PS. 1990． Enhancement of chicken lymphocyte activation and lymphokine release by avian influenza virus. Dev Comp Immunol, 14:447-55．
72． Campen H, Esterday BC, Hinshaw VS, et, al. 1989． Destruction of lymphocytes by a virulent avian influenza A virus. J Gen Virol, 70:467-72．
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95． Horisberger MA, Hallar O, Arnheiter H. 1980． Interferon-dependent genetic resistance to influenza virusin mice: virus replication in macrophages is inhibited at an early step. J Gen Virol, 50: 205-10．
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111． Robinson H L, Hunt LA, Webster RG. 1993． Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vacine, 11(9) :957-960．
112． Poon LL, Fodor E, Brownlee GG. 2000． Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear transport. J Virol,74(1) ,418-427．
2． Barnes HJ, Beard CW, McDongald LR. 1997． Disease of Poultry (Tenth edition). Iowa State niversity Press. Ames, Iowa USA. 583-606．
3． Kilbourne ED. 1987． Influenza, Plenum Press, New York.
4． Murphy BR, Webster RG. 1985． Influenza Viruses. In: Virology, Edited by Fields B. Raven Press. New York: 1179-1240．
5． Lamb RA. 1983． The influenza virus RNA segments and their encoded protein. In: Genetics of influenza virus. Edited by Pales P. Springer Verlag Vienna. 26-29
6． Lamb RA. 1989． Genes and proteins of the influenza viruses. In: The influenza viruses. Edited by King R. New York and London Plenum Press. 1-67．
7． Desselberger U. 1980． The 3' and 5'-terminal sequence of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity. Gene, 8:315-327
8． Honda A., Mukaigawa J, Yokoiyama A, et al. 1990． Purification and molecular structure of RNA polymerase from influenza virus A/PR8． J.Biochem. 107,624-628
9． Summers D F, Peng Q, et al. 1995． Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell Mrna and function only as the trimeric enzyme. Virology, 208: 38-47
10． Kiswas S K, Nayak D P. 1994 Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein. J.Virol, 68: 1819-1826
11． Asano Y, Ishihama A. 1997． Indentification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polynerase. J.Biochem, 122: 627-634
12． Nakagawa Y, Kimura N, Toyoda T, et al. 1995． The RNA polymerase PB2 subunit is not required for replication of the influenza virus genome but is involved in capped mRNA synthesis. J. Virol, 65: 245-253
13． Wiley, D.C and Skehel J J. 1987． The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu.Rev.Biochem. 56:365-394．
14． Rott R . 1992． The pathogenic determinant of avian influenza virus. Vet Microbiol,
33：303-310．
15．甘孟候．禽流感．1995．北京农业大学出版社．27-29
16．郭元吉，程小雯．流行性感冒病毒及其实验技术．中国三峡出版社．16-33．
17. Murphy BR, Webster RG. 1990. Influenza Viruses. In: Virology, Edited by Fields B. Raven Press. New York.
18. Wilson IA. 1981. Structure of the heamglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature, 289,:366-367.
19. Wiley DC, Wilson IA, Skehel JJ. 1981. Structural identification of the antibody-bindind sites of Hong Kong influenza hemaglutinin and their involvement in antigenic variation. Nature, 289: 373-378
20. Philpoot M, Easterday BC, Hinshaw VS. 1989. Neutralizing epitopes of the H5 Heamagglutinin from a virulent avian influenza virus and their relationship to pathogenicity. J Virol: 63:3453-8
21. Roges GN, Paulson JC. 1983. Receptor determinants of human and animal influenza virus isolates: difference in receptor cpecificity of the H3 hemaglutinin based on species of origin. Virology 127:361-373.
22. Toshihiro Ito and Yoshihiro Kawaoka. 2000. Host range barrier of influenza A viruses. Vet Microbiology, 74: 71-75.
23. Huang R T C, Rott R, Klenk H D. 1981. Influenza viruses cause hemolysis and fusion of cells. Virology, 110:243-247
24. White, J. M. 1994. Fusion of influenza virus in endosomes: role of hemagglutinin. In "Cellular Receptors for Animal Viruses"(E. Wimmer, ed.). Cold Spring Harbor Laboratory Press. Cold Sping Harbor, NY, 281-301.
25. Chistian F., Britta S. D., Andreas H. et al. 1998. Acylation of the Influenza Hemagglutinin Modulates Fusion Activity Virology, 248: 284-294.
26. Alexander DJ. 2000. A review of avian influenza in different bird species. Vet Microbio: 74: 3-13.
27. Wood GW, McCauley JW, Bashiruddin JB, et, al. 1993. Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtype. Arch Virol, 130: 209-217.
28. Senne D A. 1996. Survey of the hemagglutinin(HA) cleavage site sqience of H5 and H7 avian influenza: amino acid sequence at the HA cleavage site as a marker of
pathogenicity potential. Avian disease, 40:425-437．
29． Vey. M, Olich M, Adler S, et al. 1992． Haemagglutinin activation of pathogenic avian influenza viruses of subtype H7requires the recognition motif R-X-R/K-R.Virology 188,408-413
30． Horimoto T, Kawaoka Y . 1997． Biological effects of introducing additional basic amino acid residues into hemaglutinin in cleavage site of a virulent avian influenza virus. Virus Res, 50(1) : 35-40．
31． Khatchikian E, Orlich M, Rott.R. 1989． Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into hemagglutinin gene of an avian influenza virus. Nature, 340: 156-157．
32． Perdue. M L, Garcia M, Senne. D, et al. 1997． Virulence-associated sequence duplication at the haemagglutinin cleavage site of avian influenza viruses. Virus Res. 49:173-186．
33． Perdue. M L, Suarze D L. 2000． Structural features of the avian influenza virus haemagglutinin that influence virulence. Vet Micro,74:77-86
34． Perdue. M L, Latimer. J W, Crawford J M, et al. 1995． A novel carbohydrate addition site on the hemagglutinin protein of a highly pathogenic H7 subtype avian influenza virus. Virology, 213: 276-281
35． Ruarze D L, Garcia M, Latimer J, et al. 1998． Comparison of highly virulent H5N1 influenza viruses isolated from human and chickens from Hong Kong. J Virol. 72: 6678-6688．
36． Matrosovich M, Zhou N, Kawaoka Y, et, al. 1999． The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J .Virol, 73: 1146-1145．
37． Kawaoka Y and Webster RG. 1985． Evolution of the A/chicken/ Pennsylvania/83(H5N3) influenza virus. Virology, 146:130-137．
38． Reinhard U and Scholtissek C. 1988． Comparison of the nucleoprotein gene of a chicken and a mink influenza A H10 vitus. Arch Virol, 103:139-145．
39． Scholtissek C, Burger H, Kistner O, et, al. 1985． The nucleoprotein as possible factor in determining host specificity of influenza H3n2 virus. Virology, 147:287-294．
40． Snyder MH, Buckler-white AJ, London EL, et, al. 1987． The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus
polymerase genes each specify attenuation of avian-human influenza A/pintail/79 reassortant virus for monkeys. J. Virol, 61:2887-2863．
41． Ruigrok, RW., Calder LJ., Wharton SA. 1989． Electron microscopy of the influenza virus submembranal structure. Virology, 173:311-316
42． Dale B, Brown R, Miller J., et, al. 1986． nucletide and deduced amino acid sequence of the influenza neuraminidase genes of two equine serotype. Virol, 155:460-468．
43． Blok J., Air GM., Laver WG., et, al. 1982． Study on the size ,chemical composition and partial seqyence of the neuraminidase(NA) from type A influenza virus show that the N-terminal region of the NA is not processed and serves to anchor the Nain the viral membrane. Virol, 119:109-121．
44． Choppin PW. 1975． The structure of influenza virus. In: the influenza virus and influenza. Edited by Kilbourne ED. New York, Academic Press, 15-47．
45． Blok J and Air GM. 1982． Variation in the membrane-insertion and "stalk" sequences in eight subtype of influenza type A virus neuraminidase. Biochemistry, 21:4001-4007．
46． Varghes JN. 1983． Structure of the influenza virus glycoprotein antigen neuraminidase at 2． 9A resolution. Nature, 303:35-37．
47． Colman PM. 1983． Structure of the catalytic and antigen site in influenza virus neuraminidase. Nature, 303:41-43．
48． Lentz MR. 1987． Site-directed mutation of the active site of the influenza neuraminidase and implication for the catalytic mechanism. Biochemistry, 26:5351-5360．
49． Webster RG. 1987． Antigenic structure and variation in influenza virus N9 neuraminidase. J Virol,61:2910-2915．
50． 殷震，刘景华，动物病毒学．1997． 科学出版社, 671-703．
51． Fujiyoshi Y, Kume NP, Sakata K., et al. 1994． Fine structure of influenza A virus observed by electron cryo-microscope. EMBO J, 13:318-326．
52． Zhirnov OP. 1990． Solubilization of Matrix protein M1/M from virions occurs at different pH for orthomyxo and paramyxoviruses. Virology,1 76:274-279．
53． Bui M, Whittaker G, Helenius A. 1996． Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins. J Virol, 70:8391-8401．
54． Garcia-Sastre A, Egorov A, Matassov D, et,al. 1998． Influenza A virus lacking the
NS1 gene replicates in interferon-deficient systems. Virology, 252(2) :324-330．
55． O'Neill RE., Talon J., Palese P. 1998． The influenza virus NEP(NS2 protein) mediates the nuclear transport of viral ribonucleoproteins. EMBO J, 17:288-296．
56． Suarez DL and Schultz-Cherry S. 2000． Immunology of avian influenza virus: a review. Development and comparative Immunology, 24:269-283．
57． Johansson BE, Bucher DJ, Kilbourne ED. 1989． Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virol, 63: 1239-46．
58． Rott R. Becht H, Orlich M. 1974． The significance of the influenza virus neuraminidase in immunitity. J Gen Virol, 22:35-41．
59． McNulty MS, Allan GM, Adair BM. 1986． Efficacy of avian influenza neraminidase-specific vaccines in chickens. Avian Pathol, 15:107-15
60． Chen Z, Matsuo K, Asanuma H, Takahashi H, et, al. 1999． Enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin-and neuraminidase-expressing DNAs. Vaccine, 17:653-9．
61． Slepushkin VA, Katz JM, Black PA, et, al. 1995． Protection of mice against influenza Avirus challenge by vaccination with baculovirus-expressed M2 protein. Vaccine, 13:1399-402
62． Zebedee SL and Lamb RA. 1988． Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol, 62: 2762-72．
63． Frace AM, Klimov AI, Rowe T, et, al. 1999． Modified M2 proteins produce heterotypic immunity against influenza A virus. Vaccine, 17:2237-44
64． Beard CW. 1970． Avian influenza antibody detection by immunodiffussion. Avian Dis, 14:337-41．
65． Higgins DA, Shortridge KF, Ng PLK. 1987． Bile immunoglobulin of duck.Ⅱ. Antibody response in influenza A virus infections. Immunology, 62:499-504．
66． Magor KE, Warr GW, Bando Y, et, al. 1998． Secretory immune system of duck. Identification and expression of the gene encoding IgA and IgM heavy chains. Eur J Immunol, 28:1063-8．
67． Gelb JJ, Nix WA, Gellman SD. 1998． Infectious bronchitis virus antibodies in tears and their relationship to immunity. Avian Dis, 42:364-74．
68． Jayawardane GWL, Spradbrow PB. 1995． Mucosal immunity in chickens vaccinated with the V4 strain of Newcastle Disease virus. Vet Microbiol, 46:69-77．
69． Takada A, Kida H. 1996． Protective immune response of chickens against Newcastle Disease,induced by the intranasal vaccination with inactivated virus. Vet Microbiol, 50: 17-25．
70． Russell PH and Ezeifeka GO. 1995． The Hitchner B1 strain of Newcastle Disease virus induces high levels of IgA, IgG, IgM in newlyhatched chicks. Vaccine, 13:61-66
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