H5亚型禽流感DNA疫苗的细胞免疫应答及联合免疫研究
|
摘要
高致病性禽流感（HPAI）是由一些H5和H7亚型流感病毒所引起的高致病性和高死亡率的烈性禽类传染病，已对养禽业构成严重威胁，并具有重要的公共卫生意义。DNA疫苗是预防禽流感最具潜力的基因工程疫苗之一，DNA免疫不仅能诱导较好的抗体反应和特异性的细胞免疫反应，还具有安全稳定、不受宿主种属限制、可反复加强免疫、免疫持续期长等突出优点。
表达H5亚型禽流感病毒A/Goose/Guangdong/1/96(H5N1)[GS/GD/1/96]HA基因的DNA疫苗质粒pCAGGoptiHA5免疫效果良好，目前已完成临床试验，正在申请新兽药证书注册。鉴于现地免疫影响因素众多以及目前我国H5亚型禽流感不同抗原群并存的复杂状况，了解和评价DNA疫苗免疫后的细胞免疫应答状况，探讨不同抗原群DNA疫苗的联合免疫的可能性，将为DNA疫苗的推广应用提供有用的数据支撑。
为获得GS/GD/1/96的HA蛋白，将GS/GD/1/96的HA基因片段克隆于杆状病毒表达系统的转移载体pFastBacHTA中，构建重组质粒pFastBacHTA-HA，转化至DH10Bac感受态细胞，构建重组杆粒，在脂质体介导下转染Sf9昆虫细胞，获得重组杆状病毒（rBacmid-HA5）。采用间接免疫荧光（IFA)、Western blot和血凝试验对所表达的蛋白进行抗原性和生物学活性分析。结果表明，表达了分子量约为64ku的重组蛋白；IFA和western blot试验证明重组HA蛋白能与H5亚型禽流感阳性血清特异结合，具备良好的抗原性；血凝试验和淋巴细胞增殖试验显示重组蛋白具备良好生物学活性。重组蛋白的正确表达为评价DNA疫苗的细胞免疫水平、研究HA基因功能活性及制备禽流感HA基因亚单位疫苗奠定了基础。
为了解H5亚型DNA疫苗免疫后的细胞免疫应答反应，将DNA质粒pCAGGoptiHA5以15μg剂量间隔3周两次免疫SPF鸡，第二次免疫后2周用105EID50的GS/GD/1/96进行致死性攻击，免疫以及攻毒后，用流式细胞术检测试验鸡外周血及脾脏CD3+、CD4+、CD8+T淋巴细胞亚类百分含量的动态变化，利用MTT法进行T淋巴细胞增殖实验分析，同时采用ELISA方法检测血清中IFN-γ、IL-2的含量变化。结果表明：外周血及脾脏CD4+、CD8+T淋巴细胞亚类百分含量具有类似的变化趋势，免疫后CD4+、CD8+T淋巴细胞相对百分含量明显高于非免疫组（p<0.05），CD4+T细胞于第二次免疫后一周达到峰值，攻毒后CD4+含量略有下降但变化不明显；CD8+T淋巴细胞含量也于加免后一周达到峰值，攻毒后二周仍然处于上升趋势；首次免疫后CD4+/CD8+比例逐渐升高，并于加免后一周达到峰值。ConA刺激免疫组和对照组淋巴细胞后，细胞大量增殖，但两组间差异不显著（p>0.05）；表达蛋白刺激后，两组间差异显著（p<0.05）。免疫组血清中IFN-γ、IL-2的含量均高于同期对照组（p<0.05），并于第二次免疫后一周达到峰值，攻毒后略有上升。而非免疫组SPF鸡外周血中CD4+、CD8+淋巴细胞的数量、CD4+/CD8+比例以及血清中IFN-γ、IL-2的含量并没有显著变化，并于攻毒后一周内全部死亡。结果表明，DNA疫苗质粒pCAGGoptiHA5免疫后可诱导较强的细胞免疫应答。
为探讨H5亚型不同抗原群禽流感DNA疫苗联合免疫的可能性和协同免疫保护效力，将DNA疫苗质粒pCAGGoptiHA5、pCAGGoAHHA和pCAGGSXHA分别等量混合，以15μg、30μg、45μg、60μg和90μg总剂量联合免疫3周龄SPF鸡，首次免疫后3周以相同的剂量和途径进行第二次免疫，加强免疫后2周分别用105EID50的A/Goose/Guangdong/1/96(H5N1)[GS/GD/1/96]、A/Duck/Fujian/31/2007(H5N1)[DK/FJ/31/2007]及A/Chicken/Shanxi/2/2006(H5N1)[CK/SX/2/2006]通过鼻腔途径进行攻击，每天观察发病和死亡情况，并于攻毒后第3、5、7天分别采集喉头和泄殖腔拭子，进行病毒的分离和滴定。同时检测免疫后和攻毒后血清HI抗体水平。结果表明，3种质粒以60μg和90μg剂量两次联合免疫后，免疫鸡均可对不同抗原群病毒的攻击提供100%的完全保护（不发病、不排毒、不致死），15μg剂量联合免疫后可对GS/GD/1/96和DK/FJ/31/2007的攻击提供90%和80%的死亡保护；CK/SX/2/2006攻击后，15μg和30μg剂量组的免疫保护率分别为80%和90%,45μg剂量能提供100%的致死保护。结果显示，3种质粒疫苗联合免疫效果良好，可实现有效的交叉保护，减少免疫次数，DNA疫苗联合免疫有望成为抵御不同抗原群H5亚型禽流感的有效免疫策略。
本研究结果表明，DNA疫苗质粒pCAGGoptiHA5可激发SPF鸡产生较强的细胞免疫应答；3种质粒pCAGGoptiHA5、pCAGGoAHHA和pCAGGSXHA联合免疫效果良好，这将为禽流感DNA疫苗的产业化以及现地应用提供必要的科学依据和支持。
Avian Influenza is a serious disease, which causes huge economic losses for the domestic poultryin rescent years. H5subtype Highly pathogenic avian influenza virus(HPAIV) not only infect poultryand wild birds, but also threat our human health.Therefore, it is essential to research new vaccineagainst Avian Influenza. DNA vaccine has lots of advantages. It can induce both humoral and cellimmune responses. It is also easier to store, transport and manufacture. Otherwise, it will inducelong-lasting protective immune response.
DNA vaccine pCAGGoptiHA5which express HA gene has good immune effect and has completedits clinical tests. DNA vaccine is applying for a new veterinary drug registration certificate. In view ofmany influent facters of immune condition and H5subtypes of AI different antigen coexist, evalution ofcell immune response and discussion of different DNA vaccine joint immunity will provide useful datasupports for the popularization and application of DNA vaccine.
For eukaryotic expression of avian influenza virus (AIV) hemagglutinin (HA), the HA gene ofA/Goose/Guangdong/1/96(H5N1)(GS/GD/1/96)was cloned into the baculovirus transfer vectorpFastBacHTa and the resultant recombinant plasmid was transformed into DH10Bac competent cells togenerate recombinant bacmid (rBacmid-HA), which was identified by PCR and sequencing. Therecombinant baculovirus stock was prepared by transfecting rBacmid-HA into the Sf9insect cells. Theexpression of the recombinant HA in sf9cells was identified by indirect immunofluorescence assay andwestern blot, which shown that the recombinant HA were about64ku. Further identificationsdemonstrated that the HA was able to induce high titer of HI antibody and effectively stimulateproliferation of lymph cells in peripheral blood of SPF chicken detected by MTT assay. The preparationof the recombinant protein in sf9cells was facilitated the further study of HA functions, preparation ofthe subunit vaccine against AIV infection and evaluation of DNA vaccines cellular immune responses.
To verify cellular immune responses in SPF chickens after immunization, SPF chickens wereimmunized with15μg AIV HA gene DNA vaccine pCAGGoptiHA5.The percent of CD3+,CD4+andCD8+T lymphocytes and proliferation of lymph cells in periphery blood and spleen were detected byflow cytometer and MTT assay, respectively. The dynamic changes of IFN-γ、IL-2in sera werechecked with ELISA assay.The results demonstrated that the percent of CD4+and CD8+T lymphocytesin periphery blood and spleen had the same tendency. The percent of CD4+and CD8+T lymphocytes ofimmunized groups were higher than that of the control groups and had significant difference betweentwo groups(p<0.05).The amount of CD4+T lymphocytes reached the peak one week after boostingimmunization, then decreased slightly until the experiment finished. In the first immunization, thepercent of CD8+T lymphocytes consistently increased and reached the peak one week after boostingand maintained in a steady level two weeks after challenge. The ratios of CD4+/CD8+increasedgradually in the first immunization and reached the peak one week after boosting. The amount of CD4+and CD8+T lymphocytes as well as ratios of CD4+/CD8+from unchickens had increasing trend butlower than that of vaccinated chickens and then all chickens died one week after challenge. The lymphocytes in vaccinated group and control group stimulated by ConA were proliferation, but nosignificant difference between two groups(p>0.05). While the HA stimulation made significantdifference between two groups(p<0.05). The amount of IFN-γ and IL-2in sera of vaccinated groupswere higher than that of the control groups and reached the peak one week after boosting and thenincreased slightly after challenge.While the amount of IFN-γ and IL-2in control chickens hardly had nochanges and chickens died one week after challenge.The two groups had significant difference(p<0.05).These results also showed that DNA vaccine could induce strong cellular immune responses inSPF chickens.
For evaluating the protective efficacy of H5subtype avian influenza vaccine pCAGGoptiHA5、pCAGGoAHHA and pCAGGSXHA, groups of3-week-old SPF chickens were intramuscular inoculatedwith different doses of three DNA vaccines in15μg、30μg、45μg、60μg and90μg. Groups of chickenswere injected with200μl PBS as controls. Two weeks after the boost, all chickens were challenged with105EID50of highly pathogenic A/Goose/GuangDong/1/96strain, A/Duck/FuJian/31/07strain andA/Chicken/ShanXi/2/06strain, respectively. Oropharyrigeal and cloacalswab specimens were collectedfrom all chickens3,5and7days after inoculation for titration of virus in eggs and chickens wereobserved daily for disease signs and deaths for two weeks. Sera were collected weekly after vaccinationand challenged for detecting HI antibodies. Results demonstrated that60μg and90μg vaccines boostedchickens were completely protected from virus challenge (no disease sign, no virus shedding and nodeath).The protective rates in15μg groups which challenged by A/Goose/GuangDong/1/96(H5N1) orA/Duck/FuJian/31/07(H5N1) were90.0%(9/10)and80%(8/10), respectively. The protective rates in15and30μg groups which challenged by A/Chicken/ShanXi/2/06(H5N1) were80.0%(8/10)and90.0%(9/10), respectively.45μg doses of the vaccine can provide100%of the lethal protection.Resultsshow that the combination of vaccine immune can protecte chickens from different lethal H5N1viruschallenge to achieve purpose of cross protection. The joint DNA vaccine is a highly efficient geneengineering vaccine and can be used for preventing HPAIV effectively in near future.
In summary, the study imply that DNA vaccine pCAGGoptiHA5can stimulate strong cellularimmune response in SPF chickens.The effect of three plasmid joint immune is good and this willprovide necessary scientific basis and support for industrialization and application of AI DNA vaccine.
表达H5亚型禽流感病毒A/Goose/Guangdong/1/96(H5N1)[GS/GD/1/96]HA基因的DNA疫苗质粒pCAGGoptiHA5免疫效果良好，目前已完成临床试验，正在申请新兽药证书注册。鉴于现地免疫影响因素众多以及目前我国H5亚型禽流感不同抗原群并存的复杂状况，了解和评价DNA疫苗免疫后的细胞免疫应答状况，探讨不同抗原群DNA疫苗的联合免疫的可能性，将为DNA疫苗的推广应用提供有用的数据支撑。
为获得GS/GD/1/96的HA蛋白，将GS/GD/1/96的HA基因片段克隆于杆状病毒表达系统的转移载体pFastBacHTA中，构建重组质粒pFastBacHTA-HA，转化至DH10Bac感受态细胞，构建重组杆粒，在脂质体介导下转染Sf9昆虫细胞，获得重组杆状病毒（rBacmid-HA5）。采用间接免疫荧光（IFA)、Western blot和血凝试验对所表达的蛋白进行抗原性和生物学活性分析。结果表明，表达了分子量约为64ku的重组蛋白；IFA和western blot试验证明重组HA蛋白能与H5亚型禽流感阳性血清特异结合，具备良好的抗原性；血凝试验和淋巴细胞增殖试验显示重组蛋白具备良好生物学活性。重组蛋白的正确表达为评价DNA疫苗的细胞免疫水平、研究HA基因功能活性及制备禽流感HA基因亚单位疫苗奠定了基础。
为了解H5亚型DNA疫苗免疫后的细胞免疫应答反应，将DNA质粒pCAGGoptiHA5以15μg剂量间隔3周两次免疫SPF鸡，第二次免疫后2周用105EID50的GS/GD/1/96进行致死性攻击，免疫以及攻毒后，用流式细胞术检测试验鸡外周血及脾脏CD3+、CD4+、CD8+T淋巴细胞亚类百分含量的动态变化，利用MTT法进行T淋巴细胞增殖实验分析，同时采用ELISA方法检测血清中IFN-γ、IL-2的含量变化。结果表明：外周血及脾脏CD4+、CD8+T淋巴细胞亚类百分含量具有类似的变化趋势，免疫后CD4+、CD8+T淋巴细胞相对百分含量明显高于非免疫组（p<0.05），CD4+T细胞于第二次免疫后一周达到峰值，攻毒后CD4+含量略有下降但变化不明显；CD8+T淋巴细胞含量也于加免后一周达到峰值，攻毒后二周仍然处于上升趋势；首次免疫后CD4+/CD8+比例逐渐升高，并于加免后一周达到峰值。ConA刺激免疫组和对照组淋巴细胞后，细胞大量增殖，但两组间差异不显著（p>0.05）；表达蛋白刺激后，两组间差异显著（p<0.05）。免疫组血清中IFN-γ、IL-2的含量均高于同期对照组（p<0.05），并于第二次免疫后一周达到峰值，攻毒后略有上升。而非免疫组SPF鸡外周血中CD4+、CD8+淋巴细胞的数量、CD4+/CD8+比例以及血清中IFN-γ、IL-2的含量并没有显著变化，并于攻毒后一周内全部死亡。结果表明，DNA疫苗质粒pCAGGoptiHA5免疫后可诱导较强的细胞免疫应答。
为探讨H5亚型不同抗原群禽流感DNA疫苗联合免疫的可能性和协同免疫保护效力，将DNA疫苗质粒pCAGGoptiHA5、pCAGGoAHHA和pCAGGSXHA分别等量混合，以15μg、30μg、45μg、60μg和90μg总剂量联合免疫3周龄SPF鸡，首次免疫后3周以相同的剂量和途径进行第二次免疫，加强免疫后2周分别用105EID50的A/Goose/Guangdong/1/96(H5N1)[GS/GD/1/96]、A/Duck/Fujian/31/2007(H5N1)[DK/FJ/31/2007]及A/Chicken/Shanxi/2/2006(H5N1)[CK/SX/2/2006]通过鼻腔途径进行攻击，每天观察发病和死亡情况，并于攻毒后第3、5、7天分别采集喉头和泄殖腔拭子，进行病毒的分离和滴定。同时检测免疫后和攻毒后血清HI抗体水平。结果表明，3种质粒以60μg和90μg剂量两次联合免疫后，免疫鸡均可对不同抗原群病毒的攻击提供100%的完全保护（不发病、不排毒、不致死），15μg剂量联合免疫后可对GS/GD/1/96和DK/FJ/31/2007的攻击提供90%和80%的死亡保护；CK/SX/2/2006攻击后，15μg和30μg剂量组的免疫保护率分别为80%和90%,45μg剂量能提供100%的致死保护。结果显示，3种质粒疫苗联合免疫效果良好，可实现有效的交叉保护，减少免疫次数，DNA疫苗联合免疫有望成为抵御不同抗原群H5亚型禽流感的有效免疫策略。
本研究结果表明，DNA疫苗质粒pCAGGoptiHA5可激发SPF鸡产生较强的细胞免疫应答；3种质粒pCAGGoptiHA5、pCAGGoAHHA和pCAGGSXHA联合免疫效果良好，这将为禽流感DNA疫苗的产业化以及现地应用提供必要的科学依据和支持。
Avian Influenza is a serious disease, which causes huge economic losses for the domestic poultryin rescent years. H5subtype Highly pathogenic avian influenza virus(HPAIV) not only infect poultryand wild birds, but also threat our human health.Therefore, it is essential to research new vaccineagainst Avian Influenza. DNA vaccine has lots of advantages. It can induce both humoral and cellimmune responses. It is also easier to store, transport and manufacture. Otherwise, it will inducelong-lasting protective immune response.
DNA vaccine pCAGGoptiHA5which express HA gene has good immune effect and has completedits clinical tests. DNA vaccine is applying for a new veterinary drug registration certificate. In view ofmany influent facters of immune condition and H5subtypes of AI different antigen coexist, evalution ofcell immune response and discussion of different DNA vaccine joint immunity will provide useful datasupports for the popularization and application of DNA vaccine.
For eukaryotic expression of avian influenza virus (AIV) hemagglutinin (HA), the HA gene ofA/Goose/Guangdong/1/96(H5N1)(GS/GD/1/96)was cloned into the baculovirus transfer vectorpFastBacHTa and the resultant recombinant plasmid was transformed into DH10Bac competent cells togenerate recombinant bacmid (rBacmid-HA), which was identified by PCR and sequencing. Therecombinant baculovirus stock was prepared by transfecting rBacmid-HA into the Sf9insect cells. Theexpression of the recombinant HA in sf9cells was identified by indirect immunofluorescence assay andwestern blot, which shown that the recombinant HA were about64ku. Further identificationsdemonstrated that the HA was able to induce high titer of HI antibody and effectively stimulateproliferation of lymph cells in peripheral blood of SPF chicken detected by MTT assay. The preparationof the recombinant protein in sf9cells was facilitated the further study of HA functions, preparation ofthe subunit vaccine against AIV infection and evaluation of DNA vaccines cellular immune responses.
To verify cellular immune responses in SPF chickens after immunization, SPF chickens wereimmunized with15μg AIV HA gene DNA vaccine pCAGGoptiHA5.The percent of CD3+,CD4+andCD8+T lymphocytes and proliferation of lymph cells in periphery blood and spleen were detected byflow cytometer and MTT assay, respectively. The dynamic changes of IFN-γ、IL-2in sera werechecked with ELISA assay.The results demonstrated that the percent of CD4+and CD8+T lymphocytesin periphery blood and spleen had the same tendency. The percent of CD4+and CD8+T lymphocytes ofimmunized groups were higher than that of the control groups and had significant difference betweentwo groups(p<0.05).The amount of CD4+T lymphocytes reached the peak one week after boostingimmunization, then decreased slightly until the experiment finished. In the first immunization, thepercent of CD8+T lymphocytes consistently increased and reached the peak one week after boostingand maintained in a steady level two weeks after challenge. The ratios of CD4+/CD8+increasedgradually in the first immunization and reached the peak one week after boosting. The amount of CD4+and CD8+T lymphocytes as well as ratios of CD4+/CD8+from unchickens had increasing trend butlower than that of vaccinated chickens and then all chickens died one week after challenge. The lymphocytes in vaccinated group and control group stimulated by ConA were proliferation, but nosignificant difference between two groups(p>0.05). While the HA stimulation made significantdifference between two groups(p<0.05). The amount of IFN-γ and IL-2in sera of vaccinated groupswere higher than that of the control groups and reached the peak one week after boosting and thenincreased slightly after challenge.While the amount of IFN-γ and IL-2in control chickens hardly had nochanges and chickens died one week after challenge.The two groups had significant difference(p<0.05).These results also showed that DNA vaccine could induce strong cellular immune responses inSPF chickens.
For evaluating the protective efficacy of H5subtype avian influenza vaccine pCAGGoptiHA5、pCAGGoAHHA and pCAGGSXHA, groups of3-week-old SPF chickens were intramuscular inoculatedwith different doses of three DNA vaccines in15μg、30μg、45μg、60μg and90μg. Groups of chickenswere injected with200μl PBS as controls. Two weeks after the boost, all chickens were challenged with105EID50of highly pathogenic A/Goose/GuangDong/1/96strain, A/Duck/FuJian/31/07strain andA/Chicken/ShanXi/2/06strain, respectively. Oropharyrigeal and cloacalswab specimens were collectedfrom all chickens3,5and7days after inoculation for titration of virus in eggs and chickens wereobserved daily for disease signs and deaths for two weeks. Sera were collected weekly after vaccinationand challenged for detecting HI antibodies. Results demonstrated that60μg and90μg vaccines boostedchickens were completely protected from virus challenge (no disease sign, no virus shedding and nodeath).The protective rates in15μg groups which challenged by A/Goose/GuangDong/1/96(H5N1) orA/Duck/FuJian/31/07(H5N1) were90.0%(9/10)and80%(8/10), respectively. The protective rates in15and30μg groups which challenged by A/Chicken/ShanXi/2/06(H5N1) were80.0%(8/10)and90.0%(9/10), respectively.45μg doses of the vaccine can provide100%of the lethal protection.Resultsshow that the combination of vaccine immune can protecte chickens from different lethal H5N1viruschallenge to achieve purpose of cross protection. The joint DNA vaccine is a highly efficient geneengineering vaccine and can be used for preventing HPAIV effectively in near future.
In summary, the study imply that DNA vaccine pCAGGoptiHA5can stimulate strong cellularimmune response in SPF chickens.The effect of three plasmid joint immune is good and this willprovide necessary scientific basis and support for industrialization and application of AI DNA vaccine.
引文
1.顿继凤,周迎春,蒲娟,等. H5N1亚型禽流感病毒血凝素在昆虫杆状病毒系统中表达与活性鉴定[J].农业生物技术学报,2007,15(4):547-551.
2.冯保民，赵艳伟，扈丽萍，等. H5N1型流感疫苗的开发进展[J].中国预防医学杂志,2009.10-3.
3.姜永萍，张洪波，李呈军，等. H5亚型禽流感DNA疫苗质粒pCAGGoptiHA5对高致病力禽流感病毒攻击的免疫保护[J].畜牧兽医学报，2005，36(11):1178-1182.
4.姜永萍，张洪波，李呈军，等. HA基因密码子及表达载体优化的H5亚型禽流感DNA疫苗免疫保护效果比较[J].农业生物技术学报，2006,14(3):301-306.
5.李雁冰,田国斌,施建忠,等.引起我国2004年H5N1HPAI暴发的病毒特征[A].中国畜牧兽医学会禽病学分会第十二次学术研讨会论文集[C].贵阳,2004.357-360.
6.李永强，孟庆文，冉多良，等.表达带His-tag的禽流感病毒HA基因杆状病毒转移载体的构建及重组病毒的鉴定[J].新疆农业大学学报,2005,28(4):53-55.
7.卢建红，邵卫星，刘秀梵，等.禽流感感染的宿主免疫应答[J].动物医学进展，2004,25:37-40.
8.乔传玲,姜永萍,田国斌,等.表达禽流感HA和NP双基因重组禽痘病毒的免疫保护性[J].畜牧兽医学报,2004,35(2):178-181.
9.孙国庆，王纯洁，赵怀平，等.AA肉鸡生长过程中血液CD3+、CD4+和CD8+T淋巴细胞亚群的变化规律[J].内蒙古农业科技，2007,(3):59-62.
10.孙永科，田占成，王云峰，等.表达鸡γ-干扰素和传染性支气管病毒S1基因的重组鸡痘病毒疫苗对鸡外周血T淋巴细胞动态分布影响的研究[J].中国预防兽医学报,2006,28(5):540-543.
11.王国俊，熊杰，李俊平，等. H5亚型禽流感病毒A/Duck/Anhui/1/2006(Clade2.3)密码子优化的HA基因DNA疫苗的免疫保护效力研究[J].中国预防兽医学报，2010,32(2):116-120.
12.王群. H5N1亚型禽流感病毒核蛋白T细胞表位区的研究[D].中国农业科学院，2009.
13.王彦妮，曾显营，田国彬，等.AA肉鸡免疫禽流感油乳剂灭活疫苗细胞免疫应答的变化[J].中国预防兽医学报，2011,33(2):142-145.
14.温坤,丘立文,王压娣,等.禽流感病毒H5N1血凝素基因在昆虫细胞中的表达及鉴定[J].南方医科大学学报,2007,27(1):20-23.
15.张洪波，姜永萍，王全英，等.禽流感病毒HA基因DNA疫苗诱导鸡CD8+T细胞的动态变化[J].动物医学进展，2006,27(11):54-57.
16. Alam Jahangir, Sakchai Ruenphet, Dany Shoham, Masashi Okamura,Masayuki Nakamaura,Kazuaki Takehara. Phenotypic, genetic, and phylogeographical characterization of avian influenzavirus subtype H5N2isolated from northern pintail (Anas acuta) in Japan. Virus Research,2009(145):329–333.
17. B. Chaharaein. Detectio n of H5, H7and H9subtypes of avianinfluenza viruses by multiplexreverse transcription-polymerase chain reaction. Microbiological Research,2009(164):174-179.
18. Chang-Won Lee, Yehia M. Saif. Avian influenza viru.Comparative Immunology, Microbiologyand Infectious Diseases,2009(32):301–310.
19. Chengjun Li, Guobin Tian, Yanbing Li, Dandan Yu, et al. Antigenic and genetic analysis of theH9N2avian influenza viruses isolated in China. International Congress Series1263(2004):762–765.
20. Chengjun Li, Jihui Ping, Bo Jing, et al. H5N1influenza marker vaccine for serologicaldifferentiation between vaccinated and infected chickens[J]. Biochemical and BiophysicalResearch Communications,2008:293–297.
21. Chengjun Li, Kangzhen Yu, Guobin Tian, et al. Evolution of H9N2influenza viruses fromdomestic poultry in Mainland China. Virology,2005(340):70-83.
22. Chengjun Li,Masato Hatta,Shinji Watanabe, et al. Compatibility among Polymerase SubunitProteins Is a Restricting Factor in Reassortment between Equine H7N7and Human H3N2Influenza Viruses. Journal virology,2008(82):11880-11888.
23. Chi-Yuan Chen, Hung-Jen Liu, Ching-Ping Tsai, et al. Baculovirus as an avian influenza vaccinevector: Differential immune responses elicited by different vector forms. Vaccine,2010(28):7644–7651.
24. Chuan-ling Qiao, Kang-zhen Yu, Yong-ping Jiang, et al. Protection of chickens against highlylethal H5N1and H7N1avian influenza viruses with a recombinant fowlpox virus co-expressingH5haemagglutinin and N1neuraminidase genes. Avian Pathology (2003)32:25-31.
25. Chuanling Qiao, Yongping Jiang, Guobin Tian，et al. Recombinant fowlpox virus vector-basedvaccine completely protects chickens from H5N1avian influenza virus. Antiviral Research2009(81):234–238.
26. D.L. Suarez, S. Schultz-Cherry. Immunology of avian influenza virus: a review[J]. Developmentaland Comparative Immunology,2000(24):269-283.
27. Dawn Su-Yin Yeo, Sock-Hoon Ng, Chin-Wen Liaw, et al. Molecular characterization of lowpathogenic avian influenza viruses, isolated from food products imported into Singapore.Veterinary Microbiology,2009(138):304–317.
28. Deyun Wang, Xiangrui Li, Lixin Xu, Yuanliang, Baokang Zhang, Jiaguo Liu. Immunologicsynergism with IL-2and effects of cCHMIs on mRNA expression of IL-2and IFN－γ in chickenperipheral T lymphocyte. Vaccine,2006(24):7109–7114.
29. E. Johansson, K. Domeika, M. Berg, G.V. Alm, C. Fossum. Characterisation of porcinemonocyte-derived dendritic cells according to their cytokine profile. Veterinary Immunology andImmunopathology,2003(91):183–197.
30. Elena Fringuelli. Cloning and expression of pigeon IFN-γgene. Research in VeterinaryScience.2010:1-6.
31. Gao W, Tamin A, Soloff A, et al. Effects of a SARS-associated coronavirus vaccine in monkeys[J].The Lancet,2003,362(9399):1895-1896.
32. Garmory HS, Brown KA, Tiball RW. DNA vaccines: improving expression of antigens[J].GenetVaccines Ther,2003(1):2.
33. Guangxing Li,Yan Zeng, Jiechao Yin,Hyun S. Lillehoj,and Xiaofeng Ren. Cloning, ProkaryoticExpression, and Biological Analysis of Recombinant Chicken IFN-γ. Hybridoma.2010(29):1-9.
34. Guobin Tian, Suhua Zhang, Yanbing Li, et al. Protective efficacy in chickens, geese and ducks ofan H5N1-inactivated vaccine developed by reverse genetics, Virology,2005(341):153–162.
35. Gurunathan S, Chang-Yu Wu, Freidag BL, et al. DNA vaccines: a key for inducing long-termcellular immunity. Current Oplnlon in Immunology2000,12:442-447.
36. H. Chen, K. Yu, Y. Jiang, X. Tang. DNA immunization elicits high HI antibody and protectschicken from AIV challenge. International Congress Series,1219(2001)917-921.
37. Hassett DE, Slifka MK, Jie Zhang et al.Direct EX Vivo Kinetic and Phenotypic Analyses of CD8+T-Cell Responses Induced by DNA Immunization. Journal of Virology,2000,9:8286-8291.
38. Hualan Chen, Kanta Subbarao, David Swayne, et al. Generation and evaluation of a high growthreassortant H9N2influenza a virus as a pandemic vaccine candidate. Vaccine,2003(21):1974–1979.
39. Hualan Chen,Yanbing Li,Zejun Li, et al. Properties and Dissemination of H5N1Viruses Isolatedduring an Influenza Outbreak in Migratory Waterfowl in Western China, Journal virology, June2006,5976–5983.
40. Huoying Shi, Xiu Fan Liu, Xiaorong Zhang, Sujuan Chen, Lei Sun, Jianhong Lu. Generation of anattenuated H5N1avian influenza virus vaccine with all eight genes from avian viruses. Vaccine,2007(25):7379–7384.
41. Hyeok-il Kwon, Min-Suk Song, Philippe Noriel Q. Pascua, et al. Genetic characterization andpathogenicity assessment of highly pathogenic H5N1avian influenza viruses isolated frommigratory wild birds in2011,South Korea, Virus Research,2011(160):305–315.
42. Jiang YP, Yu KZ, Zhang HB,et al. Enhanced protective efficacy of H5subtype avian influenzaDNA vaccinewith codon optimized HA gene in a pCAGGS plasmid vector. Antiviral Research,2007,75:234-241.
43. Jianjun Chen, Fang Fang, Zhongdong Yang, et al. Characterization of highly pathogenic H5N1avian influenza viruses isolated from poultry markets in central China. Virus Research,2009(146):19–28.
44. Jill Taylor, Randall Weinberg, Yoshihiro Kawaokat, Robert G. Webstert and Enzo Paoletti.Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine,1988,12(6):1-5.
45. John Crawford, et al. Baculovirus-d erived hemagglutinin vaccines protect against lethal influenzainfections by avian H5and H7subtypes. Vaccine,1999(17):2265-2274.
46. John W. Shiver, Jeffrey B. Ulmer, John J. Donnelly, Margaret A. Liu. Humoral and cellularimmunities elicited by DNA vaccines: Application to the human immunodeficiency virus andinfluenza. Advanced Drug Delivery Reviews,1996(21):19-31.
47. K.D. Song, H.S. Lillehoj, K.D.Choi, Expression and functional characterization of recombinantchicken interferon-gamma. Veterinary Immunology and Immunopathology.1997(58):321-333.
48. Kanta Subbarao, Hualan Chen, David Swayne, et al. Evaluation of a Genetically ModifiedReassortant H5N1Influenza A Virus Vaccine Candidate Generated by Plasmid-Based ReverseGenetics. Virology,2003(305):192–200.
49. Kanta Subbarao, Hualan Chen, David Swayne, et al. Evaluation of a Genetically ModifiedReassortant H5N1Influenza A Virus Vaccine Candidate Generated by Plasmid-Based ReverseGenetics. Virology,2003(305):192–200.
50. Karen S. Yee, Tim E. Carpenter, Carol J. Cardona. Epidemiology of H5N1avian influenza.Comparative Immunology, Microbiology and Infectious Diseases,2009(32):325–340.
51. Kristien Van Reeth. Cytokines in the pathogenesis of influenza. Veterinary Microbiology,2000(74):109-116.
52. Lladser A,Parraga M,Quevedo L,etal.Naked DNA immunization as an approach to target thegeneric tumor antigen surviving induces humoral and cellular immune responses inmice.Immunobiology,2006,211:11-27.
53. Llilianne Ganges, Maritza Barrera, Jos′e Ignacio N′u nez, et al. A DNA vaccine expressing theE2protein of classical swine fever virus elicits T cell responses that can prime for rapid antibodyproduction and confer total protection upon viral challenge. Vaccine,2005(23):3741–3752.
54. M. Garcia, D.L. Suarez, J.M. Crawford. Evolution of H5subtype avian influenza a viruses inNorth America. Virus Research,1997(51):115-124.
55. Matthias Mueller, Sandra Renzullo, Roxann Brooks, et al. Antigenic Characterization ofRecombinant Hemagglutinin Proteins Derived from Different Avian Influenza Virus Subtypes.PlosONE,2010(5):1-9.
56. Mengcheng Luo, Pan Tao, Junwei Li, et al. Immunization with plasmid DNA encoding influenza Avirus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immuneresponses and protection against H5N1challenge in mice. Journal of Virological Methods,2008(154):121–127.
57. Mengcheng Luo, Pan Tao, Junwei Li, Siyu Zhou, Deyin Guo, Zishu Pan. Immunization withplasmid DNA encoding influenza A virus nucleoprotein fused to a tissue plasminogen activatorsignal sequence elicits strong immune responses and protection against H5N1challenge in mice.Journal of Virological Methods,2008(154):121–127.
58. Michaela Sharpe, Debbie Lynch, Simon Topham, et al. Protection of mice from H5N1influenzachallenge by prophylactic DNA vaccination using particle mediated epidermal delivery.Vaccine,2007(25):6392–6398.
59. Mookkan Prabakaran, Sumathy Velumani, Fang He, et al. Protective immunity against infl uenzaH5N1virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HAand recombinant CTB as an adjuvant. Virology380(2008)412–420.
60. Muhammad Abbas, Erica Spackman, Ron Fouchier, et al. H7avian influenza virus vaccinesprotect chickens against challenge with antigenically diverse isolates. Vaccine,29(2011):7424–7429.
61. Muhammad Athar Abbas.H7avian influenza virus vaccines protect chickens against challengewithantigenically diverse isolates, Vaccine,2011(29):7424–7429.
62. Neal Satterly, Pei-Ling Tsai,Jan van Deursen, et al. Influenza virus targets the mRNA exportmachinery and the nuclear pore complex.PNAS,2007(104):1853-1858.
63. Pan Tao, Mengcheng Luo, Ruangang Pan, et al. Enhanced protective immunity against H5N1influenza virus challenge by vaccination with DNA expressing a chimeric hemagglutinin incombination with an MHC class I-restricted epitope of nucleoprotein in mice. Antiviral Research,2009(81):253–260.
64. Peirong Jiao, Guobin Tian, Yanbing Li, A Single-Amino-Acid Substitution in the NS1ProteinChanges the Pathogenicity of H5N1Avian Influenza Viruses in Mice. JOURNAL OFVIROLOGY, Feb.2008,1146–1154.
65. Qinfang Liu, Jingjiao Ma, Zheng Kou, et al. Characterization of a highly pathogenic avianinfluenza H5N1clade2.3.4virus isolated from a tree sparrow. Virus Research,2010(147):l25–29.
66. Qunfeng Wu, Liurong Fang, Xuebao Wu, et al. A pseudotype baculovirus-mediated vaccineconfers protective immunity against lethal challenge with H5N1avian influenza virus in mice andchickens. Molecular Immunology,2009(46):2210–2217.
67. Riks Maas, Mirriam Tacken, Diana van Zoelen, Hok Oei. Dose response effects of avian influenza(H7N7) vaccination of chickens: Serology, clinical protection and reduction of virus excretion.Vaccine,2009(27):3592–3597.
68. Shufang Fan, Guohua Deng, Jiasheng Song, et al. Two amino acid residues in the matrix proteinM1contribute to the virulence difference of H5N1avian in fl uenza viruses in mice. Virology,2009(384):28–32.
69. Shufang Fan, Yuwei Gao, Kyoko Shinya, et al. Immunogenicity and Protective Efficacy of a LiveAttenuated H5N1Vaccine in Nonhuman Primates.Plos pathogens,2009(5):1-11.
70. Songlin Zhang, Lingyun Xiao, Hongbo Zhou, et al. Generation and characterization of an H5N1avian influenza virus hemagglutinin glycoprotein pseudotyped lentivirus. Journal of VirologicalMethods,2008(154):99–103.
71. Suarez D L, Schultz-Cherry S. Immunology of avian influenza virus: a review[J]. Developmentaland Comparative Immunology,2000(24):269-283.
72. Subbarao K, Chen HL, Swayne D. Evaluation of a Genetically Modified Reassortant H5N1Influenza A Virus Vaccine Candidate Generated by Plasmid-Based Reverse Genetics [J]. Virology,2003(305):192-200.
73. Tang X C, Lu H R, Ross T M. Hemagglutinin Displayed Baculovirus Protects Against HighlyPathogenic Influenza [J]. Vaccine,2010(28):6821-6831.
74. Taylor J, Weinberg R, Kawaka Y, et al. Protective immunity against avian influenza induced by afowlpox virus [J]. Vaccine,1988,6:504-508.
75. Xian-Chun Tang, Hai-Rong Lu, Ted M. Ross. Hemagglutinin Displayed Baculovirus ProtectsAgainst Highly Pathogenic Influenza. Vaccine,2010(28):6821–6831.
76. Xian-Chun Tang, Hai-Rong Lu, Ted M. Ross. Hemagglutinin Displayed BaculovirusProtectsAgainst Highly Pathogenic Influenza. Vaccine,2010(28):6821–6831.
77. Y. L i, Z.Lin, J.Shi, Q.Qi, G. Deng, Z. Li, X. Wang. Detection of Hong Kong97-like H5N1influenza viruses from eggs of Vietnamese waterfowl. Arch Virol (2006).
78. Yanbing Li, Chengjun Li, Liling Liu, et al. Characterization of an avian influenza virus of subtypeH7N2isolated from chickens in northern China. Virus Genes,(2006)33:117–122.
79. Yixin Chen, Feihai Xu, Xiaohui Fan,et al. Evaluation of a rapid test for detection of H5N1avianinfluenza virus. Journal of Virological Methods,2008(154):213–215.
80. YongpingJiang, Kangzhen Yu, Hongbo Zhang, Pingjing Zhang, Chenjun Li, Guobin Tian,YanbingLi, Xijun Wang, Jinying Ge, Zhigao Bu, Hualan Chen. Enhanced protective efficacy of H5subtypeavian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector.Antiviral Research2007(75):234–241.
81. Yu C. Hu,YuL.Luo, Wen T. Ji. Dual expression of the HA protein of H5N2avian influenza virusin a baculovirus system. Journal of Virological Methods,2006(135):43–48.
82. Zejun Li, Yongping Jiang, Peirong Jiao, et al. The NS1Gene Contributes to the Virulence of H5N1Avian Influenza Viruses. Journal virology,2006(80):111115-11123.
83. Zejun Li,Hualan Chen,Peirong Jiao,Guohua Deng, et al. Molecular Basis of Replication of DuckH5N1Influenza Viruses in a Mammalian Mouse Model. Journal virology,2005(79):12058-12064.
2.冯保民，赵艳伟，扈丽萍，等. H5N1型流感疫苗的开发进展[J].中国预防医学杂志,2009.10-3.
3.姜永萍，张洪波，李呈军，等. H5亚型禽流感DNA疫苗质粒pCAGGoptiHA5对高致病力禽流感病毒攻击的免疫保护[J].畜牧兽医学报，2005，36(11):1178-1182.
4.姜永萍，张洪波，李呈军，等. HA基因密码子及表达载体优化的H5亚型禽流感DNA疫苗免疫保护效果比较[J].农业生物技术学报，2006,14(3):301-306.
5.李雁冰,田国斌,施建忠,等.引起我国2004年H5N1HPAI暴发的病毒特征[A].中国畜牧兽医学会禽病学分会第十二次学术研讨会论文集[C].贵阳,2004.357-360.
6.李永强，孟庆文，冉多良，等.表达带His-tag的禽流感病毒HA基因杆状病毒转移载体的构建及重组病毒的鉴定[J].新疆农业大学学报,2005,28(4):53-55.
7.卢建红，邵卫星，刘秀梵，等.禽流感感染的宿主免疫应答[J].动物医学进展，2004,25:37-40.
8.乔传玲,姜永萍,田国斌,等.表达禽流感HA和NP双基因重组禽痘病毒的免疫保护性[J].畜牧兽医学报,2004,35(2):178-181.
9.孙国庆，王纯洁，赵怀平，等.AA肉鸡生长过程中血液CD3+、CD4+和CD8+T淋巴细胞亚群的变化规律[J].内蒙古农业科技，2007,(3):59-62.
10.孙永科，田占成，王云峰，等.表达鸡γ-干扰素和传染性支气管病毒S1基因的重组鸡痘病毒疫苗对鸡外周血T淋巴细胞动态分布影响的研究[J].中国预防兽医学报,2006,28(5):540-543.
11.王国俊，熊杰，李俊平，等. H5亚型禽流感病毒A/Duck/Anhui/1/2006(Clade2.3)密码子优化的HA基因DNA疫苗的免疫保护效力研究[J].中国预防兽医学报，2010,32(2):116-120.
12.王群. H5N1亚型禽流感病毒核蛋白T细胞表位区的研究[D].中国农业科学院，2009.
13.王彦妮，曾显营，田国彬，等.AA肉鸡免疫禽流感油乳剂灭活疫苗细胞免疫应答的变化[J].中国预防兽医学报，2011,33(2):142-145.
14.温坤,丘立文,王压娣,等.禽流感病毒H5N1血凝素基因在昆虫细胞中的表达及鉴定[J].南方医科大学学报,2007,27(1):20-23.
15.张洪波，姜永萍，王全英，等.禽流感病毒HA基因DNA疫苗诱导鸡CD8+T细胞的动态变化[J].动物医学进展，2006,27(11):54-57.
16. Alam Jahangir, Sakchai Ruenphet, Dany Shoham, Masashi Okamura,Masayuki Nakamaura,Kazuaki Takehara. Phenotypic, genetic, and phylogeographical characterization of avian influenzavirus subtype H5N2isolated from northern pintail (Anas acuta) in Japan. Virus Research,2009(145):329–333.
17. B. Chaharaein. Detectio n of H5, H7and H9subtypes of avianinfluenza viruses by multiplexreverse transcription-polymerase chain reaction. Microbiological Research,2009(164):174-179.
18. Chang-Won Lee, Yehia M. Saif. Avian influenza viru.Comparative Immunology, Microbiologyand Infectious Diseases,2009(32):301–310.
19. Chengjun Li, Guobin Tian, Yanbing Li, Dandan Yu, et al. Antigenic and genetic analysis of theH9N2avian influenza viruses isolated in China. International Congress Series1263(2004):762–765.
20. Chengjun Li, Jihui Ping, Bo Jing, et al. H5N1influenza marker vaccine for serologicaldifferentiation between vaccinated and infected chickens[J]. Biochemical and BiophysicalResearch Communications,2008:293–297.
21. Chengjun Li, Kangzhen Yu, Guobin Tian, et al. Evolution of H9N2influenza viruses fromdomestic poultry in Mainland China. Virology,2005(340):70-83.
22. Chengjun Li,Masato Hatta,Shinji Watanabe, et al. Compatibility among Polymerase SubunitProteins Is a Restricting Factor in Reassortment between Equine H7N7and Human H3N2Influenza Viruses. Journal virology,2008(82):11880-11888.
23. Chi-Yuan Chen, Hung-Jen Liu, Ching-Ping Tsai, et al. Baculovirus as an avian influenza vaccinevector: Differential immune responses elicited by different vector forms. Vaccine,2010(28):7644–7651.
24. Chuan-ling Qiao, Kang-zhen Yu, Yong-ping Jiang, et al. Protection of chickens against highlylethal H5N1and H7N1avian influenza viruses with a recombinant fowlpox virus co-expressingH5haemagglutinin and N1neuraminidase genes. Avian Pathology (2003)32:25-31.
25. Chuanling Qiao, Yongping Jiang, Guobin Tian，et al. Recombinant fowlpox virus vector-basedvaccine completely protects chickens from H5N1avian influenza virus. Antiviral Research2009(81):234–238.
26. D.L. Suarez, S. Schultz-Cherry. Immunology of avian influenza virus: a review[J]. Developmentaland Comparative Immunology,2000(24):269-283.
27. Dawn Su-Yin Yeo, Sock-Hoon Ng, Chin-Wen Liaw, et al. Molecular characterization of lowpathogenic avian influenza viruses, isolated from food products imported into Singapore.Veterinary Microbiology,2009(138):304–317.
28. Deyun Wang, Xiangrui Li, Lixin Xu, Yuanliang, Baokang Zhang, Jiaguo Liu. Immunologicsynergism with IL-2and effects of cCHMIs on mRNA expression of IL-2and IFN－γ in chickenperipheral T lymphocyte. Vaccine,2006(24):7109–7114.
29. E. Johansson, K. Domeika, M. Berg, G.V. Alm, C. Fossum. Characterisation of porcinemonocyte-derived dendritic cells according to their cytokine profile. Veterinary Immunology andImmunopathology,2003(91):183–197.
30. Elena Fringuelli. Cloning and expression of pigeon IFN-γgene. Research in VeterinaryScience.2010:1-6.
31. Gao W, Tamin A, Soloff A, et al. Effects of a SARS-associated coronavirus vaccine in monkeys[J].The Lancet,2003,362(9399):1895-1896.
32. Garmory HS, Brown KA, Tiball RW. DNA vaccines: improving expression of antigens[J].GenetVaccines Ther,2003(1):2.
33. Guangxing Li,Yan Zeng, Jiechao Yin,Hyun S. Lillehoj,and Xiaofeng Ren. Cloning, ProkaryoticExpression, and Biological Analysis of Recombinant Chicken IFN-γ. Hybridoma.2010(29):1-9.
34. Guobin Tian, Suhua Zhang, Yanbing Li, et al. Protective efficacy in chickens, geese and ducks ofan H5N1-inactivated vaccine developed by reverse genetics, Virology,2005(341):153–162.
35. Gurunathan S, Chang-Yu Wu, Freidag BL, et al. DNA vaccines: a key for inducing long-termcellular immunity. Current Oplnlon in Immunology2000,12:442-447.
36. H. Chen, K. Yu, Y. Jiang, X. Tang. DNA immunization elicits high HI antibody and protectschicken from AIV challenge. International Congress Series,1219(2001)917-921.
37. Hassett DE, Slifka MK, Jie Zhang et al.Direct EX Vivo Kinetic and Phenotypic Analyses of CD8+T-Cell Responses Induced by DNA Immunization. Journal of Virology,2000,9:8286-8291.
38. Hualan Chen, Kanta Subbarao, David Swayne, et al. Generation and evaluation of a high growthreassortant H9N2influenza a virus as a pandemic vaccine candidate. Vaccine,2003(21):1974–1979.
39. Hualan Chen,Yanbing Li,Zejun Li, et al. Properties and Dissemination of H5N1Viruses Isolatedduring an Influenza Outbreak in Migratory Waterfowl in Western China, Journal virology, June2006,5976–5983.
40. Huoying Shi, Xiu Fan Liu, Xiaorong Zhang, Sujuan Chen, Lei Sun, Jianhong Lu. Generation of anattenuated H5N1avian influenza virus vaccine with all eight genes from avian viruses. Vaccine,2007(25):7379–7384.
41. Hyeok-il Kwon, Min-Suk Song, Philippe Noriel Q. Pascua, et al. Genetic characterization andpathogenicity assessment of highly pathogenic H5N1avian influenza viruses isolated frommigratory wild birds in2011,South Korea, Virus Research,2011(160):305–315.
42. Jiang YP, Yu KZ, Zhang HB,et al. Enhanced protective efficacy of H5subtype avian influenzaDNA vaccinewith codon optimized HA gene in a pCAGGS plasmid vector. Antiviral Research,2007,75:234-241.
43. Jianjun Chen, Fang Fang, Zhongdong Yang, et al. Characterization of highly pathogenic H5N1avian influenza viruses isolated from poultry markets in central China. Virus Research,2009(146):19–28.
44. Jill Taylor, Randall Weinberg, Yoshihiro Kawaokat, Robert G. Webstert and Enzo Paoletti.Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine,1988,12(6):1-5.
45. John Crawford, et al. Baculovirus-d erived hemagglutinin vaccines protect against lethal influenzainfections by avian H5and H7subtypes. Vaccine,1999(17):2265-2274.
46. John W. Shiver, Jeffrey B. Ulmer, John J. Donnelly, Margaret A. Liu. Humoral and cellularimmunities elicited by DNA vaccines: Application to the human immunodeficiency virus andinfluenza. Advanced Drug Delivery Reviews,1996(21):19-31.
47. K.D. Song, H.S. Lillehoj, K.D.Choi, Expression and functional characterization of recombinantchicken interferon-gamma. Veterinary Immunology and Immunopathology.1997(58):321-333.
48. Kanta Subbarao, Hualan Chen, David Swayne, et al. Evaluation of a Genetically ModifiedReassortant H5N1Influenza A Virus Vaccine Candidate Generated by Plasmid-Based ReverseGenetics. Virology,2003(305):192–200.
49. Kanta Subbarao, Hualan Chen, David Swayne, et al. Evaluation of a Genetically ModifiedReassortant H5N1Influenza A Virus Vaccine Candidate Generated by Plasmid-Based ReverseGenetics. Virology,2003(305):192–200.
50. Karen S. Yee, Tim E. Carpenter, Carol J. Cardona. Epidemiology of H5N1avian influenza.Comparative Immunology, Microbiology and Infectious Diseases,2009(32):325–340.
51. Kristien Van Reeth. Cytokines in the pathogenesis of influenza. Veterinary Microbiology,2000(74):109-116.
52. Lladser A,Parraga M,Quevedo L,etal.Naked DNA immunization as an approach to target thegeneric tumor antigen surviving induces humoral and cellular immune responses inmice.Immunobiology,2006,211:11-27.
53. Llilianne Ganges, Maritza Barrera, Jos′e Ignacio N′u nez, et al. A DNA vaccine expressing theE2protein of classical swine fever virus elicits T cell responses that can prime for rapid antibodyproduction and confer total protection upon viral challenge. Vaccine,2005(23):3741–3752.
54. M. Garcia, D.L. Suarez, J.M. Crawford. Evolution of H5subtype avian influenza a viruses inNorth America. Virus Research,1997(51):115-124.
55. Matthias Mueller, Sandra Renzullo, Roxann Brooks, et al. Antigenic Characterization ofRecombinant Hemagglutinin Proteins Derived from Different Avian Influenza Virus Subtypes.PlosONE,2010(5):1-9.
56. Mengcheng Luo, Pan Tao, Junwei Li, et al. Immunization with plasmid DNA encoding influenza Avirus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immuneresponses and protection against H5N1challenge in mice. Journal of Virological Methods,2008(154):121–127.
57. Mengcheng Luo, Pan Tao, Junwei Li, Siyu Zhou, Deyin Guo, Zishu Pan. Immunization withplasmid DNA encoding influenza A virus nucleoprotein fused to a tissue plasminogen activatorsignal sequence elicits strong immune responses and protection against H5N1challenge in mice.Journal of Virological Methods,2008(154):121–127.
58. Michaela Sharpe, Debbie Lynch, Simon Topham, et al. Protection of mice from H5N1influenzachallenge by prophylactic DNA vaccination using particle mediated epidermal delivery.Vaccine,2007(25):6392–6398.
59. Mookkan Prabakaran, Sumathy Velumani, Fang He, et al. Protective immunity against infl uenzaH5N1virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HAand recombinant CTB as an adjuvant. Virology380(2008)412–420.
60. Muhammad Abbas, Erica Spackman, Ron Fouchier, et al. H7avian influenza virus vaccinesprotect chickens against challenge with antigenically diverse isolates. Vaccine,29(2011):7424–7429.
61. Muhammad Athar Abbas.H7avian influenza virus vaccines protect chickens against challengewithantigenically diverse isolates, Vaccine,2011(29):7424–7429.
62. Neal Satterly, Pei-Ling Tsai,Jan van Deursen, et al. Influenza virus targets the mRNA exportmachinery and the nuclear pore complex.PNAS,2007(104):1853-1858.
63. Pan Tao, Mengcheng Luo, Ruangang Pan, et al. Enhanced protective immunity against H5N1influenza virus challenge by vaccination with DNA expressing a chimeric hemagglutinin incombination with an MHC class I-restricted epitope of nucleoprotein in mice. Antiviral Research,2009(81):253–260.
64. Peirong Jiao, Guobin Tian, Yanbing Li, A Single-Amino-Acid Substitution in the NS1ProteinChanges the Pathogenicity of H5N1Avian Influenza Viruses in Mice. JOURNAL OFVIROLOGY, Feb.2008,1146–1154.
65. Qinfang Liu, Jingjiao Ma, Zheng Kou, et al. Characterization of a highly pathogenic avianinfluenza H5N1clade2.3.4virus isolated from a tree sparrow. Virus Research,2010(147):l25–29.
66. Qunfeng Wu, Liurong Fang, Xuebao Wu, et al. A pseudotype baculovirus-mediated vaccineconfers protective immunity against lethal challenge with H5N1avian influenza virus in mice andchickens. Molecular Immunology,2009(46):2210–2217.
67. Riks Maas, Mirriam Tacken, Diana van Zoelen, Hok Oei. Dose response effects of avian influenza(H7N7) vaccination of chickens: Serology, clinical protection and reduction of virus excretion.Vaccine,2009(27):3592–3597.
68. Shufang Fan, Guohua Deng, Jiasheng Song, et al. Two amino acid residues in the matrix proteinM1contribute to the virulence difference of H5N1avian in fl uenza viruses in mice. Virology,2009(384):28–32.
69. Shufang Fan, Yuwei Gao, Kyoko Shinya, et al. Immunogenicity and Protective Efficacy of a LiveAttenuated H5N1Vaccine in Nonhuman Primates.Plos pathogens,2009(5):1-11.
70. Songlin Zhang, Lingyun Xiao, Hongbo Zhou, et al. Generation and characterization of an H5N1avian influenza virus hemagglutinin glycoprotein pseudotyped lentivirus. Journal of VirologicalMethods,2008(154):99–103.
71. Suarez D L, Schultz-Cherry S. Immunology of avian influenza virus: a review[J]. Developmentaland Comparative Immunology,2000(24):269-283.
72. Subbarao K, Chen HL, Swayne D. Evaluation of a Genetically Modified Reassortant H5N1Influenza A Virus Vaccine Candidate Generated by Plasmid-Based Reverse Genetics [J]. Virology,2003(305):192-200.
73. Tang X C, Lu H R, Ross T M. Hemagglutinin Displayed Baculovirus Protects Against HighlyPathogenic Influenza [J]. Vaccine,2010(28):6821-6831.
74. Taylor J, Weinberg R, Kawaka Y, et al. Protective immunity against avian influenza induced by afowlpox virus [J]. Vaccine,1988,6:504-508.
75. Xian-Chun Tang, Hai-Rong Lu, Ted M. Ross. Hemagglutinin Displayed Baculovirus ProtectsAgainst Highly Pathogenic Influenza. Vaccine,2010(28):6821–6831.
76. Xian-Chun Tang, Hai-Rong Lu, Ted M. Ross. Hemagglutinin Displayed BaculovirusProtectsAgainst Highly Pathogenic Influenza. Vaccine,2010(28):6821–6831.
77. Y. L i, Z.Lin, J.Shi, Q.Qi, G. Deng, Z. Li, X. Wang. Detection of Hong Kong97-like H5N1influenza viruses from eggs of Vietnamese waterfowl. Arch Virol (2006).
78. Yanbing Li, Chengjun Li, Liling Liu, et al. Characterization of an avian influenza virus of subtypeH7N2isolated from chickens in northern China. Virus Genes,(2006)33:117–122.
79. Yixin Chen, Feihai Xu, Xiaohui Fan,et al. Evaluation of a rapid test for detection of H5N1avianinfluenza virus. Journal of Virological Methods,2008(154):213–215.
80. YongpingJiang, Kangzhen Yu, Hongbo Zhang, Pingjing Zhang, Chenjun Li, Guobin Tian,YanbingLi, Xijun Wang, Jinying Ge, Zhigao Bu, Hualan Chen. Enhanced protective efficacy of H5subtypeavian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector.Antiviral Research2007(75):234–241.
81. Yu C. Hu,YuL.Luo, Wen T. Ji. Dual expression of the HA protein of H5N2avian influenza virusin a baculovirus system. Journal of Virological Methods,2006(135):43–48.
82. Zejun Li, Yongping Jiang, Peirong Jiao, et al. The NS1Gene Contributes to the Virulence of H5N1Avian Influenza Viruses. Journal virology,2006(80):111115-11123.
83. Zejun Li,Hualan Chen,Peirong Jiao,Guohua Deng, et al. Molecular Basis of Replication of DuckH5N1Influenza Viruses in a Mammalian Mouse Model. Journal virology,2005(79):12058-12064.