H5亚型禽流感或新城疫不同遗传背景的毒株在鸡体的交叉保护性
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摘要
1.单表达H5亚型禽流感不同毒株HA基因的重组鸡痘病毒的构建及鉴定
通过分析不同毒株的遗传进化关系,及它们与针对H5亚型禽流感病毒(avianinfluenza virus,AIV)血凝素(Hemagglutinin,HA)蛋白的单抗的反应性,选取了4株在遗传进化上属于不同的分支,抗原性上也具有一定差异的H5亚型AIV。其中包括1株H5N2亚型北美弱毒株(WK),及3株H5N1亚型高致病性亚洲分离株:
1株属于clade 0(N36),另2株属于clade 2.3.4(SY和LK)。
以这4株AIV为供体,分别获取它们的HA基因并构建了4株重组鸡痘病毒(recombinant fowlpox virus,rFPV)。经鉴定它们中的目的基因都得到了正确的表达。本研究为进一步在rFPV水平上对H5亚型内不同AIV毒株之间的交叉保护能力进行评价,并筛选对流行株具有理想保护效力的rFPV候选疫苗株打下了基础。
2.在重组鸡痘病毒或灭活疫苗水平研究H5亚型禽流感病毒的亚型内交叉保护性
为了研究H5亚型内不同AIV毒株之间的交叉保护能力,我们选择了具有不同进化关系和抗原性的4株H5亚型AIV(SY、LK、N36和WK)作为研究对象。以SY株为参照,其它3株AIV HA蛋白的AA同源性为:WK:89.6%;N36:94.2%;LK:99.5%。分别以这4株AIV为抗原制作全病毒灭活疫苗,将它们分别免疫SPF鸡,同时将以这4株AIV为HA基因供体而构建的4株rFPVs(rFPV-SYHA、rFPV-LKHA、rFPV-N36HA及rFPV-WKHA)分别免疫SPF鸡。免疫后第21d,将各组鸡用SY株高致病性禽流感病毒(Highly pathogenic avian influenza virus,HPAIV)以1×10~5EID_(50)的剂量通过滴鼻点眼途径进行攻击。通过比较各组的死亡率,排毒率及排毒量等指标,分别在全病毒灭活苗及表达HA蛋白的rFPV水平上,对它们之间的交叉保护程度进行研究。
在全病毒灭活疫苗水平,各灭活苗均对免疫鸡提供了显著水平的死亡保护。但WK株灭活苗未能对免疫鸡提供100%的死亡保护,且死亡比例较高(11/20),而其它3组均未出现发病或死亡。WK免疫组的气管排毒率在攻毒后的第2d和第4d均显著高于同源毒株免疫组(SY),其气管的排毒量在攻毒后的第2d亦显著高于同源毒株免疫组(SY)。
在rFPV疫苗水平,各免疫组均对免疫鸡提供了显著水平的死亡保护。而rFPV-WKHA和rFPV-N36HA免疫组均出现不同程度的死亡,死亡比例分别为:12/20和5/20,均显著高于同源抗原(rFPV-SYHA)免疫组及与SY株遗传距离最小的LK抗原(rFPV-LKHA)免疫组,后两组均无发病或死亡病例发生。rFPV-WKHA免疫组在攻毒后第2d和第4d的气管排毒率,及rFPV-N36HA免疫组在攻毒后第4d的气管排毒率显著高于同源抗原(rFPV-SYHA)免疫组及与SY株遗传距离最小的LK抗原(rFPV-LKHA)免疫组。而rFPV-SYHA与rFPV-LKHA免疫组的排毒率在各个时间点上的排毒率均较低,且之间无显著差异。rFPV-WKHA免疫组的气管排毒量在攻毒后的第2d及第4d均显著高于同源抗原免疫组(rFPV-SYHA)。
这些结果表明,即使在实验室条件下的SPF鸡上,H5亚型内疫苗株与攻毒株之间在遗传距离与抗原性上的差异,不仅可以影响其对感染鸡排毒的抑制能力,而且也会影响其在死亡和发病水平上的保护。因此为了更好的控制高致病性禽流感(Highly pathogenic avian influenza,HPAI)疫情,有必要对疫苗株的保护效力进行阶段性的评估,并及时更新疫苗株或基因工程疫苗中的HA抗原,以保证其对HPAIV流行株的保护效力。
3.单表达新城疫病毒不同毒株HN基因的重组鸡痘病毒的构建与鉴定
通过分析不同毒株的遗传进化关系,及它们与针对新城疫病毒(Newcastledisease virus,NDV)血凝素神经氨酸酶蛋白(Hemagglutinin-neuraminidase,HN)的单抗的反应性,选取了4株NDV(GM、H3、F48E8和LaSota),它们在遗传进化上隶属于不同的分枝,HN蛋白的抗原性上也具有一定差异。其中2株(GM和H3)属于我国近年来的流行优势基因Ⅶ型;LaSota为常用疫苗株,属基因Ⅱ型;F48E8为中国标准强毒株,属于基因Ⅸ型。
以这4株NDV为HN基因供体,构建了4株相应的rFPV(rFPV-GMHN、rFPV-H3HN、rFPV-F48HN和rFPV-LASHN)。经鉴定它们中的目的基因都得到了正确地表达。本研究为进一步在rFPV水平上对NDV不同毒株的交叉保护能力进行研究,并为筛选对流行株具有理想保护效力的rFPV候选疫苗株打下了基础。
4.在重组鸡痘病毒或灭活疫苗水平研究新城疫病毒不同毒株之间的交叉保护性
为了研究不同遗传距离和抗原性NDV毒株之间(或其主要保护性蛋白HN之间)的交叉保护能力。选取了4株(GM、H3、F48E8和LaSota)在遗传距离和抗原性上均有差异的NDV为受试毒株,与GM株相比,LaSota、F48E8和H3在HN蛋白上的AA同源性分别为88.3%、90.0%和98.3%。将这4株NDV制作全病毒灭活疫苗,并分别免疫SPF鸡。同时将4种rFPV(分别以以上4株NDV为HN基因供体)也以相同剂量(2×10~4 PFU)分别免疫SPF鸡。免疫后21d,用GM株强毒NDV以1×10~5EID_(50)(0.1mL)的剂量经滴鼻点眼途径对以上各组进行攻击。于攻毒后第3d、5d和7d对各组采集泄殖腔及气管拭子样品;攻毒后第5d,每组取6只鸡捕杀后取不同的脏器样品。分别以鸡胚接种法和荧光实时PCR法(RRT-PCR)对棉拭或脏器样品中的病毒进行检测。
免疫后21d,各组均产生了显著水平的抗体(不论是以同源抗原进行检测还是异源抗原),各油苗组的抗体滴度均显著高于相应rFPV免疫组。各组血清的抗体均以同源性抗原检测到的滴度最高,除H3灭活苗及rFPV-H3HN免疫组例外,即由异源性抗原GM检测到的抗体滴度反而略高于同源性抗原H3的检测结果。攻毒后,未免疫对照组在攻毒后5d内全部发病死亡,其它各灭活苗或rFPV免疫组均未出现发病及死亡病例。
用鸡胚接种法对各株NDV灭活疫苗组拭子样品的检测结果显示:除LoSata灭活疫苗之外,其它3组在攻毒第3d的气管排毒率均得到了显著的降低。攻毒后第5d和第7d的气管或泄殖腔样品的病毒分离率均较低,且各组之间无显著差异。用鸡胚接种法对各rFPV免疫组的拭子样品的检测结果显示:除rFPV-GMHN免疫组外,其它3组在攻毒后第3d的气管排毒率均未得到显著降低。在攻毒后第3d,各rFPV免疫组泄殖腔样品的病毒分离率都得到了显著降低。在攻毒后第5d,rFPV-LASHN与rFPV-F48HN免疫组泄殖腔样品的病毒分离率均显著高于同源抗原组(rFPV-GMHN)及与同源抗原最相近的抗原组(rFPV-H3HN)。
用RRT-PCR法在对各组的拭子样品的检测结果显示:RRT-PCR法检测的敏感性总体高于鸡胚接种法。各rFPV免疫组在攻毒第3d的气管样品的阳性率均未得到显著降低。在攻毒后第5d,rFPV-LASHN与rFPV-F48HN免疫组气管样品的阳性率均显著高于rFPV-GMHN及rFPV-H3HN免疫组。
这些结果表明:如果说评价疫苗效力的标准只限于提供临床保护,那么在本研究中,不管各疫苗株与攻毒株之间的在抗原性和遗传距离的差异有多大,它们均能提供完全的保护。但这种在遗传距离和抗原性上的差异性会影响疫苗株对免疫鸡感染后排毒的抑制能力,而且这种在排毒水平的保护和疫苗株与攻毒株之间遗传距离的大小呈负相关,即与攻毒株的遗传距离越大,疫苗株对攻毒后抑制排毒的能力越低。
如果评价疫苗的效力只局限于对发病及死亡的保护,则不利于控制诸如ND等传染病。预防感染并杜绝或减少排毒,从而降低病毒向未感染禽群的传播机率,才能更好的控制疫情。因此,研制在遗传进化及抗原性上与流行株更接近的NDV毒株作为疫苗株,对更好地控制ND的流行有重要意义。
5.两株表达H9亚型禽流感病毒HA基因的重组鸡痘病毒的构建及其免疫效力
为获得能更好地克服母源抗体干扰的rFPV,本研究将H9亚型AIV HA基因插入到具有新复制非必需区的通用型质粒载体pP12LS中构建转移载体pP12LSH9A,再分别转染预先感染不同毒力的两种鸡痘病毒(Fowlpox virus,FPV)282E4和LP株的鸡胚成纤维细胞,经系列蓝斑筛选纯化,获得了两种重组鸡痘病毒(rFPV):rFPV_(282)-12LSH9A和rFPV_(LP)-12LSH9A。将这两株rFPV在SPF鸡和带有高水平抗H9亚型AIV母源抗体的商品鸡上分别进行免疫效力试验。结果:rFPV_(282)-12LSH9A和rFPV_(LP)-12LSH9A在SPF鸡上的排毒保护率均为100%(10/10),在商品鸡上的排毒保护率分别为88%(23/26)和92%(24/26)。结论:获得了两株在带有抗H9亚型AIV高水平母源抗体的商品鸡上免疫效力较好的rFPV。
6.三种重组鸡痘病毒联合与单独免疫效力比较
利用针对不同病原的rFPV在鸡群进行间隔性的多次免疫时,后期免疫会受到前期免疫所诱生的载体特异性免疫反应的干扰,并导致其免疫效力下降。将这些rFPV进行联合免疫或许是解决此问题的一种策略。为验证rFPV联合免疫的可行性,本研究将三种针对不同病原的rFPV以联合或单独的方式分别进行免疫,并对它们的免疫效力进行比较。rFPV-12LSH9A,rFPV-12LSH5NA及rFPV-12LSHN为分别表达H9亚型AIV HA基因、H5亚型AIV的HA和NA基因及NDV HN基因的三种rFPV。将这三种rFPV分别在SPF鸡和带有针对H9、H5亚型AIV及NDV三种病原母源抗体的商品鸡上进行联合或单独免疫,并对这两种形式的免疫效力进行比较与分析。结果显示,这三株rFPV在SPF鸡上进行联合免疫时,所诱生的体液免疫应答受到了一定程度的抑制;个别成员株的排毒及死亡保护也受到了显著的干扰。因此,在进行rFPV联合免疫时,有必要对各成员株rFPV的免疫效力进行比较分析,以确定其可行性。
7.重组鸡痘病毒首免与灭活疫苗疫苗加强免疫提高对高致病性禽流感的免疫效果
为寻找一种在有母源抗体的商品鸡上更为有效的免疫策略,本研究用表达H5亚型AIV HA与NA基因的rFPV,rFPV-12LSH5NA,对10日龄带有H5亚型HPAIV母源抗体的商品鸡进行免疫,17日龄时再用H5亚型AIV全病毒灭活疫苗加强免疫。免疫鸡分为两个批次,分别在24和31日龄用H5亚型AIV进行致死性攻击。在24日龄的攻毒中,rFPV首免与灭活苗加强免疫组的死亡比例(0/17)显著低于17日龄油苗单独免疫组(16/17)。在31日龄的攻毒中,rFPV首免与灭活苗加强免疫组的死亡比例(0/17)显著低于10日龄rFPV单独免疫组(6/17)。该结果显示,在鸡群普遍存在针对H5亚型AIV母源抗体的情况下,rFPV首免与全病毒灭活苗配合使用能使鸡体快速建立坚强的免疫保护反应,是一种非常有效的免疫策略。
1. Construction and characterization of four rFPV expressing HA gene from H5 AIVs of different genetic background respectively.
Four strains of H5 avian influenza virus (AIV) were selected for cross-protection study by phylogenetic and antigenic analysis. The HI test with a panel of HA-specific monoclonal antibodies showed different reactivity patterns for the four H5 AIV studied. Phylogenetic analysis revealed genetic diversity among these H5 viruses as well. The analysis also revealed that H5N2 low-pathogenicity WK AIV belongs to the American lineage and is located on distinct branches of the phylogenetic tree; while the other three H5N1 AIV tested belong to the Asia linage (SY and LK viruses from clade 2.3.4, and N36 from clade 0).
HA genes were amplified by RT-PCR from the four AIV stains, and then used to construct four recombinant fowlpox viruses (rFPVs). Expression of these HA genes in rFPVs were confirmed by indirect immunofluorescence assay (IFA) in secondary CEF culture using anti-H5 AIV polyclonal antibodies. These rFPVs obtained could be used to study cross-protection.
2. Cross-protection studies with H5 influenza viruses on the level of inactivated or rFPV vaccines.
The current study was conducted to determine the impact of genetic relatedness among different AIV strains on the ability to protect against clinical sings, death, and infection and shedding of challenge AIV from immunized chickens. These four avian H5 viruses mentioned above SY, LK, N36 and WK, were further characterized in cross protection studies in SPF chickens. The WK, N36 and LK strains had 89.6, 94.2 and 99.5% deduced amino acid sequence similarity to the HA protein of SY challenge strain, respectively. Chickens were immunized either with the four inactivated whole AIV viruses or with the four corresponding rFPVs, and three weeks later challenged with 1×10~5 EID_(50) of the SY strain. On the levels of inactivated or recombinant vaccines, the extent of cross protection was evaluated by comparing the mortalities, frequencies and titers of virus isolation from tracheal and cloacal swabs.
All the inactivated vaccines provided complete protection from clinical signs and death except WK inactivated vaccine with the mortality of 11/20. The virus titers in tracheal samples collected from WK inactivated group on day 2 post challenge were significantly higher than those from homologous group SY.
All the four rFPVs conferred significant protection from the lethal challenge. Both the rFPV-SYHA and rFPV-LKHA induced full protection against clinical sings and death, however, rFPV-WKHA and rFPV-N36HA immunized groups failed to provide full protection, with mortalities of 12/20 and 5/12, respectively. The frequencies of virus shedding from trachea in rFPV-WKHA group on day 2 and 4 post challenge, and rFPV-N36HA on day 4 post challenge were significantly higher than those from homologous vaccine group (rFPV-SYHA). The virus recovery rates of swab samples collected at different time points from group rFPV-SYHA and rFPV-LKHA were relatively low, and no significant difference was observed. The cloacal virus titers of rFPV-WKHA on day 2 and 4 post challenge were significantly higher than those from homologous vaccine group (rFPV-SYHA).
These data indicated that the homology of the HA between the challenge virus and the virus used as vaccine is important and can significantly influence the levels of protective immunity in terms of clinical protection and reducing virus shedding even in SPF chickens housed under experimental conditions.
Therefore, to maintain optimal protection against presently prevailing HPAIV strains by vaccination, it is necessary to select suitable new vaccine strains or HA insert in rFPV to overcome genetic drift at regular intervals. 3. Construction and characterization of four rFPVs expressing HN gene from different NDV strains respectively.
Four strains of NDV were selected for cross-protection study by phylogenitic and antigenic analysis from a pool of NDV isolates. The strains GM and H3, isolated from diseased chicken and goose flocks respectively, are phylogenetically clustered to the same genotype VII, but are phylogenetically diverged from the vaccine strain LaSota, a genotype II virus, and the standard virulent strain F48E8, a genotype IX virus isolated originally in 1946.
HN genes were amplified by RT-PCR from the four NDV strains, and then used to construct four rFPVs. Expression of these HN genes in rFPVs was confirmed by IFA in secondary CEF culture using anti-NDV polyclonal antibody. These rFPVs obtained could be used to study cross-protection.
4. Cross-protection studies with Newcastle disease viruses on the level of inactivated or rFPV vaccines.
It is widely recognized that because ND isolates are of one serotype, ND vaccine prepared with any ND lineage, given correctly, can protect poultry from clinical disease and mortality from a virulent NDV challenge. However, those ND vaccines do not protect vaccinates from virulent virus infection and viral shedding from such a challenge. The objective of this study was to compare the protection induced by ND vaccines prepared with viruses of different genotypes and antigenicity. Four NDV strains mentioned above, GM, H3, LaSota and F48E8, were used to prepare inactivated vaccines. These inactivated vaccines were inoculated into SPF chickens, together with four rFPVs, rFPV-GMHN, rFPV-H3HN, rFPV-LASHN and rFPV-F48HN. Twenty-one days after immunization, all the groups were challenged oculonasally with 100μl PBS-diluted allontoic fluid containing 1×10~5 EID_(50) of GM. Tracheal and cloacal swabs were collected on day 3, 5 and 7 post challenge. On day 5 post challenge, six birds from each group were sacrificed and six different organ samples were collected. The presences of challenge virus in swab or organ samples were determined by inoculation into 10-day-old embryonated chicken eggs or by Real-time RT-PCR.
Significant levels of HI antibodies response were induced in all the vaccinated groups regardless the antigen used. The HI titers in inactivated groups were much higher than those in corresponding rFPV groups. Each vaccine group gave the highest HI titers when the antigen used in the assay was homologous to the vaccine antigen, except for H3 and rFPV-H3HN, in which higher HI titers were determined using heterologous antigen GM rather than homologous H3. All the inactivated vaccines and rFPV immunized chickens were fully protected against morbidity and mortality after challenge, whereas unvaccinated chickens died within 5 days after challenge.
The results of virus detection by inoculation into embryonated chicken eggs of samples collected from groups immunized with inactivated vaccines showed that vaccination with Losota had no effect on tracheal shedding of virus on day 3 post-challenge when compared with controls as measured by frequencies of virus isolation. However, all the other three inactivated vaccines caused a significant reduction in virus shedding when compared with the controls. By day 5 and 7, the number of vaccinated birds shedding virus was reduced, and there was no significant difference among them. The results of virus detection by inoculation into embryonated chicken eggs of samples collected from groups immunized with rFPV showed that none of the rFPV vaccines significantly reduced the frequencies of virus shedding from trachea on day 3 post challenge with the exception of rFPV-GMHN, whereas all the rFPVs decreased the frequencies of virus shedding from cloaca. On day 5 post challenge, the frequencies of virus shedding from cloaca in rFPV-LASHN and rFPV-F48HN groups were significantly higher than those in rFPV-GMHN and rFPV-H3HN.
When the swab samples were detected by RRT-PCR, no significant decrease in the frequencies of virus shedding was observed in all the rFPV immunized groups from trachea on day 3 post challenge. On day 5 post challenge, the frequencies of virus shedding from trachea in rFPV-LASHN and rFPV-F48HN groups were significantly higher than those in rFPV-GMHN and rFPV-H3HN.
These results indicated that all of the vaccine used in the paper could provide complete protection regardless of the antigenic difference of the challenging virus in terms of preventing clinical disease. However, the antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation do affect viral shedding after a virulent challenge. There was a positive correlation in similarity between challenge virus and vaccines, and the ability to decrease the frequencies of virus recovery from trachea or cloaca.
Protection from illness and death alone cannot be a comprehensive measure of effective control against an infectious disease such as ND. Prevention of infection and elimination or reduction of virus shedding, thus reducing the likehood of spread of virus to new flocks, are necessary to achieve optimal control of an outbreak. Therefore NDV vaccines formulated to be phylogenetically and antigenically closer to potential outbreak viruses may provide better ND control.
5. Construction and protective efficacies of two recombinant fowlpox viruses expressing the HA Gene of H9 subtype AIV.
The maternal antibody interference has been an obstacle in the development of rFPV vaccines. In an earlier report (Sun Lei et al., Acta Microbiologica Sinica.45 (3):359-362, 2005), the expression vector pP12LS was used to solve the problem effectively. In the present study, we used the vector pP12LS to construct rFPV against H9 subtype AIV. The HA gene from subtype H9N2 AIV was directionally inserted into the vector pP12LS, resulting in transfer vector pP12LSH9A. Then the transfer vector pP12LSH9A was used to transfect the chicken embryo fibroblast cells, which were pre-infected with strain 282E4 or LP of FPV respectively. After serial blue plaque screening, we obtained two purified rFPVs: rFPV_(282)-12LSH9A and rFPV_(LP)-12LSH9A. The protective efficacies of the two rFPV were investigated by vaccinating antibody negative SPF and antibody positive commercial chickens. A killed vaccine was included as controls. Results showed that the protective efficacies of rFPV_(282)-12LSH9A and rFPV_(lp)-12LSH9A in SPF chickens were both 100%, while those ones in commercial chickens were 88% and 92%, respectively. We concluded that the rFPV_(282)-12LSH9A and rFPV_(LP)-12LSH9A are two effective vaccines against H9 subtype AIV even in the presence of maternal antibodies.
6. Comparison of the immunogenicity of three recombinant fowlpox viruses in the individual and combined vaccination
When rFPVs against different pathogens are used in a flock consecutively, the subsequent rFPV vaccination usually can not confer good protection due to the pre-existing vector-specific immunity. Combination of different rFPVs may be a strategy to solve the problem. However, whether the same levels of protection against each pathogen might be conferred by such combined rFPVs as those by individual rFPV alone, is unclear. Our aim in this study is to evaluate the ability of the combined rFPV vaccination to induce protection in chickens.
Three rFPVs: rFPV-12LSH9A expressing HA gene of H9N2 AIV, rFPV-12LSH5NA expressing HA and NA genes of H5N1 AIV and rFPV-12LSHN expressing HN gene of Newcastle disease virus (NDV), were constructed using FPV strain LP. These rFPV vaccines were inoculated into SPF or commercial chickens with maternal antibodies to H9, H5 subtypes of AIV and NDV, either individually or in combination. Twenty eight days after vaccination, the combination group was divided into three subgroups, which were challenged with homologous H9N2 AIV, highly pathogenic H5N1 AIV or velogenic NDV, respectively. Individual vaccination groups were also challenged with the corresponding virulent virus. Serum samples were collected at appropriate intervals after immunization. Five days after challenge, tracheal (H9N2 AIV challenged groups) or cloacal (H5N1 AIV and NDV challenged groups) swabs were collected for virus isolation. The birds were monitored for 14 days for mortality. Results showed that in SPF chickens, administration of rFPVs in combination induced significant antibody responses to H9, H5 subtype AIV and NDV, but the HI titers were lower than those of individually vaccinated chickens. No death or virus shedding were seen after respective challenge except for the groups challenged with NDV, in which combined rFPV vaccinated group showed significantly higher virus recovery rate than rFPV-12LSHN immunized group(12/16 Vs 3/16). In commercial chickens, no obvious increase in HI titers after inoculation was detected in all the rFPV vaccinated groups. Among the groups challenged with H5N1 AIV, the combined vaccination group experienced a higher mortality (6/25) than the rFPV-12LSH5NA group (4/25), but the difference was not significant. The mortality in combined vaccination group (13/25) was significantly higher than that of the rFPV-12LSHN group (5/25), suggesting that the protective efficacy of rFPV-12LSHN was influenced by the other two rFPVs when being used in combination. No significant difference in the rate of virus recovery was observed between the combined group (17/25) and rFPV-12LSH9A group (13/25) after being challenged with H9N2 AIV.
The results indicated that the protective efficacies of some rFPVs, when vaccinated in combination, might be influenced by other rFPVs. Therefore, it is wise to test each rFPV for efficacies when using multiple rFPVs in combination.
7. A vaccination strategy primed with recombinant fowlpox virus and boosted with inactivated vaccine provided significantly better protection in chickens against H5 subtype avian influenza virus
Ten-day-old chickens with maternal antibodies to H5 subtype AIV were primarily vaccinated with a rFPV, rFPV-12LSH5NA, followed by a boost vaccination with H5 subtype AIV inactivated vaccine 1 week later. Ten-day-old chickens inoculated with rFPV-12LSH5NA and 17-day-old chickens inoculated with inactivated vaccine also served as control groups. One or two week after the boost vaccination, all the groups were challenged intranasally with lethal doses of homologous H5 AIV. All birds in the prime-boost group survived and the virus shedding rate was also significantly reduced, comparing to the groups with a single vaccination with rFPV or inactivated vaccine. These results indicate that the prime-boost strategy is very effective to protect chickens against lethal challenge of HPAI virus, even in the presence of maternal antibodies.
通过分析不同毒株的遗传进化关系,及它们与针对H5亚型禽流感病毒(avianinfluenza virus,AIV)血凝素(Hemagglutinin,HA)蛋白的单抗的反应性,选取了4株在遗传进化上属于不同的分支,抗原性上也具有一定差异的H5亚型AIV。其中包括1株H5N2亚型北美弱毒株(WK),及3株H5N1亚型高致病性亚洲分离株:
1株属于clade 0(N36),另2株属于clade 2.3.4(SY和LK)。
以这4株AIV为供体,分别获取它们的HA基因并构建了4株重组鸡痘病毒(recombinant fowlpox virus,rFPV)。经鉴定它们中的目的基因都得到了正确的表达。本研究为进一步在rFPV水平上对H5亚型内不同AIV毒株之间的交叉保护能力进行评价,并筛选对流行株具有理想保护效力的rFPV候选疫苗株打下了基础。
2.在重组鸡痘病毒或灭活疫苗水平研究H5亚型禽流感病毒的亚型内交叉保护性
为了研究H5亚型内不同AIV毒株之间的交叉保护能力,我们选择了具有不同进化关系和抗原性的4株H5亚型AIV(SY、LK、N36和WK)作为研究对象。以SY株为参照,其它3株AIV HA蛋白的AA同源性为:WK:89.6%;N36:94.2%;LK:99.5%。分别以这4株AIV为抗原制作全病毒灭活疫苗,将它们分别免疫SPF鸡,同时将以这4株AIV为HA基因供体而构建的4株rFPVs(rFPV-SYHA、rFPV-LKHA、rFPV-N36HA及rFPV-WKHA)分别免疫SPF鸡。免疫后第21d,将各组鸡用SY株高致病性禽流感病毒(Highly pathogenic avian influenza virus,HPAIV)以1×10~5EID_(50)的剂量通过滴鼻点眼途径进行攻击。通过比较各组的死亡率,排毒率及排毒量等指标,分别在全病毒灭活苗及表达HA蛋白的rFPV水平上,对它们之间的交叉保护程度进行研究。
在全病毒灭活疫苗水平,各灭活苗均对免疫鸡提供了显著水平的死亡保护。但WK株灭活苗未能对免疫鸡提供100%的死亡保护,且死亡比例较高(11/20),而其它3组均未出现发病或死亡。WK免疫组的气管排毒率在攻毒后的第2d和第4d均显著高于同源毒株免疫组(SY),其气管的排毒量在攻毒后的第2d亦显著高于同源毒株免疫组(SY)。
在rFPV疫苗水平,各免疫组均对免疫鸡提供了显著水平的死亡保护。而rFPV-WKHA和rFPV-N36HA免疫组均出现不同程度的死亡,死亡比例分别为:12/20和5/20,均显著高于同源抗原(rFPV-SYHA)免疫组及与SY株遗传距离最小的LK抗原(rFPV-LKHA)免疫组,后两组均无发病或死亡病例发生。rFPV-WKHA免疫组在攻毒后第2d和第4d的气管排毒率,及rFPV-N36HA免疫组在攻毒后第4d的气管排毒率显著高于同源抗原(rFPV-SYHA)免疫组及与SY株遗传距离最小的LK抗原(rFPV-LKHA)免疫组。而rFPV-SYHA与rFPV-LKHA免疫组的排毒率在各个时间点上的排毒率均较低,且之间无显著差异。rFPV-WKHA免疫组的气管排毒量在攻毒后的第2d及第4d均显著高于同源抗原免疫组(rFPV-SYHA)。
这些结果表明,即使在实验室条件下的SPF鸡上,H5亚型内疫苗株与攻毒株之间在遗传距离与抗原性上的差异,不仅可以影响其对感染鸡排毒的抑制能力,而且也会影响其在死亡和发病水平上的保护。因此为了更好的控制高致病性禽流感(Highly pathogenic avian influenza,HPAI)疫情,有必要对疫苗株的保护效力进行阶段性的评估,并及时更新疫苗株或基因工程疫苗中的HA抗原,以保证其对HPAIV流行株的保护效力。
3.单表达新城疫病毒不同毒株HN基因的重组鸡痘病毒的构建与鉴定
通过分析不同毒株的遗传进化关系,及它们与针对新城疫病毒(Newcastledisease virus,NDV)血凝素神经氨酸酶蛋白(Hemagglutinin-neuraminidase,HN)的单抗的反应性,选取了4株NDV(GM、H3、F48E8和LaSota),它们在遗传进化上隶属于不同的分枝,HN蛋白的抗原性上也具有一定差异。其中2株(GM和H3)属于我国近年来的流行优势基因Ⅶ型;LaSota为常用疫苗株,属基因Ⅱ型;F48E8为中国标准强毒株,属于基因Ⅸ型。
以这4株NDV为HN基因供体,构建了4株相应的rFPV(rFPV-GMHN、rFPV-H3HN、rFPV-F48HN和rFPV-LASHN)。经鉴定它们中的目的基因都得到了正确地表达。本研究为进一步在rFPV水平上对NDV不同毒株的交叉保护能力进行研究,并为筛选对流行株具有理想保护效力的rFPV候选疫苗株打下了基础。
4.在重组鸡痘病毒或灭活疫苗水平研究新城疫病毒不同毒株之间的交叉保护性
为了研究不同遗传距离和抗原性NDV毒株之间(或其主要保护性蛋白HN之间)的交叉保护能力。选取了4株(GM、H3、F48E8和LaSota)在遗传距离和抗原性上均有差异的NDV为受试毒株,与GM株相比,LaSota、F48E8和H3在HN蛋白上的AA同源性分别为88.3%、90.0%和98.3%。将这4株NDV制作全病毒灭活疫苗,并分别免疫SPF鸡。同时将4种rFPV(分别以以上4株NDV为HN基因供体)也以相同剂量(2×10~4 PFU)分别免疫SPF鸡。免疫后21d,用GM株强毒NDV以1×10~5EID_(50)(0.1mL)的剂量经滴鼻点眼途径对以上各组进行攻击。于攻毒后第3d、5d和7d对各组采集泄殖腔及气管拭子样品;攻毒后第5d,每组取6只鸡捕杀后取不同的脏器样品。分别以鸡胚接种法和荧光实时PCR法(RRT-PCR)对棉拭或脏器样品中的病毒进行检测。
免疫后21d,各组均产生了显著水平的抗体(不论是以同源抗原进行检测还是异源抗原),各油苗组的抗体滴度均显著高于相应rFPV免疫组。各组血清的抗体均以同源性抗原检测到的滴度最高,除H3灭活苗及rFPV-H3HN免疫组例外,即由异源性抗原GM检测到的抗体滴度反而略高于同源性抗原H3的检测结果。攻毒后,未免疫对照组在攻毒后5d内全部发病死亡,其它各灭活苗或rFPV免疫组均未出现发病及死亡病例。
用鸡胚接种法对各株NDV灭活疫苗组拭子样品的检测结果显示:除LoSata灭活疫苗之外,其它3组在攻毒第3d的气管排毒率均得到了显著的降低。攻毒后第5d和第7d的气管或泄殖腔样品的病毒分离率均较低,且各组之间无显著差异。用鸡胚接种法对各rFPV免疫组的拭子样品的检测结果显示:除rFPV-GMHN免疫组外,其它3组在攻毒后第3d的气管排毒率均未得到显著降低。在攻毒后第3d,各rFPV免疫组泄殖腔样品的病毒分离率都得到了显著降低。在攻毒后第5d,rFPV-LASHN与rFPV-F48HN免疫组泄殖腔样品的病毒分离率均显著高于同源抗原组(rFPV-GMHN)及与同源抗原最相近的抗原组(rFPV-H3HN)。
用RRT-PCR法在对各组的拭子样品的检测结果显示:RRT-PCR法检测的敏感性总体高于鸡胚接种法。各rFPV免疫组在攻毒第3d的气管样品的阳性率均未得到显著降低。在攻毒后第5d,rFPV-LASHN与rFPV-F48HN免疫组气管样品的阳性率均显著高于rFPV-GMHN及rFPV-H3HN免疫组。
这些结果表明:如果说评价疫苗效力的标准只限于提供临床保护,那么在本研究中,不管各疫苗株与攻毒株之间的在抗原性和遗传距离的差异有多大,它们均能提供完全的保护。但这种在遗传距离和抗原性上的差异性会影响疫苗株对免疫鸡感染后排毒的抑制能力,而且这种在排毒水平的保护和疫苗株与攻毒株之间遗传距离的大小呈负相关,即与攻毒株的遗传距离越大,疫苗株对攻毒后抑制排毒的能力越低。
如果评价疫苗的效力只局限于对发病及死亡的保护,则不利于控制诸如ND等传染病。预防感染并杜绝或减少排毒,从而降低病毒向未感染禽群的传播机率,才能更好的控制疫情。因此,研制在遗传进化及抗原性上与流行株更接近的NDV毒株作为疫苗株,对更好地控制ND的流行有重要意义。
5.两株表达H9亚型禽流感病毒HA基因的重组鸡痘病毒的构建及其免疫效力
为获得能更好地克服母源抗体干扰的rFPV,本研究将H9亚型AIV HA基因插入到具有新复制非必需区的通用型质粒载体pP12LS中构建转移载体pP12LSH9A,再分别转染预先感染不同毒力的两种鸡痘病毒(Fowlpox virus,FPV)282E4和LP株的鸡胚成纤维细胞,经系列蓝斑筛选纯化,获得了两种重组鸡痘病毒(rFPV):rFPV_(282)-12LSH9A和rFPV_(LP)-12LSH9A。将这两株rFPV在SPF鸡和带有高水平抗H9亚型AIV母源抗体的商品鸡上分别进行免疫效力试验。结果:rFPV_(282)-12LSH9A和rFPV_(LP)-12LSH9A在SPF鸡上的排毒保护率均为100%(10/10),在商品鸡上的排毒保护率分别为88%(23/26)和92%(24/26)。结论:获得了两株在带有抗H9亚型AIV高水平母源抗体的商品鸡上免疫效力较好的rFPV。
6.三种重组鸡痘病毒联合与单独免疫效力比较
利用针对不同病原的rFPV在鸡群进行间隔性的多次免疫时,后期免疫会受到前期免疫所诱生的载体特异性免疫反应的干扰,并导致其免疫效力下降。将这些rFPV进行联合免疫或许是解决此问题的一种策略。为验证rFPV联合免疫的可行性,本研究将三种针对不同病原的rFPV以联合或单独的方式分别进行免疫,并对它们的免疫效力进行比较。rFPV-12LSH9A,rFPV-12LSH5NA及rFPV-12LSHN为分别表达H9亚型AIV HA基因、H5亚型AIV的HA和NA基因及NDV HN基因的三种rFPV。将这三种rFPV分别在SPF鸡和带有针对H9、H5亚型AIV及NDV三种病原母源抗体的商品鸡上进行联合或单独免疫,并对这两种形式的免疫效力进行比较与分析。结果显示,这三株rFPV在SPF鸡上进行联合免疫时,所诱生的体液免疫应答受到了一定程度的抑制;个别成员株的排毒及死亡保护也受到了显著的干扰。因此,在进行rFPV联合免疫时,有必要对各成员株rFPV的免疫效力进行比较分析,以确定其可行性。
7.重组鸡痘病毒首免与灭活疫苗疫苗加强免疫提高对高致病性禽流感的免疫效果
为寻找一种在有母源抗体的商品鸡上更为有效的免疫策略,本研究用表达H5亚型AIV HA与NA基因的rFPV,rFPV-12LSH5NA,对10日龄带有H5亚型HPAIV母源抗体的商品鸡进行免疫,17日龄时再用H5亚型AIV全病毒灭活疫苗加强免疫。免疫鸡分为两个批次,分别在24和31日龄用H5亚型AIV进行致死性攻击。在24日龄的攻毒中,rFPV首免与灭活苗加强免疫组的死亡比例(0/17)显著低于17日龄油苗单独免疫组(16/17)。在31日龄的攻毒中,rFPV首免与灭活苗加强免疫组的死亡比例(0/17)显著低于10日龄rFPV单独免疫组(6/17)。该结果显示,在鸡群普遍存在针对H5亚型AIV母源抗体的情况下,rFPV首免与全病毒灭活苗配合使用能使鸡体快速建立坚强的免疫保护反应,是一种非常有效的免疫策略。
1. Construction and characterization of four rFPV expressing HA gene from H5 AIVs of different genetic background respectively.
Four strains of H5 avian influenza virus (AIV) were selected for cross-protection study by phylogenetic and antigenic analysis. The HI test with a panel of HA-specific monoclonal antibodies showed different reactivity patterns for the four H5 AIV studied. Phylogenetic analysis revealed genetic diversity among these H5 viruses as well. The analysis also revealed that H5N2 low-pathogenicity WK AIV belongs to the American lineage and is located on distinct branches of the phylogenetic tree; while the other three H5N1 AIV tested belong to the Asia linage (SY and LK viruses from clade 2.3.4, and N36 from clade 0).
HA genes were amplified by RT-PCR from the four AIV stains, and then used to construct four recombinant fowlpox viruses (rFPVs). Expression of these HA genes in rFPVs were confirmed by indirect immunofluorescence assay (IFA) in secondary CEF culture using anti-H5 AIV polyclonal antibodies. These rFPVs obtained could be used to study cross-protection.
2. Cross-protection studies with H5 influenza viruses on the level of inactivated or rFPV vaccines.
The current study was conducted to determine the impact of genetic relatedness among different AIV strains on the ability to protect against clinical sings, death, and infection and shedding of challenge AIV from immunized chickens. These four avian H5 viruses mentioned above SY, LK, N36 and WK, were further characterized in cross protection studies in SPF chickens. The WK, N36 and LK strains had 89.6, 94.2 and 99.5% deduced amino acid sequence similarity to the HA protein of SY challenge strain, respectively. Chickens were immunized either with the four inactivated whole AIV viruses or with the four corresponding rFPVs, and three weeks later challenged with 1×10~5 EID_(50) of the SY strain. On the levels of inactivated or recombinant vaccines, the extent of cross protection was evaluated by comparing the mortalities, frequencies and titers of virus isolation from tracheal and cloacal swabs.
All the inactivated vaccines provided complete protection from clinical signs and death except WK inactivated vaccine with the mortality of 11/20. The virus titers in tracheal samples collected from WK inactivated group on day 2 post challenge were significantly higher than those from homologous group SY.
All the four rFPVs conferred significant protection from the lethal challenge. Both the rFPV-SYHA and rFPV-LKHA induced full protection against clinical sings and death, however, rFPV-WKHA and rFPV-N36HA immunized groups failed to provide full protection, with mortalities of 12/20 and 5/12, respectively. The frequencies of virus shedding from trachea in rFPV-WKHA group on day 2 and 4 post challenge, and rFPV-N36HA on day 4 post challenge were significantly higher than those from homologous vaccine group (rFPV-SYHA). The virus recovery rates of swab samples collected at different time points from group rFPV-SYHA and rFPV-LKHA were relatively low, and no significant difference was observed. The cloacal virus titers of rFPV-WKHA on day 2 and 4 post challenge were significantly higher than those from homologous vaccine group (rFPV-SYHA).
These data indicated that the homology of the HA between the challenge virus and the virus used as vaccine is important and can significantly influence the levels of protective immunity in terms of clinical protection and reducing virus shedding even in SPF chickens housed under experimental conditions.
Therefore, to maintain optimal protection against presently prevailing HPAIV strains by vaccination, it is necessary to select suitable new vaccine strains or HA insert in rFPV to overcome genetic drift at regular intervals. 3. Construction and characterization of four rFPVs expressing HN gene from different NDV strains respectively.
Four strains of NDV were selected for cross-protection study by phylogenitic and antigenic analysis from a pool of NDV isolates. The strains GM and H3, isolated from diseased chicken and goose flocks respectively, are phylogenetically clustered to the same genotype VII, but are phylogenetically diverged from the vaccine strain LaSota, a genotype II virus, and the standard virulent strain F48E8, a genotype IX virus isolated originally in 1946.
HN genes were amplified by RT-PCR from the four NDV strains, and then used to construct four rFPVs. Expression of these HN genes in rFPVs was confirmed by IFA in secondary CEF culture using anti-NDV polyclonal antibody. These rFPVs obtained could be used to study cross-protection.
4. Cross-protection studies with Newcastle disease viruses on the level of inactivated or rFPV vaccines.
It is widely recognized that because ND isolates are of one serotype, ND vaccine prepared with any ND lineage, given correctly, can protect poultry from clinical disease and mortality from a virulent NDV challenge. However, those ND vaccines do not protect vaccinates from virulent virus infection and viral shedding from such a challenge. The objective of this study was to compare the protection induced by ND vaccines prepared with viruses of different genotypes and antigenicity. Four NDV strains mentioned above, GM, H3, LaSota and F48E8, were used to prepare inactivated vaccines. These inactivated vaccines were inoculated into SPF chickens, together with four rFPVs, rFPV-GMHN, rFPV-H3HN, rFPV-LASHN and rFPV-F48HN. Twenty-one days after immunization, all the groups were challenged oculonasally with 100μl PBS-diluted allontoic fluid containing 1×10~5 EID_(50) of GM. Tracheal and cloacal swabs were collected on day 3, 5 and 7 post challenge. On day 5 post challenge, six birds from each group were sacrificed and six different organ samples were collected. The presences of challenge virus in swab or organ samples were determined by inoculation into 10-day-old embryonated chicken eggs or by Real-time RT-PCR.
Significant levels of HI antibodies response were induced in all the vaccinated groups regardless the antigen used. The HI titers in inactivated groups were much higher than those in corresponding rFPV groups. Each vaccine group gave the highest HI titers when the antigen used in the assay was homologous to the vaccine antigen, except for H3 and rFPV-H3HN, in which higher HI titers were determined using heterologous antigen GM rather than homologous H3. All the inactivated vaccines and rFPV immunized chickens were fully protected against morbidity and mortality after challenge, whereas unvaccinated chickens died within 5 days after challenge.
The results of virus detection by inoculation into embryonated chicken eggs of samples collected from groups immunized with inactivated vaccines showed that vaccination with Losota had no effect on tracheal shedding of virus on day 3 post-challenge when compared with controls as measured by frequencies of virus isolation. However, all the other three inactivated vaccines caused a significant reduction in virus shedding when compared with the controls. By day 5 and 7, the number of vaccinated birds shedding virus was reduced, and there was no significant difference among them. The results of virus detection by inoculation into embryonated chicken eggs of samples collected from groups immunized with rFPV showed that none of the rFPV vaccines significantly reduced the frequencies of virus shedding from trachea on day 3 post challenge with the exception of rFPV-GMHN, whereas all the rFPVs decreased the frequencies of virus shedding from cloaca. On day 5 post challenge, the frequencies of virus shedding from cloaca in rFPV-LASHN and rFPV-F48HN groups were significantly higher than those in rFPV-GMHN and rFPV-H3HN.
When the swab samples were detected by RRT-PCR, no significant decrease in the frequencies of virus shedding was observed in all the rFPV immunized groups from trachea on day 3 post challenge. On day 5 post challenge, the frequencies of virus shedding from trachea in rFPV-LASHN and rFPV-F48HN groups were significantly higher than those in rFPV-GMHN and rFPV-H3HN.
These results indicated that all of the vaccine used in the paper could provide complete protection regardless of the antigenic difference of the challenging virus in terms of preventing clinical disease. However, the antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation do affect viral shedding after a virulent challenge. There was a positive correlation in similarity between challenge virus and vaccines, and the ability to decrease the frequencies of virus recovery from trachea or cloaca.
Protection from illness and death alone cannot be a comprehensive measure of effective control against an infectious disease such as ND. Prevention of infection and elimination or reduction of virus shedding, thus reducing the likehood of spread of virus to new flocks, are necessary to achieve optimal control of an outbreak. Therefore NDV vaccines formulated to be phylogenetically and antigenically closer to potential outbreak viruses may provide better ND control.
5. Construction and protective efficacies of two recombinant fowlpox viruses expressing the HA Gene of H9 subtype AIV.
The maternal antibody interference has been an obstacle in the development of rFPV vaccines. In an earlier report (Sun Lei et al., Acta Microbiologica Sinica.45 (3):359-362, 2005), the expression vector pP12LS was used to solve the problem effectively. In the present study, we used the vector pP12LS to construct rFPV against H9 subtype AIV. The HA gene from subtype H9N2 AIV was directionally inserted into the vector pP12LS, resulting in transfer vector pP12LSH9A. Then the transfer vector pP12LSH9A was used to transfect the chicken embryo fibroblast cells, which were pre-infected with strain 282E4 or LP of FPV respectively. After serial blue plaque screening, we obtained two purified rFPVs: rFPV_(282)-12LSH9A and rFPV_(LP)-12LSH9A. The protective efficacies of the two rFPV were investigated by vaccinating antibody negative SPF and antibody positive commercial chickens. A killed vaccine was included as controls. Results showed that the protective efficacies of rFPV_(282)-12LSH9A and rFPV_(lp)-12LSH9A in SPF chickens were both 100%, while those ones in commercial chickens were 88% and 92%, respectively. We concluded that the rFPV_(282)-12LSH9A and rFPV_(LP)-12LSH9A are two effective vaccines against H9 subtype AIV even in the presence of maternal antibodies.
6. Comparison of the immunogenicity of three recombinant fowlpox viruses in the individual and combined vaccination
When rFPVs against different pathogens are used in a flock consecutively, the subsequent rFPV vaccination usually can not confer good protection due to the pre-existing vector-specific immunity. Combination of different rFPVs may be a strategy to solve the problem. However, whether the same levels of protection against each pathogen might be conferred by such combined rFPVs as those by individual rFPV alone, is unclear. Our aim in this study is to evaluate the ability of the combined rFPV vaccination to induce protection in chickens.
Three rFPVs: rFPV-12LSH9A expressing HA gene of H9N2 AIV, rFPV-12LSH5NA expressing HA and NA genes of H5N1 AIV and rFPV-12LSHN expressing HN gene of Newcastle disease virus (NDV), were constructed using FPV strain LP. These rFPV vaccines were inoculated into SPF or commercial chickens with maternal antibodies to H9, H5 subtypes of AIV and NDV, either individually or in combination. Twenty eight days after vaccination, the combination group was divided into three subgroups, which were challenged with homologous H9N2 AIV, highly pathogenic H5N1 AIV or velogenic NDV, respectively. Individual vaccination groups were also challenged with the corresponding virulent virus. Serum samples were collected at appropriate intervals after immunization. Five days after challenge, tracheal (H9N2 AIV challenged groups) or cloacal (H5N1 AIV and NDV challenged groups) swabs were collected for virus isolation. The birds were monitored for 14 days for mortality. Results showed that in SPF chickens, administration of rFPVs in combination induced significant antibody responses to H9, H5 subtype AIV and NDV, but the HI titers were lower than those of individually vaccinated chickens. No death or virus shedding were seen after respective challenge except for the groups challenged with NDV, in which combined rFPV vaccinated group showed significantly higher virus recovery rate than rFPV-12LSHN immunized group(12/16 Vs 3/16). In commercial chickens, no obvious increase in HI titers after inoculation was detected in all the rFPV vaccinated groups. Among the groups challenged with H5N1 AIV, the combined vaccination group experienced a higher mortality (6/25) than the rFPV-12LSH5NA group (4/25), but the difference was not significant. The mortality in combined vaccination group (13/25) was significantly higher than that of the rFPV-12LSHN group (5/25), suggesting that the protective efficacy of rFPV-12LSHN was influenced by the other two rFPVs when being used in combination. No significant difference in the rate of virus recovery was observed between the combined group (17/25) and rFPV-12LSH9A group (13/25) after being challenged with H9N2 AIV.
The results indicated that the protective efficacies of some rFPVs, when vaccinated in combination, might be influenced by other rFPVs. Therefore, it is wise to test each rFPV for efficacies when using multiple rFPVs in combination.
7. A vaccination strategy primed with recombinant fowlpox virus and boosted with inactivated vaccine provided significantly better protection in chickens against H5 subtype avian influenza virus
Ten-day-old chickens with maternal antibodies to H5 subtype AIV were primarily vaccinated with a rFPV, rFPV-12LSH5NA, followed by a boost vaccination with H5 subtype AIV inactivated vaccine 1 week later. Ten-day-old chickens inoculated with rFPV-12LSH5NA and 17-day-old chickens inoculated with inactivated vaccine also served as control groups. One or two week after the boost vaccination, all the groups were challenged intranasally with lethal doses of homologous H5 AIV. All birds in the prime-boost group survived and the virus shedding rate was also significantly reduced, comparing to the groups with a single vaccination with rFPV or inactivated vaccine. These results indicate that the prime-boost strategy is very effective to protect chickens against lethal challenge of HPAI virus, even in the presence of maternal antibodies.
引文
[1]Lee C W,Suarez D L.Avian influenza virus:prospects for prevention and control by vaccination.Anim Hel Res Rev 2005;6:1-15.
[2]Chen W,Calvo P A,Malide D,et al.A novel influenza A virus mitochondrial protein that induces cell death.Nat Med 2001;7:1306-1312.
[3]Lamb R A,Krug R M.Orthomyxoviridae:The viruses and their replication.In Field B N,Knipe D M,Howley E M.Fields Virology.Lippincott-Raven:NY,1996,1353-1395.
[4]Klenk H D,Keil W,Niemann R.The characterization of influenza A viruses by carbohydrate analysis.Curt Top Microbiot Immunol 1983;104:247-257
[5]Palese E,Garcia A.Influenza Viruses(orthomyxoviruses):Molecular Biology.In Granoff,Webster R G(eds.).Encyclopedia of Virology:1999 Volume 2,2nd ed.Academics Press:San Diego,830-836.
[6]Cox N J,Fuller F,Kaverin N.Orthornyxoviridae 2000.In Van M H,Fauquet C M,Bishop D H L,et al.Virus taxomony.Seventh report of the international committee on taxonomy of viruses.Academic Press:San Deigo,585-597.
[7]Easterday B C,Hinshaw V S,Halvorson D A.Influenza 1997.In Calnek B W,Barnes H J,Beard C W et al.Diease of Poultry,10th ed.Iowa State University Press:Ames,IA,583-605
[8]Kaiser J.A one-size-fits-all Flu vaccine?Sciene 2006;312:380-382.
[9]Franklin R M,Wecker E.Inactivation of some animal viruses by hydroxylamine and the structure of ribonucleic acid.Nature 1959;84:343-345.
[10]Lavr G.The structure of influenza viruses,Ⅱ.Disruption of the virus particles and separation of the neuraminidase activity.Virology 1963;20:523-526.
[11] King D J. Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum. Avian Dis 1991; 35: 505-514.
[12] Beard C W, Brugh M, Johnson D C. Laboratory studies with the Pennsylvania avian influenza viruses (H5N2). Proceedings of the U S Animal Health Association 1984; 88: 462-473.
[13] Fichtner G J. The Pennsylvania/Virginia experience in eradication of avian influenza (H5N2). 1987. In Easterday B C. Proceedings of the second international symposium on avian influenza U S Animal Health Association: Richmond, VA, 33-38.
[14] Webster R G, Yakhno M, Hinshaw V S, et al. Intestinal influenza: Replication and characterization of influenza viruses in ducks. Virology 1978; 84:268-278.
[15] Fouchier R A M, Munster V, Wallensten A, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology 2005; 79: 2814-2822.
[16] WHO Expert committee. A revision of the system of nomenclature for influenza viruses: A WHO memorandum. Bull WHO 1980; 585-591.
[17] Bean W J, Kawaoka Y, Wood J M, et al. Characterization of virulent and avirulent A/Chicken/Pennsylvania/83 influenza viruses: Potential role of defective interfering RNAs in nature. J Virol 1985; 54: 151-160.
[18] Alexander D J, Allan W H, Parsons DG,et al. The pathogenicity of four avian influenza viruses for fowls , turkeys and ducks. Res Vet Sci 1978; 24: 242-247.
[19] Alexander D J. Avian influenza. Recent developments. Vet Bulls 1982. 341-359.
[20] Easterday B C, Tumova B. 1972. Avian influenza. In Hofstad M S, Calnek B W, Helmbolt C F, et al. (eds.). Disease of poultry, 6th ed. Iowa State University Press: Ames, IA, 670-700.
[21] Smithies L K, Emerson E G, Robertson S M, et al. Two different type A influenza virus infections in turkeys in Wisconsin. Ⅱ. 1968 outbreak. Avian Dis 1969; 13: 606-610.
[22] Beard C W, Easterday B C. A-Turkey-Oregon-71, an avirulent influenza isolate with the hemagglutinin of fowl plaque virus, Aivan Dis 1973; 17: 173-181.
[23] Humberd J, Boyd K, Webster R G. Emergence of influenza virus variants after prolonged shedding from pheasants. J Virol 2007; 81: 4044-4051.
[24] Lee C W, Senne D A, Suarez D L. Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J Virol 2004; 78: 8372-8381.
[25] Suarez D L, Senne D A. Sequence analysis of related low-pathogenic and highly pathogenic H5N2 avian influenza isolates from United States live bird markets and poultry farms from 1983 to 1989. Avian Dis 2000; 44: 356-364.
[26] Spackman E, Senne D A, Davison S. Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States. J Virol 2003; 77: 13399-13402.
[27] Tsuckiya E, Sugawara K, Hongo S, et al. Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus. J Gen Virol 2001; 82: 2475-2484.
[28] Kaverin N V. Rudneva I A, Ilyushina N A, et al. Stnicture of antigenic sites on the heamagglutinin molecule of H5 avian influenza virus and phenotvpic variation of escape mutants. J Gen Virol 2002; 83: 2497-2505.
[29] Murakami S, Iwasa A, Iwatsuke-Horimoto K, et al. Cross-clade protective immunity of H5N1 influenza vaccines in a mouse model. Vaccine 2008; 26: 6398-6404.
[30] Austin E J, Webster R G. Antigenic mapping of an avian H1 influenza virus haemagglutinin and interrelationships of H1 viruses from humans, pigs and birds . J Gen Virol 1986; 67: 983-992.
[31] Hinshaw V S, Alexander D J, Ayrnand M, ei al. Antigenic comparisons of swine-influenza-like H1N1 isolates from pigs, birds and humans: an international collaborative study. Bull WHO1984; 62: 871-878.
[32] Wu W L, Chen Y X, Wang P, et al. Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007. J Virol 2008; 82: 1798-1807.
[33] Kaverin N V, Rudneva I A, Govorkova E A, et al. Epitope mapping of the hemagglitinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies. J Virol 2007; 81:12911-12917.
[34] Ada G L, Jones P D. The immune response to influenza infection. Curr Top Microbial Immunol 1986; 128: 1-54.
[35] Tamura S I, Kurata T. Defence mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn J Infect Dis 2004; 57: 236-247.
[36] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001; 2: 675-680.
[37] Lund J M, Alexopoulou L, Sato A, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA; 101:5598-5603.
[38] Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004; 303: 1526-1529.
[39] Diebold S S, Kaisho T, Hemmi H, et al. Innate antival responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004; 303: 1529-1531.
[40] Scherle P A, Palladino G, Gerhard W. Mice can recover from pulmonary influenza virus infection in the absence of class-Ⅰ-restricted cytotoxic T cells. J Immunol 1992; 148: 212-217.
[41] Gerhard W, Mozdzanowska K, Furchner M, et al. Role of the B-cell response in the recovery of mice from primary influenza virus infection. Immunol Rev 1997; 159: 95-103.
[42] Tamura S I, Iwasaki T, Thompson A M, et al. Antibody-forming cells in the nasal-associated lymphoid tissue during primary influenza virus infection. J Gen Virol 1998; 79: 291-299.
[43] Zuercher A W, Coffin S E, Thurnheer M C, et al. Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses. J Immunol 2002; 168: 1796-1803.
[44] McMichael A. Cytotoxic T lymphocytes specific for influenza virus. Cuur Top Microb Immunol 1994; 189: 75-91.
[45] Flynn K J, Belz G T, Alman J D, et al. Virus-specific CD8~+ T cells in primary and secondary influenza pneumonia. Immunity 1998; 8: 683-691.
[46] Flynn K J, Riberdy J M, Christensen J M, et al. Iin vivo proliferation of naive and memory influenza-specific CD8~+ T cells. Proc Natl Acad Sci USA 1999; 96: 8597-8602.
[47] Epstein S L, Lo Chia-Yun, Misplon, et al. Mechanism of protective immunity against influenza virus infection in mice without antibodies. J Immunol 1998; 160: 322-329.
[48] Wiley J A, Hogan R J, Woodland D L, et al. Antigen-specific CD8 (+) T cells persist in the upper respiratory tract following influenza virus infection. J Immunol 2001; 167: 3293-3299.
[49] Yoshikawa T, Matsuo Ke, Matsuo Ka, et al. Total viral genome copies and virus-Ig complexes after infection with influenza virus in the nasal secretions of immunized mice. J Gen Virol 2004; 85: 2339-2346.
[50] Liew F Y, Russell S M, Appleyard G, et al. Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity. Eur J Immunol 1984; 14: 350-356.
[51] Brandtzaeg P, Krajci P, Lamm M E, et al. Epithelial and hepatobiliary transport of polymeric immunoglobulins. 1994 .P. 113-126. In Ogra P W, Lamm M E, McGhee J R, et al. Handbook of Mucosal Immunology. Academic Press, San Diego, Calif.
[52] Asahi Y, Yoshikawa T, Watanabe I, et al. Protection against influenza virus infection in ploy Ig receptor-knockout mice immunized intranasally with adjuvant-combined vaccine. J Immunol 2002; 168: 2930-2938.
[53] Ramphal R, Cogliano R C, Shands J W, et al. Serum antibody prevents lethal murine influenza pneumonitis but not tracheitis. Infect Immun 1979; 25: 992-997.
[54] Palladino G, Mozdzanowska K, Washko G, et al. Virus-neutrilizing antiodies of immunoglobulin G (IgG) but not IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice. J Virol 1995; 69: 2075-2081.
[55] Ito R, Yoshikawa T, Ozaki Y, et al. Roles of anti-hemagglutinin IgA and IgG antibodies in different sites of the respiratory tract of vaccinated mice in preventing lethal influenza pneumonia. Vaccne 2003; 21: 2362-2371.
[56] Murphy B R, Clements M L. The systemic and mucosal immune response of humans to influenza A virus. Curr Top Microb Immunol 1989; 146:107-116.
[57] Murphy B R. Mucosal immunity to viruses. P. 333-343. In Ogra P L, Lamm M E, McGhee J R, et al. Handbook of Mucosal Immunology. Academic Press, San Diego, Calif.
[58] Mazanec M B, Kaetzel C S, Lamm M E, et al. Intracellular neutralization of virus by immunoglobin A antibodies. Proc Natl Acad Sci USA 1992; 89: 6901-6905.
[59] Mazanec M B, Coulder C L, Fletcher D R. Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J Virol 1995; 69:1339-1343.
[60] Fujioka H, Emancipator S N, Aikawa M, et al. Immunocytochemical colocalization of specific immunoglobulin A with sendai virus protein in infected polarized epithelium. J Exp Med 1998; 188: 1223-1229.
[61] Tamura S I, Miyata K, Matsuo K, et al. Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein. J Immunol 1996; 156: 3892-3900.
[62] Tamura S I, Tanimoto T, Kurata T. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn J Infect Dis 2005; 58: 195-207.
[63] Portner A, Murti K G. Localization of P, NP, and M protein on Sendai virus nucleocapsid using immunogold labeling. Virology 1986; 150:469-478.
[64] Wraith D C. The recognition of influenza A virus-infected cells by cytotoxic lymphocytes. Immunol Today 1987; 8: 239-246.
[65] Yewdell J W, Bennink J R, Smith G L, et al. Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes. Proc Natle Aca Sci USA 1985; 82: 1785-1789.
[66] Tulp A, Verwoerd D, Dobberstein B. et al. Isolation and characterization of the intracellular MHC class II compartment. Nature 1994; 369:660-662.
[67] Johansson B E, Bucher D J, Kilbourne E D. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce constrasting types of immunity to infection. J Virol 1989; 63: 1239-1246.
[68] Black R A, Rota P A, Gorodkova N, et al. Antibody response to the M2 protein of influenza A virus expressed in insect cells. J Gen Virol 1993; 74: 143-146.
[69] Murphy B R, Clement M L. The systemic and mucosal immune response of humans to influenza A virus. Top Microb Immunol 1989; 146: 107-116.
[70] Chen Z, Sahashi Y, Matsuo K. Comparison of the ability of viral protein-expressing plasmid DNAs to protect against influenza A virus. Vaccine 1998; 16: 1544-1549.
[71] Chen Z, Kadowaki S, Hagiwara Y. Protection against influenza B virus infection by immunization with DNA vaccines. Vaccine 2001; 19: 1446-1455.
[72] Chen Z, Kurata T, Tamura S-I. Identification of effective constituents of influenza vaccine by immunization with plasmid DNAs encoding viral protein. Jpn J Infect Dis 2000; 53: 219-228.
[73] Ulmer J B, Donnelly J J, Parker S E, et al. Heterogous protection against influenza by infection of DNA encoding a viral protein. Science 1993; 259: 1745-1749.
[74] Grebe K M, Yewdell J W, Bennink J R. Heterosubtypic immunity to influenza A virus: Where do we Stand? Microbes and infection 2008; 10: 1024-1029.
[75] Tumpey T M, Clements J D, Katz J M. Mucosal vaccination protects mice against influenza A heterosubtypic challenge. International Congress Series 2001; 1219: 985-992.
[76] Yetter R A, Shoshana Lehrer, Reuben Ramphal, et al. Outcome of influenza infection: Effect of site of initial infection and hererotypic immunity. Infect Immun 1980; 29 (2): 654-662.
[77] Richt J A, Porntippa Lekcharoensuk, Lager K M, et al. Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine. J Virol 2006; 80(22): 11009-11018.
[78] Heinen P P, Els A. de Boer-Luijtze, Andre T. J. Bianchi. Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection. J gen virol 2001; 82: 2697-2707.
[79] Webster G G, Askonas B A. Cross-protection and cross-reactive cytotoxic T cells induced by influenza virus vaccines in mice. Eur J Immunol 1980; 10: 396-401.
[80] Liang S, Mozdzanowska K, Palladino G, et al. Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity. J Immunol 1994; 152: 1653-1661.
[81] Benton K A, Misplon J A, Lo C-Y, et al. Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or γδT cells. J Immunol 2001; 166: 7437.7445.
[82] Erstein S L, Lo C-Y, Miplon J A, et al. Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell-depleted, P2-microglobulin-deficient, and J chain-deficient mice. J Immunol 1997; 158: 1222-1230.
[83] Scherle P A, Gerhard W. Functional analysis of influenza-specific helper T cell clones in vivo: T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin. J Exp Med 1986; 164,1114-1128.
[84] Swayne D E, Perdue M L, Beck J R, et al. Vaccines protect chickens against H5 highly pathogenic avian influenza in the face of genetic changes in field viruses over multiple years. Vet Microbiol 2000; 74: 165-172.
[85] Swayne D E, Maricarmen Garcia, Beck J R, et al. Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 2000; 18: 1088-1095.
[86] Swayne D E, Lee Chang-won, Erica Spackman. Influenza North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus. Avian Pathol 2006; 35 (2): 141-146.
[87] Smirnov Y A, Kaverin N V, Govorkova E A, et al. Cross-protection studies with H5 influenza viruses. International Congress Series 2001; 1219: 767-773.
[88] Lu X H, Edwards L E, Desheva J A, et al. Cross-protective immunity in mice induced by live-attenuated or inactivated vaccines against highly pathogenic influenza A (H5N1) viruses. Vaccne 2006; 24: 6588-6593.
[89] Ninomiya A, Imai M, Tashiro M, et al. Inactivated influenza H5N1 whole-virus vaccine with aluminum adjuvant induces homologous and heterologous protective immunities against lethal challenge with highly pathogenic H5N1 avian influenza viruses in a mouse model. Vaccine 2007; 25: 3554-3560.
[90] Jeanet A. van der Goot, Michiel van Boven, Arjan Stegeman, et al. Transmission of highly pathogenic avian influenza H5N1 virus in Pekin ducks is significantly reduced by a genetically distant H5N2 vaccine. Virol 2008; 382: 91-97.
[91] Asahi-Ozaki Y, Yoshikawa T, Iwakura Y, et al. Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus. J Med Virol 2004; 74:328-335.
[92] Hoskins T W, Davies J R, Smith A J et al. Influenza at Christ's Hospital: March, 1974. Lancet 1976; 1: 105-108.
[93] Hoskins T W, Davies J R, Smith A J et al. Assessment of inactivated influenza-A vaccine after three outbreaks of influenza A at Christ's Hospital. Lancet 1979; 1: 33-35.
[94] Couch R B, Kasel J A. Immunity to influenza in man . Ann Rev Microbiol 1983; 37: 529-549.
[95] Tamura S I, Funato H, Hirabayashi Y, et al. Cross-protection against influenza A virus infection by passively transferred RT IgA antibodies to defferent hemagglutinin molecules. Eur J Immunol 1991; 21: 1337-1344.
[96] Renegar K B, Jackson G D, Mestecky J. In vitro comparison of the biologic activities of monoclonal monomeric IgA, polymeric IgA, and secretory IgA. J Immunol 1998; 160: 1219-1223.
[1]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005,45(3):359-362.
[2]朱爱华,彭大新,吴艳涛,等.鸡痘病毒载体强启动子的构建和筛选.扬州大学学报,1999,2(2):25-28.
[3]郝贵杰.抗H5亚型禽流感病毒血凝素蛋白特异性单克隆抗体的研制及初步应用.扬州大学硕士学位论文.2005.
[4]龙进学,郝贵杰,唐应华,等.H5N1亚型禽流感病毒血凝素单克隆抗体的制备及其应用.中国兽医科技.2005,35(6):450-454.
[5]Hoffmann E,Stech J,Guan Y,et al.Universal primer set for the full-length amplification of all influenza A viruses.Arch Virol 2001;146:2275-2289.
[6]Kozak M.Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.Cell 1986;44:283-292.
[7]Kozak M.At least six nucleotides proceeding the AUG initiator codon enhance translation in mammalian cells.J Mol Biol 1987;196:947-950.
[8]Shuman S,Moss B.Factor-dependent transcription termination by vaccina virus RNA polymerase.J Biol Chem 1988;263:6220-6225.
[9]Mohamed R M,Edward G N.UUUUUNU Oligonucleotide Stimulation of Vaccinia Virus Early Gene Transcription Termination,in Trans.J Biol Chem 2003;278:11794-11801.
[10]Bernard M.Genetically engineered poxviruses for recombinant gene expression,vaccination,and safety.Proc Natl Acad Sci USA 1996;93:11341-11348.
[11]Yuen L,Moss B.Oligonucleotide Sequence Signaling Transcriptional Termination of Vaccinia Virus Early Genes.Proc Natl Acad Sci USA 1987;84:6417-6421.
[12]Earl P L,Hugin A W,Moss B.Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus.J Virol 1990;64:2448-2451.
[13]Afonso C L,Tulman E R,Lu Z,et al.The genome of fowlpox virus.J Virol 2000;74:3815-3813.
[14]孙蕾.外源基因插入不同复制非必需区影响重组鸡痘病毒抵抗母源抗体干扰的能力.扬州大学博士学位论文.2005.
[15]Humberd J,Boyd K,Webster R G.Emergence of influenza virus variants after prolonged shedding from pheasants.J Virol 2007;81:4044-4051.
[16]Lee C W,Senne D A,Suarez D L.Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus.J Virol 2004;78:8372-8381.
[17]Wu W L,Chen Y X,Wang P,et al.Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007.J Virol 2008;82:1798-1807.
[18]Kaverin N V,Rudneva I A,Govorkova E A,et al.Epitope mapping of the hemagglitinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies.J Virol 2007;81:12911-12917.
[19]Smith G J D,Fan X H,Wang J,et al.Emergence and predominance of an H5N 1influenza variant in China.Proc Natl Acad Sci USA 2006;103:16936-16941.
[1]Ada G L,Jones P D.The immune response to influenza infection.Curr Top Microbiol Immunol 1986;128:1-54.
[2]Bean W J,Schell M,Katz J,et al.Evolution of the H3 influenza virus hemagglutinin from human and monhuman hosts.J Virol 1992;66:1129-1138.
[3]Swayne D E,Perdue M L,Beck J R,et al.Vaccines protect chickens against highly pathogenic avian influenza in the face of genetic changes in field viruses over multiple years.Vet Microbiol 2000;74:165-172.
[4]Swayne D E,Beck J R,Perdue M L,et al.Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza.Avian Dis 2001;45:355-365.
[5]Smirnov Y A,Kaverin N V,Govorkova E A,et al.Cross-protection studies with H5 influenza viruses.Internation Congress Series 1219 2001;767-773.
[6]Lee C W,Suarez D L.Avian influenza virus:prospects for prevention and control by vaccination.Anim Hel Res Rev 2005;6:1-15.
[7]Swayne D E,Lee Chang-won,Erica Spackman.Influenza North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N 1high pathogenicity avian influenza virus.Avian Pathol 2006;35(2):141-146.
[8]Swayne D E,Maricarmen Garcia,Beck J R,et al.Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert.Vaccine 2000;18:1088-1095.
[1]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005,45(3):359-362.
[2]朱爱华,彭大新,吴艳涛,等.鸡痘病毒载体强启动子的构建和筛选.扬州大学学报,1999,2(2):25-28.
[3]胡顺林,吴艳涛,刘文博,等.鹅源新城疫病毒单克隆抗体的研制及其与不同NDV毒株的反应性.中国兽医科技,2005,35(5):341-345.
[4]Czegledi A,Ujvari D,Somogyi E,et al.Third genome size category of avian paramyxovirus serotype 1(Newcastle disease virus)and evolutionary implications.Virus Res 2006;120:36-48.
[5]Kozak M.Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.Cell 1986;44:283-292.
[6]Kozak M.At least six nucleotides proceeding the AUG initiator codon enhance translation in mammalian cells.J Mol Biol 1987;196:947-950.
[7]Shuman S,Moss B.Factor-dependent transcription termination by vaccina virus RNA polymerase.J Biol Chem 1988;263:6220-6225.
[8]Mohamed R M,Edward G N.UUUUUNU Oligonucleotide Stimulation of Vaccinia Virus Early Gene Transcription Termination,in Trans.J Biol Chem 2003;278:11794-11801.
[9]Bernard M.Genetically engineered poxviruses for recombinant gene expression,vaccination,and safety.Proc Natl Acad Sci USA 1996;93:11341-11348.
[10]Yuen L,Moss B.Oligonucleotide Sequence Signaling Transcriptional Termination of Vaccinia Virus Early Genes.Proc Natl Acad Sci USA 1987;84:6417-6421.
[11]Earl P L,Hugin A W,Moss B.Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus.J Virol 1990;64:2448-2451.
[12]Afonso C L,Tulman E R,Lu Z,et al.The genome of fowlpox virus.J Virol 2000;74:3815-3813.
[13]孙蕾.外源基因插入不同复制非必需区影响重组鸡痘病毒抵抗母源抗体干扰的能力.扬州大学博士学位论文.2005.
[1]Liu H L,Wang Z L,Wu Y G,et al.Molecular epidemiological analysis of Newcastle disease virus isolated in China in 2005.J Virol Methods 2007;140:206-211.
[2]Liang R,Cao D J,Li J Q,et al.Newcastle disease outbreaks in western China were caused by the genotypes Ⅶa and Ⅷ.Vet Microbiol 2002;87:193-203.
[3]Wang Z L,Liu H L,Xu J T,et al.Genotyping of Newcastle disease viruses isolated from 2002 to 2004 in Chinal.Ann NY Acad Sci 2006;108:228-239.
[4]Li Y,Wang Z L,Jiang Y H,et al.Characterization of Newly Emerging Newcastle Disease Virus Isolates from the People's Republic of China and Taiwan.J Clin Microbiol 2001;39:3512-3519.
[5]Miller P J,King D J,Afonso C L,et al.Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge.Vaccine 2007;41:7238-7246.
[6]Veits J,Romer-Oberdorfer A,Helferich D,et al.Protective efficacy of several vaccines against highly pathogenic H5N 1 avian influenza virus under experiment conditions.Vaccine 2008;26:1688-1696.
[1]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005;45(7):359-362.
[2]冀德君,刘红旗,彭大新,等.H9亚型禽流感重组鸡痘病毒疫苗的免疫保护试验.扬州大学学报(农业与生命科学版),2002;23(2):13-16.
[3]高璐,胡仁莉,陈祥,等.SPF鸡人工感染禽源大肠杆菌、H9亚型禽流感病毒后的体液免疫应答和死亡率分析.畜牧兽医学报,2006;37(9):899-902.
[4]Katz J M.The impact of avian influenza viruses on public health.Avian Dis 2003;47:914-20.
[5]Wong S S Y,Yuen K Y.Avian influenza virus infections in humans.Chest 2006;129:156-68.
[6]Boyle D B,Pre A D,Coupar B E.Comparison of field and vaccine strains of Australian fowlpox viruses.Arch Virol 1997;142:737-748.
[7]邵卫星,卢建红,刘玉良,等.表达鸡白细胞介素1β基因重组鸡痘病毒的构建.中国兽医科技,2004;34(1):12-16.
[8]邵卫星 彭大新 卢建红,等.表达鸡白细胞介素2重组鸡痘病毒的构建及其体外表达产物生物活性的检测.生物工程学报,2004;20(1):136-9.
[9]Ma M X,Jin N Y,Wang Z G,et al.Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18.Vaccine 2006;24:4304-4311.
[10]王振国,金宁一,马明潇,等.共表达H5、H7亚型AIV HA基因与鸡IL-18基因的重组鸡痘病毒的构建.高技术通讯,2006;16(4):408-413.
[11]程坚,刘秀梵,彭大新,等.表达H9亚型禽流感病毒血凝素基因的重组鸡痘病毒及其免疫效力.微生物学报,2002;42(4):442-447.
[12]陈平,陈素娟,彭大新,等.高效表达H9亚型禽流感病毒HA基因的重组鸡痘病毒的构建及免疫效力试验.中国兽医学报,2004;24(3):216-218.
[13]朱爱华,彭大新,吴艳涛,等.鸡痘病毒载体强启动子的构建和筛选.扬州大学学报,1999;2(2):25-28.
[14]Afonso C L,Tulman E R,Lu Z,et al.The genome of fowlpox virus.J Virol 2000; 74:3815-3831.
[15] Kozak M. Point mutation define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 1986; 44: 283-292.
[16] Kozak M. At least six nucleotides proceeding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 1987; 196: 947-950.
[17] Shuman S, Moss B. Factor-dependant transcription termination by vaccinia virus RNA polymerase. J Biol Chem 1988; 263: 6220-6225.
[1]Swayne D E,Beck J R,Kinney N.Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine.Avian Dis 2000;44:132-137.
[2]陈素娟,孙蕾,刘武杰,等.共表达H5亚型禽流感病毒HA和NA基因的重组鸡痘病毒及其免疫效力.微生物学报,2006;45:111-114.
[3]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005;45:359-362.
[4]Kutinova L,Ludvikova V,Maresova L,et al.Effect of virulence on immunogenicity of single and double vaccinia virus recombinants expressing differently immunogenic antigens:antibody-response inhibition induced by immunization with a mixture of recombinants differing in virulence.J Gen Virol 1999;80:2901-2908.
[5]Xiang Z Q,Gao G P,Reyes-Sandoval A,et al.Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier.J Virol 2003;77:10780-10789.
[6]Yang Z Y,Wyatt L S,Kong W P,et al.Overcoming immunity to a viral vaccine by DNA priming before vector boosting.J Virol 2003;77:799-803.
[7]Belyakov I M,Moss B,Strober W et al.Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity.Proc Natl Acad Sci USA 1999;96:4512-4517.
[1]陈素娟,孙蕾,刘武杰,等.共表达H5亚型禽流感病毒HA和NA基因的重组鸡痘病毒及其免疫效力.微生物学报,2006,46.111-114.
[2]Swayne D E,Beck J R,Kinney N.Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine.Avian Dis 2000;44:132-137.
[3]Woodland D L.Jump-starting the immune system:prime-boosting comes of age.Trends Immunol 2004;25:98-104.
[4]Ramshaw I A,Ramsay A J.The prime-boost strategy:exciting prospects for improved vaccination.Immunol Today,2000,21(4):163-165.
[5]Zavala F,Rodrigues M,Rodriguez D,et al.A striking property of recombinant poxviruses:efficient inducers of in vivo expansion of primed CD8~+ T cells.Virology 2001;280:155-159.
[6]HE Xiao-wen,SUN Shu-han,HU Zhen-lin,et al.Augmented humoral and cellular immune responses of a hepatitis B DNA vaccine encoding HBsAg by protein boosting.Vaccine 2005;23:1649-1656.
[7]Reber A J,Tanner M,Okinaga T,et al.Evaluation of multiple immune parameters after vaccination with modified live or killed bovine viral diarrhea virus vaccines.Comp Immun Microbiol Infect Dis 2006;29:61-77.
[8]Haygreen E A,Kaiser P,Burgess S C,et al.In ovo DNA immunization followed by a recombinant fowlpox boost is fully protective to challenge with virulent IBDV.Vaccine 2006;24:4951-4961.
[9]Estcourt M J,Ramsay A J,Brooks A,et al.Prime-boost immunization generates a high frequency,high-avidity CD8~+ cytotoxic T lymphocyte population.Int Immunol 2001;14:31-37.
[10]Wong P,Lara-Tejero M,Ploss A,et al.Rapid development of T cell memory.J Immunol 2004;172:7239-7245.
[11]Toussaint J F,Letellier C,Paquet D,et al.Prime-boost strategies combining DNA and inactivated vaccines confer high immunity and protection in cattle against bovine herpes virus-1.Vaccine 2005;23:5073-5081.
[12]Steensels M,Bublot M,Bonn S V,et al.Prime-boost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with Asia H5N1 HPAI.Vaccine 2009;27:646-654.
[13]Lee C W,Suarez D L.Avian influenza virus:prospects for prevention and control by vaccination.Anim Health Res Rev 2005;6:1-15.
[2]Chen W,Calvo P A,Malide D,et al.A novel influenza A virus mitochondrial protein that induces cell death.Nat Med 2001;7:1306-1312.
[3]Lamb R A,Krug R M.Orthomyxoviridae:The viruses and their replication.In Field B N,Knipe D M,Howley E M.Fields Virology.Lippincott-Raven:NY,1996,1353-1395.
[4]Klenk H D,Keil W,Niemann R.The characterization of influenza A viruses by carbohydrate analysis.Curt Top Microbiot Immunol 1983;104:247-257
[5]Palese E,Garcia A.Influenza Viruses(orthomyxoviruses):Molecular Biology.In Granoff,Webster R G(eds.).Encyclopedia of Virology:1999 Volume 2,2nd ed.Academics Press:San Diego,830-836.
[6]Cox N J,Fuller F,Kaverin N.Orthornyxoviridae 2000.In Van M H,Fauquet C M,Bishop D H L,et al.Virus taxomony.Seventh report of the international committee on taxonomy of viruses.Academic Press:San Deigo,585-597.
[7]Easterday B C,Hinshaw V S,Halvorson D A.Influenza 1997.In Calnek B W,Barnes H J,Beard C W et al.Diease of Poultry,10th ed.Iowa State University Press:Ames,IA,583-605
[8]Kaiser J.A one-size-fits-all Flu vaccine?Sciene 2006;312:380-382.
[9]Franklin R M,Wecker E.Inactivation of some animal viruses by hydroxylamine and the structure of ribonucleic acid.Nature 1959;84:343-345.
[10]Lavr G.The structure of influenza viruses,Ⅱ.Disruption of the virus particles and separation of the neuraminidase activity.Virology 1963;20:523-526.
[11] King D J. Evaluation of different methods of inactivation of Newcastle disease virus and avian influenza virus in egg fluids and serum. Avian Dis 1991; 35: 505-514.
[12] Beard C W, Brugh M, Johnson D C. Laboratory studies with the Pennsylvania avian influenza viruses (H5N2). Proceedings of the U S Animal Health Association 1984; 88: 462-473.
[13] Fichtner G J. The Pennsylvania/Virginia experience in eradication of avian influenza (H5N2). 1987. In Easterday B C. Proceedings of the second international symposium on avian influenza U S Animal Health Association: Richmond, VA, 33-38.
[14] Webster R G, Yakhno M, Hinshaw V S, et al. Intestinal influenza: Replication and characterization of influenza viruses in ducks. Virology 1978; 84:268-278.
[15] Fouchier R A M, Munster V, Wallensten A, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology 2005; 79: 2814-2822.
[16] WHO Expert committee. A revision of the system of nomenclature for influenza viruses: A WHO memorandum. Bull WHO 1980; 585-591.
[17] Bean W J, Kawaoka Y, Wood J M, et al. Characterization of virulent and avirulent A/Chicken/Pennsylvania/83 influenza viruses: Potential role of defective interfering RNAs in nature. J Virol 1985; 54: 151-160.
[18] Alexander D J, Allan W H, Parsons DG,et al. The pathogenicity of four avian influenza viruses for fowls , turkeys and ducks. Res Vet Sci 1978; 24: 242-247.
[19] Alexander D J. Avian influenza. Recent developments. Vet Bulls 1982. 341-359.
[20] Easterday B C, Tumova B. 1972. Avian influenza. In Hofstad M S, Calnek B W, Helmbolt C F, et al. (eds.). Disease of poultry, 6th ed. Iowa State University Press: Ames, IA, 670-700.
[21] Smithies L K, Emerson E G, Robertson S M, et al. Two different type A influenza virus infections in turkeys in Wisconsin. Ⅱ. 1968 outbreak. Avian Dis 1969; 13: 606-610.
[22] Beard C W, Easterday B C. A-Turkey-Oregon-71, an avirulent influenza isolate with the hemagglutinin of fowl plaque virus, Aivan Dis 1973; 17: 173-181.
[23] Humberd J, Boyd K, Webster R G. Emergence of influenza virus variants after prolonged shedding from pheasants. J Virol 2007; 81: 4044-4051.
[24] Lee C W, Senne D A, Suarez D L. Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J Virol 2004; 78: 8372-8381.
[25] Suarez D L, Senne D A. Sequence analysis of related low-pathogenic and highly pathogenic H5N2 avian influenza isolates from United States live bird markets and poultry farms from 1983 to 1989. Avian Dis 2000; 44: 356-364.
[26] Spackman E, Senne D A, Davison S. Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States. J Virol 2003; 77: 13399-13402.
[27] Tsuckiya E, Sugawara K, Hongo S, et al. Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus. J Gen Virol 2001; 82: 2475-2484.
[28] Kaverin N V. Rudneva I A, Ilyushina N A, et al. Stnicture of antigenic sites on the heamagglutinin molecule of H5 avian influenza virus and phenotvpic variation of escape mutants. J Gen Virol 2002; 83: 2497-2505.
[29] Murakami S, Iwasa A, Iwatsuke-Horimoto K, et al. Cross-clade protective immunity of H5N1 influenza vaccines in a mouse model. Vaccine 2008; 26: 6398-6404.
[30] Austin E J, Webster R G. Antigenic mapping of an avian H1 influenza virus haemagglutinin and interrelationships of H1 viruses from humans, pigs and birds . J Gen Virol 1986; 67: 983-992.
[31] Hinshaw V S, Alexander D J, Ayrnand M, ei al. Antigenic comparisons of swine-influenza-like H1N1 isolates from pigs, birds and humans: an international collaborative study. Bull WHO1984; 62: 871-878.
[32] Wu W L, Chen Y X, Wang P, et al. Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007. J Virol 2008; 82: 1798-1807.
[33] Kaverin N V, Rudneva I A, Govorkova E A, et al. Epitope mapping of the hemagglitinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies. J Virol 2007; 81:12911-12917.
[34] Ada G L, Jones P D. The immune response to influenza infection. Curr Top Microbial Immunol 1986; 128: 1-54.
[35] Tamura S I, Kurata T. Defence mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn J Infect Dis 2004; 57: 236-247.
[36] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001; 2: 675-680.
[37] Lund J M, Alexopoulou L, Sato A, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA; 101:5598-5603.
[38] Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004; 303: 1526-1529.
[39] Diebold S S, Kaisho T, Hemmi H, et al. Innate antival responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004; 303: 1529-1531.
[40] Scherle P A, Palladino G, Gerhard W. Mice can recover from pulmonary influenza virus infection in the absence of class-Ⅰ-restricted cytotoxic T cells. J Immunol 1992; 148: 212-217.
[41] Gerhard W, Mozdzanowska K, Furchner M, et al. Role of the B-cell response in the recovery of mice from primary influenza virus infection. Immunol Rev 1997; 159: 95-103.
[42] Tamura S I, Iwasaki T, Thompson A M, et al. Antibody-forming cells in the nasal-associated lymphoid tissue during primary influenza virus infection. J Gen Virol 1998; 79: 291-299.
[43] Zuercher A W, Coffin S E, Thurnheer M C, et al. Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses. J Immunol 2002; 168: 1796-1803.
[44] McMichael A. Cytotoxic T lymphocytes specific for influenza virus. Cuur Top Microb Immunol 1994; 189: 75-91.
[45] Flynn K J, Belz G T, Alman J D, et al. Virus-specific CD8~+ T cells in primary and secondary influenza pneumonia. Immunity 1998; 8: 683-691.
[46] Flynn K J, Riberdy J M, Christensen J M, et al. Iin vivo proliferation of naive and memory influenza-specific CD8~+ T cells. Proc Natl Acad Sci USA 1999; 96: 8597-8602.
[47] Epstein S L, Lo Chia-Yun, Misplon, et al. Mechanism of protective immunity against influenza virus infection in mice without antibodies. J Immunol 1998; 160: 322-329.
[48] Wiley J A, Hogan R J, Woodland D L, et al. Antigen-specific CD8 (+) T cells persist in the upper respiratory tract following influenza virus infection. J Immunol 2001; 167: 3293-3299.
[49] Yoshikawa T, Matsuo Ke, Matsuo Ka, et al. Total viral genome copies and virus-Ig complexes after infection with influenza virus in the nasal secretions of immunized mice. J Gen Virol 2004; 85: 2339-2346.
[50] Liew F Y, Russell S M, Appleyard G, et al. Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity. Eur J Immunol 1984; 14: 350-356.
[51] Brandtzaeg P, Krajci P, Lamm M E, et al. Epithelial and hepatobiliary transport of polymeric immunoglobulins. 1994 .P. 113-126. In Ogra P W, Lamm M E, McGhee J R, et al. Handbook of Mucosal Immunology. Academic Press, San Diego, Calif.
[52] Asahi Y, Yoshikawa T, Watanabe I, et al. Protection against influenza virus infection in ploy Ig receptor-knockout mice immunized intranasally with adjuvant-combined vaccine. J Immunol 2002; 168: 2930-2938.
[53] Ramphal R, Cogliano R C, Shands J W, et al. Serum antibody prevents lethal murine influenza pneumonitis but not tracheitis. Infect Immun 1979; 25: 992-997.
[54] Palladino G, Mozdzanowska K, Washko G, et al. Virus-neutrilizing antiodies of immunoglobulin G (IgG) but not IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice. J Virol 1995; 69: 2075-2081.
[55] Ito R, Yoshikawa T, Ozaki Y, et al. Roles of anti-hemagglutinin IgA and IgG antibodies in different sites of the respiratory tract of vaccinated mice in preventing lethal influenza pneumonia. Vaccne 2003; 21: 2362-2371.
[56] Murphy B R, Clements M L. The systemic and mucosal immune response of humans to influenza A virus. Curr Top Microb Immunol 1989; 146:107-116.
[57] Murphy B R. Mucosal immunity to viruses. P. 333-343. In Ogra P L, Lamm M E, McGhee J R, et al. Handbook of Mucosal Immunology. Academic Press, San Diego, Calif.
[58] Mazanec M B, Kaetzel C S, Lamm M E, et al. Intracellular neutralization of virus by immunoglobin A antibodies. Proc Natl Acad Sci USA 1992; 89: 6901-6905.
[59] Mazanec M B, Coulder C L, Fletcher D R. Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J Virol 1995; 69:1339-1343.
[60] Fujioka H, Emancipator S N, Aikawa M, et al. Immunocytochemical colocalization of specific immunoglobulin A with sendai virus protein in infected polarized epithelium. J Exp Med 1998; 188: 1223-1229.
[61] Tamura S I, Miyata K, Matsuo K, et al. Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein. J Immunol 1996; 156: 3892-3900.
[62] Tamura S I, Tanimoto T, Kurata T. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn J Infect Dis 2005; 58: 195-207.
[63] Portner A, Murti K G. Localization of P, NP, and M protein on Sendai virus nucleocapsid using immunogold labeling. Virology 1986; 150:469-478.
[64] Wraith D C. The recognition of influenza A virus-infected cells by cytotoxic lymphocytes. Immunol Today 1987; 8: 239-246.
[65] Yewdell J W, Bennink J R, Smith G L, et al. Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes. Proc Natle Aca Sci USA 1985; 82: 1785-1789.
[66] Tulp A, Verwoerd D, Dobberstein B. et al. Isolation and characterization of the intracellular MHC class II compartment. Nature 1994; 369:660-662.
[67] Johansson B E, Bucher D J, Kilbourne E D. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce constrasting types of immunity to infection. J Virol 1989; 63: 1239-1246.
[68] Black R A, Rota P A, Gorodkova N, et al. Antibody response to the M2 protein of influenza A virus expressed in insect cells. J Gen Virol 1993; 74: 143-146.
[69] Murphy B R, Clement M L. The systemic and mucosal immune response of humans to influenza A virus. Top Microb Immunol 1989; 146: 107-116.
[70] Chen Z, Sahashi Y, Matsuo K. Comparison of the ability of viral protein-expressing plasmid DNAs to protect against influenza A virus. Vaccine 1998; 16: 1544-1549.
[71] Chen Z, Kadowaki S, Hagiwara Y. Protection against influenza B virus infection by immunization with DNA vaccines. Vaccine 2001; 19: 1446-1455.
[72] Chen Z, Kurata T, Tamura S-I. Identification of effective constituents of influenza vaccine by immunization with plasmid DNAs encoding viral protein. Jpn J Infect Dis 2000; 53: 219-228.
[73] Ulmer J B, Donnelly J J, Parker S E, et al. Heterogous protection against influenza by infection of DNA encoding a viral protein. Science 1993; 259: 1745-1749.
[74] Grebe K M, Yewdell J W, Bennink J R. Heterosubtypic immunity to influenza A virus: Where do we Stand? Microbes and infection 2008; 10: 1024-1029.
[75] Tumpey T M, Clements J D, Katz J M. Mucosal vaccination protects mice against influenza A heterosubtypic challenge. International Congress Series 2001; 1219: 985-992.
[76] Yetter R A, Shoshana Lehrer, Reuben Ramphal, et al. Outcome of influenza infection: Effect of site of initial infection and hererotypic immunity. Infect Immun 1980; 29 (2): 654-662.
[77] Richt J A, Porntippa Lekcharoensuk, Lager K M, et al. Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine. J Virol 2006; 80(22): 11009-11018.
[78] Heinen P P, Els A. de Boer-Luijtze, Andre T. J. Bianchi. Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection. J gen virol 2001; 82: 2697-2707.
[79] Webster G G, Askonas B A. Cross-protection and cross-reactive cytotoxic T cells induced by influenza virus vaccines in mice. Eur J Immunol 1980; 10: 396-401.
[80] Liang S, Mozdzanowska K, Palladino G, et al. Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity. J Immunol 1994; 152: 1653-1661.
[81] Benton K A, Misplon J A, Lo C-Y, et al. Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or γδT cells. J Immunol 2001; 166: 7437.7445.
[82] Erstein S L, Lo C-Y, Miplon J A, et al. Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell-depleted, P2-microglobulin-deficient, and J chain-deficient mice. J Immunol 1997; 158: 1222-1230.
[83] Scherle P A, Gerhard W. Functional analysis of influenza-specific helper T cell clones in vivo: T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin. J Exp Med 1986; 164,1114-1128.
[84] Swayne D E, Perdue M L, Beck J R, et al. Vaccines protect chickens against H5 highly pathogenic avian influenza in the face of genetic changes in field viruses over multiple years. Vet Microbiol 2000; 74: 165-172.
[85] Swayne D E, Maricarmen Garcia, Beck J R, et al. Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 2000; 18: 1088-1095.
[86] Swayne D E, Lee Chang-won, Erica Spackman. Influenza North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus. Avian Pathol 2006; 35 (2): 141-146.
[87] Smirnov Y A, Kaverin N V, Govorkova E A, et al. Cross-protection studies with H5 influenza viruses. International Congress Series 2001; 1219: 767-773.
[88] Lu X H, Edwards L E, Desheva J A, et al. Cross-protective immunity in mice induced by live-attenuated or inactivated vaccines against highly pathogenic influenza A (H5N1) viruses. Vaccne 2006; 24: 6588-6593.
[89] Ninomiya A, Imai M, Tashiro M, et al. Inactivated influenza H5N1 whole-virus vaccine with aluminum adjuvant induces homologous and heterologous protective immunities against lethal challenge with highly pathogenic H5N1 avian influenza viruses in a mouse model. Vaccine 2007; 25: 3554-3560.
[90] Jeanet A. van der Goot, Michiel van Boven, Arjan Stegeman, et al. Transmission of highly pathogenic avian influenza H5N1 virus in Pekin ducks is significantly reduced by a genetically distant H5N2 vaccine. Virol 2008; 382: 91-97.
[91] Asahi-Ozaki Y, Yoshikawa T, Iwakura Y, et al. Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus. J Med Virol 2004; 74:328-335.
[92] Hoskins T W, Davies J R, Smith A J et al. Influenza at Christ's Hospital: March, 1974. Lancet 1976; 1: 105-108.
[93] Hoskins T W, Davies J R, Smith A J et al. Assessment of inactivated influenza-A vaccine after three outbreaks of influenza A at Christ's Hospital. Lancet 1979; 1: 33-35.
[94] Couch R B, Kasel J A. Immunity to influenza in man . Ann Rev Microbiol 1983; 37: 529-549.
[95] Tamura S I, Funato H, Hirabayashi Y, et al. Cross-protection against influenza A virus infection by passively transferred RT IgA antibodies to defferent hemagglutinin molecules. Eur J Immunol 1991; 21: 1337-1344.
[96] Renegar K B, Jackson G D, Mestecky J. In vitro comparison of the biologic activities of monoclonal monomeric IgA, polymeric IgA, and secretory IgA. J Immunol 1998; 160: 1219-1223.
[1]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005,45(3):359-362.
[2]朱爱华,彭大新,吴艳涛,等.鸡痘病毒载体强启动子的构建和筛选.扬州大学学报,1999,2(2):25-28.
[3]郝贵杰.抗H5亚型禽流感病毒血凝素蛋白特异性单克隆抗体的研制及初步应用.扬州大学硕士学位论文.2005.
[4]龙进学,郝贵杰,唐应华,等.H5N1亚型禽流感病毒血凝素单克隆抗体的制备及其应用.中国兽医科技.2005,35(6):450-454.
[5]Hoffmann E,Stech J,Guan Y,et al.Universal primer set for the full-length amplification of all influenza A viruses.Arch Virol 2001;146:2275-2289.
[6]Kozak M.Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.Cell 1986;44:283-292.
[7]Kozak M.At least six nucleotides proceeding the AUG initiator codon enhance translation in mammalian cells.J Mol Biol 1987;196:947-950.
[8]Shuman S,Moss B.Factor-dependent transcription termination by vaccina virus RNA polymerase.J Biol Chem 1988;263:6220-6225.
[9]Mohamed R M,Edward G N.UUUUUNU Oligonucleotide Stimulation of Vaccinia Virus Early Gene Transcription Termination,in Trans.J Biol Chem 2003;278:11794-11801.
[10]Bernard M.Genetically engineered poxviruses for recombinant gene expression,vaccination,and safety.Proc Natl Acad Sci USA 1996;93:11341-11348.
[11]Yuen L,Moss B.Oligonucleotide Sequence Signaling Transcriptional Termination of Vaccinia Virus Early Genes.Proc Natl Acad Sci USA 1987;84:6417-6421.
[12]Earl P L,Hugin A W,Moss B.Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus.J Virol 1990;64:2448-2451.
[13]Afonso C L,Tulman E R,Lu Z,et al.The genome of fowlpox virus.J Virol 2000;74:3815-3813.
[14]孙蕾.外源基因插入不同复制非必需区影响重组鸡痘病毒抵抗母源抗体干扰的能力.扬州大学博士学位论文.2005.
[15]Humberd J,Boyd K,Webster R G.Emergence of influenza virus variants after prolonged shedding from pheasants.J Virol 2007;81:4044-4051.
[16]Lee C W,Senne D A,Suarez D L.Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus.J Virol 2004;78:8372-8381.
[17]Wu W L,Chen Y X,Wang P,et al.Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007.J Virol 2008;82:1798-1807.
[18]Kaverin N V,Rudneva I A,Govorkova E A,et al.Epitope mapping of the hemagglitinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies.J Virol 2007;81:12911-12917.
[19]Smith G J D,Fan X H,Wang J,et al.Emergence and predominance of an H5N 1influenza variant in China.Proc Natl Acad Sci USA 2006;103:16936-16941.
[1]Ada G L,Jones P D.The immune response to influenza infection.Curr Top Microbiol Immunol 1986;128:1-54.
[2]Bean W J,Schell M,Katz J,et al.Evolution of the H3 influenza virus hemagglutinin from human and monhuman hosts.J Virol 1992;66:1129-1138.
[3]Swayne D E,Perdue M L,Beck J R,et al.Vaccines protect chickens against highly pathogenic avian influenza in the face of genetic changes in field viruses over multiple years.Vet Microbiol 2000;74:165-172.
[4]Swayne D E,Beck J R,Perdue M L,et al.Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza.Avian Dis 2001;45:355-365.
[5]Smirnov Y A,Kaverin N V,Govorkova E A,et al.Cross-protection studies with H5 influenza viruses.Internation Congress Series 1219 2001;767-773.
[6]Lee C W,Suarez D L.Avian influenza virus:prospects for prevention and control by vaccination.Anim Hel Res Rev 2005;6:1-15.
[7]Swayne D E,Lee Chang-won,Erica Spackman.Influenza North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N 1high pathogenicity avian influenza virus.Avian Pathol 2006;35(2):141-146.
[8]Swayne D E,Maricarmen Garcia,Beck J R,et al.Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert.Vaccine 2000;18:1088-1095.
[1]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005,45(3):359-362.
[2]朱爱华,彭大新,吴艳涛,等.鸡痘病毒载体强启动子的构建和筛选.扬州大学学报,1999,2(2):25-28.
[3]胡顺林,吴艳涛,刘文博,等.鹅源新城疫病毒单克隆抗体的研制及其与不同NDV毒株的反应性.中国兽医科技,2005,35(5):341-345.
[4]Czegledi A,Ujvari D,Somogyi E,et al.Third genome size category of avian paramyxovirus serotype 1(Newcastle disease virus)and evolutionary implications.Virus Res 2006;120:36-48.
[5]Kozak M.Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.Cell 1986;44:283-292.
[6]Kozak M.At least six nucleotides proceeding the AUG initiator codon enhance translation in mammalian cells.J Mol Biol 1987;196:947-950.
[7]Shuman S,Moss B.Factor-dependent transcription termination by vaccina virus RNA polymerase.J Biol Chem 1988;263:6220-6225.
[8]Mohamed R M,Edward G N.UUUUUNU Oligonucleotide Stimulation of Vaccinia Virus Early Gene Transcription Termination,in Trans.J Biol Chem 2003;278:11794-11801.
[9]Bernard M.Genetically engineered poxviruses for recombinant gene expression,vaccination,and safety.Proc Natl Acad Sci USA 1996;93:11341-11348.
[10]Yuen L,Moss B.Oligonucleotide Sequence Signaling Transcriptional Termination of Vaccinia Virus Early Genes.Proc Natl Acad Sci USA 1987;84:6417-6421.
[11]Earl P L,Hugin A W,Moss B.Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type 1 envelope gene enhances expression and immunogenicity of a recombinant vaccinia virus.J Virol 1990;64:2448-2451.
[12]Afonso C L,Tulman E R,Lu Z,et al.The genome of fowlpox virus.J Virol 2000;74:3815-3813.
[13]孙蕾.外源基因插入不同复制非必需区影响重组鸡痘病毒抵抗母源抗体干扰的能力.扬州大学博士学位论文.2005.
[1]Liu H L,Wang Z L,Wu Y G,et al.Molecular epidemiological analysis of Newcastle disease virus isolated in China in 2005.J Virol Methods 2007;140:206-211.
[2]Liang R,Cao D J,Li J Q,et al.Newcastle disease outbreaks in western China were caused by the genotypes Ⅶa and Ⅷ.Vet Microbiol 2002;87:193-203.
[3]Wang Z L,Liu H L,Xu J T,et al.Genotyping of Newcastle disease viruses isolated from 2002 to 2004 in Chinal.Ann NY Acad Sci 2006;108:228-239.
[4]Li Y,Wang Z L,Jiang Y H,et al.Characterization of Newly Emerging Newcastle Disease Virus Isolates from the People's Republic of China and Taiwan.J Clin Microbiol 2001;39:3512-3519.
[5]Miller P J,King D J,Afonso C L,et al.Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge.Vaccine 2007;41:7238-7246.
[6]Veits J,Romer-Oberdorfer A,Helferich D,et al.Protective efficacy of several vaccines against highly pathogenic H5N 1 avian influenza virus under experiment conditions.Vaccine 2008;26:1688-1696.
[1]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005;45(7):359-362.
[2]冀德君,刘红旗,彭大新,等.H9亚型禽流感重组鸡痘病毒疫苗的免疫保护试验.扬州大学学报(农业与生命科学版),2002;23(2):13-16.
[3]高璐,胡仁莉,陈祥,等.SPF鸡人工感染禽源大肠杆菌、H9亚型禽流感病毒后的体液免疫应答和死亡率分析.畜牧兽医学报,2006;37(9):899-902.
[4]Katz J M.The impact of avian influenza viruses on public health.Avian Dis 2003;47:914-20.
[5]Wong S S Y,Yuen K Y.Avian influenza virus infections in humans.Chest 2006;129:156-68.
[6]Boyle D B,Pre A D,Coupar B E.Comparison of field and vaccine strains of Australian fowlpox viruses.Arch Virol 1997;142:737-748.
[7]邵卫星,卢建红,刘玉良,等.表达鸡白细胞介素1β基因重组鸡痘病毒的构建.中国兽医科技,2004;34(1):12-16.
[8]邵卫星 彭大新 卢建红,等.表达鸡白细胞介素2重组鸡痘病毒的构建及其体外表达产物生物活性的检测.生物工程学报,2004;20(1):136-9.
[9]Ma M X,Jin N Y,Wang Z G,et al.Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18.Vaccine 2006;24:4304-4311.
[10]王振国,金宁一,马明潇,等.共表达H5、H7亚型AIV HA基因与鸡IL-18基因的重组鸡痘病毒的构建.高技术通讯,2006;16(4):408-413.
[11]程坚,刘秀梵,彭大新,等.表达H9亚型禽流感病毒血凝素基因的重组鸡痘病毒及其免疫效力.微生物学报,2002;42(4):442-447.
[12]陈平,陈素娟,彭大新,等.高效表达H9亚型禽流感病毒HA基因的重组鸡痘病毒的构建及免疫效力试验.中国兽医学报,2004;24(3):216-218.
[13]朱爱华,彭大新,吴艳涛,等.鸡痘病毒载体强启动子的构建和筛选.扬州大学学报,1999;2(2):25-28.
[14]Afonso C L,Tulman E R,Lu Z,et al.The genome of fowlpox virus.J Virol 2000; 74:3815-3831.
[15] Kozak M. Point mutation define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 1986; 44: 283-292.
[16] Kozak M. At least six nucleotides proceeding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 1987; 196: 947-950.
[17] Shuman S, Moss B. Factor-dependant transcription termination by vaccinia virus RNA polymerase. J Biol Chem 1988; 263: 6220-6225.
[1]Swayne D E,Beck J R,Kinney N.Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine.Avian Dis 2000;44:132-137.
[2]陈素娟,孙蕾,刘武杰,等.共表达H5亚型禽流感病毒HA和NA基因的重组鸡痘病毒及其免疫效力.微生物学报,2006;45:111-114.
[3]孙蕾,刘武杰,陈素娟,等.鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响.微生物学报,2005;45:359-362.
[4]Kutinova L,Ludvikova V,Maresova L,et al.Effect of virulence on immunogenicity of single and double vaccinia virus recombinants expressing differently immunogenic antigens:antibody-response inhibition induced by immunization with a mixture of recombinants differing in virulence.J Gen Virol 1999;80:2901-2908.
[5]Xiang Z Q,Gao G P,Reyes-Sandoval A,et al.Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier.J Virol 2003;77:10780-10789.
[6]Yang Z Y,Wyatt L S,Kong W P,et al.Overcoming immunity to a viral vaccine by DNA priming before vector boosting.J Virol 2003;77:799-803.
[7]Belyakov I M,Moss B,Strober W et al.Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity.Proc Natl Acad Sci USA 1999;96:4512-4517.
[1]陈素娟,孙蕾,刘武杰,等.共表达H5亚型禽流感病毒HA和NA基因的重组鸡痘病毒及其免疫效力.微生物学报,2006,46.111-114.
[2]Swayne D E,Beck J R,Kinney N.Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine.Avian Dis 2000;44:132-137.
[3]Woodland D L.Jump-starting the immune system:prime-boosting comes of age.Trends Immunol 2004;25:98-104.
[4]Ramshaw I A,Ramsay A J.The prime-boost strategy:exciting prospects for improved vaccination.Immunol Today,2000,21(4):163-165.
[5]Zavala F,Rodrigues M,Rodriguez D,et al.A striking property of recombinant poxviruses:efficient inducers of in vivo expansion of primed CD8~+ T cells.Virology 2001;280:155-159.
[6]HE Xiao-wen,SUN Shu-han,HU Zhen-lin,et al.Augmented humoral and cellular immune responses of a hepatitis B DNA vaccine encoding HBsAg by protein boosting.Vaccine 2005;23:1649-1656.
[7]Reber A J,Tanner M,Okinaga T,et al.Evaluation of multiple immune parameters after vaccination with modified live or killed bovine viral diarrhea virus vaccines.Comp Immun Microbiol Infect Dis 2006;29:61-77.
[8]Haygreen E A,Kaiser P,Burgess S C,et al.In ovo DNA immunization followed by a recombinant fowlpox boost is fully protective to challenge with virulent IBDV.Vaccine 2006;24:4951-4961.
[9]Estcourt M J,Ramsay A J,Brooks A,et al.Prime-boost immunization generates a high frequency,high-avidity CD8~+ cytotoxic T lymphocyte population.Int Immunol 2001;14:31-37.
[10]Wong P,Lara-Tejero M,Ploss A,et al.Rapid development of T cell memory.J Immunol 2004;172:7239-7245.
[11]Toussaint J F,Letellier C,Paquet D,et al.Prime-boost strategies combining DNA and inactivated vaccines confer high immunity and protection in cattle against bovine herpes virus-1.Vaccine 2005;23:5073-5081.
[12]Steensels M,Bublot M,Bonn S V,et al.Prime-boost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with Asia H5N1 HPAI.Vaccine 2009;27:646-654.
[13]Lee C W,Suarez D L.Avian influenza virus:prospects for prevention and control by vaccination.Anim Health Res Rev 2005;6:1-15.