【作者】 童俊容；

【导师】 陈政良； 童光志；

【作者基本信息】 第一军医大学 ， 免疫学， 2006， 博士

【摘要】 高致病性禽流感（Highly pathogenic avian influenza A，HPAI）是由高致病性禽流感病毒中的H5或H7亚型引起的一种禽类的急性、高度接触性烈性传染病。自2003年以来，东南亚多个国家暴发了家禽感染H5N1亚型禽流感疫情，在6个国家出现人感染禽流感，且死亡率非常高。2005年夏天，HPAI逐渐往西扩散，哈萨克斯坦、俄罗斯、罗马尼亚、土耳其、克罗地亚和乌克兰暴发了家禽H5N1亚型HPAI疫情或野生候鸟中出现了H5N1亚型禽流感感染。迄今，H5N1亚型禽流感疫情已在欧洲大陆十几个国家出现，并蔓延到了非洲。H5N1亚型HPAI不仅给养禽业造成了巨大的经济损失，同时也对人类的健康构成严重的威胁。高致病性禽流感病毒属于正黏病毒科A型流感病毒，是有包膜的单股负链RNA病毒。其包膜表面的血凝素（hemagglutinin，HA）和神经氨酸酶（neuraminidase，NA）是主要的免疫原性物质。大量研究表明，HA在病毒入侵宿主细胞的过程中起重要作用。HA也是诱导体液免疫应答的主要保护性抗原，抗HA抗体能抑制病毒的血凝活性并能中和病毒，是病毒诱导机体产生的主要保护性抗体。因此，HA是目前研制流感疫苗的关键成分。全群捕杀和生物安全防护是及时扑灭和控制HPAI的主要措施。然而，禽流感疫情大范围流行时，这些措施可能难以奏效，而疫苗接种可能是一个可靠而经济的方法。作为疫苗的理想病毒株应该是与流行病毒抗原性匹配、致病性低而又能在鸡胚中良好生长的毒株。自1996年以来，中国分离的所有H5N1亚型毒株HA裂解位点都有几个碱性氨基酸残基，都是高致病性的，因而不适于作为疫苗。采用传统方法构建H5亚型禽流感疫苗株非常困难，而流感病毒反向遗传学操作技术为禽流感疫苗的研制提供了新的技术手段。本研究利用RT-PCR技术克隆了H9N2亚型禽源高产病毒株的6个内部基因、H5N1亚型高致病性禽流感病毒的血凝素（HA）基因及H2N3亚型参考毒株的神经氨酸酶基因。对血凝素HA1和HA2连接处的5个碱性氨基酸残基（R-R-R-K-K）进行基因缺失与修饰，消除了病毒基因的毒力相关序列。分别构建8个基因的转录与表达载体，利用反向遗传学技术拯救出了全部基因都源于禽的重组禽流感病毒疫苗株rH5N3。rH5N3疫苗株对鸡和鸡胚均无致病性，在鸡胚尿囊液的HA效价极大提高，是其亲本H5N1毒株的8倍。该禽流感疫苗免疫鸡后2周即可诱导产生高效价的HI抗体，免疫后18周依然保持高水平的HI抗体，不论是对于国内早期分离的禽流感病毒强毒株A／Goose／Guangdong／1／96还是近期分离的强毒株A／Goose／HLJ／QFY／04都能够产生完全的免疫保护作用。一、H5亚型禽流感高产疫苗株的构建采用RT-PCR技术，从鹅源高产禽流感病毒A／Goose／Dalian／3／01（H9N2）扩增6条内部基因片段，从高致病性禽流感病毒A／Goose／HLJ／QFY／04（H5N1）扩增HA基因片段，从禽流感参考株A／Duck／Germany／1215／73（H2N3）扩增NA基因片段。将其分别与质粒pHW2000连接，获得含8个目的基因PB2、PB1、PA、NP、M、NS、HA和NA的重组质粒pML-PB2、pML-PB1、pML-PA、pML-NP、pML-M、pML-NS、pML-HA和pML-NA。其中HA基因已按实验设计删除了与毒力相关的连续5个碱性氨基酸残基（R-R-R-K-K）。将8个重组质粒转染293T／MDCK混合细胞单层，72h后出现明显细胞病变。收集细胞培养上清获得重组禽流感病毒，其血凝效价为1：32。结果表明，我们已构建成功重组禽流感疫苗株，命名为rH5N3。二、重组禽流感疫苗株rH5N3的生物学特性（一）抗原性分析以血凝抑制（hemagglutination inhibition，HI）试验和神经氨酸酶抑制（neuraminidase inhibition，NI）试验分析重组病毒rH5N3的抗原性，发现其血凝活性可为鸡抗A／Goose／Guangdong／1／96（H5N1）和A／Goose／HLJ／QFY／04（H5N1）抗血清完全抑制，HI效价分别为1：512和1：1024，但是不能够被抗亲本毒株A／Goose／Dalian／01（H9N2）抗血清抑制；抗参考毒株A／Duck／Germany／1215／73（H2N3）抗血清对rH5N3的NI效价为1：800，抗A／Goose／HLJ／QFY／2004抗血清对rH5N3的NI效价小于1：10。结果表明，rH5N3毒株具有符合实验设计的H5和N3亚型基因表型。（二）致病性分析用10^-1～10^-10稀释的重组株rH5N3接种SPF鸡胚（0.1ml／枚），37℃孵化48h，收集尿囊液测血凝（hemagglutination，HA）活性，按Reed-Muench法计算鸡胚半数感染剂量(EID₅₀)为2×10⁹／mL；以1×10⁸ EID₅₀剂量rH5N3接种鸡胚，培养96h鸡胚仍然存活。将1：10稀释的重组毒株rH5N3尿囊液静脉接种4周龄SPF鸡（0.1ml／只），每日观察鸡的临床表现，分别计算静脉接种致病指数（intravenous pathogenicity index，IVPI），发现10天观察期内感染鸡无任何异常或临床发病，IVPI指数等于0。结果表明，重组病毒rH5N3对鸡胚无致死性，对于鸡属于低致病性毒株。（三）生长特性分析将1：10稀释的rH5N3及其亲本毒株A／Goose／HLJ／QFY／04尿囊液各0.1ml分别接种MDCK细胞单层，37℃培养箱培养72h，血凝试验测定rH5N3、A／Goose／HLJ／QFY／04培养上清液的血凝效价分别为1：512和1：64。经绒毛尿囊腔途径分别接种10日龄SPF鸡胚，0.1mL／枚，置37℃恒温箱中孵育，无菌收集48h尿囊液，rH5N3和A／Goose／HLJ／QFY／04尿囊液血凝价分别为1：2048和1：256。表明重组病毒rH5N3哺乳细胞系和鸡胚中生长良好，为一株高产禽流感疫苗株。（四）遗传稳定性分析将1：10稀释的rH5N3尿囊液0.1ml接种10日龄SPF鸡胚，置37℃恒温箱中孵育48h，收集尿囊液继续传代，连续鸡胚传10代。对第10代rH5N3病毒进行核酸序列测定，发现其与rH5N3亲本毒株核酸序列相同，证明该疫苗株具有很好的遗传稳定性。三、重组禽流感疫苗株rH5N3的免疫效果（一）免疫效力6周龄SPF鸡随机分为不同疫苗剂量（0.1、0.3、0.6、0.9 mL／只）组和对照组（0.6 mL PBS／只），每组8只。重组病毒rH5N3经甲醛灭活后按矿物油：含毒尿囊液1：2比例制成油包水型油乳剂灭活疫苗，给SPF鸡颈部皮下注射，免疫后不同时间采血，测定其HI抗体滴度，计算抗体效价的几何均数。免疫后7天个别鸡出现低效价HI抗体；免疫后2周所有鸡都产生明显的免疫应答，即使0.1mL低剂量免疫组HI抗体效价也可达到1：128（7 log2）以上；免疫后4周HI抗体水平达到高峰，HI抗体效价可达1：256（8 log2）以上；免疫后18周HI抗体几何均数仍然大于1：16这一抗体保护临界值。表明rH5N3具有良好的免疫原性。（二）保护作用3周龄SPF鸡免疫前采血测HI抗体全部阴性。rH5N3灭活疫苗经鸡颈部皮下接种，免疫后3周采血测定HI抗体滴度，同时采用滴鼻、点眼方式进行H5N1亚型强毒株（A／Goose／Guangdong／1／96和A／Goose／HLJ／QFY／04）攻击。攻毒后的第3和第7天分别采取咽喉和泄殖腔拭子进行病毒分离与滴定，攻毒后14天采血测定攻毒后存活鸡的HI抗体水平。发现0.3 mL疫苗即能诱导产生高效价的HI抗体，几何均数在1：512（9 log2）以上。100 CLD₅₀ HPAIV攻击，rH5N3疫苗免疫鸡无临床发病或死亡，病毒分离全部阴性。对照组鸡在攻毒后2～5天全部死亡，病毒分离均为阳性。结果表明，rH5N3疫苗免疫不但能够保护鸡抵抗HPAI病毒的致死性攻击，也能够起到减少或防止病毒在鸡群中的传播的作用。本研究选择2004年国内流行的H5N1亚型HPAIV代表株作为疫苗株HA基因供体，利用反向遗传学技术拯救出全部基因片段都来自禽源的重组禽流感疫苗株rH5N3。由于删除HA基因中与毒力相关的5个连续碱性氨基酸，而使疫苗株对鸡和鸡胚的毒力完全消除；选取国内流感病毒中较少见的神经氨酸酶亚型N3作为疫苗株的分子标记，为疫苗免疫家禽和野毒感染家禽的鉴别诊断提供方便；rH5N3疫苗株具有安全、低毒的特性，与野毒株相比不仅抗原性完全一致，而且鸡胚生长性能大大改善；基因重组疫苗株不仅具有很好的遗传稳定性，而且具有良好的免疫原性，免疫动物完全能够抵抗强毒株的致死性攻击。该疫苗株的成功研制为H5N1亚型HPAI的防控提供了新的技术保障。更多还原

【Abstract】 Highly pathogenic avian influenza （HPAI） is a severe form of generalized avian influenza among domestic poultry and various other birds which is characterized by a rapid and severe course of disease and a very high mortality, which is caused by highly pathogenic avian influenza virus （HPAIV）, avian influenza virus strains of the subtypes H5 or H7. Since 2003, a widespread occurrence of HPAI has been registered in Southeast Asia, and in six countries HPAIV has also caused fatal human infections. During summer 2005, the disease has slowly spread westward. Isolated outbreaks have been reported from Kazakhstan, Russia, Romania, Turkey, Croatia and Ukraine. Avian influenza viruses are continuing to spread in waterfowl in Eurasia, even to Africa. The disease has caused enormous economical loss and threatened the health of humans.HPAIVs belong to the family of Orthomyxoviridae, which are pleomorphic, enveloped RNA viruses. Protruding from the lipid envelope are two distinct glycoproteins, the hemagglutinin （HA） and neuraminidase （NA）. HA attaches to cell surface sialic acid receptors, thereby facilitating entry of the virus into host cells. Since it is the most important antigenic determinant to which neutralizing antibodies are directed, HA represents a crucial component of current vaccines.The culling of infected poultry is the time-honored method to control or eradicate the highly pathogenic avian influenza outbreaks and also the best-known way to prevent transmission to humans. However, when the viruses are widely spread over a huge area, culling and physical containment are highly unlikely to be successful. The vaccination program is a reliable strategy in the control of prevalence of these disastrous diseases. The ideal seed virus for vaccine production is a strain of low pathogenicity that is wellmatched antigenically with the prevailing virus and capable of growing well in eggs. All H5N1 viruses isolated in China since 1996 have multiple basic amino acids in the cleavage site of HA and therefore are all either highly pathogenic or potentially highly pathogenic avian influenza viruses unsuitable for vaccine production. It is difficult to construct avian influenza virus H5 subtype vaccine by traditional methods. Plasmid-based reverse genetics is a powerful tool to generate ideal reassortant influenza vaccine candidates.In this study, the six internal genes of the high yield influenza virus A/Goose/Dalian/3/01 （H9N2）, the hemagglutinin （HA） gene of A/Goose/HLJ/QFY/ 04 （H5N1） strain and the neuraminidase gene from A/Duck/Germany/1215/73 （H2N3） reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting site between HA1 and HA2. Eight gene expressing plasmids were constructed and the recombinant virus H5N3 was reassorted by cells transfection. The chickens embryos infection and chickens challenge tests demonstrated the recombinant H5N3 （rH5N3） influenza virus is non-virulent. The rH5N3 oil-emulsified vaccine could induce hemagglutination inhibition （HI） antibodies in chickens in two weeks post-vaccination and maximum geometric mean HI titer were observed on 4～5 weeks post-vaccination and were kept during the eighteen weeks observation. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, both A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04.ⅠConstruction of the high yield avian influenza virus H5 vaccine strainThe six internal genes of the high yield influenza virus A/Goose/Dalian/3/01 （H9N2）, the hemagglutinin （HA） gene of A/Goose/HLJ/QFY/04 （H5N1） strain and the neuraminidase （NA） gene from A/Duck/Germany/1215/73 （H2N3） reference strain were amplified by RT-PCR technique. These amplified gene fragments, PB2, PB1, PA, NP, M, NS, HA and NA, were linked with pHW2000 plasmids respectively, and eight recombinant plasmids, named pML-PB2, pML-PB1, pML-PA, pML-NP, pML-M, pML-NS, pML-HA and pML-NA, were obtained. The virulence associated five continuous basic amino acids （R-R-R-K-K） of the HA gene were removed. The eight recombinant plasmids were transfected into 293T/MDCK mixed cell monolayer. Obvious cellular pathological changes could be observed after 72 hours. The hemagglutination （HA） titer of recombinant virus H5N3 （rH5N3） in the collected supernatant was 1:32. The results indicated that the recombinant vaccine strain rH5N3 whose all gene segments derived from avian influenza viruses was successfully constructed.ⅡThe biological characteristics of the recombinant avian influenza vaccine strain rH5N3Analysis of antigenicityAnalyzing the antigenicity by the hemagglutination inhibition（HI） assay and neuraminidase inhibition（NI） assay, it was found that the antibodies from chickens against A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04 could inhibit the hemagglutination activity of rH5N3 and the HI titers were 1:512 and 1:1 024 respectively, while the antibody against the parent avian influenza virus strain, A/Goose/Dalian/01（H9N2）, couldn’t inhibit the hemagglutination activity. The antibody against the reference avian influenza virus strain, A/Duck/Germany/1215/ 73（H2N3）, could acted with rH5N3, whose NI titer was 1:800, but the NI titer of the anti-A/Goose/HLJ/QFY/2004 antibody was less than 1:10. The results demonstrated that the phenotype of the recombinant avian influenza virus strain is H5 and N3 subtypes, as same as that of the experimental design.Analysis of pathogenicitySPF chicken embryonated eggs were inoculated with attenuated (10^-1～10^-10) rH5N3 virus （0.1 ml/each）. The allantoic liguids were collected and analyzed for HA activity. According to Reed-Muench method, in this study, the EID₅₀ （50% embryo infective dose） of rH5N3 is 2×10⁹/mL. The HA titer reached 1:2048 at 48 h post-vaccination （p.v.）. Embryonated SPF eggs inoculated with rH5N3 virus of 1×10⁸ EID₅₀ dose were still alive at 96 h. 4-mouth-old SPF chickens were inoculated with attenuated （1: 10） rH5N3 virus （0.1ml/each） by the intravenous route and observed for disease signs and death for 10 days. It was found that the chickens were completely normal and the IVPI is naught. This results indicated that rH5N3 virus is non-pethogenic to embryonated eggs and less pathogenic to chickens.Characteristics of growthMDCK cells and SPF chicken embryonated eggs were inoculated with attenuated allantoic fluids (10^-1) of the recombinant virus strain rH5N3 and its parent H5N1 virus strain A/Goose/HLJ/QFY/04 （0.1 ml/each）. The HA titers of rH5N3 in the collected supernatants and allantoic fluids were 1:512 and 1:2048 respectively, which are eight times of that parental virus strain A/Goose/HLJ/QFY/04. It was showed that the recombinant virus strain rH5N3 grows well in culture of mammalian cell lines and embryonated eggs, and is a high yield avian influenza vaccine strain.Genetic stability10-day-old SPF chicken embryonated eggs were inoculated with attenuated (10^-1) rH5N3 virus （0.1 ml/each）, and the allantoic fluid from infected eggs was harvested. After ten sequential passage in chicken embryonated eggs, the nucleic acid sequence recombinant virus rHSN3 didn’t change, which indicated that the recombinant virus strain rH5N3 displays high levels of genotypic stability.ⅢThe immune efficacy of the recombinant avian Influenza vaccine strain rH5N3Immune efficacySixteen six-week-old SPF chickens were disparted into two groups on average randomly. The chickens in one were injected with the vaccine of different doses （0.1, 0.3, 0.6, 0.9 mL/each） and the chickens in the other were only injected with allantoic fluid from NS-vaccinated chickens’ embryos （0.6 mL/each）. SPF chicken were vaccinated hypodermically with formalin-inactivated oil-emulsion rH5N3 vaccine, which was prepared by mixing mineral oil and allantoic fluid in the light of 1:2 proportion, and the blood of the chickens was drew at different time and detected for HI antibody titer and calculated the geometric mean of the HI antibody. The HI antibody became detectable but the titers were low in several chickens on day 7. All chickens had obvious antibody response at 2 weeks, even in the group were injected with the vaccine of low dose （0.1mL）, with the HI titer exceeding 1:128 （7 log2）. The HI antibody titer increased to 1:256 （8 log2） or higher at 4 weeks after vaccination. The geometric mean of the HI antibody titer still exceeded 1:16, which is the critical level in antibody protection.Protective efficacy3-week-old SPF chickens with negative HI antibody before immunizing were hypodermically injected with formalin-inactivated oil-emulsion rH5N3 vaccine. After 3 weeks, the chickens were detected for the titer of HI antibody in sera, and attacked with virulent avian influenza virus H5N1 subtype strains （A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04） using the method of dripping them into their noses and eyes. Oropharyneal and cloacal swabs were collected for virus isolation and titration at 3 and 7 days respectively post-attacking and detected the surviving chickens’ HI antibody titer on days 14 post-attacking. It indicated 0.3mL vaccine dose could induce the chickens to produce high titer HI antibody and the geometric mean of the HI antibody titer exceeded 1:512 （9 log2）. There was no clinic disease and death appearance and no virus separation among the chickens immunized with rH5N3 vaccine after attacking with 100 CLD₅₀ HPAI virus, but all chickens in the blank comparison group died at 2～5 days after attacking and virus separation were positive. These results indicated that rH5N3 vaccine can not only protect chickens from HPAI virus’ fatal attacking but reduce and prevent the transmission of HPAI virus among chickens.By using plasmid-based reverse genetics, the recombinant vaccine strain rH5N3, whose all gene segments derived from avian influenza viruses, was generated. Because of deletion of the virulence associated five continuous basic amino acids of the HA gene, the resulting virus is attenuated for chickens and chicken eggs. N3 neuraminidase subtype, which is infrequent in China, was selected as the molecular marker of the vaccine strain in order to distinguish vaccinated poultry and infected poultry with wild viruses conveniently. The vaccine strain rH5N3 with safety, low virulence and high yield properties improves it’s growth property in chickens’ embryos. This vaccine strain appears not only good inheritance stability but also favorable immunogenicity, and it can absolutely protect poultry from strong virulent viruses’fatal attack. The development of this vaccine strain provides us with a new tool to control and prevent the infection of avian influenza virus H5N1 subtype.更多还原