【作者】 时景伟；

【导师】 李凡；

【作者基本信息】 吉林大学 ， 病原生物学， 2011， 博士

【摘要】 麻疹是由麻疹病毒（Measles Virus,MV）引起的一种呼吸道传染病,是少数可以用疫苗有效控制的病毒性传染病之一。虽然麻疹疫苗的使用使麻疹的发病率和死亡率大幅度降低,但是至今,麻疹仍是严重威胁儿童健康和生命安全的全球（特别是发展中国家）公共卫生问题。麻疹病毒为单一血清型,一直被认为遗传性较为稳定,但是近年来分离的麻疹野毒株与上个世纪60年代分离的麻疹病毒比较,在基因水平上已经有了较大的变异,麻疹疫情的回升是否与病毒的变异有关?麻疹野毒株的变异是否会造成麻疹疫苗保护能力的降低?是否需要开发研制新型麻疹疫苗以达到最终在全世界消除麻疹的目标?这些已经成为世界各国学者广泛关注的话题。为探讨麻疹野毒株的变异情况、现行麻疹减毒活疫苗的免疫效果以及麻疹减毒活疫苗的减毒机制等问题,本研究开展了下述研究工作:1.麻疹野毒株变异情况研究:收集2008～2009年麻疹疑似病例标本40份（咽拭子32份,尿液8份）,用Vero/SLAM细胞成功分离到4株病毒。经典型的细胞融合病变、麻疹单克隆抗体和N基因羧基末端450个核苷酸扩增检测,鉴定4株病毒全部为麻疹病毒。应用逆转录-聚合酶链反应（reverse transcript polymerase chain reaction,RT-PCR）对16株麻疹野毒株（本实验分离的4株麻疹野毒株和本实验室保存的2005～2007年吉林省麻疹野毒株12株）的N基因羧基（-COOH）末端450个核苷酸片段进行扩增,测序后使用MEGA 4.0软件对测定结果与国际标准株Edmonston株、疫苗株沪-191株和长-47株的对应序列进行基因亲缘性分析。结果显示:16株麻疹野毒株之间N基因羧基端450个核苷酸和150个氨基酸同源性分别为99.11%和99.33%,其组内遗传距离小于0.009;16株麻疹野毒株与标准株Edmonston株比较,组间遗传距离介于0.069～0.074之间,核苷酸和氨基酸的差异率分别为6.00%～7.55%和8.00%～8.67%;16株麻疹野毒株与疫苗株沪-191株比较,遗传距离为0.084～0.091,核苷酸和氨基酸的差异率分别为6.67%～8.22%和10.00%～10.67%;16株麻疹野毒株与疫苗株长-47株比较,遗传距离为0.071～0.076,核苷酸和氨基酸的差异率分别为6.22%～6.67%和8.67%～9.33%。此结果说明16株麻疹野病毒基因序列差异较小,其组内遗传距离上差异不大,而与标准株Edmonston株、疫苗株沪-191株和长-47株相比,组间遗传距离差异较大,核苷酸水平和氨基酸水平上都出现了较大的变异。通过以上实验研究证明,现今的麻疹野毒株与标准株Edmonston株、麻疹疫苗株相比,在基因水平上发生了较大的变化,变异逐渐被累积并且有可能影响到现行麻疹疫苗的免疫效果。2.麻疹疫苗减毒机制的研究应用病毒蚀斑实验的方法对所分离的麻疹野毒株进行纯化,观察病毒蚀斑的形态特征和出斑速度,并对挑斑后的病毒克隆株进行病毒扩增,观察产生的细胞病变特点,测定其毒力。模拟麻疹疫苗制备的减毒技术,将分离于麻疹患者标本的麻疹野毒株和经纯化后的麻疹克隆株进行减毒培养,观察减毒效果。实验结果显示,同一株麻疹野毒株所产生的蚀斑,其斑块的大小、形状、中心和边缘特征以及出斑的速度均存在明显的差异,经过挑斑后得到的各个克隆株,其毒力从6.17 lgTCID₅₀/0.1ml～7.40 lgTCID₅₀/0.1ml之间不等,挑斑扩增后出现细胞病变的时间和病变的特点也有较大的差别。通过减毒实验对2株野毒株和来源于同一麻疹野毒株的6株克隆株进行减毒培养,结果显示,麻疹野毒株的毒力有所下降,而克隆株的毒力未见明显变化。麻疹病毒的毒力下降只发生在野毒株的减毒过程,克隆株的毒力l并无变化,提示野毒株与克隆株之间存在差异。野毒株毒力的降低主要发生在病毒由原代人羊膜细胞（HAM）向原代鸡胚细胞（CEC）的适应过程中,而病毒由原代人胚肾细胞（HK）适应HAM的过程和病毒在CEC上连续传代的过程均未见毒力的明显变化。结果提示,麻疹野毒株可能是一个混合体,其中包含了大量的强毒株和少量的弱毒株,CEC对病毒起到筛选作用,将能适应CEC的弱毒株保留下来,强毒株由于不能很好的适应CEC而被淘汰,弱毒株在CEC上的传代起到了将弱毒株大量扩增的目的。3.疫苗接种与麻疹流行病学研究本研究收集了1988年至今我国麻疹的流行病学资料,采用描述性流行病学方法分析了1990～2007年我国麻疹疫苗接种率与全国发病率变化的情况和2003～2009年我国31个省的发病率差异情况,并且分析了2004～2010年全国麻疹的发病季节分布特点及吉林省强化免疫后麻疹的发病情况。结果显示,虽然我国麻疹疫苗的接种率一直维持较高的水平,但麻疹的发病率与死亡率在我国法定传染病中仍居于前列,每隔2～3年就会出现一次小规模的暴发流行,并且各个省市自治区的麻疹发病率相差悬殊;麻疹患病出现高峰的时间明显推迟,麻疹发病的季节性已经发生了较大的变化;加强免疫可以在短期内收到较好的预防效果,但麻疹的流行周期并没有被强化免疫所打破,强化免疫的长期效果有待进一步观察和研究。本研究提示,有较好的计划免疫基础并不能有效的避免麻疹的暴发,为达到消除麻疹的目标,除继续加强麻疹野病毒的血清学和分子流行病学监测,扎实开展儿童免疫规划基础工作外,还需对我国现行的麻疹免疫策略及免疫程序进行调整,适时研究开发更有效的新型麻疹疫苗提高疫苗的免疫效力。更多还原

【Abstract】 Measles is a kind of respiratory infectious diseases caused by the measles virus, and it is one of limited kinds of infectious diseases that could be effectively controlled via vaccination. Although the morbidity and mortality caused by measles were significantly reduced by the widely use of measles vaccines, measles is still a problem of public health and still threats to the children’s health and safety all over the world. Measles virus is single serotype, and its heredity has been considered relatively stable. However, measles strains isolated in recent years had a great genetic variation compared to the ones which were isolated in the 60s of last century. Whether the elevation of prevalence of measles has significant correlations with the variations of measles? Whether the variations of wild measles strains will result in reduction in protection of measles vaccine? Is there a need of developing new measles vaccines to achieve the ultimate goal of measles elimination in the world? All of these topics above have attracted wide attention from scholars around the world.To investigate the variations of wild strains of measles, the effects of current live attenuated measles vaccine and the mechanisms of live attenuated measles vaccine and other issues, this research carried out the following works:1. Variations of wild measles strains:40 samples of suspected cases of measles from 2008 to 2009 (32 of throat swabs and 8 of urine samples) were collected and 4 virus strains were successfully isolated by Vero / SLAM cells. According to results of cross-neutralization assays and the sequences of the 450 nucleotides carboxyl(-COOH) terminus of N gene, all of 4 strains of the virus were identified as measles virus.The 450 nucleotides fragments of carboxyl end of N gene on 16 wild measles strains(4 wild strains isolated in this research and 12 wild strains stored in our laboratory which were isolated in Jilin Province from 2005 to 2007) were amplified with RT-PCR. Software MEGA4.0 was applied to analyze the genetic affinity of corresponding sequences between test results and international standard strain Edmonston strain, Shanghai-191 and Changchun-47 vaccine strains. The results showed that the sequence homologies of 450 nucleotides and 150 amino acid in carboxyl-terminal of N gene were respectively 99.11% and 99.33%, the group genetic distance is less than 0.009; The genetic distance among 16 wild strains and Edmonston standard strain was 0.069 ~ 0.074, and the differences in nucleotide and amino acid were 6.00% ~ 7.55% and 8.00%~8.67%, respectively; The genetic distance among 16 wild strains and Shanghai-191 vaccine strain was 0.084 ~ 0.091, and the differences in nucleotide and amino acid were 6.67% ~ 8.22% and 10.00% ~ 10.67%, respectively; The genetic distance among 16 wild strains and Changchun-47 vaccine strain was 0.071 ~ 0.076 and the differences in nucleotide and amino acid were 6.22%~ 6.67% and 8.67%~9.33%, respectively.The results showed that the 16 strains of measles virus had neither significant genome sequences differences nor evident group genetic distance differences each other. But the 16 strains of measles virus had evident differences in group genetic distance and great variations in nucleotide and amino acid levels compared with Edmonston standard strain, Shanghai -191 and Changchun-47 vaccine strains.These experiments above showed that great variations in genetic level existed in the wild type strains compared with Edmonston strain and measles vaccine strain The variations accumulated gradually and might affect the effects of the current measles vaccine.2. Research of the mechanism of measles vaccine attenuation.Wild type measles strains were purified via the method of virus plaque experiments, and we observed the morphological characteristics of the virus plaque and the spot rates, then picked the spot and amplified the clone of the virus. Consequently we observed the characteristics of produced cytopathic and determined their virulence. In this research, we simulated the attenuated measles vaccine cultivation technology, cultured the attenuated measles wild strains which was separated from the specimens of the patients and the purified measles cloning strains to see the attenuation result.The results showed that the plaques generated from the same wild strains of measles had different size, shape, center and edge features as well as the spot rate. After picking out from the spot of each clone strains, different strains had different virulence ranged, which is 6.17lgTCID50/0.1ml~7.40lgTCID50/0.1ml. The characteristics and appears time of cell lesions were different. These results showed that the virulence of wild strains of measles had declined, while the clone strains had no significant change in virulence, which revealed that there were differences between clone strains and wild-type strains.Reduction of the virulence of wild strains occurred in the process of adaptation of virus from Primary human amnion cells(HAM) to Primary chick embryo cells(CEC). But there were no obvious changing neither in the process of virus adapts primary human embryonic kidney cells(HK) to HAM nor in the process of continuous reproduction on CEC. The result showed that one wild type measles strain may be a hybrid, which contained a large number of virulent strains and a small amount of attenuated strains, CEC played a selection role of the virus, the attenuated strains which were adapted to CEC would be retained while virulent strain which were not well adapted to CEC were eliminated. The reproduction of attenuated strain on CEC had amplified the attenuated strain to a large number.3. Vaccination and Epidemiological studies of measlesWe collected the epidemiological data of measles since 1988 in China and then analysed the circumstances of the rate of measles vaccination and the changing of national incidence rates in 1990~2007 and the differences of incidence in 31 provinces in China in 2003~2009 by the way of descriptive epidemiology. And then, we analysed the characteristics of distributions of measles’seasonal incidence of nationwide in 2004~2010 and the incidence of measles after immunization in Jilin Province.The results indicated that although the rate of measles vaccination had remained high level in China, however measles was still one of the leading legal infectious diseases in China. There would be a small-scale outbreak every 2 to 3 years, and there were significant differences in the incidence of measles in different provinces. The peak time of the measles disease was delayed obviously and seasonal incidence of the measles disease had changed greatly. Strengthening the vaccination might soon receive nice preventive effect; however the epidemic cycle of measles had not been broken through the Strengthened vaccination, long-term effects of vaccination needed further observation and research.This study suggests that good basis of immunization through vaccination can’t effectively prevent the outbreaks of measles. To achieve the goal of the elimination of measles, we should continue strengthening the epidemiological surveillance of the serological and molecular epidemiology of measles virus, as well as carry out the basic works of children’s immunization and vaccination. More over, checking and adjusting the current strategies and immunization programs in our country, developing new measles vaccines in time are needed to improve the efficacy of the measles vaccine.更多还原