麻疹疫苗减毒机制及疫苗接种后我国麻疹流行病学特点的研究

发布时间：2018-05-28 22:54

本文选题：麻疹病毒 + 核苷酸变异　；参考：《吉林大学》2011年博士论文

【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 lgTCID_(50)/0.1ml～7.40 lgTCID_(50)/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。
[Abstract]:measles is a respiratory infectious disease caused by measles virus ( MV ) . It is one of the few viral infectious diseases that can be effectively controlled by vaccine . Although measles vaccine has been used to reduce the morbidity and mortality of measles , measles virus is a single serotype and has been considered to be genetically stable .

In order to explore the variation of measles wild strain , the effect of measles attenuated live vaccine and the anti - toxic mechanism of measles attenuated live vaccine , the following research work has been carried out :

1 . Study on variation of measles wild strain :

Forty samples of suspected measles cases from 2008 to 2009 were collected ( 32 parts of pharyngeal swab and 8 parts of urine ) . Four strains of virus were isolated successfully by Vero / SLAM cells .

The results showed that 450 nucleotides and 150 amino acid sequences were 99.11 % and 99.33 % , respectively , and the genetic distance was between 0.069 and 0.074 , and the difference of nucleotide and amino acids was 6.67 % ~ 8.22 % and 10.00 % ~ 10.67 % , respectively .

The results showed that there were no significant differences in the genetic distance between the 16 measles virus gene sequences , but there was a large variation in the genetic distance between the two groups , compared with that of the standard strain , which was similar to that of the standard strain , which showed a large variation in the genetic distance , the nucleotide level and the amino acid level .

Through the above experimental studies , it has been proved that the measles wild strain has a large change in gene level compared with the standard strain , and the measles vaccine strain is gradually accumulated and has the potential to affect the immune effect of the current measles vaccine .

2 . Research on the Anti - Virus Mechanism of measles Vaccine

Using the method of virus plaque assay , the isolated measles wild strain was purified to observe the morphological characteristics and spot speed of the virus plaque , and the virus clone strain was amplified by virus . The characteristics of the produced cell lesion were observed and the virulence of the virus was observed . The attenuated technique was used to simulate the attenuated measles vaccine prepared by measles vaccine .

The results showed that there were significant differences in the size , shape , center and edge of the plaque , and the rate of plaque formation . The results showed that the virulence of the strain decreased and the virulence of the cloned strain was not changed . The virulence of the cloned strain was not changed . The difference between wild strain and clone was suggested .

The results showed that the attenuated strain could be a mixture containing a large number of virulent strains and a small number of attenuated strains .

3 . Epidemiological study on vaccination and measles

The epidemiological data of measles in China from 1988 to present were collected . The incidence of measles vaccination in China from 1990 to 2007 and the incidence of measles in 31 provinces in China from 2003 to 2009 were analyzed by descriptive epidemiological methods , and the seasonal distribution characteristics of measles in 2004 - 2010 and the incidence of measles in Jilin Province were analyzed .

The results showed that , although the rate of measles vaccination in China has been maintained at a high level , the incidence and mortality rate of measles is still at the forefront of the notifiable infectious diseases in our country , and the incidence of measles in every province and city has been greatly delayed . The time of measles occurrence is obviously delayed , and the epidemic period of measles has not been broken , and the long - term effect of enhanced immunity needs to be further observed and studied .

In order to achieve the goal of eliminating measles , we should adjust the current measles immune strategy and immune procedure in our country , and study and develop more effective new measles vaccine to improve the immune efficacy of the vaccine .
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R186

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