北京地区流行性腮腺炎野病毒基因特征及疫苗效果评价

发布时间：2018-05-02 16:21

本文选题：流行性腮腺炎病毒 + 基因特征　；参考：《中国协和医科大学》2009年硕士论文

【摘要】： 流行性腮腺炎(腮腺炎)是由流行性腮腺炎病毒(mumps virus)引起的急性呼吸道传染病,属于我国法定的丙类传染病。患者主要是5～15岁的儿童和青少年。腮腺炎的并发症发生率较高,脑膜炎的发生率为1%～10%,青春期后男性睾丸附睾炎发生率为25%。腮腺炎病毒只有一个血清型,但有多个基因型,腮腺炎病毒SH基因的变异程度最大,因此国际上将其作为分型依据。目前已发现了12个基因型,分别命名为A～L。中国特有的流行株为F基因型。腮腺炎病毒的血凝素一神经氨酸酶(HN)是病毒主要的免疫原性蛋白。不同基因型的HN蛋白抗原性不同,其特异性抗原位点存在差异。本研究用Vero细胞和Vero/SLAM细胞从北京地区2004-2007年腮腺炎暴发和散发的107个病例的含漱液和尿液标本中分离到34株阳性分离物,经过逆转录.聚合酶链反应(RT-PCR)法等方法鉴定,共得到33株腮腺炎病毒,均来自于含漱液标本。血清学检测中,5例病毒分离阳性病例的急性期血标本IgM抗体检测阴性,证实了仅用IgM阳性作为腮腺炎实验室确诊指标会有病例漏检。北京市2000年将腮腺炎疫苗列入计划免疫,2006年开始免费接种麻疹、腮腺炎、风疹(MMR)三联疫苗,并在2006年对中小学未接种过腮腺炎疫苗的学生进行了大规模的MMR的补种。但大规模接种后,2007年的发病率没有明显下降,有免疫史的病例大幅增加。实验室2007年的病毒分离率和IgM抗体阳性率都明显低于往年。统计分析,病毒分离率和IgM抗体阳性率与病例的免疫史状况密切相关,因此提出疫苗免疫效果的问题。用RT-PCR方法扩增33株腮腺炎病毒SH基因316个核苷酸片段,进行核苷酸序列测定和分析。结果33株腮腺炎病毒均属于F基因型,疫苗株VAC-S79属于A基因型。说明近年来北京地区流行的优势株为F基因型,没有发现其他基因型的野病毒流行,也没发现基因型间的变异。但同源性分析发现,北京株与F基因型代表株氨基酸差异为4.9%,己存在到达分型临界值(5.O%)的型内差异。同时,北京株与上海株的核苷酸差异明显大于兰州株,即北京株与兰州株亲缘关系更近。在对SH蛋白氨基酸序列进行分析时发现北京株的变异程度较高,部分保守的氨基酸位点发生了变化,8位L→F,11位T→K,23位I→N,45位O→H＼L,个别在48位L＼R→P,57位L→P。提示SH蛋白结构有可能发生变化。同时,与基因分型有关的氨基酸三联体也出现变异,北京株虽都属于F基因型,却有6株为IIL,为D,I,K,L和部分基因型G特征;另有3株为ILL,2株为IVL。北京株与F基因型代表株之间氨基酸最大差异为6个氨基酸,证明F基因型属于变异程度高的基因型。同时发现部分氨基酸位点随时间逐渐出现变异的倾向。 HN蛋白基因共计1749个核苷酸,编码582个氨基酸,为病毒主要结构抗原,保守性高。本研究阳性分离株HN基因测序成功的为31株。转换为氨基酸序列后,分析它们的同源性发现不同分离株之间的差异为0～2.3%。流行株与Jeryl-Lvnn疫苗株之间的差异为4.2～5.3%,差异较大。做北京地区流行株与疫苗株的亲缘关系进化树(图18),发现流行株与Urabe株属于同一分支,而JL2、JL5和Rubini属于另一分支。所有北京地区流行株在HN蛋白基因具有降低交叉中和能力的2个位点上与疫苗株为不同的氨基酸。354位:流行株为Q(谷酰胺),疫苗株为P(脯氨酸)。356位:流行株为D(天冬氨酸),疫苗株为E(谷氨酸)。在329～340位置也有个别流行株存在差异,如:334位V→F,338位R→C/R→K。HN蛋白上有9个潜在的N-糖基化位点,实验发现北京株在464～466位置上存在NCS的N-糖基化位点,Jeryl-Lynn疫苗株此位置为NCR。本研究证实疫苗株和北京地区流行株在SH和HN蛋白上都存在较大差异,可能导致蛋白结构发生改变,从而对抗原性造成影响,因此可以初步解释现用疫苗株在人群中产生的免疫效果不好的问题。
[Abstract]:Epidemic parotitis (mumps) is an acute respiratory infection caused by mumps virus (mumps). It belongs to the legal class C infectious disease in China. The patients are mainly 5~15 year old children and adolescents. The incidence of mumps is high, the incidence of meningitis is 1% to 10%, and the incidence of testicular epididymitis in postpuberty males. 25%. mumps virus has only one serotype, but there are many genotypes, the SH gene of mumps virus has the greatest variation, so it is considered as the basis of the genotyping. 12 genotypes have been found, respectively named A to L., endemic to the F genotype in China. The hemagglutinin one neuraminidase (HN) of mumps virus is a kind of genotype. The main immunogenicity proteins of the virus. Different genotypes of HN protein have different antigenicity, and their specific antigen sites are different.
In this study, Vero cells and Vero/SLAM cells were used to separate 34 positive isolates from 107 cases of mouthwash and urine from 2004-2007 years' mumps in Beijing, and 33 parotitis viruses were obtained by reverse transcriptase polymerase chain reaction (RT-PCR) method, all from the gargle specimens. Serological examination. In the test, the IgM antibody test was negative in the acute phase of 5 cases of virus isolation, which proved that only IgM positive as the laboratory diagnosis of mumps could be missed.
In 2000, the mumps vaccine was included in the planned immunization of Beijing, and the measles, mumps and rubella (MMR) triple vaccine were vaccinated free of charge in 2006. In 2006, a massive MMR replanting was carried out for students who had not been vaccinated with mumps in primary and middle schools. However, after large-scale inoculation, the incidence of 2007 was not significantly decreased and the history of immunization was large. The virus isolation rate and the positive rate of IgM antibody in the laboratory in 2007 were significantly lower than those in previous years. Statistical analysis showed that the rate of virus isolation and the positive rate of IgM antibody were closely related to the history of the cases, so the problem of the immune effect of the vaccine was raised.
The RT-PCR method was used to amplify 316 nucleotide fragments of the 33 parotitis virus SH gene, and the nucleotide sequence was determined and analyzed. Results 33 strains of mumps virus belong to the F genotype, and the vaccine strain VAC-S79 belongs to the A genotypes. It shows that the dominant strain in Beijing area is F genotypes in recent years, and there is no other genotypic wild virus epidemic and no other genotypes. The variation between the genotypes was found, but the homology analysis found that the difference in the amino acid difference between the Beijing and the F genotypes was 4.9%, and there was a difference in the typing critical value (5.O%). At the same time, the nucleotide difference between the Beijing strain and the Shanghai strain was significantly greater than that of the Lanzhou strain, that is, the relationship between the Beijing strain and the Lanzhou plant was closer.
In the analysis of the amino acid sequence of the SH protein, it was found that the variation in the Beijing strain was high, and some conserved amino acid loci were changed, 8 bit L, F, 11 T to K, 23 I to N, 45 O to H / L, and 48 bits of L, R, and L, 57 bits of protein structure may change. At the same time, the amino acid triad related to genotyping was found. There are also variations in the body, although all Beijing strains belong to the F genotype, but there are 6 strains of IIL, which are D, I, K, L and partial genotype G; the other 3 are ILL, 2 are IVL. Beijing and F genotypes with the maximum amino acid difference, which proves that the F genotype belongs to the genotype with high variation, and some amino acid sites are found with time. A tendency to change gradually.
The HN protein gene totalling 1749 nucleotides, encoding 582 amino acids, is the main structural antigen of the virus, and has high conservatism. 31 strains of HN gene were sequenced successfully in this study. After converting to amino acid sequences, the difference between different isolates was found to be the difference between 0 2.3%. epidemic strains and Jeryl-Lvnn vaccine strains. The difference was 4.2 to 5.3%. The phylogenetic tree of the epidemic strains and the vaccine strains in Beijing region (Figure 18) found that the epidemic and Urabe strains belonged to the same branch, while JL2, JL5 and Rubini belonged to another branch.
All Beijing epidemic strains in the 2 loci of the HN protein gene have the ability to reduce cross neutralization with the vaccine strain of different amino acid.354: the epidemic strain is Q (Gu Xianan), the vaccine strain is P (proline).356 bit: the epidemic strain is D (aspartic acid), the vaccine strain is E (glutamic acid). In the 329~340 position, there are also individual strains, such as 33 There were 9 potential N- glycosylation sites on 4 V - F and 338 R - C/R - K.HN proteins. The experiment found that there was a N- glycosylation site of NCS in the 464~466 position of Beijing strain, and the Jeryl-Lynn vaccine strain was NCR..
This study confirmed that there was a great difference between the SH and the HN proteins in the vaccine strain and the Beijing area. It may lead to the change of protein structure and the effect on the progenicity. Therefore, the problem of the immune effect produced by the present vaccine strain in the population can be explained preliminarily.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R450;R186

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