昆明地区麻疹病毒的分子流行病学研究

发布时间：2018-05-12 22:44

本文选题：麻疹病毒 + 基因型　；参考：《昆明医学院》2008年硕士论文

【摘要】： 目的麻疹是一种世界性分布的、主要感染儿童的急性、高度传染性的疾病。尽管在过去40多年具备了完全和有效的疫苗,但是麻疹仍是造成发展中国家儿童死亡的重要原因。2004年(具有统计数字的最近年份)全球有454000人死于麻疹,其中多数是儿童。WTO认为,通过核查麻疹病毒的基因型,可以判断新发病例是否预示新的流行。因此,对于麻疹病毒的分子流行病学监测研究,是麻疹控制工作中实验室方面最重要的任务之一。本课题通过对昆明地区麻疹病毒的基因型和分子流行病学研究,了解本研究中麻疹病毒的变异情况,以及与麻疹患者临床表现的相关性,为麻疹监测和防治提供依据。方法采集38例麻疹患儿急性传染期静脉血2ml,应用酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测麻疹病毒血清IgM抗体;同时收集其中30份麻疹患儿的尿液标本,运用逆转录—聚合酶链反应(ReverseTranscription-Polymerase Chain Reaction,RT-PCR)对尿液标本中所分离到的麻疹野病毒株的核蛋白(N)基因羧基末端的516个核苷酸进行双向扩增,对扩增产物进行核苷酸序列测定和分析,并与麻疹病毒基因库中的参考株进行比较,然后以核蛋白基因羧基末端的543个核苷酸片段构建基因亲缘性关系树,进行核苷酸、氨基酸同源性分析。同时收集麻疹患儿的临床流行病资料,进行临床流行病学分析。结果 1.38例麻疹患儿的血清麻疹IgM抗体均为阳性,阳性率100%。从30份麻疹患儿尿液标本中分离到7株麻疹野病毒,阳性检出率为23.33%。麻疹患儿出疹后6天的尿液标本中仍可检出麻疹病毒。 2.从分离到的7株麻疹野病毒株中随机选取4株,对这4株麻疹野病毒的核蛋白(N)羧基末端的516个核苷酸进行序列测定和分析,并与Genebank中的麻疹病毒的23个参考株进行基因亲缘性关系比较,证实该4株麻疹野病毒均为H基因型。 3.将所分离到的4株麻疹野病毒N基因羧基末端的543个核苷酸序列与WHO中国流行的H_1型参考株China93-7、H_(1a)型参考株China93-2、H_(1b)型参考株China94-7、H_2型参考株China94-1的对应序列做基因亲缘性关系分析,分别构建核苷酸序列亲缘关系树和氨基酸序列亲缘关系树,所分离到的昆明地区麻疹野病毒均为H_(1a)基因型。 4.4株麻疹病毒核苷酸序列与WHO的H_1、H_2基因型参考株China93-7、China94-1所对应的核苷酸序列的遗传距离分别为0.031～0.047和0.332～0.398,而与H_(1a)的遗传距离为0.010～0.033。4株麻疹野病毒组内核苷酸遗传距离为0.005～0.040。与WHO的H_1、H_2基因型参考株China93-7、China94-1所对应的氨基酸序列的遗传距离分别为0.088～0.149和0.205～0.282,而与H_(1a)的遗传距离为0.003～0.066。4株麻疹野病毒组内氨基酸遗传距离为0.010～0.073。说明昆明地区的麻疹野病毒株主要为H_(1a)基因型。 5.4株麻疹野病毒N基因羧基末端543个核苷酸的同源性为96%～98.9%,核苷酸的差异为6～22个碱基;氨基酸的同源性为95.6%～99.6%。与H_1标准株China93—7的核苷酸同源性为95.9%～99.7%,氨基酸同源性为89.9%～94.8%。与H_(1a)标准株China93—2的核苷酸同源性为96.9%～99.8%,氨基酸同源性为94.8%～99.7%。 6.本次研究中,麻疹患儿最小年龄为51天,最大11岁,≤2岁的患儿共30人,占本次研究总病人数的78.95%。说明麻疹高发年龄是≤2岁的婴幼儿;男女患儿的比例为1.53:1,男性患儿多于女性患儿。 7.从麻疹患儿尿液标本中分离到的7株麻疹野病毒株中随机选取4株,进行序列测定和分析表明均为H_(1a)基因型,但这4名患儿的临床表现差异很大(见表8),表明麻疹患儿的临床表现和麻疹病毒的基因型无明显相关性。 8.本次研究中的麻疹患儿均为散发病例,未发现爆发流行的病例。所收集的病例中昆明市区以五华区和西山区病例最多,周边地区以禄劝患者病例数最多,病人数均为7例。 9.38例麻疹患儿中,未免疫和免疫史不祥的病例数占总病人数的81.58%。结论 1.目前昆明地区流行的麻疹病毒优势基因型为H_(1a)基因型,与云南省2005年的本土流行株基因型一致,未发现有明显的核苷酸和氨基酸变异。未检测到H_(1b)和H_2基因型。 2.患儿的临床表现与麻疹病毒的基因型无明显相关性。 3.麻疹患儿的年龄跨度大,但以≤2岁的婴幼儿为主,且以未接种过麻疹疫苗的患儿居多。
[Abstract]:Objective measles is a worldwide distribution of acute, highly contagious diseases that mainly infect children. Although a complete and effective vaccine has been available over the past 40 years, measles is still an important cause of death in children in developing countries.2004 (in the latest year of Statistics) 454000 people have died of measles in the world and many of them have died of measles. The number of children.WTO believes that by checking the genotypes of the measles virus, it is possible to determine whether new cases predict a new epidemic. Therefore, the molecular epidemiological monitoring of measles virus is one of the most important tasks in the laboratory of measles control. This topic is based on the genotype and molecular flow of measles virus in the Kunming region. In order to provide basis for measles surveillance and prevention, we studied the variation of measles virus and its correlation with clinical manifestations in measles patients.
Methods the venous blood 2ml of 38 cases of measles was collected and the serum IgM antibody of measles virus was detected by Enzyme-linked immunosorbent assay (ELISA), and the urine samples of 30 children with measles were collected, and the reverse transcription polymerase chain reaction (ReverseTranscription-Polymerase Chain Reaction, RT) was used. -PCR) double amplification of 516 nucleotides at the end of the nucleoprotein (N) gene of the wild virus strain of the measles virus strains isolated from the urine specimen, assay and analyze the nucleotide sequence of the amplified products, and compare with the reference strain of the measles virus gene bank, and then construct the 543 nucleotide fragments of the carboxyl terminal of the nuclear protein gene. The genetic relationship tree was used to carry out nucleotide and amino acid homology analysis. Meanwhile, the clinical epidemiological data of measles children were collected, and clinical epidemiological analysis was carried out.
Result
The serum measles IgM antibody in 1.38 cases of measles was positive, and the positive rate was 100%. from 30 samples of measles children's urine, 7 measles wild viruses were isolated. The positive rate of the positive rate was the measles virus in the urine specimens of the measles children 6 days after the eruption of 23.33%. measles.
2. randomly selected 4 strains of measles wild virus strains isolated from 7 strains of measles virus, and analyzed 516 nucleotides of the nucleoprotein (N) carboxyl terminal of the 4 measles virus, and compared the genetic relationship with 23 reference strains of measles virus in Genebank. The 4 measles wild viruses were all H genotypes.
3. nucleotide sequence of the 543 nucleotide sequences of the N gene of 4 measles wild viruses isolated from WHO, China93-7, H_ (1a) reference strain China93-2, H_ (1b) type reference strain China94-7, and H_2 type reference strain China94-1 were analyzed, and the genetic relationship tree of nucleotide sequence was constructed. The amino acid sequence phylogenetic tree was isolated from the wild measles virus in Kunming area of H_ (1a) genotype.
The genetic distance between 4.4 nucleotide sequences of measles virus and WHO H_1, H_2 genotype reference strain China93-7 and China94-1 was 0.031 to 0.047 and 0.332 to 0.398 respectively, while the genetic distance from H_ (1a) was 0.005 to 0.040. and H_1, H_2 genotypes were 0.005 to 0.040. and WHO. The genetic distance of the amino acid sequence corresponding to the reference strain China93-7, China94-1 was 0.088 to 0.149 and 0.205 to 0.282 respectively, while the genetic distance from H_ (1a) from 0.003 to 0.066.4 strain of the wild virus group was 0.010 to 0.073., indicating that the Ma Zhenye strain of the Kunming region was mainly the H_ (1a) genotype in the Kunming region.
The homology of the carboxyl terminal 543 nucleotides of the 5.4 measles wild virus N gene is 96% to 98.9%, and the nucleotide difference is 6~22 bases. The homology of the amino acid is 95.9% to 99.7% and the amino acid homology is 89.9% to 94.8%. and H_ (1a) standard strain China93 2 nucleotides, the homology of amino acids is 95.6% to the H_1 standard strain. The homology is 96.9% to 99.8%, and the amino acid homology is 94.8% ~ 99.7%..
6. in this study, the minimum age of children with measles was 51 days, the maximum age of 11 years old and less than 2 years old, which accounted for 30 children, accounting for the total number of diseases in this study, 78.95%. showed that the age of high incidence of measles was less than 2 years old; the proportion of children in boys and girls was 1.53:1, and the male children were more than those of women.
7. of the 7 measles wild virus strains isolated from the urine specimens of measles children, 4 strains were selected randomly. Sequence determination and analysis showed that all of them were H_ (1a) genotypes, but the clinical manifestations of these 4 children were very different (see Table 8), indicating that there was no significant correlation between the clinical manifestations of measles and the genotypes of measles.
8. in this study, all cases of measles were sporadic cases, and no outbreak of epidemic cases were found. Among the cases in Kunming, the most cases were in Wuhua and Xishan District, and the number of patients in the surrounding area was the most in Luquan, and the number of patients were 7 cases.
In 9.38 measles children, the number of unimmunized and immunomomies accounts for 81.58%. of the total number of measles.
conclusion
1. the epidemic of measles virus dominant genotypes in Kunming area is H_ (1a) genotype, which is consistent with the genotypes of local epidemic strains in Yunnan province in 2005. There is no obvious nucleotide and amino acid variation. H_ (1b) and H_2 genotypes are not detected.
2. there was no significant correlation between clinical manifestations and genotype of measles virus.
3. measles children have a large age span, but mainly infants and young children younger than 2 years old.

【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R181.3;R725.1

【参考文献】