本文選題：麻疹病毒 + 基因型　； 參考：《昆明醫(yī)學(xué)院》2008年碩士論文

【摘要】： 目的麻疹是一種世界性分布的、主要感染兒童的急性、高度傳染性的疾病。盡管在過去40多年具備了完全和有效的疫苗,但是麻疹仍是造成發(fā)展中國家兒童死亡的重要原因。2004年(具有統(tǒng)計數(shù)字的最近年份)全球有454000人死于麻疹,其中多數(shù)是兒童。WTO認為,通過核查麻疹病毒的基因型,可以判斷新發(fā)病例是否預(yù)示新的流行。因此,對于麻疹病毒的分子流行病學(xué)監(jiān)測研究,是麻疹控制工作中實驗室方面最重要的任務(wù)之一。本課題通過對昆明地區(qū)麻疹病毒的基因型和分子流行病學(xué)研究,了解本研究中麻疹病毒的變異情況,以及與麻疹患者臨床表現(xiàn)的相關(guān)性,為麻疹監(jiān)測和防治提供依據(jù)。 方法采集38例麻疹患兒急性傳染期靜脈血2ml,應(yīng)用酶聯(lián)免疫吸附試驗(Enzyme-linked immunosorbent assay,ELISA)檢測麻疹病毒血清IgM抗體;同時收集其中30份麻疹患兒的尿液標(biāo)本,運用逆轉(zhuǎn)錄—聚合酶鏈反應(yīng)(ReverseTranscription-Polymerase Chain Reaction,RT-PCR)對尿液標(biāo)本中所分離到的麻疹野病毒株的核蛋白(N)基因羧基末端的516個核苷酸進行雙向擴增,對擴增產(chǎn)物進行核苷酸序列測定和分析,并與麻疹病毒基因庫中的參考株進行比較,然后以核蛋白基因羧基末端的543個核苷酸片段構(gòu)建基因親緣性關(guān)系樹,進行核苷酸、氨基酸同源性分析。同時收集麻疹患兒的臨床流行病資料,進行臨床流行病學(xué)分析。 結(jié)果 1.38例麻疹患兒的血清麻疹I(lǐng)gM抗體均為陽性,陽性率100%。從30份麻疹患兒尿液標(biāo)本中分離到7株麻疹野病毒,陽性檢出率為23.33%。麻疹患兒出疹后6天的尿液標(biāo)本中仍可檢出麻疹病毒。 2.從分離到的7株麻疹野病毒株中隨機選取4株,對這4株麻疹野病毒的核蛋白(N)羧基末端的516個核苷酸進行序列測定和分析,并與Genebank中的麻疹病毒的23個參考株進行基因親緣性關(guān)系比較,證實該4株麻疹野病毒均為H基因型。 3.將所分離到的4株麻疹野病毒N基因羧基末端的543個核苷酸序列與WHO中國流行的H_1型參考株China93-7、H_(1a)型參考株China93-2、H_(1b)型參考株China94-7、H_2型參考株China94-1的對應(yīng)序列做基因親緣性關(guān)系分析,分別構(gòu)建核苷酸序列親緣關(guān)系樹和氨基酸序列親緣關(guān)系樹,所分離到的昆明地區(qū)麻疹野病毒均為H_(1a)基因型。 4.4株麻疹病毒核苷酸序列與WHO的H_1、H_2基因型參考株China93-7、China94-1所對應(yīng)的核苷酸序列的遺傳距離分別為0.031～0.047和0.332～0.398,而與H_(1a)的遺傳距離為0.010～0.033。4株麻疹野病毒組內(nèi)核苷酸遺傳距離為0.005～0.040。與WHO的H_1、H_2基因型參考株China93-7、China94-1所對應(yīng)的氨基酸序列的遺傳距離分別為0.088～0.149和0.205～0.282,而與H_(1a)的遺傳距離為0.003～0.066。4株麻疹野病毒組內(nèi)氨基酸遺傳距離為0.010～0.073。說明昆明地區(qū)的麻疹野病毒株主要為H_(1a)基因型。 5.4株麻疹野病毒N基因羧基末端543個核苷酸的同源性為96%～98.9%,核苷酸的差異為6～22個堿基;氨基酸的同源性為95.6%～99.6%。與H_1標(biāo)準(zhǔn)株China93—7的核苷酸同源性為95.9%～99.7%,氨基酸同源性為89.9%～94.8%。與H_(1a)標(biāo)準(zhǔn)株China93—2的核苷酸同源性為96.9%～99.8%,氨基酸同源性為94.8%～99.7%。 6.本次研究中,麻疹患兒最小年齡為51天,最大11歲,≤2歲的患兒共30人,占本次研究總病人數(shù)的78.95%。說明麻疹高發(fā)年齡是≤2歲的嬰幼兒;男女患兒的比例為1.53:1,男性患兒多于女性患兒。 7.從麻疹患兒尿液標(biāo)本中分離到的7株麻疹野病毒株中隨機選取4株,進行序列測定和分析表明均為H_(1a)基因型,但這4名患兒的臨床表現(xiàn)差異很大(見表8),表明麻疹患兒的臨床表現(xiàn)和麻疹病毒的基因型無明顯相關(guān)性。 8.本次研究中的麻疹患兒均為散發(fā)病例,未發(fā)現(xiàn)爆發(fā)流行的病例。所收集的病例中昆明市區(qū)以五華區(qū)和西山區(qū)病例最多,周邊地區(qū)以祿勸患者病例數(shù)最多,病人數(shù)均為7例。 9.38例麻疹患兒中,未免疫和免疫史不祥的病例數(shù)占總病人數(shù)的81.58%。 結(jié)論 1.目前昆明地區(qū)流行的麻疹病毒優(yōu)勢基因型為H_(1a)基因型,與云南省2005年的本土流行株基因型一致,未發(fā)現(xiàn)有明顯的核苷酸和氨基酸變異。未檢測到H_(1b)和H_2基因型。 2.患兒的臨床表現(xiàn)與麻疹病毒的基因型無明顯相關(guān)性。 3.麻疹患兒的年齡跨度大,但以≤2歲的嬰幼兒為主,且以未接種過麻疹疫苗的患兒居多。
[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.

【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R181.3;R725.1

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