本文選題：流行性腮腺炎病毒 + 基因特征�。� 參考：《中國協(xié)和醫(yī)科大學》2009年碩士論文

【摘要】： 流行性腮腺炎(腮腺炎)是由流行性腮腺炎病毒(mumps virus)引起的急性呼吸道傳染病,屬于我國法定的丙類傳染病�；颊咧饕�5～15歲的兒童和青少年。腮腺炎的并發(fā)癥發(fā)生率較高,腦膜炎的發(fā)生率為1%～10%,青春期后男性睪丸附睪炎發(fā)生率為25%。腮腺炎病毒只有一個血清型,但有多個基因型,腮腺炎病毒SH基因的變異程度最大,因此國際上將其作為分型依據(jù)。目前已發(fā)現(xiàn)了12個基因型,分別命名為A～L。中國特有的流行株為F基因型。腮腺炎病毒的血凝素一神經(jīng)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,其特異性抗原位點存在差異。 本研究用Vero細胞和Vero/SLAM細胞從北京地區(qū)2004-2007年腮腺炎暴發(fā)和散發(fā)的107個病例的含漱液和尿液標本中分離到34株陽性分離物,經(jīng)過逆轉錄.聚合酶鏈反應(RT-PCR)法等方法鑒定,共得到33株腮腺炎病毒,均來自于含漱液標本。血清學檢測中,5例病毒分離陽性病例的急性期血標本IgM抗體檢測陰性,證實了僅用IgM陽性作為腮腺炎實驗室確診指標會有病例漏檢。 北京市2000年將腮腺炎疫苗列入計劃免疫,2006年開始免費接種麻疹、腮腺炎、風疹(MMR)三聯(lián)疫苗,并在2006年對中小學未接種過腮腺炎疫苗的學生進行了大規(guī)模的MMR的補種。但大規(guī)模接種后,2007年的發(fā)病率沒有明顯下降,有免疫史的病例大幅增加。實驗室2007年的病毒分離率和IgM抗體陽性率都明顯低于往年。統(tǒng)計分析,病毒分離率和IgM抗體陽性率與病例的免疫史狀況密切相關,因此提出疫苗免疫效果的問題。 用RT-PCR方法擴增33株腮腺炎病毒SH基因316個核苷酸片段,進行核苷酸序列測定和分析。結果33株腮腺炎病毒均屬于F基因型,疫苗株VAC-S79屬于A基因型。說明近年來北京地區(qū)流行的優(yōu)勢株為F基因型,沒有發(fā)現(xiàn)其他基因型的野病毒流行,也沒發(fā)現(xiàn)基因型間的變異。但同源性分析發(fā)現(xiàn),北京株與F基因型代表株氨基酸差異為4.9%,己存在到達分型臨界值(5.O%)的型內(nèi)差異。同時,北京株與上海株的核苷酸差異明顯大于蘭州株,即北京株與蘭州株親緣關系更近。 在對SH蛋白氨基酸序列進行分析時發(fā)現(xiàn)北京株的變異程度較高,部分保守的氨基酸位點發(fā)生了變化,8位L→F,11位T→K,23位I→N,45位O→H＼L,個別在48位L＼R→P,57位L→P。提示SH蛋白結構有可能發(fā)生變化。同時,與基因分型有關的氨基酸三聯(lián)體也出現(xiàn)變異,北京株雖都屬于F基因型,卻有6株為IIL,為D,I,K,L和部分基因型G特征;另有3株為ILL,2株為IVL。北京株與F基因型代表株之間氨基酸最大差異為6個氨基酸,證明F基因型屬于變異程度高的基因型。同時發(fā)現(xiàn)部分氨基酸位點隨時間逐漸出現(xiàn)變異的傾向。 HN蛋白基因共計1749個核苷酸,編碼582個氨基酸,為病毒主要結構抗原,保守性高。本研究陽性分離株HN基因測序成功的為31株。轉換為氨基酸序列后,分析它們的同源性發(fā)現(xiàn)不同分離株之間的差異為0～2.3%。流行株與Jeryl-Lvnn疫苗株之間的差異為4.2～5.3%,差異較大。做北京地區(qū)流行株與疫苗株的親緣關系進化樹(圖18),發(fā)現(xiàn)流行株與Urabe株屬于同一分支,而JL2、JL5和Rubini屬于另一分支。 所有北京地區(qū)流行株在HN蛋白基因具有降低交叉中和能力的2個位點上與疫苗株為不同的氨基酸。354位:流行株為Q(谷酰胺),疫苗株為P(脯氨酸)。356位:流行株為D(天冬氨酸),疫苗株為E(谷氨酸)。在329～340位置也有個別流行株存在差異,如:334位V→F,338位R→C/R→K。HN蛋白上有9個潛在的N-糖基化位點,實驗發(fā)現(xiàn)北京株在464～466位置上存在NCS的N-糖基化位點,Jeryl-Lynn疫苗株此位置為NCR。 本研究證實疫苗株和北京地區(qū)流行株在SH和HN蛋白上都存在較大差異,可能導致蛋白結構發(fā)生改變,從而對抗原性造成影響,因此可以初步解釋現(xiàn)用疫苗株在人群中產(chǎn)生的免疫效果不好的問題。
[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.

【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R450;R186

【參考文獻】

相關期刊論文 前10條

1 楊文震,陳志慧;腮腺炎病毒的基因分型及當前疫苗的免疫效果[J];國外醫(yī)學(預防、診斷、治療用生物制品分冊);2005年02期

2 崔霞,李香玉;流行性腮腺炎病人并發(fā)癥相關因素的調(diào)查[J];護理學雜志;2002年01期

3 仲兆軍;一起接種腮苗后發(fā)生的流行性腮腺炎暴發(fā)疫情分析[J];江蘇預防醫(yī)學;2002年03期

4 ;Molecular Epidemiological Analysis of Echovirus 19 Isolated From an Outbreak Associated With Hand, Foot, and Mouth Disease (HFMD) in Shandong Province of China[J];Biomedical and Environmental Sciences;2007年04期

5 劉松友;腮腺炎病毒研究進展[J];微生物學免疫學進展;2002年04期

6 王樹巧,顧祖萬,張亞達,劉惠珍,謝廣中,方天郎,孫木興,朱建慧;國產(chǎn)腮腺炎和進口麻風腮疫苗免后腮腺炎HI抗體比較[J];微生物學免疫學進展;1998年02期

7 武力,寧小軍,李益民,白植生;用逆轉錄套式PCR檢測臨床標本中的腮腺炎病毒RNA[J];中國病毒學;1998年01期

8 陳萌;陳麗娟;張鐵鋼;楊潔;和京果;張偉;;北京地區(qū)流行性腮腺炎病毒流行株基因型分析[J];中國公共衛(wèi)生;2006年09期

9 陳志慧;流行性腮腺炎病毒及其疫苗[J];中國計劃免疫;2004年02期

10 孫美平,范晨陽,劉東磊,劉維祥;北京市風疹 流行性腮腺炎免疫策略的探索與實踐[J];中國計劃免疫;2004年06期

，

本文編號：1834626