本文選題：風疹病毒 + 基因特征�。� 參考：《中國協(xié)和醫(yī)科大學》2008年碩士論文

【摘要】： 風疹是由風疹病毒引起的急性呼吸道傳染病,在我國目前規(guī)定為丙類傳染病。一般感染后臨床癥狀輕微,并發(fā)癥較少,但如果妊娠期婦女缺乏對風疹的免疫力而感染了風疹病毒,可導致流產(chǎn)、死胎或嬰兒出生后出現(xiàn)以多器官嚴重損傷為主要表現(xiàn)的先天性風疹綜合征(CRS),是風疹病毒引起的最嚴重的危害。風疹病毒只有一個血清型,但有多個基因型,世界衛(wèi)生組織(WHO)將全球流行的風疹病毒分為兩個進化枝,一共13個基因型,兩個進化枝在核苷酸水平上差異為8～10%。 本研究用Vero細胞或Vero/SLAM細胞從我國10省(自治區(qū)、直轄市)2003～2007年風疹暴發(fā)和散發(fā)病例的咽拭子標本進行病毒分離,并對分離物使用間接免疫熒光法(IFA)、比色法免疫學實驗(ICA)以及短片段逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)法進行鑒定,結(jié)果分離到57株風疹病毒。用RT-PCR方法擴增57株風疹病毒E1基因的1107個核苷酸片段,并對該PCR產(chǎn)物進行核苷酸序列測定和分析,獲得用于分子流行病學研究的靶核苷酸——E1基因的739個核苷酸的(nt8731～nt9469)。結(jié)果提示,55株風疹病毒株屬于1E基因型,相對于其它國家的1E基因型,形成一個獨立分支;另外2株風疹病毒屬于2B基因型。 基于E1基因的739個核苷酸(nt8731～nt9469),55株1E基因型風疹病毒之間的核苷酸同源性為97.8%～100%,氨基酸同源性為98.3%～100%;2株2B基因型風疹病毒的核苷酸和氨基酸同源性均為100%;1E與2B基因型風疹病毒之間的核苷酸同源性為89.1%～90.1%,氨基酸為98.3%～100%。55株1E基因型風疹病毒分離株的組內(nèi)遺傳距離為0.014,與WHO的1E基因型參考株(中國株T14-CH-02和M1-MAL-01株)的組間遺傳距離分別為0.013和0.026,與其它國家和地區(qū)的10株1E基因型風疹病毒分離株的組間遺傳距離為0.019,大于組內(nèi)遺傳距離。從時間縱向分析可以看出,于2003～2007間,同一時期的毒株間差異無明顯規(guī)律性變化,這說明我國仍有多個風疹病毒傳播鏈持續(xù)傳播。 57株風疹病毒大部分核苷酸的突變?yōu)闊o義突變,氨基酸序列高度保守,除了2株1E基因型風疹病毒在E1蛋白血凝抑制和中和位點區(qū)域第212位氨基酸由Thr變?yōu)镾er,其它病毒株均無重要抗原位點的改變;所有我國已分離到的1E基因型風疹病毒在E1蛋白第338位氨基酸共享突變位點(Leu~(338)→Phe~(338)),而其它基因型以及其它國家的1E基因型風疹病毒在該位點均未發(fā)生突變,該氨基酸(Phe~(338))可能是我國1E基因型風疹病毒所特有。 2003～2007年在我國10省市均分離到1E基因型,而2B基因型只在2006年四川省從越南輸入病例中分離到,提示1E為中國大陸絕對優(yōu)勢基因型,2B基因型為輸入基因型,我國近年風疹的流行是由1E基因型為主的風疹野病毒的多個傳播鏈引起。在此期間沒有分離到1979～1984年間我國曾經(jīng)流行的1A、2A和2B基因型風疹病毒;也沒有分離到1999～2002年間我國曾經(jīng)發(fā)現(xiàn)的1F和2A基因型風疹病毒,說明我國流行的風疹野病毒的基因型隨年代發(fā)生了更替。 2007年,風疹疫苗納入了我國國家免疫規(guī)劃,這給我國風疹控制工作提供了前所未有的機遇。目前,我國要盡早實現(xiàn)麻疹與風疹實驗室網(wǎng)絡(luò)一體化,加強風疹病毒的分子流行病學研究,建立風疹病毒毒株庫和基因數(shù)據(jù)庫,了解我國風疹病毒基因型的分布,鑒別病毒的來源和傳播途徑,提供評估風疹控制策略效果的方法來科學地阻斷病毒的傳播,評估不同來源疫苗對不同基因型的保護效果,對于全球風疹的控制乃至消除也有著重要意義。
[Abstract]:Rubella is an acute respiratory infectious disease caused by rubella virus. In China, it is now prescribed as a class C infectious disease. The clinical symptoms are mild and the complications are less after the infection. However, if pregnant women lack the immunity to rubella, it can lead to abortion, death or babies are seriously injured by multiple organs after birth. The main manifestation of the congenital rubella syndrome (CRS) is the most serious damage caused by the rubella virus. The rubella virus has only one serotype, but there are multiple genotypes. The WHO (WHO) divides the global epidemic of rubella virus into two evolutionary branches, a total of 13 genotypes, and two evolutionary branches at the nucleotide level of 8 to 10%..
In this study, Vero cells or Vero/SLAM cells were used to isolate the virus from the pharynx swabs from 10 provinces (autonomous regions and municipalities) for 2003~2007 years of eruption and sporadic cases, and the isolates were identified by indirect immunofluorescence (IFA), colorimetric immunoassay (ICA) and short segment reverse transcription polymerase chain reaction (RT-PCR). Results 57 strains of rubella virus were isolated. 1107 nucleotide fragments of 57 strains of rubella virus E1 gene were amplified by RT-PCR method, and the nucleotide sequence of the PCR product was determined and analyzed to obtain 739 nucleotides (nt8731 to nt9469) of the target nucleotide of the molecular epidemiology study, the E1 gene. The results suggested that 55 strains of rubella virus It belongs to the 1E genotype, which forms an independent branch relative to the 1E genotype in other countries. The other 2 strains of rubella virus belong to the 2B genotype.
Based on 739 nucleotides (nt8731 to nt9469) of the E1 gene, the nucleotide homology between 55 1E genotypes of rubella virus was 97.8% to 100%, the amino acid homology was 98.3% to 100%, and 2 strains of 2B genotype rubella virus had the nucleotide and amino acid homology of 100%, and the nucleotide homology between 1E and 2B genotypes was 89.1% to 90.1%. The intra group genetic distance of 98.3% to 100%.55 strains of 1E genotypes was 0.014, and the genetic distance between the 1E genotype reference strain (T14-CH-02 and M1-MAL-01 strain) of WHO was 0.013 and 0.026 respectively, and the genetic distance between the 10 strains of 1E genotype rash virus isolates from other countries and regions was 0.019. From the time longitudinal analysis, there was no obvious change in the difference between the 2003~2007 strains of the same period. This shows that there are still a number of rubella virus transmission chains in China.
The mutation of most nucleotides of 57 strains of rubella virus was nonsense mutation and the amino acid sequence was highly conserved. Except for the 2 1E genotypes of rubella virus, 212nd amino acids changed from Thr to Ser in the E1 protein hemagglutination inhibition and neutralization loci. All the other virus strains had no major antigen loci changes; all the isolated 1E genotypes in China were isolated. The E1 protein 338th - bit amino acid shared the mutation site (Leu~ (338) - Phe~ (338)), while other genotypes and other countries' 1E genotypic rubella viruses were not mutated at this site. The amino acid (Phe~ (338)) might have been found in our country's 1E genotype rubella virus.
In 2003~2007 years, the 1E genotypes were isolated in 10 provinces and cities in China, and the 2B genotype was isolated from the Vietnamese imported cases in Sichuan province in 2006, suggesting that 1E is the dominant genotype in the mainland of China, and the 2B genotype is the input genotype. In recent years, the epidemic of rubella in China is caused by the multiple transmission chain of the rash wild virus, which is the main 1E genotype. The 1A, 2A and 2B genotypes of rubella virus, which was once popular in China during the period of 1979~1984, were not separated, and the 1F and 2A genotypes that were found in China in the past 1999~2002 years were not separated, indicating that the genotypes of the epidemic rash wild virus in our country were changed with age.
In 2007, the rubella vaccine was brought into our national immunization program, which provided an unprecedented opportunity for the control work of rubella in China. At present, our country should realize the integration of measles and rubella laboratory network as soon as possible, strengthen the molecular epidemiological study of rubella virus, establish the virus library and gene database of rubella disease, and understand the rubella virus in our country The distribution of genotypes, the identification of the source and transmission of the virus, the method of assessing the effectiveness of the rash control strategy to scientifically block the spread of the virus, and evaluate the protective effects of different sources of vaccines on different genotypes, is also of great significance to the control and even elimination of the global rash.
【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】：2008
【分類號】：R181.3;R511.2

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