2003～2007年中国大陆风疹病毒分子流行病学研究

发布时间：2018-05-30 17:12

本文选题：风疹病毒 + 基因特征　；参考：《中国协和医科大学》2008年硕士论文

【摘要】： 风疹是由风疹病毒引起的急性呼吸道传染病,在我国目前规定为丙类传染病。一般感染后临床症状轻微,并发症较少,但如果妊娠期妇女缺乏对风疹的免疫力而感染了风疹病毒,可导致流产、死胎或婴儿出生后出现以多器官严重损伤为主要表现的先天性风疹综合征(CRS),是风疹病毒引起的最严重的危害。风疹病毒只有一个血清型,但有多个基因型,世界卫生组织(WHO)将全球流行的风疹病毒分为两个进化枝,一共13个基因型,两个进化枝在核苷酸水平上差异为8～10%。本研究用Vero细胞或Vero/SLAM细胞从我国10省(自治区、直辖市)2003～2007年风疹暴发和散发病例的咽拭子标本进行病毒分离,并对分离物使用间接免疫荧光法(IFA)、比色法免疫学实验(ICA)以及短片段逆转录-聚合酶链反应(RT-PCR)法进行鉴定,结果分离到57株风疹病毒。用RT-PCR方法扩增57株风疹病毒E1基因的1107个核苷酸片段,并对该PCR产物进行核苷酸序列测定和分析,获得用于分子流行病学研究的靶核苷酸——E1基因的739个核苷酸的(nt8731～nt9469)。结果提示,55株风疹病毒株属于1E基因型,相对于其它国家的1E基因型,形成一个独立分支;另外2株风疹病毒属于2B基因型。基于E1基因的739个核苷酸(nt8731～nt9469),55株1E基因型风疹病毒之间的核苷酸同源性为97.8%～100%,氨基酸同源性为98.3%～100%;2株2B基因型风疹病毒的核苷酸和氨基酸同源性均为100%;1E与2B基因型风疹病毒之间的核苷酸同源性为89.1%～90.1%,氨基酸为98.3%～100%。55株1E基因型风疹病毒分离株的组内遗传距离为0.014,与WHO的1E基因型参考株(中国株T14-CH-02和M1-MAL-01株)的组间遗传距离分别为0.013和0.026,与其它国家和地区的10株1E基因型风疹病毒分离株的组间遗传距离为0.019,大于组内遗传距离。从时间纵向分析可以看出,于2003～2007间,同一时期的毒株间差异无明显规律性变化,这说明我国仍有多个风疹病毒传播链持续传播。 57株风疹病毒大部分核苷酸的突变为无义突变,氨基酸序列高度保守,除了2株1E基因型风疹病毒在E1蛋白血凝抑制和中和位点区域第212位氨基酸由Thr变为Ser,其它病毒株均无重要抗原位点的改变;所有我国已分离到的1E基因型风疹病毒在E1蛋白第338位氨基酸共享突变位点(Leu~(338)→Phe~(338)),而其它基因型以及其它国家的1E基因型风疹病毒在该位点均未发生突变,该氨基酸(Phe~(338))可能是我国1E基因型风疹病毒所特有。 2003～2007年在我国10省市均分离到1E基因型,而2B基因型只在2006年四川省从越南输入病例中分离到,提示1E为中国大陆绝对优势基因型,2B基因型为输入基因型,我国近年风疹的流行是由1E基因型为主的风疹野病毒的多个传播链引起。在此期间没有分离到1979～1984年间我国曾经流行的1A、2A和2B基因型风疹病毒;也没有分离到1999～2002年间我国曾经发现的1F和2A基因型风疹病毒,说明我国流行的风疹野病毒的基因型随年代发生了更替。 2007年,风疹疫苗纳入了我国国家免疫规划,这给我国风疹控制工作提供了前所未有的机遇。目前,我国要尽早实现麻疹与风疹实验室网络一体化,加强风疹病毒的分子流行病学研究,建立风疹病毒毒株库和基因数据库,了解我国风疹病毒基因型的分布,鉴别病毒的来源和传播途径,提供评估风疹控制策略效果的方法来科学地阻断病毒的传播,评估不同来源疫苗对不同基因型的保护效果,对于全球风疹的控制乃至消除也有着重要意义。
[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.
【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】：2008
【分类号】：R181.3;R511.2

【参考文献】