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目的对1989年8月分离自云南省盈江县蚊虫的2株登革病毒进行生物学及分子特征的鉴定,明确这两株病毒的血清型及基因型。方法蚊虫用BHK21细胞和乳鼠法分离病毒,用间接免疫荧光试验、RT-PCR等方法进行鉴定,并对这两株病毒的分子生物学特征进行分析。结果从白纹伊蚊和达勒姆阿蚊中各分离到一株病毒,分别命名为M110和M113,这两株病毒对BHK21细胞能产生明显的细胞病变,脑内接种乳鼠4d左右导致乳鼠死亡。该病毒经血凝、血凝抑制和间接免疫荧光试验提示为黄病毒。分别用黄病毒属特异引物、登革4型病毒NS1和NS2a基因片段特异引物进行RT-PCR扩增、序列测定,证实为登革4型病毒。NS1和NS2a基因序列片段分析表明,M110和M113的NS1核苷酸序列与登革4型病毒基因Ⅰ型的H241株(Y19176)同源性最高,分别为99%、98%;NS2a基因片段与H241的核苷酸序列的同源性分别为96.5%、97.1%。结论从盈江县蚊虫分离到的M110和M113病毒为登革4型病毒基因Ⅰ型,表明云南省西部边境地区在20世纪80年代发生过登革4型病毒的流行。
Objective To identify the biological and molecular features of two dengue viruses isolated from mosquitoes in Yingjiang County, Yunnan Province in August 1989, and to identify the serotypes and genotypes of the two viruses. Methods Mosquitoes were used to isolate the virus by BHK21 cells and neonatal rat and identified by indirect immunofluorescence assay and RT-PCR. The molecular biological characteristics of the two viruses were also analyzed. Results A virus was isolated from Aedes albopictus and Durham mosquito respectively and named as M110 and M113 respectively. These two viruses could produce obvious cytopathic effect on BHK21 cells. Rat died. The virus by hemagglutination, hemagglutination inhibition and indirect immunofluorescence test prompted the flavivirus. The specific primers of flavivirus and dengue virus type 4 NS1 and NS2a were respectively amplified by RT-PCR and sequenced, which was confirmed as dengue type 4 virus. Sequence analysis of NS1 and NS2a revealed that the NS1 nucleotide sequence of M110 and M113 shared the highest homology with that of H241 (Y19176) of dengue virus type 4 (99% and 98%, respectively). The NS2a gene fragment H241 nucleotide sequence homology was 96.5%, 97.1%. Conclusion The M110 and M113 viruses isolated from Yingjiang mosquitoes are of genotype Ⅰ of dengue virus type 4, indicating the prevalence of dengue type 4 virus in the western border area of Yunnan Province in the 1980s.