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目的研究SARS患者中多病原感染状况,测定并分析新分离呼肠病毒基因组序列,从病毒的分子生物学角度研究其分类学位置。方法SARS患者咽拭子标本经处理后接种Hep-2细胞,提取分离到的病毒RNA,以随机引物逆转录PCR扩增,PCR产物经克隆、测序,将其核苷酸序列及推断的氨基酸序列与GenBank数据库中基因进行比较分析并建立系统进化树。结果从4份SARS患者咽拭子标本中分离到3株呼肠病毒,部分基因片段测序结果显示为同一病毒,其病毒基因与呼肠病毒1、2、3型有较高的同源性,尤其是与呼肠病毒1、3型,相应片段的核苷酸序列同源性达82%~92%,推断相应氨基酸序列同源性高达94%~99%,而与其他病毒不存在有意义的同源性;系统进化树分析显示新分离呼肠病毒属于呼肠病毒科呼肠病毒属,且可能为哺乳动物呼肠病毒中的新血清型。结论SARS患者咽拭子标本中存在呼肠病毒,新分离呼肠病毒核苷酸和氨基酸序列与呼肠病毒1、3型同源性最高,由于尚未得到决定病毒血清型的S1片段基因序列,故该病毒的确切分型有待进一步确定,与SARS患者病情的关系有待进一步研究。
Objective To study the multi-pathogen infection in SARS patients, to determine and analyze the genome sequence of the newly isolated reovirus and to study its taxonomic position from the perspective of molecular biology of the virus. Methods The throat swab samples from SARS patients were inoculated into Hep-2 cells after treatment, and the isolated viral RNAs were extracted and amplified by random primer reverse transcription PCR. The PCR products were cloned and sequenced. The nucleotide sequence and deduced amino acid sequence The gene was compared with the GenBank database and phylogenetic tree was constructed. Results Three strains of reovirus were isolated from throat swab specimens of four SARS patients. The results of sequencing of some of the gene fragments showed the same virus, and their viral genes showed high homology with reovirus type 1, 2 and 3, Especially with the reovirus type 1, 3, the nucleotide sequence homology of the corresponding fragment was 82% -92%, deduced that the homology of the corresponding amino acid sequence was as high as 94% -99%, but not significant with other viruses Phylogenetic tree analysis showed that the newly isolated reovirus belongs to the genus Rehoviridae and may be a new serotype in mammalian reovirus. Conclusion The reovirus exists in the throat swab specimens of SARS patients. The nucleotide and amino acid sequences of the newly isolated reovirus have the highest homology with the reovirus type 1 and 3. Since the S1 gene sequence of the virus serotype has not yet been determined, Therefore, the exact typing of the virus needs to be further confirmed, and SARS patients the relationship needs further study.