用原位杂交法检测肠道病毒基因组同源性的研究

人类肠道病毒属于微小RNA病毒科,包括脊髓灰质炎病毒(PV)、柯萨奇A组(CAV)、B组病毒(CBV),埃可病毒(EV)4组病毒和肠道病毒68-72型。72型为肠道病毒的新成员甲型肝炎病毒(HAV)。肠道病毒是一组与人类多种疾病密切相关较为复杂的病毒。属内各组病毒基因同源关系尚未完全清楚。而HAV与传统肠道病毒的

Study on Genomic Honiology of Enteroviruses by in Situ Hybridization

Three recombinant plasmid cDNA, pCBIII/51,35, and 29 derived from Coxsackie B3 virus (CBV3) genome, were employed as probes. After E. coli transformation and plasmid preparation, a biotin labeled in situ hybridization technique has been established for the detection of enterovirus genomes. Enterovirus gecomic RNA can be detected under a light microscope within 24 hours by the in situ hybridization. Most hybridization signals were localized in the cytoplasm of virus infected Vero cells as brown colours. The probes did not hybridize with uninfected cells, neither did labeled plasmid with infected cells. This method has the advantages of simplicity, specificity reproducibility and stability. Hybridizations between these three probes and thirteen enteroviruses, CBV1-6, Coxsackie A virus 1, 9, Poliovirus 1, E.chovirus 1, 7, 11 and Hepatitis A virus (HAV) were performed. The results showed that probes 51 and 35 hybridized with all the enteroviruses except HAV, while probe 29 hybridized only with CBV3, indicating a various homologous relationship of the viruses with CBV3 genomic cDNA. HAV showed no hybridization signals with all three probes representing more than 90% of CBV3 genome. It therefore, failed to show any significent genomic homology with CBV3 cDNA. These data joined by the accumulated information in this field suggests that HAV may not be suitable in enterovirus genus and might be required to modify its taxonomy. This in situ hybridization method will play an important role also in the relationship between Coxsackie B virus and clinical diseases.