2株不同基因型树鼩呼肠孤病毒的分离和鉴定

目的 从腹泻树鼩的粪便样本中分离和鉴定病毒。方法 树鼩腹泻粪便样本分别接种Vero、LLC-MK2和KMB17细胞，经连续传代，观察记录细胞病变，并对培养上清进行透射电镜检查、病毒RNA-PAGE胶电泳分析、轮状病毒鉴别筛查、S1全长基因片段扩增和生物信息学分析。结果 树鼩腹泻粪便样品在KMB17、Vero 和LLC-MK2细胞上经连续3代次传代后，均能产生细胞病变。进一步经电镜检查、病毒RNA的聚丙烯酰胺凝胶电泳分析和轮状病毒鉴别筛查，推测其为呼肠孤病毒。病毒基因组全长S1基因扩增、序列测定和分析结果表明，KMB17培养上清中获得的病毒与I型原型株T1L同源性最高，核苷酸和氨基酸同源性分别为85%和90%，因此该病毒定义为呼肠孤病毒I型。而LLC-MK2和Vero细胞上清中的病毒S1基因与III型原型株T3D核苷酸和氨基酸同源性分别为85%和92 %，因此为III型病毒。S1基因的遗传进化分析表明，本研究的分离株已经进化适应了树鼩。结论 本研究首次通过3种基质细胞和S1基因片段特异性引物从同一粪便样品中分离和鉴定了2个不同基因型呼肠孤病毒，对今后树鼩和其它宿主呼肠孤病毒的分离鉴定有一定的指导意义。

Isolation and identification of 2 different genotypes of Tupaia Orthoreoviruses

Objective To isolate and identify viruses from fecal samples of tree shrew with diarrhea. Methods Three lines of cells, Vero, LLC-MK2 and KMB17 were inoculated with fecal samples of tree shrew with diarrhea and cytopathic effects were observed. The infectious particles in the culture supernatant were further analyzed by transparent electron microscopy (TEM), genomic RNA-PAGE, rotavirus detection kit, amplification of s1 complete segment and bioinformatics analysis. Results Constant cytopathic effects were induced on Vero, LLC-MK2 and KMB17 cell lines after 3 passages of culturing. The results from TEM, RNA-PAGE and rotavirus analysis indicated that they belong to reoviruses. Analysis of S1 segments revealed that the S1 sequence from KMB17 culture has the highest homology with that of prototype isolate T1L (85% nucleotide homology and amino acid homology 90%), therefore this isolate was named as type I reovirus. The other two S1 sequences from LLC-MK2 and Vero culture were identical and have 85% nucleotide homology and 92% amino acid homology with the prototype isolate T3D, named as type III reovirus. Phylogenetic analysis indicated that isolates in this study were evolutionally adapted to tree shrews. Conclusion It is the first report here that 2 genotypes of Tupaia Orthoreoviruses are isolated and identified from one fecal sample via 3 cell lines and viral S1-specific primers, which provides useful guidelines for the isolation and identification of other reoviruses.