目的  对轮状病毒（rotavirus, RV）阳性临床粪便标本进行毒株分离、纯化及鉴定。方法  将感染RV的腹泻患儿粪便标本在单层恒河猴肾细胞MA104上传代培养，分离毒株，通过噬斑法克隆纯化病毒，电子显微镜（电镜）观察病毒形态和大小；采用SDS-PAGE、免疫印迹法和间接免疫荧光法（indirect immunofluorescence assay, IFA）、聚丙烯酰胺凝胶电泳（polyacrylamide gel electrophoresis，PAGE）、反转录PCR及全基因组测序对病毒进行鉴定。结果 对大量临床标本进行传代筛选和噬斑纯化，得到1株RV野毒株。50%蔗糖超速离心纯化病毒在电镜下可见直径约75 nm的典型RV颗粒；SDS-PAGE 和免疫印迹法结果显示在预期位置出现目的条带；IFA结果显示所分离病毒为RV；RNA-PAGE显示分离株电泳型为长型，电泳图谱中11个条带呈现4∶2∶3∶2排列；PCR扩增片段及全基因组测序结果表明分离毒株为G1P[8]型。结论 成功分离出1株人G1P[8]型RV野毒株，并命名为HN15D2。

Objective  To isolate, purify and identify of a human wild rotavirus (RV) strain from RV-positive clinical stool samples. Methods  The stool samples of infants with diarrhea infected by RV were cultured and isolated in monolayer rhesus monkey kidney cell MA104, cloned and purified by plaque method. The virus morphology and size was observed by electron microscope (EM), and identified by, SDS-PAGE, Western blot, indirect immunofluorescence assay (IFA), polyacrylamide gel electrophoresis (PAGE), reverse transcription PCR and whole genome sequencing. Results  A wild strain of RV was isolated and purified by passage screening and plaque cloning from a large number of clinical samples. Typical RV particles with a diameter of ~75 nm were observed under EM after 50% sucrose ultracentrifugation. SDS-PAGE and Western blotting results showed that target bands appeared at the expected position. IFA result showed that the isolated virus was RV. RNA-PAGE showed that the isolate virus had a long electrophoretic pattern with 11 bands arranged by 4∶2∶3∶2 of the group A RV. Amplified PCR fragments and whole genome sequencing showed that the isolated strain was G1P[8] human RV. Conclusion  A wild human RV strain G1P[8] has been successfully isolated and named HN15D2.