CAJ | 학술논문

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。经电镜检查、病毒RNA-PAGE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T1 L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％，因此为呼肠孤病毒III型。结论对今后树鼩和其他宿主呼肠孤病毒的分离鉴定有一定的指导意义。
목적：종복사수구적분편양본중분리화감정병독。방법수구복사분편양본분별접충Vero、LLC-MK2화KMB17세포，경련속전대，관찰기록세포병변，병대배양상청진행투사전경검사、병독RNA-PAGE전영분석、륜상병독감별사사、S1전장기인편단확증화생물신식학분석。결과수구복사분편양품재KMB17、Vero 화LLC-MK2세포상경련속3대차전대후，균능산생세포병변。경전경검사、병독RNA-PAGE전영분석화륜상병독감별사사，추측기위호장고병독。병독기인조전장S1기인확증、서렬측정화분석결과표명，KMB17배양상청중획득적병독여I형원형주T1 L동원성최고，핵감산화안기산동원성분별위85％화90％，인차해병독정의위호장고병독I형。이LLC-MK2화Vero세포상청중적병독S1기인여III형원형주T3D핵감산화안기산동원성분별위85％화92％，인차위호장고병독III형。결론대금후수구화기타숙주호장고병독적분리감정유일정적지도의의。
Objective To isolate and identify viruses from fecal samples of tree shrew with diarrhea.Methods Fecal sample supernatant of tree shrew with diarrhea was inoculated to three cell lines ( Vero, LLC-MK2 and KMB17 ) , and the cytopathic effects on the cells were observed.The infectious particles in the culture supernatant were further ana-lyzed by transmission electron microscopy ( TEM) , genomic RNA-PAGE, rotavirus detection kit, amplification of S1 com-plete segment and bioinformatics analysis.Results Constant cytopathic effects were induced in Vero, LLC-MK2 and KMB17 cell lines after three passages of culture.The results from TEM, RNA-PAGE and rotavirus analysis indicated that they belong to reoviruses.Analysis of the S1 segments revealed that the S1 sequence from KMB17 cell culture had the high-est homology with that of prototype isolate T1L (85%nucleotide homology and 90%amino acid homology), therefore this isolate was named as type I reovirus.The other two S1 sequences from LLC-MK2 and Vero cell culture were identical to have 85%nucleotide homology and 92%amino acid homology with the prototype isolate T3D, named as type III reovirus. Phylogenetic analysis indicated that the isolates in this study are evolutionally adapted to tree shrews.Conclusions It is the first report here that 2 genotypes of Tupaia orthoreovirus are isolated and identified from one fecal sample via three cell lines and viral S1-specific primers, which provides useful guidelines for the isolation and identification of other reoviruses from tree shrew or other hosts.