Isolation and identification of two different genotypes of Tupaia orthoreoviruses / 中国实验动物学报

ABSTRACT

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.