Characterization of a novel orthoreovirus isolated from fruit bat, China

Hu, Tingsong; Qiu, Wei; He, Biao; Zhang, Yan; Yu, Jing; Liang, Xiu; Zhang, Wendong; Chen, Gang; Zhang, Yingguo; Wang, Yiyin; Zheng, Ying; Feng, Ziliang; Hu, Yonghe; Zhou, Weiguo; Tu, Changchun; Fan, Quanshui; Zhang, Fuqiang

Virus isolation was carried out in Vero E6 cells. To refer [23] , kohl et al [26] , Vero E6 cells were maintained at 37°C with 5% CO 2 unless stated otherwise. Cells were seeded into 24-well cell-culture plates and incubated overnight until 80-90% confluent. For infection, homogenates of the intestine samples for each bat were centrifuged at low-speed (6,000 × g) for 10 min at 4°C, treated with 100,000 U/ml penicillin and 100 μg/ml streptomycin, and then inoculated into Vero E6 cells for 1 hour under standard cellculture conditions and washed the cells three times with PBS (phosphate buffer saline) after inoculation. The infected cells were sub-cultured three times and observed daily for the occurrence of cytopathic effects (CPE). Upon CPE in the third subcultivation, the supernatant was passaged in 80-90% confluent fresh Vero cells in a 175 cm 2 flask for one week. Aliquots were stored at -80°C. One aliquot was titrated on Vero E6 cells to estimate the viral titer. After the second passage, Virus titrations were performed by end-point titration in VeroE6 cells and the TCID50/0.1 ml was calculated from 5 replicates by the method of Spearman-Karber.