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

Culture supernatants of Cangyuan virus was analyzed by VIDISCR. Virus particles were harvested from cells by three freeze-thaw cycles and the resulting suspension purified from cell debris by low-speed centrifugation. Nucleic acids were extracted using the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, Inc.). Reverse transcription of the viral RNA was performed by using the RevertAidâ„¢ First Strand cDNASynthesis Kit (Fermentas, Inc). The VIDISCR assay was performed as previously described [27] . To further characterize the virus and its phylogeny primers were designed with Primer Premier 5.0 based on published sequences selective for the 10 genome segments of Melaka virus and other orthoreoviruses (Additional file 4: Table S4 ) [9, 16] . Each of the 10 genome segments were amplified using the pfu PCR Polymerase Kit (Fermentas, Inc) with the primers as listed in Additional file 4: Table S4 . Reverse transcription PCR was performed as described [9, 16] . The amplicons were visualized using 2% agarose gel electrophoresis. PCR products were sequenced after cloning by using CloneJETâ„¢ PCR cloning kit (Fermentas, Inc