Virome Profiling of Bats from Myanmar by Metagenomic Analysis of Tissue Samples Reveals More Novel Mammalian Viruses

He, Biao; Li, Zuosheng; Yang, Fanli; Zheng, Junfeng; Feng, Ye; Guo, Huancheng; Li, Yingying; Wang, Yiyin; Su, Nan; Zhang, Fuqiang; Fan, Quanshui; Tu, Changchun

Briefly, bat organs and tissues (laryngopharynx, trachea, lung, heart, liver, spleen, stomach, gut, kidney and bladder) of each of the 4 groups were pooled and homogenized in a blender (Waring, Torrington, CT) with SM buffer (50 mM Tris, 10 mM MgSO 4 , 0.1 M NaCl, pH7.5). Following centrifugation at 80006g at 4uC for 30 min to remove cell debris and foreign materials, the supernatants were immediately filtered through 0.45-mm and 0.22-mm Pellicon II filters (Millipore, Billerica, MA). Filtrates were then concentrated in a 100-kDa Pellicon II filter (Millipore), and filtered once again through 0.22-mm syringe filters (Millipore). To eliminate contamination by host genome and other free nucleic acids, 14 U Turbo DNase (Ambion, Austin, TX), 25 U Benzonase Nuclease (Novagen, San Diego, CA) and 20 U RNase I (Fermentas, Ontario, Canada), ddH 2 O and 106DNase buffer (Ambion) were added to 116 ml filtrate to a final volume of 140 ml followed by digestion at 37uC for 1 h. The viral RNAs and DNAs were then extracted immediately using TRIzol (TaKaRa, Dalian, China) according to the manufacturer's protocol. Total viral nucleic acids were dissolved in RNase-free H 2 O (TaKaRa) and used immediately for the following reverse transcription.