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

To validate the results of Solexa sequencing, primers to amplify identified viral sequences were synthesized according to publications, or designed based on the Solexa sequences obtained in this study using Primer 5 (Premier Biosoft International, Palo Alto, CA) and Genefisher (http://bibiserv.techfak.uni-bielefeld.de/bibi/ Tools.html). Primer sequences used in the validation are available upon request. Viral RNA and DNA were automatically extracted with RNeasy and QIAamp DNA mini kits respectively (Qiagen, Hilden, Germany) in a QIAcube (Qiagen) from organ tissues of each bat and subjected to all nucleic acid amplification tests. Viral RNA was amplified using RT-PCR or RT-nested PCR, while viral DNA was subjected directly to PCR with negative but no positive control to avoid false positive results. The amplification of nucleic acids was conducted with PCR Master Mix (Tiangen, Beijing, China) according to the manufacturer's protocol. Positive PCR products were sequenced in both directions commercially by an ABI 3730 DNA Analyzer (Invitrogen, Beijing, China). Two near full genomes of bat bocaviruses were constructed with a genome walking kit (TaKaRa, Dalian, China) following the manufacturer's protocol.