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

By Solexa sequencing, 20 and 4 contigs were found closely related to bocavirus (BoV) and adeno-associated virus (AAV) respectively. In this viral metagenomic analysis, bocavirus sequence was detected in groups XM and WM, but not in XO and WO (Table 3 ). The finding by specific PCR of a partial VP1 ORF gene sequence using degenerated primers designed based on available bocavirus sequences in GenBank was consistent with the metagenomic analysis, and analysis of all bat samples further showed that 2% (6/320) of guts of M. fuliginosus in XM and 6% (20/320) of M. fuliginosus in WM were bocavirus positive (Table 1 ). Ten amplicons (five from each group, accession numbers: JX863718 -JX863727) with lengths of 620 nt were sequenced and phylogenetically analyzed against representative sequences of bocaviruses identified to date. Results showed that the newly identified bat bocaviruses were significantly divergent from known bocaviruses, with 39-53% aa identity. The highest aa identity (53%) observed was with canine minute virus and only 43% aa identity with M. myotis bocavirus, the first bat bocavirus found in China [10] (Figure 3B ). Further phylogenetic analysis clustered our ten sequences into two independent groups. The highest amino acid identity between two groups was 85%, while that within a group was 97% ( Figure 3B ). This indicates the discovery of two new bocavirus species from bats. To further characterize the bocaviruses, nearly full length genomic sequences of Bt BoV XM30 in group 1 and Bt BoV WM40 in group 2 were obtained by gene walking. As shown in figure 3A , the genomes of Bt BoV XM30 with an incomplete 59 end (accession number: KC339250) and Bt BoV WM40 (accession number: KC339251) were 4,832 and 4,995 nt containing four ORFs: NS1, NP1, VP1 and VP2. ( Figure 3A ). The aa sequences of NS1 were used to construct the phylogenetic tree with other representatives ( Figure 3C ). Multiple alignments showed that Bt BoV XM30 and WM40 shared 76% aa identity, with 35-54% aa and 44-56% nt identities with other bocaviruses. The highest (54%) aa identity was with canine bocavirus HK882U but only 40% to Myotis myotis bocavirus 1, with the same results obtained by analysis of VP1 (data not shown).