Identification of a novel bat papillomavirus by metagenomics

Abstract

The discovery of novel viruses in animals expands our knowledge of viral diversity and potentially emerging zoonoses. High-throughput sequencing (HTS) technology gives millions or even billions of sequence reads per run, allowing a comprehensive survey of the genetic content within a sample without prior nucleic acid amplification. In this study, we screened 156 rectal swab samples from apparently healthy bats (n = 96), pigs (n = 9), cattles (n = 9), stray dogs (n = 11), stray cats (n = 11) and monkeys (n = 20) using a HTS metagenomics approach. The complete genome of a novel papillomavirus (PV), Miniopterus schreibersii papillomavirus type 1 (MscPV1), with L1 of 60% nucleotide identity to Canine papillomavirus (CPV6), was identified in a specimen from a Common Bent-wing Bat (M. schreibersii). It is about 7.5kb in length, with a G+C content of 45.8% and a genomic organization similar to that of other PVs. Despite the higher nucleotide identity between the genomes of MscPV1 and CPV6, maximum-likelihood phylogenetic analysis of the L1 gene sequence showed that MscPV1 and Erethizon dorsatum papillomavirus (EdPV1) are most closely related. Estimated divergence time of MscPV1 from the EdPV1/MscPV1 common ancestor was approximately 60.2-91.9 millions of years ago, inferred under strict clocks using the L1 and E1 genes. The estimates were limited by the lack of reliable calibration points from co-divergence because of possible host shifts. As the nucleotide sequence of this virus only showed limited similarity with that of related animal PVs, the conventional approach of PCR using consensus primers would be unlikely to have detected the novel virus in the sample. Unlike the first bat papillomavirus RaPV1, MscPV1 was found in an asymptomatic bat with no apparent mucosal or skin lesions whereas RaPV1 was detected in the basosquamous carcinoma of a fruit bat Rousettus aegyptiacus. We propose MscPV1 as the first member of the novel Dyolambda-papillomavirus genus.

Figure 1. MEGAN tree with taxonomic assignments.

The distribution of the sequence reads through blastx analysis against the nr database. Size of circles located next to taxa are proportional to the total number of reads identified. Not assigned contains those reads that are not assigned by the least common ancestor algorithm. No hits contains those reads that did not return any significant alignments to the nr database.

Figure 2. Circular and linear genome maps of Miniopterus schreibersii papillomavirus type 1 (MscPV1).

Characteristic features of the long control region of MscPV1, showing genomic locations of E2 binding sites (bold), polyadenylation sites (underlined), and TATA box (boxed).

Figure 3. Maximum likelihood phylogenetic tree of the L1 nucleotide sequences of 79 PVs.

The PV genus of each strain is indicated. PVs with putative PV genera that are currently unclassified are marked by asterisks. The PV discovered in this study is shown in bold. Scale bar indicates 0.2 inferred substitutions per site. AaPV, Alces alces papillomavirus; BpPV, Bettongia penicillata papillomavirus; BPV, Bovine papillomavirus; CcaPV, Capreolus capreolus papillomavirus; CcPV, Caretta caretta papillomavirus; CgPV, Colobus guereza papillomavirus; ChPV, Capra hircus papillomavirus; CPV, Canine papillomavirus; EcPV, Equus caballus papillomavirus; EdPV, Erethizon dorsatum papillomavirus; EePV, Erinaceus europaeus papillomavirus; FcPV, Fringilla coelebs papillomavirus; FdPV, Felis domesticus papillomavirus; FlPV, Francolinus leucoscepus papillomavirus; HPV, Human papillomavirus; LrPV, Lynx rufus papillomavirus; MaPV, Mesocricetus auratus papillomavirus; MfPV, Macaca fascicularis papillomavirus; MmiPV, Micromys minutus papillomavirus; MmPV, Macaca mulatta papillomavirus; MnPV, Mastomys natalensis papillomavirus; MscPV, Miniopterus schreibersii papillomavirus; MsPV, Morelia spilota spilota papillomavirus; OaPV, Ovis aries papillomavirus; OcPV, Oryctolagus cuniculus papillomavirus; OvPV, Odocoileus virginianus papillomavirus; PcPV, Puma concolor papillomavirus; PePV, Psittacus erithacus timneh papillomavirus; PlpPV, Panthera leo persica papillomavirus; PlPV, Procyon lotor papillomavirus; PpPV, Pygmy chimpanzee papillomavirus; PsPV, Phocoena spinipinnis papillomavirus; RaPV, Rousettus aegyptiacus papillomavirus; SfPV, Sylvilagus floridanus papillomavirus; SsPV, Sus scrofa papillomavirus; TmPV, Trichechus manatus latirostris papillomavirus; TtPV, Tursiops truncatus papillomavirus; UmPV, Ursus maritimus papillomavirus; UuPV, Uncia uncia papillomavirus; ZcPV, Zalophus californianus papillomavirus.