Detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in China

Abstract

Background: In recent years, novel hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses have been discovered in various species of bat around the world, indicating that bats may act as natural reservoirs for these hepatitis viruses. In order to further assess the distribution of hepatitis viruses in bat populations in China, we tested the presence of these hepatitis viruses in our archived bat liver samples that originated from several bat species and various geographical regions in China.

Methods: A total of 78 bat liver samples (involving two families, five genera, and 17 species of bat) were examined using nested or heminested reverse transcription PCR (RT-PCR) with degenerate primers. Full-length genomic sequences of two virus strains were sequenced followed by phylogenetic analyses.

Results: Four samples were positive for hepadnavirus, only one was positive for hepevirus, and none of the samples were positive for hepatovirus or hepacivirus. The hepadnaviruses were discovered in the horseshoe bats, Rhinolophus sinicus and Rhinolophus affinis, and the hepevirus was found in the whiskered bat Myotis davidii. The full-length genomic sequences were determined for one of the two hepadnaviruses identified in R. sinicus (designated BtHBVRs3364) and the hepevirus (designated BtHEVMd2350). A sequence identity analysis indicated that BtHBVRs3364 had the highest degree of identity with a previously reported hepadnavirus from the roundleaf bat, Hipposideros pomona, from China, and BtHEVMd2350 had the highest degree of identity with a hepevirus found in the serotine bat, Eptesicus serotinus, from Germany, but it exhibited high levels of divergence at both the nucleotide and the amino acid levels.

Conclusions: This is the first study to report that the Chinese horseshoe bat and the Chinese whiskered bat have been found to carry novel hepadnaviruses and a novel hepevirus, respectively. The discovery of BtHBVRs3364 further supports the significance of host switches evolution while opposing the co-evolutionary theory associated with hepadnaviruses. According to the latest criterion of the International Committee on Taxonomy of Viruses (ICTV), we hypothesize that BtHEVMd2350 represents an independent genotype within the species Orthohepevirus D of the family Hepeviridae.

Keywords: Bat; Genome characterization; Hepadnavirus; Hepevirus; Natural reservoir.

Fig. 1

Phylogenetic analysis of bat hepadnavirus based on the full-length genomic sequences. Maximum likelihood phylogenetic tree was constructed based on the complete genomes of BtHBVRs3364 (in bold) and representative members of the family Hepadnaviridae using the Hasegawa-Kishino-Yano substitution model and complete deletion option in MEGA version 7. The values at the nodes indicate the bootstrap values (using 1,000 replications). The branches are labeled with the strain designation, the host species, and the GenBank accession number. The classification of the family Hepadnaviridae is indicated on the right

Fig. 2

Phylogenetic analysis of bat hepevirus based on the full-length genomic sequences. Neighbor joining phylogenetic tree was constructed based on the alignment of the complete genomes of BtHEVMd2350 (in bold) and representative members of the family Hepeviridae using nucleotide percentage distance substitution matrix and complete deletion option implemented in MEGA version 7. The values at nodes indicate the bootstrap values (using 1,000 replications). The branches are labeled with the strain designation, the host species, and the GenBank accession number. The classification of the family Hepeviridae is indicated on the right

Fig. 3

Representation map of China and Yunnan province. Circle indicates the sampling site where the hepevirus (BtHEVMd2350) was detected. Triangles indicates the sampling sites where the hepadnaviruses were detected

Fig. 4

Tissue distribution of bat hepevirus and hepadnaviruses. Virus concentrations assessed by virus-specific one-step real-time RT-PCR using quantified in vitro-transcribed RNA controls