Detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in China
© The Author(s). 2017
Received: 17 August 2016
Accepted: 4 February 2017
Published: 22 February 2017
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.
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.
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.
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.
KeywordsBat Natural reservoir Hepadnavirus Hepevirus Genome characterization
Nearly 60% of emerging infectious diseases in humans are zoonotic, with up to 70% of them being found to originate from wildlife . Bats have been identified as natural reservoirs of many viruses. Some of these viruses cause outbreaks of severe disease in humans , including the Ebola virus, the lyssavirus, the severe acute respiratory syndrome coronavirus, and henipaviruses . Interestingly, these viruses rarely cause apparent clinical signs in bats . Bats possess unique characteristics that may contribute to their ability to act as a major natural reservoir for viruses, including a high level of species diversity, a long lifespan, a high population density, and high levels of spatial mobility .
Previous studies mainly focused on bat-borne viruses that are transmitted via respiratory droplets . However, in recent years, several hepatitis virus-related sequences, including those associated with hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses, have been found in bats across the globe, indicating the importance of bats as the natural reservoirs of these viruses [5–9].
Hepatitis virus-like sequences detected in bats
Sampling site (s) (year)
Ghana (2011), Gabon (2009)
Costa Rica (2010)
Romania (2008/2009), Bulgaria (2008/2009), Luxemburg (2011)
Bulgaria (2008/2009), Spain (2010)
Côte d’Ivoire (2013)
M. cf. manavi
Romania (2008), Germany (2008/2010)
Romania (2008/2009), Germany (2009)
H. cf. ruber
Cameroon (2010), Kenya (2010)
Guatemala (2010), Mexico (2011)
Cameroon (2010), DRC (2011)
Kenya (2010), Nigeria (2008/2010)
Nigeria (2008/2010), Mexico (2010/2011)
There are around 120 species of bat in China; however, only limited information has been reported regarding the hepatitis viruses, a novel Orthohepadnavirus in pomona roundleaf bats from Yunnan province was identified in 2015 . In this study, we report the discovery of four novel hepadnaviruses and a hepevirus in our archived bat liver samples that had been collected from several bat species and various geographical regions in China.
Detection of hepadnavirus and hepevirus in bats in China between 2008 and 2013
No. of samples
No. of hepadnavirus positive samples
No. of hepevirus positive samples
Sampling site (s) (year)
Hubei (2011), Yunnan (2011)
Yunan (2009), Hubei (2011)
Hubei (2008/2011), Sichuan (2011),
Hubei (2011), Chongqing (2011)
Henan (2010), Hubei (2011) Yunnan (2012/2013),
RNA extraction and PCR
Primers used for virus RT-PCR screening and virus quantification
The complete genomic sequences of one hepadnavirus strain and one hepevirus strain were amplified using PCR with degenerate primers (the primers are available upon request). The genome ends were amplified using a 5′-Full RACE Kit (TaKaRa, Japan). The PCR products underwent gel purification with MinElute Gel Extraction Kit (Qiagen, Germany) and they were sequenced with both forward and reverse primers using the 3100 Sequencer. The sequencing chromatograms were inspected for overlapping multicolor peaks, which are an indicator of sequence heterogeneity in the amplicons. The PCR products were cloned using the pGEM-T Easy Vector System (Promega, Germany) and at least three clones for each PCR fragment were sequenced to obtain a consensus sequence.
Quantification real-time PCR
Virus load of bat hepevirus and hepadnaviruese of different tissues was measured by using photometrically quantified in vitro RNA transcripts and specific real-time RT-PCR primers (Table 3). Quantification was done by using 5 μL of RNA extract, 300 nM each primer, using the One Step SYBR PrimeScript™ PLUS RT-PCR Kit (TaKaRa, Japan). Cycling in a Biorad CFX Connect instrument involved the following steps: 42 °C for 5 min, 95 °C for 10 s, and 40 cycles of 95 °C 5 s and 60 °C 20 s with measurement of fluorescence.
Detection of four hepadnaviruses and a hepevirus in bat liver samples
Sequence analysis of the bat hepadnavirus
All four of the hepadnavirus-positive samples were from horseshoe bats, two each from R. sinicus (designated BtHBVRs3364 and BtHBVRs3366) and R. affinis (designated BtHBVRa4325 and BtHBVRa4328) (Table 2). The four partial polymerase gene sequences had 92.1–97.5% nucleotide sequence identity and they were found to be closely related to the roundleaf bat hepadnavirus from Yunnan province, China, with nucleotide identities of 88.8–95.5% .
Nucleotide and amino acid sequence identity between BtHBVRs3364 and representative orthohepadnavirus strainsa
Hepadnavirus (no. of strains compared)
Degree of identity (%)
S gene (1–672)
X gene (1217–1645)
C gene (1657–2301)
P gene (2147–1475)
Asian roundleaf bat hepadnavirus (3)
African horseshoe bat hepadnavirus (1)
African roundleaf bat hepadnavirus (4)
Long-fingered bat hepadnavirus (3)
Tent-making bat hepadnavirus (4)
Primate HBV (15)
Woolly monkey hepadnavirus (1)
Ground squirrel hepadnavirus (2)
Woodchuck hepadnavirus (1)
Duck hepadnavirus (1)
Phylogenetic analysis of the bat hepadnavirus
A phylogenetic tree was constructed based on the alignment of the full-length genomic sequence of BtHBVRs3364 with those of representative hepadnavirus strains available in GenBank. As shown in Fig. 1, the previously reported bat hepadnaviruses formed three clusters, with clear specificities for particular hosts. Although BtHBVRs3364 clustered with the bat hepadnaviruses, it formed an independent branch. Interestingly, the BtHBVRs3364 detected in the horseshoe bat is phylogenetically closer to viruses from the Asian roundleaf bat compared to viruses from the African horseshoe bat, despite the fact that it was found in an Asian horseshoe bat.
Sequence analysis of the bat hepevirus
Nucleotide and amino acid sequence identity between BtHEVMd2350 and representative hepevirus strainsa
Hepevirus (no. of strains compared)
Degree of identity (%)
ORF1 (genome positions 56–4699)
ORF2 (genome positions 4700–6607)
ORF3 (genome positions 4779–5192)
Bat hepevirus (1)
Avian hepevirus (4)
HEV genotype 3 (22)
HEV genotype 4 (5)
HEV genotype 1 (3)
HEV genotype 2 (1)
Rodent hepevirus (3)
Ferret hepevirus (1)
Trout hepevirus (1)
Phylogenetic analysis of the bat hepevirus
A phylogenetic tree was constructed based on the alignment of the full-length genomic sequence of BtHEVMd2350 with those of representative full-length hepevirus genomic sequences (Fig. 2). The results showed that bat hepeviruses (BtHEVMd2350 and BS7) cluster into a separate monophyletic clade within the family Hepeviridae.
Quantification of novel viruses
Conclusions and discussion
Since the discovery of genetically diverse hepatitis virus-related sequences in bats, bats have been considered to be important natural reservoirs for hepatitis viruses, and potential sources of human diseases . However, these hypotheses need to be proved by screening more bat samples from across the globe for hepatitis viruses. In this study, we screened for hepatitis viruses in bats from China and discovered four novel hepadnaviruses circulating in two species of horseshoe bat in Jinning city, Yunnan province and one hepevirus in the whiskered bat M. davidii in Xianning city, Hubei province. The full-length genomic sequences of one of the two hepadnaviruses from R. sinicus and the hepevirus from M. davidii were determined.
The phylogenetic analysis indicates that the bat hepadnavirus found in this study is closely related to roundleaf bat hepadnaviruses, which were discovered in Pu’er city, Yunnan province in 2011 , but shows remarkable divergence when compared to the African horseshoe bat, despite the fact that it was found in an Asian horseshoe bat. A similar phylogenetic relationship was found between hepadnaviruses from the African roundleaf bat and the African horseshoe bat , indicating the separate evolution of these viruses and their hosts.
Regarding the bat hepevirus, the phylogenetic analysis indicates that the known bat hepeviruses are highly divergent from other mammalian hepeviruses and that they form an independent branch in the family Hepeviridae. According to the latest proposal of the ICTV in 2016, amino acid distances of concatenated ORF1 and ORF2 (lacking hypervariable regions) greater than 0.088 could then act as threshold to demarcate intra- and inter- genotype distances . The hepevirus detected in the whiskered bat, M. davidii, and that found in the German serotine bat, E. serotinus (the only reported bat hepevirus with a full-length genome) shared significant diversity from both nucleotide and amino acid levels, we propose that they can be grouped into the species Orthohepevirus D which is divided into two genotypes: D1 and D2.
Our results provide further evidence to support the theory regarding the long-term co-evolution of hepadnaviruses and hepeviruses with their hosts, and the theory that bats act as major natural reservoirs for these hepatitis viruses. Our results have limitations due to the small sample size used, which was a result of the protection of bat populations in China, as bats play important roles in the pollination of plants and in pest control, as they feed on insects. However, based on our discovery of hepatitis viruses in bats, it is expected that there are many more hepatitis viruses circulating in numerous bat species and in various geographic regions. In order to obtain larger sample sizes, non-invasive methods of virus detection should be considered for future studies.
Hepatitis A virus
Hepatitis B virus
Hepatitis C virus
Hepatitis D virus
Hepatitis E virus
Open reading frames
Reverse transcription polymerase chain reaction
We appreciate Wei Zhang, Bei Li and Yu-Tao Gao (all Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China) for the excellent technical assistance.
This work was jointly funded by the National Natural Science Foundation of China (81290341), China Mega-Project for Infectious Disease (2014ZX10004001), the Scientific and Technological Basis Special Project (2013FY113500) and Funds for Environment Construction & Capacity Building of GDAS’ Research Platform (2016GDASPT-0215). BW was supported by the China Scholarship Council (CSC), Beijing, China.
Availability of data and materials
BW conducted the experiments and drafted the manuscript. X-LY, WL and YZ conducted molecular studies. BW, X-LY, X-YG, L-BZ, and Y-ZZ performed the sampling. L-ZS devised the study design and provided scientific oversight. The manuscript was revised by X-LY, C-TB, and L-ZS with input from all the contributing authors. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study protocol was reviewed and approved by the Ethics Committee of the Yunnan Institute of Endemic Disease Control and Prevention. All the animals were treated in strict accordance with the Guidelines for the Use and Care of Laboratory Animals from the Chinese CDC and the Rules for the Implementation of Laboratory Animal Medicine (1998) from the Ministry of Health, China. The protocols followed for the use of the animals were approved by the National Institute for Communicable Disease Control and Prevention, China. All surgery was performed under ether anesthesia, and all efforts were made to minimize suffering.
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- Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, Daszak P. Global trends in emerging infectious diseases. Nature. 2008;451:990–3.View ArticlePubMedGoogle Scholar
- Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006;19:531–45.View ArticlePubMedPubMed CentralGoogle Scholar
- Shi ZL. Emerging infectious diseases associated with bat viruses. Sci China-Life Sci. 2013;56:678–82.View ArticlePubMedGoogle Scholar
- Wibbelt G, Moore MS, Schountz T, Voigt CC. Emerging diseases in Chiroptera: why bats? Biol Lett. 2010;6:438–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Drexler JF, Corman VM, Lukashev AN, van den Brand JM, Gmyl AP, Brunink S, Rasche A, Seggewibeta N, Feng H, Leijten LM, et al. Evolutionary origins of hepatitis A virus in small mammals. Proc Natl Acad Sci U S A. 2015;112:15190–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Drexler JF, Geipel A, Konig A, Corman VM, van Riel D, Leijten LM, Bremer CM, Rasche A, Cottontail VM, Maganga GD, et al. Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc Natl Acad Sci U S A. 2013;110:16151–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Drexler JF, Seelen A, Corman VM, Tateno AF, Cottontail V, Zerbinati RM, Gloza-Rausch F, Klose SM, Adu-Sarkodie Y, Oppong SK, et al. Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae. J Virol. 2012;86:9134–47.View ArticlePubMedPubMed CentralGoogle Scholar
- He B, Fan Q, Yang F, Hu T, Qiu W, Feng Y, Li Z, Li Y, Zhang F, Guo H, et al. Hepatitis virus in long-fingered bats, Myanmar. Emerg Infect Dis. 2013;19:638–40.View ArticlePubMedPubMed CentralGoogle Scholar
- He B, Zhang F, Xia L, Hu T, Chen G, Qiu W, Fan Q, Feng Y, Guo H, Tu C. Identification of a novel Orthohepadnavirus in pomona roundleaf bats in China. Arch Virol. 2015;160:335–7.View ArticlePubMedGoogle Scholar
- Rasche A, Souza BF, Drexler JF. Bat hepadnaviruses and the origins of primate hepatitis B viruses. Curr Opin Virol. 2016;16:86–94.View ArticlePubMedGoogle Scholar
- Quan PL, Firth C, Conte JM, Williams SH, Zambrana-Torrelio CM, Anthony SJ, Ellison JA, Gilbert AT, Kuzmin IV, Niezgoda M, et al. Bats are a major natural reservoir for hepaciviruses and pegiviruses. Proc Natl Acad Sci U S A. 2013;110:8194–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, Mazet JK, Hu B, Zhang W, Peng C, et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013;503:535–8.View ArticlePubMedGoogle Scholar
- Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66.View ArticlePubMedPubMed CentralGoogle Scholar
- Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4.View ArticlePubMedGoogle Scholar
- Smith DB, Simmonds P, members of the International Committee on the Taxonomy of Viruses Hepeviridae Study G, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA. Consensus proposals for classification of the family Hepeviridae. J Gen Virol. 2015;96:1191–2.View ArticlePubMedGoogle Scholar