Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses

Abstract

Hepadnaviruses (hepatitis B viruses [HBVs]) are the only animal viruses that replicate their DNA by reverse transcription of an RNA intermediate. Until recently, the known host range of hepadnaviruses was limited to mammals and birds. We obtained and analyzed the first amphibian HBV genome, as well as several prototype fish HBVs, which allow the first comprehensive comparative genomic analysis of hepadnaviruses from four classes of vertebrates. Bluegill hepadnavirus (BGHBV) was characterized from in-house viral metagenomic sequencing. The African cichlid hepadnavirus (ACHBV) and the Tibetan frog hepadnavirus (TFHBV) were discovered using in silico analyses of the whole-genome shotgun and transcriptome shotgun assembly databases. Residues in the hydrophobic base of the capsid (core) proteins, designated motifs I, II, and III, are highly conserved, suggesting that structural constraints for proper capsid folding are key to capsid protein evolution. Surface proteins in all vertebrate HBVs contain similar predicted membrane topologies, characterized by three transmembrane domains. Most striking was the fact that BGHBV, ACHBV, and the previously described white sucker hepadnavirus did not form a fish-specific monophyletic group in the phylogenetic analysis of all three hepadnaviral genes. Notably, BGHBV was more closely related to the mammalian hepadnaviruses, indicating that cross-species transmission events have played a major role in viral evolution. Evidence of cross-species transmission was also observed with TFHBV. Hence, these data indicate that the evolutionary history of the hepadnaviruses is more complex than previously realized and combines both virus-host codivergence over millions of years and host species jumping.

Importance: Hepadnaviruses are responsible for significant disease in humans (hepatitis B virus) and have been reported from a diverse range of vertebrates as both exogenous and endogenous viruses. We report the full-length genome of a novel hepadnavirus from a fish and the first hepadnavirus genome from an amphibian. The novel fish hepadnavirus, sampled from bluegills, was more closely related to mammalian hepadnaviruses than to other fish viruses. This phylogenetic pattern reveals that, although hepadnaviruses have likely been associated with vertebrates for hundreds of millions of years, they have also been characterized by species jumping across wide phylogenetic distances.

FIG 1

Genome organization of the hepadnaviruses. Open reading frames encoding the polymerase (Pol), core, surface (PreS/S), and X proteins are indicated by colors. Circular genomes are linearized, with the exception of the partial sequence of ACHBV.

FIG 2

Coverage map for BGHBV, TFHBV, and ACHBV. The circular genomes of BGHBV and TFHBV are linearized, and sequence coverages over 15 reads are collapsed for display purpose. The overlapping sequences confirming the circular nature of the genomes are indicated by small orange triangles.

FIG 3

Conserved motifs in the polymerase proteins of mammalian, avian, amphibian, and piscine hepadnaviruses. An expanded reverse transcriptase domain is evident in mammalian orthohepadnaviruses and BGHBV. The expansion of the viral DNA polymerase N-terminal domain was observed only in mammalian orthohepadnaviruses. The degrees of sequence conservation are highlighted in grayscale. PHBV, parrot HBV; HHBV, heron HBV; ShHBV, sheldgoose HBV; SGHBV, snow goose HBV; RGHBV, Ross's goose HBV; HBHBV, horseshoe bat HBV; RBHBV, roundleaf bat HBV; BtHBV, bat HBV; TBHBV, tent-making bat HBV; GSHBV, ground squirrel HBV; WHBV, woodchuck HBV.

FIG 4

Conserved motifs in the core proteins of mammalian, avian, amphibian, and piscine hepadnaviruses. (A) Amino acid sequence alignment of the three conserved motifs in the core proteins. The positions in the HuHBV protein are indicated (42), and the degrees of sequence conservation are highlighted in grayscale. The accession numbers of the included HBV protein sequences are listed in Table 4. (B) Motifs I, II, and III (red, blue, and green, respectively) in the capsid protein dimer using HuHBV as a model. (C) Motif locations in the surface representation of the capsid dimer. (D and E) Homohexamer representation showing the proximity of the motifs between subunits.

FIG 5

Membrane protein analysis. (A) TMHMM analysis of the fish hepadnaviruses, BGHBV and WSHBV, as well as TFHBV, with known models for orthohepadnaviruses (HuHBV) and avihepadnaviruses (DHBV) (35). TMHMM probability was plotted against the length of the protein. Predicted transmembrane regions were highlighted in green. (B) Transmembrane topologies of the L protein (curved line) in the ER membrane (double horizontal lines), compared to the established model of orthohepadnavirus (45). Predicted transmembrane regions were highlighted in green. Since alternative start codon positions resulting in envelope proteins with different lengths were detected for TFHBV, the analysis was performed on both.

FIG 6

Maximum-likelihood phylogenetic trees of the polymerase (A), core (B), and surface (C) genes of exogenous and endogenous vertebrate hepadnaviruses. The viruses are color coded to reflect their host groups of origin. All the trees are drawn to a scale of amino acid substitutions (subs) per site and rooted on the fish (WSHBV and, where available, ACHBV) sequences, as (i) they are the most divergent and (ii) this rooting position maximizes the extent of virus-host codivergence. Bootstrap support values of >70% are shown for relevant nodes.