Novel hepatitis D-like agents in vertebrates and invertebrates

Abstract

Hepatitis delta virus (HDV) is the smallest known RNA virus, encoding a single protein. Until recently, HDV had only been identified in humans, where it is strongly associated with co-infection with hepatitis B virus (HBV). However, the recent discovery of HDV-like viruses in metagenomic samples from birds and snakes suggests that this virus has a far longer evolutionary history. Herein, using additional meta-transcriptomic data, we show that highly divergent HDV-like viruses are also present in fish, amphibians, and invertebrates, with PCR and Sanger sequencing confirming the presence of the invertebrate HDV-like viruses. Notably, the novel viruses identified here share genomic features characteristic of HDV, such as a circular genome of only approximately 1.7 kb in length, and self-complementary, unbranched rod-like structures. Coiled-coil domains, leucine zippers, conserved residues with essential biological functions, and isoelectronic points similar to those in the human hepatitis delta virus antigens (HDAgs) were also identified in the putative non-human viruses. Importantly, none of these novel HDV-like viruses were associated with hepadnavirus infection, supporting the idea that the HDV-HBV association may be specific to humans. Collectively, these data not only broaden our understanding of the diversity and host range of HDV, but also shed light on its origin and evolutionary history.

Keywords: evolution; fish; hepatitis D virus; meta-transcriptomics; phylogeny; termites.

Figure 1.

Genome organization of the novel HDV-like agents in diverse animal taxa described here. In each metadata ring, the external circles indicate the percentage GC content (blue), percentage nucleotide polymorphism (orange), and read coverage (yellow) of the genomes. The inner gray circle represents the genome, and the black region shows the predicted ORF of the HDAg. Additional predicted ORFs over 200 nt in length and that do not overlap with the predicted HDAg are shown below the genomes.

Figure 2.

Circle graphs indicating each circular RNA genome structure of the HDV-like agents folding into unbranched rod-like structures. The circle circumference represents the genome sequence, while the arcs represent base pairing. The coloring of arcs is as follows: red for G–C pairing, blue for A–U pairing, green for G–U pairing, and yellow for other types.

Figure 3.

Phylogenetic relationships among the amino acid sequences of the HDAg proteins from human HDV and the HDV-like viruses newly determined here and previously. The phylogeny is rooted on the most divergent sequence from the termite. All branch lengths are scaled to the number of amino acid sequences per site. Bootstrap support values are shown for key nodes.

Figure 4.

Characterization of the putative HDAg proteins in the HDV-like viruses newly determined here. The translated HDAg genomes of three (human) HDV genotypes were compared with the putative HDAg proteins. The potential coiled-coil region is highlighted, also including the presence of leucine residues in the correct spacing for a leucine zipper (red arrow). Post-translationally modified arginine residues (methylation), lysine residues (acetylation), and serine residues (phosphorylation) that are conserved between different HDV genotypes are indicated with blue asterisks. The conserved regions shared similar signatures between different HDAgs are marked with purple frames.

Figure 5.

Schematic representation of the assembled overlapping PCR products for the termite HDV-like virus.