Identification of novel avian and mammalian deltaviruses provides new insights into deltavirus evolution

Abstract

Hepatitis delta virus (HDV) is a satellite virus that requires hepadnavirus envelope proteins for its transmission. Although recent studies identified HDV-related deltaviruses in certain animals, the evolution of deltaviruses, such as the origin of HDV and the mechanism of its coevolution with its helper viruses, is unknown, mainly because of the phylogenetic gaps among deltaviruses. Here, we identified novel deltaviruses of passerine birds, woodchucks, and white-tailed deer by extensive database searches and molecular surveillance. Phylogenetic and molecular epidemiological analyses suggest that HDV originated from mammalian deltaviruses and the past interspecies transmission of mammalian and passerine deltaviruses. Further, metaviromic and experimental analyses suggest that the satellite-helper relationship between HDV and hepadnavirus was established after the divergence of the HDV lineage from non-HDV mammalian deltaviruses. Our findings enhance our understanding of deltavirus evolution, diversity, and transmission, indicating the importance of further surveillance for deltaviruses.

Keywords: deltavirus; inter-species transmission; virome.

Figure 1.

Genome organization of novel deltaviruses. Genomes of (a) tgDeV, mmDeV, and ovDeV (complete genomes) and (b) scDeV and egDeV (partial genomes). Annotations (ORF, poly-A signal, and ribozymes) are shown by colored arrow pentagons. The numbers indicate nucleotide positions. (c) Self-complementarities of novel deltaviruses. The predicted RNA structures were visualized using the Mfold web server (Zuker 2003). Red, blue, and green arcs indicate G–C, A–U, and G–U pairs, respectively.

Figure 2.

Amino acid sequence characterization of putative delta antigens of novel deltaviruses. (a) Alignment and functional features of the putative S-HDAg and DAgs of representative HDVs and novel deltaviruses. (Putative) functional domains are shown by colored boxes. Me: arginine methylation site, Ac: lysine acetylation site, P: Serine phosphorylation site. (b) ovDeV mRNA (upper panel) and a possible A-to-I RNA-editing site (lower panel). Consensus ovDeV-DAg mRNA sequence and mapped read sequence with potential RNA-edited nucleotides (blue boxes). Pink boxes indicate the ORF of ovDeV DAg. (c) Deduced amino acid sequences of ovDeV-DAg proteins translated from the viral mRNA with or without RNA-editing. The blue letter shows the possible RNA-editing site.

Figure 3.

Mapping coverages of original short reads of each contig. Mapped read graphs of (a) tgDeV, (b) mmDeV, and (c) ovDeV. Lines, arrow pentagons, and arrowheads indicate viral genomes, ribozymes, and poly(A) signals, respectively. The numbers above the graphs show nucleotide positions. The light pink box indicates a low read depth region in the putative transcript of tgDeV.

Figure 4.

Interfamily transmission of deltaviruses among passerine birds. (a–c) RT-PCR detection of a deltavirus from Lonchura striata. (a) Plasmid used for the establishment of real-time PCR detection system for tgDeV and (b) the tgDeV circular genome. The blue arrows indicate the primers used for endo-point RT-PCR detection. (c) Endo-point RT-PCR for detection of the circular deltavirus genome. M, 100-bp ladder marker. (d) Pairwise nucleotide identities between deltaviruses detected in passerine birds. (e) Phylogenetic tree of passerine birds positive for deltaviruses. Phylogenetic tree of birds and deltavirus infections are indicated. MYA: million years ago.

Figure 5.

Phylogenetic analysis of deltaviruses. (a) Heat map of pairwise amino acid sequence identities between deltaviruses. (b) The phylogenetic tree was inferred by the maximum likelihood method using an amino acid sequence alignment of representative deltaviruses. Known phenotypes (RNA-editing and expression of the large isoform of DAg protein) and helper virus(es) of each virus are shown on the right. Note that the SDeV phenotypes are shown in gray letters, because there is insufficient information, evidence, or both for the RNA-editing and L-DAg expression. The deltaviruses identified in this study are indicated by the blue circles. Bootstrap values >70 are shown. SDeV: snake deltavirus, RDeV: rodent deltavirus.

Figure 6.

Detection of tgDeV DAg and mmDeV DAg in cells ectopically expressing the tgDeV or mmDeV dimer genome. (a, b) Western blotting analysis of Huh7 or WCH-17 cells transfected with a tgDeV or mmDeV dimer-sequence expression plasmid. The numbers on the left side of panels indicate the size marker of protein (kDa). (c–f) Indirect immunofluorescence analysis of the expression of tgDeV or mmDeV DAg protein. The cells were observed using fluorescent microscopy (c, d) or a confocal microscopy (e, f). Blue; DAPI, Red; tgDeV or mmDeV DAg. Scale bars = 50 μm (c, d) and 5 μm (e, f).

Figure 7.

No infectious particle of tgDeV and mmDeV was produced by supplementation of HBV envelop proteins. (a) Quantification of deltavirus RNAs in culture supernatants. HDV, tgDeV, or mmDeV expression plasmid was transfected with or without plasmid expressing HBV envelope proteins into Huh7 cells. Viral RNA levels in supernatants were quantified using quantitative RT-PCR (n = 3). (b, c) HepG2-NTCP cells were incubated with the culture supernatants of the transfectants for 24 h in the presence or absence of 500 nM Myrcludex B (MyrB), an inhibitor of HBV envelope-dependent viral entry. The cells were cultured for an additional 6 days, and viral RNA levels and protein expression were analyzed using quantitative RT-PCR (n = 3) (b) and IFA (c), respectively. The numbers in (c) correspond to those of (b). Blue, DAPI; Red, HDV; tgDeV, or mmDeV DAg. Scale bar = 50 μm.