Molecular phylogenetic analyses indicate a wide and ancient radiation of African hepatitis delta virus, suggesting a deltavirus genus of at least seven major clades

Abstract

Hepatitis D virus (HDV) is a satellite of hepatitis B virus (HBV) for transmission and propagation and infects nearly 20 million people worldwide. The HDV genome is a compact circular single-stranded RNA genome with extensive intramolecular complementarity. Despite its different epidemiological and pathological patterns, the variability and geographical distribution of HDV are limited to three genotypes and two subtypes that have been characterized to date. Phylogenetic reconstructions based on the delta antigen gene and full-length genome sequence data show an extensive and probably ancient radiation of African lineages, suggesting that the genetic variability of HDV is much more complex than was previously thought, with evidence of additional clades. These results relate the geographic distribution of HDV more closely to the genetic variability of its helper HBV.

FIG. 1.

Phylogenetic trees inferred from delta virus nucleotide sequences. (a) NJ phylogram obtained from the R0 data set; bootstrap values obtained for NJ and MP are indicated above the branches, whereas ML posterior probabilities inferred from 400 metaGA samplings are indicated below the branches. (b) ML cladogram obtained from the full genome data set (capital-lettered taxon); numbers at the branches indicate ML posterior probabilities inferred from 400 metaGA samplings. Of the 25 viral HDV sequences reported here, 15 form new clades (boxed in red), characterizing a variability range larger than that of type I or type III sequences. The remaining 10 new sequences cluster with type I. Asterisks indicate newly characterized HDV sequences. Arrows correspond to HDV genome sequences within a family, indicating that horizontal, rather than vertical, transmission from the mother (dFr-45) occurred. Scale is in percent expected substitution per position.

FIG. 2.

ML analyses of 33 sHD cDNA sequences. Analyses were conducted using PAUP4b6 and MetaPIGA. Numbers above the horizontal lines indicate bootstrap values (100 replicates with PAUP*, parameters constrained to those obtained from the ML analysis of the original data set), and those below the lines indicate metaGA posterior probabilities (10,000 samples). Scale is in percent expected substitution per position. Suggested clade names are indicated.

FIG. 3.

Alignment of HDV amino acid sequences from African (dFr-910, dFr-47, dFr-73, dFr-48, dFr-45, and dFr-644,), Italian (A20, genotype I), Japanese (-S, genotype IIA), Peruvian (-1, genotype III), and Taiwanese (TW-2b, genotype IIB) isolates. Dots indicate identities to dFr-910, whereas asterisks indicate positions conserved across all aligned sequences. Bold type indicates polymorphisms. Motifs corresponding to functional properties are boxed (NLS, nuclear localization signal; ARM, arginine-rich motifs; PKR, protein kinase R). The carboxy-terminal part of LHD constitutes a highly variable proline-rich domain corresponding to the packaging signal except for three conserved residues, a tryptophan, a cysteine, and a glutamine, corresponding, respectively, to the UAG-to-UGG editing site, the farnesylation box CXXX, and the C-terminal residue. Letters on the bottom line indicate when all (capital letters) or 50 to 99% (lowercase letters) of the 10 aligned sequences yielded identical secondary-structure consensus predictions (H and h, helix; C and c, coiled; E and e, beta-sheet).

FIG. 4.

Pseudo-double-strand antigenomic HDV RNA structure, as inferred by the mfold program, in the vicinity of the editing site for representatives of HDV-1, HDV-2, HDV-3, and HDV-4 and three representatives of the new African clades (dFr-47, dFr-48, and dFr-644). Boxes indicate the stop codons for the small hepatitis delta protein. The base-paired structure required for edition (4 bp on each side of the central adenosine of the amber codon) is observed only for clade HDV-1, strain A20.