Species association of hepatitis B virus (HBV) in non-human apes; evidence for recombination between gorilla and chimpanzee variants

Abstract

Hepatitis B virus (HBV) infections are widely distributed in humans, infecting approximately one third of the world's population. HBV variants have also been detected and genetically characterised from Old World apes; Gorilla gorilla (gorilla), Pan troglodytes (chimpanzee), Pongo pygmaeus (orang-utan), Nomascus nastusus and Hylobates pileatus (gibbons) and from the New World monkey, Lagothrix lagotricha (woolly monkey). To investigate species-specificity and potential for cross species transmission of HBV between sympatric species of apes (such as gorillas and chimpanzees in Central Africa) or between humans and chimpanzees or gorillas, variants of HBV infecting captive wild-born non-human primates were genetically characterised. 9 of 62 chimpanzees (11.3%) and two from 11 gorillas (18%) were HBV-infected (15% combined frequency), while other Old world monkey species were negative. Complete genome sequences were obtained from six of the infected chimpanzee and both gorillas; those from P. t .ellioti grouped with previously characterised variants from this subspecies. However, variants recovered from P. t. troglodytes HBV variants also grouped within this clade, indicative of transmission between sub-species, forming a paraphyletic clade. The two gorilla viruses were phylogenetically distinct from chimpanzee and human variants although one showed evidence for a recombination event with a P.t.e.-derived HBV variant in the partial X and core gene region. Both of these observations provide evidence for circulation of HBV between different species and sub-species of non-human primates, a conclusion that differs from the hypothesis if of strict host specificity of HBV genotypes.

Figure 1. Phylogenetic analysis based on the HBV genome and identified recombinant region 1560–2120 bp.

Phylograms displaying phylogenetic trees based on (a) complete HBV genome; (c) HBV recombinant region 1560–2120 bp and equivalent fragments immediately preceding; (b) 999–1559 bp and succeeding; (d) 2121–2681 bp this region; with HBV reference sequences from human genotypes (A–H). Relative species and sub-species HBV variants are identified as follows Pan troglodytes troglodytes▴, Pan troglodytes ellioti▾, Pan troglodytes verus▪, Pan troglodytes schweinfurthii♦, Gorilla gorilla○ and Hylobates spp. □, and the host specific cluster is identified by ]. The trees were rooted with the woolly monkey HBV sequence, NC_001896▿. Sequences from this study are in bold and underlined and while recombinant HBV variants have bold branches.

Figure 2. Tree Order Scan of HBV sequences.

Figure 2(a). TreeOrder Scan of HBV sequences, indicating positions of individual sequences (y axis) in Phylogenetic trees generated from sequential 250-base sequence fragments, incrementing by 50 bases. Changes in sequence order as a result of changes in phylogeny at the 70% bootstrap level are shown. Sequences are colour coded by genotype and host species, as indicated by the labels in left and right margin: genotype A, purple; B, light blue; C, wine; D, emerald; E, royal blue; F, orange; G, pale green; H, navy; Gorilla, blue (Gor); Chimpanzee, green (Pan); and Woolly monkey (WM-out-group on line 1), red. For comparison the Tree Order Scan has been aligned with scale genome of HBV (top panel). Recombinant sequences are highlighted as by dashed lines; black gorilla/P.t.e ECO50003LIP3, green FJ798099 P.t.e/P.t.t, pink FJ798098 P.t.e/P.t.t, orange AB046525 P.t.t and purple AF498266 P.t.s 2(b). Tree Order Scan of HBV sequences, indicating positions of individual sequences (y axis) in phylogenetic trees generated from sequential 250-base sequence fragments, incrementing by 50 bases. Changes in sequence order as a result of changes in phylogeny at the 70% bootstrap level are shown. Sequences are colour coded by host species and sub-species of chimpanzee, as indicated by the labels in left and right margin: Gorilla gorilla, blue (Gor); Pan troglodytes troglodytes, yellow (Ptt); Pan troglodytes ellioti, green (Pte); Pan troglodytes verus, purple (Ptv); Pan troglodytes schweinfurthii, violet (Pts); and Hylobates pileatus (Hyl) (out-group-line 1-GII), red. For comparison the Tree Order Scan has been aligned with scale genome of HBV (top panel). Recombinant sequences are highlighted as by dashed lines; black gorilla/P.t.e ECO50003LIP3, green FJ798099 P.t.e/P.t.t, brown FJ798098 P.t.e/P.t.t, orange AB046525 P.t.t and blue AF498266 P.t.s.

Figure 3. Grouping Scan analysis.

Sequence fragments of 250 bases incrementing by 100 bases with 100 bootstrap replicates, were used to compare and analyse (a) P.t.troglodytes/P.t.ellioti recombinant FJ98098.1 (b) P.t.ellioti/P.t.troglodytes recombinant FJ98099.1 (c) P.t.schweinfurthii isolate A498266; (d) P.t.troglodytes AM117396 (e) P.t.troglodytes recombinant AB046525 (f) study recombinant Gorilla gorilla HBV sequence (ECO50003); to sequence groups from Gorilla gorilla (red), Pan troglodytes ellioti (blue), Pan troglodytes troglodytes (green), Pan troglodytes verus (yellow), Pan troglodytes schweinfurthii (purple) and human genotype HBV/C (light blue) with respect to A498266. Values >0.5 indicate clustering within the indicated group.