Origins and Evolution of the Primate Hepatitis B Virus

Abstract

Recent interest in the origins and subsequent evolution of the hepatitis B virus (HBV) has strengthened with the discovery of ancient HBV sequences in fossilized remains of humans dating back to the Neolithic period around 7,000 years ago. Metagenomic analysis identified a number of African non-human primate HBV sequences in the oldest samples collected, indicating that human HBV may have at some stage, evolved in Africa following zoonotic transmissions from higher primates. Ancestral genotype A and D isolates were also discovered from the Bronze Age, not in Africa but rather Eurasia, implying a more complex evolutionary and migratory history for HBV than previously recognized. Most full-length ancient HBV sequences exhibited features of inter genotypic recombination, confirming the importance of recombination and the mutation rate of the error-prone viral replicase as drivers for successful HBV evolution. A model for the origin and evolution of HBV is proposed, which includes multiple cross-species transmissions and favors subsequent recombination events that result in a pathogen and can successfully transmit and cause persistent infection in the primate host.

Keywords: ancient DNA; evolution; genotype; hepatitis B virus; human migration.

Figure 1

Schematic representation of the HBV genome. The inner circles represent the partial double-stranded DNA molecule and location of the direct repeat (DR) sequences, while the solid gray arrows illustrate the four open reading frames in the HBV genome: polymerase (P), precore (preC), core (C), hepatitis b X protein (X), and envelope (preS1, preS2, and S).

Figure 2

Phylogenetic tree of Hepadnaviridae. Phylogenetic analysis of representative hepadnavirus full-length genome sequences from each of the host species known to harbor hepatitis viruses including the HBV genotypes from humans. In general, all HBVs from the same host species cluster together with strong bootstrap support (>80%; with the exception of WSHBV, an enveloped HBV from fish). Based on the available HBV genome sequences, the enveloped and non-enveloped HBVs had distinct evolutionary histories following divergence from their most recent common ancestor (MRCA). The enveloped HBVs are further divided into two major clusters, mammals and fish and amphibians, reptiles, and birds sharing another MRCA. Of these genome sequences, 34 were sourced from GenBank and 14 were annotated sequences from the supplementary data files of Lauber et al. (2017), representing new isolates. The maximum-likelihood (ML) phylogenetic tree was estimated using IQ-TREE v1.6.5 (Nguyen et al., 2015), which is packaged with ModelFinder (Kalyaanamoorthy et al., 2017) and UFBoot (Hoang et al., 2018). Clade support was assessed using 1,000 pseudo replicates generated with the UFBoot non-parametric bootstrap procedure. Branch lengths are scaled to the number of nucleotide substitutions per sequence site. (From (Revill et al., 2020); modified, with permission from the author).

Figure 3

Maximum likelihood (ML) phylogenetic tree of full-length HBV genome sequences extracted from non-human primates (A) in Africa and (B) Southeast Asia, and reference HBV subgenotype sequences obtained from GenBank. The natural habitat locations of the non-human primates are shown in the corresponding maps, with same the color code used on the ML trees for the different host species. The trees were inferred using IQ-TREE v1.6.5 (Nguyen et al., 2015). The best-fit models determined by ModelFinder (Kalyaanamoorthy et al., 2017) were GTR+F+R2 and GTR+F+R4, respectively. Clade support was assessed using 1,000 pseudo replicates generated with the UFBoot non-parametric bootstrap procedure (Hoang et al., 2018), and bootstrap values >70% are shown by a color scale on the tree branches. The ML tree was annotated using iTOL v5.7 (Letunic and Bork, 2019). Branch lengths are scaled to the number of nucleotide substitutions per sequence site.

Figure 4

Maximum likelihood (ML) phylogenetic tree of full-length HBV genome sequences (aHBV has shown in bold red font) extracted from human and non-human primate hosts, and reference HBV subgenotype sequences obtained from GenBank. For aHBV, the number after ID represents the approximate age of the source fossil samples. The tree was inferred using IQ-TREE v1.6.5 (Nguyen et al., 2015), which is packaged with ModelFinder (Kalyaanamoorthy et al., 2017) and UFBoot (Hoang et al., 2018). The best-fit model determined by ModelFinder was GTR+F+R5. Clade support was assessed using 1,000 pseudo replicates generated with the UFBoot non-parametric bootstrap procedure, and bootstrap values >70% are shown by a color scale on the tree branches. The ML tree was annotated using iTOL v5.7 (Letunic and Bork, 2019). Branch lengths are scaled to the number of nucleotide substitutions per sequence site.

Figure 5

Global distribution of HBV. The map shows the predominant genotype and subgenotypes that have been identified in different countries around the world.

Figure 6

Schematic representation of known HBV genotype C recombinants, showing the conserved regions of the major and minor components. Sequences are color coded by genotype: genotype B (green), C (orange), D (blue), G (pink), J (red), and chimpanzee (purple). The space between the conserved regions is due to uncertainty about the precise recombination breakpoints and is indicated in gray.

Figure 7

A schematic representation of the genus Homo evolution timeline over the last 700,000 years, showing the admixture relationships between Homo sapiens, during migration out of Africa, and other archaic humans. At the end of each lineage, circle marks represent modern humans and rod marks represent archaic humans who are now extinct.