Early Mesozoic Coexistence of Amniotes and Hepadnaviridae

Abstract

Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs) are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs) have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic “fossil” is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote–HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts.

Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses

“…The study describing a hepadnavirus in the white sucker fish (16) and our discovery of the second fish hepadnavirus and the first amphibian hepadnavirus are evidence that hepadnaviruses have a broader host range than previously appreciated. Indeed, the analysis of these new genomes, as well as previously described exogenous (HBV) and endogenous (eHBV) hepadnavirus (14,15,46) sequences, indicates that the Hepadnaviridae have been able to infect all five major groups of vertebrates, namely, mammals, birds, reptiles, amphibians, and fish (Table 6 and Fig. 6).…”

“…Un- fortunately, a lack of tissue specimens precluded verification of the presence or absence of the virus in additional frogs. The genome organizations of the fish and amphibian hepadnaviruses are similar to those of orthohepadnaviruses and avihepadnaviruses, although, with the exception of the highly conserved functional domains (14,15), the sequence identities between these virus groups are very low ( Table 4). The polymerases in BGHBV, TFHBV, and ACHBV also contained conserved domains, including the viral DNA polymerase C and N termini and the reverse transcriptase LTR.…”

“…To be as broad as possible, the background GenBank data set included exogenous Avihepadnavirus, Orthohepadnavirus, and white sucker hepatitis B virus (WSHBV) sequences, as well as available avian and reptilian (crocodilian) endogenous hepadnavirus (eHBV) sequences that were of sufficient length to conduct phylogenetic analyses, although sequence availability differed by gene. Although a number of snake eHBVs have been documented (14,15), they are highly fragmentary and contain multiple stop codons and hence were of insufficient length to be included in our phylogenetic analysis, which was based on amino acid sequences (see below). A full list of the sequences utilized is provided in Table 3).…”

“…Hepadnaviral EVEs have been documented in turtles, crocodiles, snakes, and birds (14,(17)(18)(19), although no mammalian, amphibian, or fish endogenous hepadnaviruses have yet been detected. The presence of EVEs has helped provide a time scale of hepadnavirus evolution, particularly as some of the endogenization events may have occurred as early as 200 million years ago (15). Hence, although there is clear evidence for some cross-species transmission (20), current data suggest that hepadnavirus evolution largely follows a pattern of virus-host codivergence that extends to at least the origin of the ray-finned fish.…”

“…In the genus Orthohepadnavirus, a fourth ORF encodes protein X. Despite these similar genome organizations, nucleotide sequence identity between hepadnavirus genera is limited, with the exception of some highly conserved functional domains (14,15). Human hepatitis B virus (HuHBV) affects more than onethird of the human population, and infections have the potential to cause both severe chronic liver disease and hepatocellular carcinoma (1)(2)(3).…”