Ancient origins and global spread of domestic cat hepatitis B virus

Abstract

Mammalian hepadnaviruses have likely been evolving alongside their hosts for millions of years. Domestic cat HBV (DCHBV) has been detected in cats from several countries, but its genealogy, epidemiology, and host range remain unclear. Besides DCHBV, the only hepadnavirus identified among carnivores is the ringtail HBV (RtHBV). Because there is a gap in the felid fossil record of approximately 5-7 million years between the late Oligocene and the early Miocene, carnivore-derived viruses might help to shed light on Felidae evolution. Here, we screened 2260 sera and 154 paraffin-embedded liver samples from cats and 2123 sera from dogs sampled in Europe and South and Central America between 2018 and 2020 by PCR for DCHBV. We identified DCHBV genotype A (GtA) in 0.6% (7/1,195; 95% CI, 0.2-1.2) of cats sampled in Germany, France, Croatia, and Bulgaria and a genetically divergent DCHBV genotype B (GtB; 10.8% genomic sequence distance) in 0.2% of cats (2/1,065; 95% CI, 0.0-0.7) from Brazil. The detection rates of the two genotypes did not differ significantly (Fisher, P = .19). Viral loads ranged from 4 × 10¹-6 × 10⁶ for DCHBV GtA to 5-7 × 10³ for DCHBV GtB DNA copies per milliliter of serum. None of the cat livers or dog sera tested positive by PCR. Immunoglobulin G against the DCHBV core antigen (anti-DCHBc) was detected in 8/504 cat sera (1.6%; 95% CI, 0.7-3.1), without significant variation between countries (χ², P = .17), and in none of 180 dog sera by indirect immunofluorescence assay (IFA). Neither IFA (Fisher, P = .11; n = 311) nor PCR (Fisher, P = .63; n = 699) positivity was significantly associated with increased liver enzymes in cats, respectively. Coevolutionary reconciliations of virus and host phylogenies and Bayesian hypothesis testing suggested evolutionary origins of DCHBV during the Miocene, ∼8-17 million years ago (mya) from ancestral carnivores, consistent with long-term evolution. The long-term association of DCHBV with felines aids in elucidating orthohepadnaviral infection patterns and felid genealogy.

Keywords: animal models; carnivores; hepatitis b; neotropics; viral evolution.

Figure 1.

Molecular epidemiology of DCHBV. (a) Worldwide distribution of DCHBV genotypes. (b) Maximum-likelihood phylogenetic tree based on the complete genome of mammalian hepadnaviruses. (c) Maximum-likelihood phylogenetic tree of DCHBV without recombinant sequences using the white sucker hepatitis B virus as an outgroup (see Appendix for detailed information). Sequences identified in this study are highlighted in bold(d) Pairwise comparison of DCHBV and related hepadnaviruses. (e) Nucleotide-level intra- and inter-genotypic pairwise comparison of DCHBV sequences . (f) Genome characteristics of DCHBV genotype B. (g) Translated open reading frame phylogenies of DCHBV genotypes and other hepadnaviruses. (h) Immunofluorescence assay co-staining of the DCHBV core transfected into Vero E6 cells using rabbit anti-woodchuck hepatitis virus and a seropositive domestic cat serum.

Figure 2.

Clinical characteristics of domestic cats. (a) Reasons for collecting samples from cats in Brazil and Europe. The lack of more detailed information on the reasons for sampling was due to local data protection legislation. (b) Serum alanine transaminase (ALT) levels and the presence of DCHBV DNA or antibodies against DCHBV in domestic cats. (c) Serum aspartate aminotransferase (AST) levels and the presence of DCHBV DNA or antibodies against DCHBV in domestic cats.

Figure 3.

Evolutionary characteristics of DCHBV. (a) Representation of the most parsimonious reconciliation of host and hepadnavirus coevolutionary events. (b) Distance-based coevolutionary analysis. (c) Bayesian ASR. Pie charts indicate the posterior probabilities of ancestral traits for mammalian orders. Black dots indicate posterior probability support of grouping > 0.9. Trees (a-c) were built from nucleotide alignments of host and viral sequences.

Figure 4.

Evolutionary origins of DCHBV. (a) Hypothesis testing. The priors used for calibrating the hypothesis testing are labeled with numbers to improve visualization. Bayes factors (BF) allow the comparison of two priors. Abbreviations: kya, thousand years ago; mya, million years ago. (b) Bayesian phylogenetic tree and the timeline of selected events in domestic cat evolution. Asterisks indicate that the estimation of the DCHBV genotypes' TMRCA estimates should be carefully considered since there may be other DCHBV and related carnivore-derived sequences to be discovered.