A hepatitis B virus causes chronic infections in equids worldwide

Abstract

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 10⁹ mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na⁺/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection.

Keywords: HBV; animal model; coinfection; equids; evolution.

Fig. 1.

Phylogeny and epidemiology of EqHBV. (A) Organization of HBV and EqHBV genomes. (B) Complete genome-based maximum-likelihood phylogeny. (C) Immunofluorescence costaining using an anti-EqHBV antibody⁺ donkey serum (Do-15, Germany) and a rabbit anti-HBc serum. (D) EqHBV DNA and antibody detection in donkeys and zebras. (E–G) Seroprevalence per country (E), sex (F), and age group (G). Ab, antibodies; EqHBV, Equid HBV; EqHCV, Equid HCV; S, surface protein; Pol, polymerase; HBx, hepatitis B virus X protein.

Fig. 2.

Hepatotropism and pathogenicity of EqHBV. (A) EqHBV concentrations in Ghanaian donkey samples. Black bars, medians. (B and C) Pathological analyses. H&E-stained (B) and Sirius red-stained (C) liver tissue from donkeys. Three representatives are shown, sample IDs (B, left to right) Li-40, Li-34, Li-35, and (C, left to right) Li-09, Li-10, Li-41. EqHBV, Equid HBV; neg., negative; pos., positive.

Fig. 3.

Cell entry studies of EqHBV and HBV. (A) preS1-encoding region. Highlighted, essential NTCP-binding domain in human HBV. Dots, identical amino acids. The translated EqHBV sequence was identical in all strains. Additional sequence elements in HBV genotype A are not shown for clarity of presentation. (B) Infection of HepG2 cells expressing the human or the donkey NTCP ortholog. (C) Infection of PHHs and (D) Infection of PEHs with HDV pseudotyped with EqHBV-derived (HDV_Do-EqHBV/HDV_Ze-EqHBV) or HBV-derived HBs (HDV_HBV). Red, infected cells detected via Delta antigen staining using a patient-derived polyclonal HDV-antiserum. Blue, nuclei. (E) HBeAg production in PEHs (Architect; Abbott). S/CO, signal to cutoff ratio. anti-EqHBV, EqHBV-antibody⁺ donkey serum; anti-HBs, vaccine-derived antiserum; CSHBV, crowned shrew HBV; WHV, woodchuck hepatitis B virus; WMHBV, woolly monkey HBV.

Fig. 4.

(A–G) EqHBV infection markers in longitudinally sampled donkeys. Alkaline phosphatase (AP), aspartate aminotransferase (AST), γ-glutamyltransferase (γ-GT) units per liter (U/L). Upper limit in five EqHBV⁻ animals (SI Appendix, Fig. S6): AP, 140 U/L; AST, 180 U/L; γ-GT, 35 U/L. n.d., no data. LOD, limit of detection.

Fig. 5.

Evolutionary origin of EqHBV. (A) Simplified cladogram of mammals (47). Red, taxa positive for hepadnaviruses. (B) Bootscan graph of EqHBV and reference sequences. Below, phylogenetic trees of genome regions as indicated by black bars. (C) Ancestral state and TMRCA reconstruction using EqHBV complete genome sequences. Filled dots, posterior probability of grouping >0.90. (D) Hypothesis testing. (Left) Cladogram of Perissodactyla (48). Priors used for hypothesis testing are labeled with numbers. (Right) Bayes factors (BF) supporting the comparison of two priors. Details provided in the SI Appendix. kya, thousand years ago; mya, million years ago; ya, years ago. 2 ln BF, twice the natural logarithm of the BF.