A novel hepatovirus identified in wild woodchuck Marmota himalayana

Abstract

Hepatitis A virus (HAV) is a hepatotropic picornavirus that causes acute liver disease worldwide. Here, we report on the identification of a novel hepatovirus tentatively named Marmota Himalayana hepatovirus (MHHAV) in wild woodchucks (Marmota Himalayana) in China. The genomic and molecular characterization of MHHAV indicated that it is most closely related genetically to HAV. MHHAV has wide tissue distribution but shows tropism for the liver. The virus is morphologically and structurally similar to HAV. The pattern of its codon usage bias is also consistent with that of HAV. Phylogenetic analysis indicated that MHHAV groups with known HAVs but forms an independent branch, and represents a new species in the genus Hepatovirus within the family Picornaviridae. Antigenic site analysis suggested MHHAV has a new antigenic property to other HAVs. Further evolutionary analysis of MHHAV and primate HAVs led to a most recent common ancestor estimate of 1,000 years ago, while the common ancestor of all HAV-related viruses including phopivirus can be traced back to 1800 years ago. The discovery of MHHAV may provide new insights into the origin and evolution of HAV and a model system with which to explore the pathogenesis of HAV infection.

Figure 1. MHHAV genome organization and cleavage sites.

(a). Structure map of the MHHAV genome. P1, encoding the viral structural proteins VP4-VP2-VP3-VP1; P2 and P3 are nonstructural proteins, of which, P2 contains the 2A–2C regions and P3 the 3A–3D regions. (b). Amino acid sequences of MHHAV, the prototypic human and simian HAVs and phopivirus adjacent to the predicted protease cleavage sites (10 aa on each side are shown). The amino acids in red indicated by an arrow represent cleavage sites.

Figure 2. Predicted partial secondary structure of MHHAV.

(a) Secondary structure of the MHHAV 5′ UTR; domains are labeled I to V. The putative initiator codon (AUG) and UUUCC sequence are indicated in red. (b) Cis-acting secondary structures of MHHAV in the 3D pol-coding region.

Figure 3. Positive-strand and negative-strand viral RNA copy number determined by real-time PCR.

(a) Numbers of positive-strand viral RNA copies in feces, blood and other tissues. The highest viral RNA load was in the liver, and the lowest in the trachea. T-test showed the viral loads in liver and other tissues were statistically significant (blood, p = 0.000; spleen, p = 0.022; lung, p = 0.000; trachea, p = 0.000). (b) Negative-strand RNA in the liver. The amplification curves of the negative-strand viral RNA were not observed in all collected tissues except for the liver. Note: “ID” stands for “identifier”.

Figure 4. Phylogenetic analysis of sequences of MHHAV: (a) VP1 region (nucleotide); (b) polyprotein (amino acid).

The tree was constructed using the neighbor-joining method by MEGA ver. 5 with 1,000 bootstrap replicates. The virus in this study is indicated by the red “•.” Bootstrap values are shown on the branches. Results showed that MHHAV formed a distinct lineage to the known primate HAVs and phopivirus.

Figure 5. The Bayesian Markov Chain Monte Carlo (MCMC) tree of the VP1 regions of MHHAV and the known primate HAVs and phopivirus.

Horizontal branches are drawn to scale of estimated year of divergence, with tip times reflecting sampling data (year). The estimated time for the most recent common ancestors of the major nodes of the lineages are shown. IA, IB, IIIB: subgenotypes of HAV genotypes I and III.

Figure 6. Spherical, non-enveloped virus particles of ~27 nm in diameter visualized by negative-staining electron microscopy: (a) unclumped MHHAV; (b) immune-complexed MHHAV.

The black arrow indicates an intact particle; the white arrow indicates a potential empty particle.