A Novel Marsupial Hepatitis A Virus Corroborates Complex Evolutionary Patterns Shaping the Genus Hepatovirus
Authors: de Oliveira Carneiro Ianei , Sander Anna-Lena , Silva Namá , Moreira-Soto Andres , Normann Andrea , Flehmig Bertram , Lukashev Alexander N. , Dotzauer Andreas , Wieseke Nicolas , Franke Carlos Roberto , Drexler Jan Felix 
Published: April 2018

The discovery of highly diverse nonprimate hepatoviruses illuminated the evolutionary origins of hepatitis A virus (HAV) ancestors in mammals other than primates. Marsupials are ancient mammals that diverged from other Eutheria during the Jurassic. Viruses from marsupials may thus provide important insight into virus evolution. To investigate
Hepatovirus
macroevolutionary patterns, we sampled 112 opossums in northeastern Brazil. A novel marsupial HAV (MHAV) in the Brazilian common opossum (
Didelphis aurita
) was detected by nested reverse transcription-PCR (RT-PCR). MHAV concentration in the liver was high, at 2.5 × 10
9
RNA copies/g, and at least 300-fold higher than those in other solid organs, suggesting hepatotropism. Hepatovirus seroprevalence in
D. aurita
was 26.6% as determined using an enzyme-linked immunosorbent assay (ELISA). Endpoint titers in confirmatory immunofluorescence assays were high, and marsupial antibodies colocalized with anti-HAV control sera, suggesting specificity of serological detection and considerable antigenic relatedness between HAV and MHAV. MHAV showed all genomic hallmarks defining hepatoviruses, including late-domain motifs likely involved in quasi-envelope acquisition, a predicted C-terminal pX extension of VP1, strong avoidance of CpG dinucleotides, and a type 3 internal ribosomal entry site. Translated polyprotein gene sequence distances of at least 23.7% from other hepatoviruses suggested that MHAV represents a novel
Hepatovirus
species. Conserved predicted cleavage sites suggested similarities in polyprotein processing between HAV and MHAV. MHAV was nested within rodent hepatoviruses in phylogenetic reconstructions, suggesting an ancestral hepatovirus host switch from rodents into marsupials. Cophylogenetic reconciliations of host and hepatovirus phylogenies confirmed that host-independent macroevolutionary patterns shaped the phylogenetic relationships of extant hepatoviruses. Although marsupials are synanthropic and consumed as wild game in Brazil, HAV community protective immunity may limit the zoonotic potential of MHAV.

IMPORTANCE
Hepatitis A virus (HAV) is a ubiquitous cause of acute hepatitis in humans. Recent findings revealed the evolutionary origins of HAV and the genus
Hepatovirus
defined by HAV in mammals other than primates in general and in small mammals in particular. The factors shaping the genealogy of extant hepatoviruses are unclear. We sampled marsupials, one of the most ancient mammalian lineages, and identified a novel marsupial HAV (MHAV). The novel MHAV shared specific features with HAV, including hepatotropism, antigenicity, genome structure, and a common ancestor in phylogenetic reconstructions. Coevolutionary analyses revealed that host-independent evolutionary patterns contributed most to the current phylogeny of hepatoviruses and that MHAV was the most drastic example of a cross-order host switch of any hepatovirus observed so far. The divergence of marsupials from other mammals offers unique opportunities to investigate HAV species barriers and whether mechanisms of HAV immune control are evolutionarily conserved.