A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains

Zoonotic hepatitis E virus (HEV) infection is an emerging cause of acute viral hepatitis in developed countries. Known reservoirs of zoonotic genotype 3 (HEV-3) are mainly pigs and wild boar, and to a lesser extent rabbits and deer. Rabbit hepatitis E virus (HEV-3ra) is prevalent in rabbits worldwide and represents a particular risk for zoonotic infection. Current understanding of the molecular mechanisms of HEV pathogenesis is incomplete, particularly due to the limited availability of efficient and reliable cell culture systems. In order to identify genomic regions responsible for HEV propagation in cell culture, we developed a modular chimeric reporter replicon system based on cell culture-adapted (Kernow-C1/p6 and 47832mc) and rabbit-derived HEV strains. Replication in HepG2 cells was monitored on the basis of a Gaussia luciferase reporter gene that was inserted in place of the open reading frame (ORF) 2 of the HEV genome. Luciferase activity of rabbit HEV-derived replicons was significantly lower than that of Kernow-C1/p6 and 47832mc replicons. Serial exchanges of defined ORF1 segments within the Kernow-C1/p6 replicon backbone indicated that HEV replication in HepG2 cells is not determined by a single domain but rather by an interplay of longer segments of the ORF1-derived nonstructural polyprotein. This implies that a specific combination of viral factors is required for efficient HEV propagation in cell culture.