Background and Aim: Hepatitis E virus (HEV) is one of the most common causes of acute viral hepatitis worldwide. Genotype (gt) 1 and 2 are restricted to humans and are transmitted via the fecal- oral route, leading to epidemics and sporadic cases in emerging countries. However, in developed countries the transmission of gt3 and 4 occur through the consumption of raw/undercooked meat and contaminated fruits and vegetables. Recently, a HEV infection transmitted by consumption of a raw sausage called “mortadella di fegato cruda” was described in Switzerland. Surprisingly, the identified Swiss HEV gt3 strain showed only 88% similarity with other gt3 strains. Full-length genome sequencing of HEV then suggested the existence of a Swiss-specific HEV subcluster 3s, recently reassigned within the subtype 3h. Therefore, the aim of this study was to further characterize HEV infections in Switzerland at the molecular level.
Methods: Various plasmids containing the full-length HEV sequence or subgenomic constructs consisting of ORF1 harboring a selection gene or Gaussia luciferase marker were transfected into human hepatocellular carcinoma cell lines using electroporation or a transfection reagent. Cell culture acquired mutations in the subgenomic constructs were identified by PCR amplification of ORF1 using primers designed for 6 overlapping fragments, followed by Zero Blunt cloning and sequencing. The effect of the acquired mutations on RNA replication was analyzed by cloning the mutations into the subgenomic WT constructs and performing luciferase assays. Furthermore, immunofluorescence staining, western blotting and RT qPCR were used to analyze the expression levels of ORF2 and ORF3 of the HEV full- length constructs.
Results: Three missense and one silent mutation were identified in the subgenomic RS23-Neo construct, which were constitutively replicating in Hep293TT cells. Surprisingly, these acquired mutations had no positive effect on RNA replication. Furthermore, expression analysis of ORF2 from RS01 and RS23 full-length constructs, revealed an initial localization to the nucleus and cytoplasm and was later only detected in the cytoplasm. Moreover, the expression level of ORF3 was much lower compared to ORF2. However, at later times points, it was mostly detected in the cytoplasm with only few concentrated spots that localized also in the nucleus (day 4 or 5).
Conclusion: Initial expression of ORF2 in the nucleus and cytoplasm and later detection in the cytoplasm, corresponds with other publications, where it was reported that ORF2 transits through the nucleus and plays an important role in the control of antiviral responses.1 Furthermore, our results raise the hypothesis that ORF3 transits also through the nucleus as ORF2.