06: Visualization of hepatitis E virus RNA and proteins in the human liver

Background: Although hepatitis E constitutes a substantial disease burden worldwide, surprisingly little is known about the localization of hepatitis E Virus (HEV) in the human liver. We therefore aimed to visualize HEV RNA and proteins in situ. Methods: Twelve antibodies against HEV open reading frame (ORF) 1–3 proteins for immunohistochemistry (IHC) and two probes for in situ hybridization (ISH) were tested on formalin-fixed, paraffin-embedded (FFPE) Huh-7 cells transfected with HEV ORF1-3 Expression vectors. IHC (and partly ISH) were then applied to Hep293TT cells replicating infectious HEV and liver specimens from patients with hepatitis E (n=20) and controls (n=134). Results: Whereas ORF1-3 proteins were all detectable in HEV protein expressing cells, only ORF2 and 3 proteins were traceable in cells replicating infectious HEV. In liver specimens from patients with hepatitis E, only the ORF2encoded capsid protein was unequivocally detectable. IHC for ORF2 protein revealed a patchy expression in individual or grouped hepatocytes, generally stronger in cases of chronic compared to acute hepatitis. Besides cytoplasmic and canalicular, ORF2 protein also displayed a hitherto not described nuclear localization. Furthermore, positivity for ORF2 protein in defined areas correlated with HEV RNA detection by ISH. IHC was specific and comparably sensitive as PCR for HEV RNA. Conclusions: In livers from patients with hepatitis E, the ORF2 protein can be visualized reliably by IHC, allowing sensitive and specific detection of HEV in FFPE samples. Therefore, HEV ORF2 IHC can be used as a rapid, handy and not expensive ancillary tool in histopathologic diagnostics of HEV. In addition, its variable subcellular distribution in individual hepatocytes of the same liver suggests an interaction with nuclear components and thus a redistribution of ORF2 protein during infection.