Daniela Lenggenhager1, Jérôme Gouttenoire2, Mohsen Malehmir1, Marion Bawohl1, Hanna Honcharova-Biletska1, Susanne Kreutzer1, David Semela3, Jörg Neuweiler4, Sandra Hürlimann5, Patrick Aepli6, Montserrat Fraga2, Roland Sahli7, Luigi Terracciano8, Laura Rubbia-Brandt9, Beat Müllhaupt10, Christine Sempoux11, Darius Moradpour2, Achim Weber12. 1. Department of Pathology and Molecular Pathology, University Zurich and University Hospital Zurich, Zurich, Switzerland. 2. Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. 3. Division of Gastroenterology and Hepatology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland. 4. Institute of Pathology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland. 5. Institute of Pathology, Cantonal Hospital Lucerne, Lucerne, Switzerland. 6. Gastroenterology and Hepatology Unit, Cantonal Hospital Lucerne, Lucerne, Switzerland. 7. Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. 8. Department of Pathology, University Hospital Basel, Basel, Switzerland. 9. Service de Pathologie Clinique Geneva University Hospitals Faculté de Médecine Geneva, Switzerland. 10. Clinics of Hepatology and Gastroenterology, University and University Hospital Zurich, Zurich, Switzerland. 11. Institut Universitaire de Pathologie, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. 12. Department of Pathology and Molecular Pathology, University Zurich and University Hospital Zurich, Zurich, Switzerland. Electronic address: achim.weber@usz.ch.
Abstract
BACKGROUND & AIMS: 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: A panel of 12 different antibodies against HEV open reading frame (ORF) 1-3 proteins was evaluated for immunohistochemistry (IHC) and two probes for in situ hybridization (ISH) in formalin-fixed, paraffin-embedded (FFPE) HuH7 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 transfected, HEV protein-expressing cells, only ORF2 and 3 proteins were traceable in cells replicating infectious HEV. Only the ORF2-encoded capsid protein was also unequivocally detectable in liver specimens from patients with hepatitis E. IHC for ORF2 protein revealed a patchy expression in individual or grouped hepatocytes, generally stronger in chronic compared to acute hepatitis. Besides cytoplasmic and canalicular, ORF2 protein also displayed a hitherto unknown nuclear localization. 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: ORF2 protein can be reliably visualized in the liver of patients with hepatitis E, allowing for sensitive and specific detection of HEV in FFPE samples. Its variable subcellular distribution in individual hepatocytes of the same liver suggests a redistribution of ORF2 protein during infection and interaction with nuclear components. LAY SUMMARY: The open reading frame (ORF) 2 protein can be used to visualize the hepatitis E virus (HEV) in the human liver. This enabled us to discover a hitherto unknown localization of the HEV ORF2 protein in the nucleus of hepatocytes and to develop a test for rapid histopathologic diagnosis of hepatitis E, the most common cause of acute hepatitis worldwide.
BACKGROUND & AIMS: 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: A panel of 12 different antibodies against HEV open reading frame (ORF) 1-3 proteins was evaluated for immunohistochemistry (IHC) and two probes for in situ hybridization (ISH) in formalin-fixed, paraffin-embedded (FFPE) HuH7 cells transfected with HEVORF1-3 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 transfected, HEV protein-expressing cells, only ORF2 and 3 proteins were traceable in cells replicating infectious HEV. Only the ORF2-encoded capsid protein was also unequivocally detectable in liver specimens from patients with hepatitis E. IHC for ORF2 protein revealed a patchy expression in individual or grouped hepatocytes, generally stronger in chronic compared to acute hepatitis. Besides cytoplasmic and canalicular, ORF2 protein also displayed a hitherto unknown nuclear localization. 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:ORF2 protein can be reliably visualized in the liver of patients with hepatitis E, allowing for sensitive and specific detection of HEV in FFPE samples. Its variable subcellular distribution in individual hepatocytes of the same liver suggests a redistribution of ORF2 protein during infection and interaction with nuclear components. LAY SUMMARY: The open reading frame (ORF) 2 protein can be used to visualize the hepatitis E virus (HEV) in the human liver. This enabled us to discover a hitherto unknown localization of the HEVORF2 protein in the nucleus of hepatocytes and to develop a test for rapid histopathologic diagnosis of hepatitis E, the most common cause of acute hepatitis worldwide.
Authors: Andrea Beer; Heidemarie Holzmann; Sven Pischke; Patrick Behrendt; Fritz Wrba; Jerome Schlue; Uta Drebber; Barbara Neudert; Emina Halilbasic; Hans Kreipe; Ansgar Lohse; Martina Sterneck; Heiner Wedemeyer; Michael Manns; Hans P Dienes Journal: Liver Int Date: 2019-06-17 Impact factor: 5.828