Claire Montpellier1, Czeslaw Wychowski2, Ibrahim M Sayed3, Jean-Christophe Meunier4, Jean-Michel Saliou1, Maliki Ankavay1, Anne Bull4, André Pillez1, Florence Abravanel5, François Helle6, Etienne Brochot6, Hervé Drobecq7, Rayan Farhat1, Cécile-Marie Aliouat-Denis1, Juliano G Haddad1, Jacques Izopet5, Philip Meuleman8, Anne Goffard1, Jean Dubuisson1, Laurence Cocquerel9. 1. University of Lille, CNRS, INSERM, CHU Lille, Pasteur Institute of Lille, U1019-UMR8204-CIIL-Center for Infection and Immunity of Lille, Lille, France. 2. University of Lille, CNRS, INSERM, CHU Lille, Pasteur Institute of Lille, U1019-UMR8204-CIIL-Center for Infection and Immunity of Lille, Lille, France. Electronic address: czeslaw.wychowski@ibl.cnrs.fr. 3. Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium; Microbiology and Immunology Department, Faculty of Medicine, Assiut University, Assiut, Egypt. 4. Inserm-U966, University F. Rabelais, Tours, France. 5. CHU Toulouse, Hôpital Purpan, Laboratoire de virologie, National Reference Center for Hepatitis E, Toulouse, France. 6. EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France. 7. University of Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Target Therapies, Lille, France. 8. Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium. 9. University of Lille, CNRS, INSERM, CHU Lille, Pasteur Institute of Lille, U1019-UMR8204-CIIL-Center for Infection and Immunity of Lille, Lille, France. Electronic address: laurence.cocquerel@ibl.cnrs.fr.
Abstract
BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. METHODS: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. RESULTS: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. CONCLUSIONS: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.
BACKGROUND & AIMS:Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. METHODS: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. RESULTS: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. CONCLUSIONS: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.
Authors: Mohamed A El-Mokhtar; Haidi Karam-Allah Ramadan; Muhamad R Abdel Hameed; Ayat M Kamel; Sahar A Mandour; Maha Ali; Mohamed A Y Abdel-Malek; Doaa M Abd El-Kareem; Sara Adel; Eman H Salama; Khaled Abo Bakr Khalaf; Ibrahim M Sayed Journal: Virulence Date: 2021-12 Impact factor: 5.882