Authors G. SARI (1), X. YIN (2), A. BOONSTRA (3), Z. FENG (2), T. VANWOLLEGHEM (4) / [1] Erasmus MC, Rotterdam, Netherlands, Rotterdam, Netherlands (the), Gastroenterology and Hepatology, [2] The Research Institute, Ohio (Columbus), United States (the), Center for Vaccines and Immunity, [3] Erasmus Medical Center, Rotterdam, Netherlands (the), Gastroenterology and Hepatology, [4] UZA, Universitair Ziekenhuis Antwerpen, Edegem, Belgium, Gastroenterology&Hepatology Introduction Hepatitis E viruses (HEV) are an important enterically transmitted cause of viral hepatitis. The HEV RNA genome is single-stranded, positive-sensed encoding 3 ORFs(ORF1- 3). Although ORF3 has a viroporin structure and is required for viral propagation in macaques, its exact role remains unclear. Aim In the present study, we dissect the function of ORF3 via reverse genetics and infectivity studies, both in vitro as in the liver humanized mouse model. Methods A genotype 3 HEV strain (Kernow C1, P6) was used as backbone to construct ORF3 mutant viruses. Virus stocks were produced by transfecting in vitro transcripts into Huh-7 (clone S10-3) cells followed by gradient ultracentrifugation to purify intracellular non-enveloped virions. uPA-NOG and TK-NOG mice (n=15) were transplanted with human hepatocytes from a single donor and inoculated iv with above mentioned ORF3 mutant viruses (6 log geq/mouse) upon establishment of a stable graft. Weekly feces and serum samples were obtained during the 6-weeks infection course, after which animals were sacrificed and liver and bile were collected for viral load determination by multiplex qPCR. Results Three ORF3 mutants were generated: an ORF3del mutant that contains a mutation in the start codon, a PSAP mutant that contains mutations in the C terminal late domain, and a CCC mutant that contains mutations in the N terminal cysteines 11-13 (a putative palmitoylation site). Replication and infectivity of all 3 mutants were comparable to the wild type (WT) HEV in vitro. WT ORF3 and the PSAP mutant predominantly localized to the apical membrane of HepG2 cells, whereas the CCC mutant was predominantly cytoplasmic. At sacrifice, HEV RNA was detectable in liver of 9/14 animals (1 animal died at 4 weeks pi), with HEV RNA titers (4-7 log IU/gr tissue) comparable between WT (n=4/4), PSAP (n=4/4) and CCC (n=1/2) inoculated mice. None of the ORF3del mutants proved infectious in vivo (n=4/4). While all WT inoculated mice had detectable HEV RNA in bile (5-7 log IU/mL) and feces (3-6 log IU/gr), only 1/4 PSAP mutant inoculated mice showed quantifiable HEV RNA in bile and feces. The HEV CCC mutant was barely detectable in bile <3.75 log IU/mL and negative in fecal samples (n=3/3). Despite this, serum titers between WT-HEV PSAP and HEV CCC mutant viruses were comparable. Conclusions Overall this corroborates the importance of ORF3 in HEV propagation in vivo and suggests a role of a putative palmitoylation site at the N-terminus of ORF3 in secretion of HEV into the biliary canaliculi.
