Raphael Wolfisberg1,2, Caroline E Thorselius1,2, Eduardo Salinas3,4, Elizabeth Elrod3,4, Sheetal Trivedi5, Louise Nielsen1,2, Ulrik Fahnøe1,2, Amit Kapoor5, Arash Grakoui3,4, Charles M Rice6, Jens Bukh1,2, Kenn Holmbeck1,2, Troels K H Scheel1,2,6. 1. Copenhagen Hepatitis C Program, Department of Infectious Diseases, Hvidovre Hospital, Copenhagen, Denmark. 2. Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. 3. Emory Vaccine Center, Division of Microbiology and Immunology, Yerkes Research Primate Center, Emory University School of Medicine, Atlanta, Georgia, USA. 4. Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. 5. Center for Vaccines and Immunity, Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA. 6. Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA.
Abstract
BACKGROUND AND AIMS: Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. APPROACH AND RESULTS: We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. CONCLUSIONS: The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.
BACKGROUND AND AIMS: Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. APPROACH AND RESULTS: We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. CONCLUSIONS: The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.
Authors: Eva Billerbeck; Raphael Wolfisberg; Ulrik Fahnøe; Jing W Xiao; Corrine Quirk; Joseph M Luna; John M Cullen; Alex S Hartlage; Luis Chiriboga; Kalpana Ghoshal; W Ian Lipkin; Jens Bukh; Troels K H Scheel; Amit Kapoor; Charles M Rice Journal: Science Date: 2017-07-14 Impact factor: 47.728
Authors: Valerie J Kinchen; Guido Massaccesi; Andrew I Flyak; Madeleine C Mankowski; Michelle D Colbert; William O Osburn; Stuart C Ray; Andrea L Cox; James E Crowe; Justin R Bailey Journal: J Clin Invest Date: 2019-08-13 Impact factor: 14.808
Authors: William O Osburn; Anna E Snider; Brittany L Wells; Rachel Latanich; Justin R Bailey; David L Thomas; Andrea L Cox; Stuart C Ray Journal: Hepatology Date: 2014-04-29 Impact factor: 17.425
Authors: Søren U Nielsen; Margaret F Bassendine; Alastair D Burt; Caroline Martin; Wanna Pumeechockchai; Geoffrey L Toms Journal: J Virol Date: 2006-03 Impact factor: 5.103