Towards a small animal model for hepatitis C Alexander Ploss & Charles M. Rice

1. Towards a small animal model for hepatitis CAlexander Ploss& Charles M. Rice EMBO reports (2009) 10, 1220 - 1227 Abteilung und/oder Titel Abteilung und/oder Titel Datum

2. Hepatitis C virus • Hepatitis C virus (HCV) causes chronic liver diseases. • Can lead to cirrhosis, liver failure and liver cancer. • 130 million people infected worldwide • Current Interferon treatment is often ineffective and poorly tolerated • No vaccine available till date. Abteilung und/oder Titel Abteilung und/oder Titel Datum

3. HCV life cycle

4. Drawbacks to HCV research Chimpanzee are the only available immunocompetent experimental system. Use limited by ethical constraint, restricted availability and high costs Preferred animal model due to easy availability and handling, economical and complete genome information available

5. Approaches towards animal model • Viral adaptation to infect non human cells • Genetic host humanization to adapt the rodent tissues • Xenotransplation of human tissues

6. Viral adaptation • Sera inoculated from HCV infected individual resisted due to block in HCV entry • CD81 and OCLN must be of human origin to permit HCV infection • Expression of mouse CD81 in human hepatoma cells causes very low infection probably due to reduced affinity • Mouse CD81 is 90% homologous to human; 4 unconserved key residues • HCV can be adapted in vitro to use mouse CD81; • Selection of three mutations in E1 and E2 enhanced mouse CD81 dependent uptake to levels comparable to human orthologue

7. Viral adaptation • Studies show HCV entry and replication in murine environment; assembly and release of infectious virions are unclear • Increasing viral replication can allow detection of infectious virus by expression of cDNA transgene encoding entire HCV genome • Generation of proper 5’ and 3’ ends of HCV genome crucial, production of infectious HCV from stably HCV cDNA-transfected HepG2 has been reported • Additional adaptation steps necessary to select for genomes with assembly competence in a mouse cell environment

8. Viral adaptation • The efficiency of murine tropic HCV infection to that of human cells expressing the mouse entry factors is uncertain • Post entry additional restriction might exist, thus virally encoded replication machinery might not adapt to murine counterpart • Modifications affecting species tropism might have significant efforts on immune responses and pathogenesis

9. Xenotranplantation model • Chimeric mice harboring HCV permissive tissue can be obtained, susceptible to human hepatotropic pathogens • Alb-uPa transgene overexpression results in severe liver damage rescued by transplanting non transgenic (human) hepatocytes • Xenotransplanted humanized mice are potential for studying drug and vaccine efficacy as well as pharmacokinetics and toxicity • Application limited by less production, substantial variability and logistical constraints. • Pathogenesis and immunity studies impeded by absence of functional immune system-required to avoid xenograft rejection

10. Xenotransplantation model • Combination of humanized liver model with mice harbouring a human haematolymphoid system • Generation by engrafting suspensions of human haematopoetic progenitor stem cells into immunodeficient animals, virus-specific immune response can be elicited on successful human immune reconstitution • Merging hepatic and haematolymphoid reconstitution in a single recipient will allow studies of pathogenesis, immune correlates and mechanisms of persistence of HCV and other human hepatotropic pathogens • The generation of these types of humanized mice requires substantial infrastructure and advanced technical skills

11. Host adaptation • An inbred mouse model with inheritable susceptibility to HCV would overcome the technical difficulties of the xenotransplantation model • Efforts for genetic adaptation of human CD81-transgenic mice were resistant to HCV infection • Functional cDNA complementation screen approach could identify critical human factors required for efficient HCV RNA replication in mouse cells. • Liver specific miRNAs- miR122 and miR199a have shown to affect HCV RNA translation and replication • Dominant-negative restriction factors causing altered innate antiviral responses or inability of HCV proteins to overcome murine defences could impair HCV replication in mouse.

12. Host adaptation • Several genetic adaptations to humanize the murine host are required to create an inbred mouse model for HCV infection which could be a platform for further improvements designed to mimic more closely the clinical features of human hepatitis. • Studies to identify crucial human factors is dubious due to minimal overlap and relevance of HCV interactions yet to be demonstrated • Sequence divergence of identified factors might reduce the infectious capacity and affect tropism.

14. Conclusion • HCV infection aggravated by insufficient therapy and lack of proper vaccine • A robust animal model easily propagated and reproducing the viral life cycle would be instrumental in understanding HCV pathogenesis and to guide drug development • Deciphering the barriers to HCV replication in mouse cells will provide rich insights into virus host interactions directing towards a sustainable mouse model of HCV infection.