Title: Intravital Imaging of the Liver Microenvironment in The Woodchuck Model of Hepatitis B Virus Infection

Background: The hepatitis B virus (HBV) is a non-cytopathic virus with a dynamic disease course determined by virus-host interactions. HBV can precipitate an acute, self-limited hepatitis coinciding with a robust innate and adaptive immune response to achieve virus control. In contrast, chronic liver injury develops due to virus-induced disruptions in host immunity and immune-mediated inflammation leading to progressive liver damage and fibrosis. Uncovering the virus-host interactions during the initial phase of infection, including viral trafficking, cells interacting with virus and the time of its uptake by hepatocytes leading to early resolution of hepatitis as opposed to chronic inflammation is essential for understanding HBV immunopathogenesis. The eastern North American woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV), is a valuable immunocompetent model that well recapitulate the natural history and liver disease pathogenesis and pathology in HBV infection.

Methods: Intravital microscopy (IVM) is a powerful and novel tool for studying in vivo immune cell behaviour and for identifying the dynamic pathogen-host cell interactions in a living host. For the first time, we have developed and optimized surgery, anaesthesia, and physiological supports to perform IVM in this animal model.

Results: By optimizing species-independent markers and cross-reactive antibodies, we were able to develop an effective imaging protocol to capture in situ innate and adaptive immune cellular events at high resolution and specificity. We visualized very early cellular events after WHV infection including intrahepatic sinusoidal vascular virus trafficking and virus capture by liver macrophages (Kupffer cells) and hepatocytes 20 minutes following intravenous injection with fluorescently labelled virus.

Conclusion: This advanced model will further our understanding of hepadnaviral biology and associated hepatic immunological dysfunction leading to either resolution or progression to chronic hepatitis B.