Hepatitis B virus (HBV) is a prototype hepadnavirus with 400 million chronic carriers worldwide. Chronic HBV infection is a main risk factor of hepatocellular carcinoma (HCC), causing >50% of all HCCs. Early in infection, HBV spreads virtually throughout the entire liver, but during chronic infection, areas of apparently virus-free hepatocytes (20-90% of the entire hepatocyte population) appear regardless of the ongoing viremia. These findings were made in HBV-infected humans and chimpanzees and in woodchucks infected with the woodchuck hepatitis virus (WHV), which is closely related to HBV. These observations, along with the analysis of kinetics of the emergence of drug-resistant HBV mutants, laid the foundation for a long-standing unresolved argument in the HBV field that in chronic infection, cell-to-cell spread of a hepadnavirus is at least very inefficient (if it occursat all), and super-infection is an unlikely event. It was proposed that chronic hepadnavirus infection can be maintained exclusively via division of the infected hepatocytes in the absence of the spread. Super-infection exclusion was shown for HBV-related duck hepatitis B virus (DHBV), and was suggested for HBV and WHV. However, the absence of the viral cell-to-cell spread and super-infection in chronic infections with either WHV or HBV has not been confirmed or dismissed. Therefore, in Aim 1, we propose to determine directly whether the cell-to-cell spread of hepadnavirus and super-infection of already infected cells occur during chronic infection, which would indicate that spread could be an essential factor for maintenance of chronic infection and thus could also be a so far unrecognized risk factor of HCC. In addition, HCCs are also frequently reported as being apparently virus-free in HBV carrier humans and WHV carrier woodchucks, suggesting that HCCs arise from altered hepatocytes that have lost the ability to support efficient hepadnavirus replication, and thus have a selective advantage for a clonal expansion since they are no longer targeted by the immune system. This has led us to Aim 2 that will determine (i) if hepadnavirus-induced HCC is still susceptible to infection with a hepadnavirus, and (ii) what controls the apparent virus-free status of HCC. We propose to use an invaluable surrogate model to study HBV infection and especially HCC development - WHV carrier woodchucks. We will super-infect WHV carriers with early HCCs with a different natural infectious WHV strain, WHVNY, which has a unique deletion and thus can be easily discriminated from the strain WHV7 used for the primary infection. We will (i) directly determine the susceptibility of normal hepatocytes and HCCs to WHVNY super-infection;and (ii) address if the immune system controls the ability of cells to get re-infected. Overall, the proposed study will greatly improve our understanding of the mechanism of chronic hepadnavirus infection in relation to HCC development. It has the potential to identify infectivity of virions and virus spred as important factors of pathogenesis and HCC risk, and may facilitate the use of entry inhibitors that bind the HBV receptor and block virus spread as new therapeutics for battling chronic HBV infection and reducing HCC risk.

Public Health Relevance

Our study will determine if virus spread and super-infection of hepatocytes and HCC cells take place in chronic hepadnaviral infection in vivo, which will significantly advance our understanding of the mechanism of hepadnavirus-related pathogenesis, and may facilitate use of entry inhibitors as antiviral and anti-HCC drugs.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-VIRB-P (09))
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Read-Connole, Elizabeth Lee
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University of Kansas
Schools of Medicine
Kansas City
United States
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Freitas, Natalia; Lukash, Tetyana; Gunewardena, Sumedha et al. (2018) Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers. J Virol 92:
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Freitas, Natalia; Lukash, Tetyana; Dudek, Megan et al. (2015) Capacity of a natural strain of woodchuck hepatitis virus, WHVNY, to induce acute infection in naive adult woodchucks. Virus Res 205:12-21
Freitas, Natalia; Lukash, Tetyana; Rodrigues, Louise et al. (2015) Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection. J Virol 89:8749-63
Rodrigues, Louise; Freitas, Natalia; Kallakury, Bhaskar V et al. (2015) Superinfection with woodchuck hepatitis virus strain WHVNY of livers chronically infected with strain WHV7. J Virol 89:384-405
Cunha, Celso; Tavanez, João Paulo; Gudima, Severin (2015) Hepatitis delta virus: A fascinating and neglected pathogen. World J Virol 4:313-22
Freitas, Natalia; Abe, Kenji; Cunha, Celso et al. (2014) Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different genotypes of hepatitis B virus. J Virol 88:6255-67
Freitas, Natalia; Cunha, Celso; Menne, Stephan et al. (2014) Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles. J Virol 88:5742-54
Freitas, Natalia; Salisse, Jessica; Cunha, Celso et al. (2012) Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks. Hepatology 56:76-85