BackgroundHuman pluripotent stem cell (hPSC)‐derived hepatocyte‐like cells (HLCs) are a valuable model to investigate host‐pathogen interactions of hepatitis viruses in a mature and authentic environment. Here, we investigate the susceptibility of HLCs to the hepatitis delta virus (HDV).MethodsWe differentiated hPSC into HLCs, and inoculated them with infectious HDV produced in Huh7NTCP. HDV infection and cellular response was monitored by RTqPCR and immunostaining.ResultsCells undergoing hepatic differentiation become susceptible to HDV after acquiring expression of the viral receptor Na+‐taurocholate co‐transporting polypeptide (NTCP) during hepatic specification. Inoculation of HLCs with HDV leads to detection of intracellular HDV RNA and accumulation of the HDV antigen in the cells. Upon infection, the HLCs mounted an innate immune response based on induction of the interferons IFNB and L, and upregulation of interferon‐stimulated genes. The intensity of this immune response positively correlated with the level of viral replication and was dependant on both the JAK/STAT and NFκB pathway activation. Importantly, this innate immune response did not inhibit HDV replication. However, pre‐treatment of the HLCs with IFNα2b reduced viral infection, suggesting that ISGs may limit early stages of infection. Myrcludex efficiently abrogated infection and blocked innate immune activation. Lonafarnib treatment of HDV mono infected HLCs on the other hand led to exacerbated viral replication and innate immune response.ConclusionThe HDV in vitro mono‐infection model represents a new tool to study HDV replication, its host‐pathogen interactions and evaluate new antiviral drugs in cells displaying mature hepatic functions.

Human pluripotent stem cell-derived hepatocyte-like cells (HLCs) are a valuable model to investigate host-pathogen interactions of hepatitis viruses in a mature and authentic environment. Here, we investigated the susceptibility of HLCs to the Hepatitis D Virus (HDV), a virus that in co-infection with HBV is responsible for the most severe form of viral hepatitis. Cells undergoing hepatic differentiation became susceptible to HDV infection after acquiring expression of the Na+-taurocholate cotransporting polypeptide (NTCP), the receptor mediating HBV and HDV entry. Inoculation of mature HLCs with HDV lead to increasing amounts of intracellular HDV RNA and accumulation of the HDV antigen in the cells. The infection was abrogated when using known entry inhibitors targeting NTCP or by disrupting genome replication using the nucleoside analogue Ribavirin. Upon infection, the HLCs mounted an innate immune response based on induction of the interferons IFNB and L, but not IFNA, and were associated with an upregulation of interferon-stimulated genes. The intensity of this immune response positively correlated with the level of viral replication and was dependant on both the JAK/STAT and NFκB pathway activation. Importantly, neither this innate immune response nor an exogenous treatment of IFNα2b inhibited HDV replication. However, pre-treatment of the HLCs with IFNα2b reduced viral infection, suggesting that ISGs may limit early stages of infection. This novel HDV in vitro infection model represents a valuable tool for studying HDV replication and investigating candidate antiviral drugs in cells displaying mature hepatic functions.