Woodchuck and deer Hepatitis Delta-like agents show distinct innate immune activation and IFN-resistance compared to human Hepatitis D Virus

Abstract Replication of human hepatitis delta virus (HDV) is sensed by the pattern recognition receptors (PRRs) melanoma differentiation antigen 5 (MDA5) and Laboratory of Genetics and Physiology 2 (LGP2), activating interferon (IFN) responses. Both HDV-induced IFNs and exogenously administered IFN-α and -λ suppress HDV RNA spread via cell division (CDMS) but have only minor effects on replication in resting cells. With the discovery of HDV-like agents (DLAs) in diverse animal species an opportunity arose to comparatively study the interplay between these evolutionarily related replicons with respect to innate immune responses. To overcome the lack of authentic in vitro infection models for the viroid-like agents, we established a Hepatitis B virus (HBV)-envelope protein (HBsAg) pseudotype-based infection system for woodchuck (WoDV) and deer (DeDV) DLA. Infection of human hepatic cell lines with WoDV/HBsAg and DeDV/HBsAg pseudo-typed virions allowed initiation of replication in HuH7 NTCP cells. To assess the interdependence of the innate immune system and viral persistence, innate immunocompetent HepaRG NTCP cells were infected with HDV, WoDV/HBsAg and DeDV/HBsAg. Consistent with previous findings, HDV replication robustly activated IFN responses, while replication of WoDV and, to a lesser extent, DeDV induced weaker IFN responses, as measured by IFN stimulated gene expression. Moreover, while CDMS of HDV was abrogated by IFN (either endogenous activation or exogenous administration), CDMS of WoDV and DeDV was not majorly affected, indicating different resistance mechanisms to IFN treatment, compared to HDV. Importantly, this phenotype of IFN resistance during cell division was confirmed for WoDV in woodchuck-derived hepatoma cells (WCH17 NTCP ), a model closer to the natural host. Furthermore, we demonstrated that the large delta antigen of HDV does not contribute to interferon sensitivity during cell division, suggesting that other viral or cellular factors mediate this process. Together, these results revealed that non-human DLAs display immune evasion strategies distinct from HDV, providing new insights into deltavirus biology and its evolutionary diversity.