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Abstract

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to form infectious virions. For the past 40 years, the evolutionary history of HDV has remained largely unknown. Recent discoveries have revolutionized our knowledge of HDV biology. HDV-like agents were identified in a vast and heterogeneous group of vertebrates and invertebrates, highlighting that the evolution of HDV is more complex than previously foreseen and not restricted to humans as primary hosts. Here, I focused on the characterization of HDV-like agents recently discovered in the woodchuck (Marmota monax), the white-tailed deer (Odocoileus virginianus), the lesser dog-like bat (Peropteryx macrotis) and several species of duck (Anas gracilis, Anas castanea, Anas superciliosa) in terms of replication, viral spreading pathways and cellular permissiveness. Viral replication was initiated by transfecting constructs encoding 1.1-fold over-length antigenomic HDV-like agents (DLA) RNA into human and non-human hepatic and non-hepatic cell lines. A cell-division-mediated viral amplification assay demonstrated the capability of the novel HDV-like agents to replicate and propagate not exclusively in hepatic tissues and without the requirement of envelopment. To elucidate whether the non-human HDV-like agents can exploit the envelope glycoproteins of hepadna- and other viruses to form infectious particles, I co-transfected cells with the respective expression constructs and plasmids encoding envelope proteins from different viruses. Strikingly, secretion of pseudo-typed virions capable of establishing infection in susceptible target cells was observed. HDV replication activates interferon (IFN) responses via activation and sensing by MDA5 and LGP2. HDV-induced IFNs and exogenous IFN-α and -γ profoundly suppressed cell division-mediated HDV spread (CDMS) but had only a minor effect on already ongoing HDV replication in resting cells. The discovery of HDV-like agents provides the chance to investigate the interplay between HDV and IFN response from an evolutionary perspective. However, appropriate cell culture models to investigate replication, host factor dependence and modes of spreading, are lacking. In my study, I established a robust infection system for the HDV-like agents found in woodchuck (WoDV) and deer (DeDV), overcoming the challenge that they do not express a farnesylated large delta antigen (L-HDAg) and cannot be packaged by HBsAg. After verifying that these agents, as HDV, can propagate efficiently via CDMS, I packaged their genomes with HBsAg by trans-complementation of the HDV L-HDAg. The supernatant was used to infect HepaRGNTCP non-targeted control (NT) and HepaRGNTCP MDA5 and/or LGP2 knock-out (KO) cells always in comparison with HDV. As expected, the HDV replication led to IFN activation in HepaRGNTCPNT but not MDA5/LGP2 KO cells. Accordingly, restriction of CDMS in HepaRGNTCP NT was observed. In contrast, infection with WoDV and DeDV induced minor IFN response in HepaRGNTCPNT cells, and the CDMS of these agents remained efficient independently of MDA5/LGP2 expression. Furthermore, the CDMS of WoDV and DeDV was not significantly affected by exogenous IFN treatment. Therefore, both agents not only lack a strong IFN activation but also display resistance to IFN treatment. My doctoral thesis on the replication, assembly, transmission, and host tropism of novel HDV-like agents provides insights into the molecular biology, evolution, and virus-host interaction of this unique group of agents.