Short '1.2x genome' infectious clone initiates deltavirus replication in Boa constrictor cells

Abstract

Human hepatitis D virus (HDV), discovered in 1977, represented the sole known deltavirus for decades. The dependence on hepatitis B virus (HBV) co-infection and its glycoproteins for infectious particle formation led to the assumption that deltaviruses are human-only pathogens. However, since 2018, several reports have described identification of HDV-like agents from various hosts but without co-infecting hepadnaviruses. Indeed, we demonstrated that Swiss snake colony virus 1 (SwSCV-1) uses arenaviruses as the helper for infectious particle formation, thus shaking the dogmatic alliance with hepadnaviruses for completing deltavirus life cycle. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2x genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used SwSCV-1 as the model to compare the ability of the previously reported "2x genome" and the "1.2x genome" plasmid constructs/infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2x and 1.2x genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. We hypothesize that duplicating the ribozymes facilitates the cleavage of genome multimers into unit-length pieces during the initial round of replication. The 1.2x genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions. IMPORTANCEHepatitis D virus (HDV) is a satellite virus infecting humans with strict association to hepatitis B virus (HBV) co-infection because HBV glycoproteins can mediate infectious HDV particle formation. For decades, HDV was the sole representative of deltaviruses, which had led to hypotheses suggesting that it evolved in humans, the only known natural host. Recent sequencing studies have led to the discovery of HDV-like sequences across a wide range of species, representing a paradigm shift in deltavirus evolution. Molecular biology tools such as infectious clones, which enable initiation of deltavirus infection without helper virus, are key to demonstrate that the recently found deltaviruses are capable of independent replication. Such tools will enable identification of the potential helper viruses. Here, we report a 1.2x genome copy strategy for designing plasmid-based infectious clones to study deltaviruses and to demonstrate that plasmid delivery into cultured snake cells sufficiently initiates replication of different deltaviruses.