Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

Abstract

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. 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 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported “2× genome” and the “1.2× genome” infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.

Unraveling human, rodent and snakeKolmioviridaereplication to anticipate cross-species transmission

“…While SDeV produces a single form of SDAg in cultured snake cells (Fig. 1E) and in infected snakes 13,14 , we observed two forms of the SDAg in human Huh7.5 and HEK293T cells (Fig. 1C).…”

“…The findings led to the creation of the novel realm Ribozyviria with the family Kolmioviridae to host the genus Deltavirus and seven other genera 16 . As only a minority of the studies on novel kolmiovirids made efforts towards molecular characterization of their replication 9,11,14 , our understanding of the kolmiovirid biology relies on the seminal research performed on HDV. Importantly, the cross-species transmission potential of kolmiovirids 10 coupled to their ability to form infectious particles with envelope proteins of a variety of helper viruses 13,32 raises an important question: can these minimal RNA viruses replicate in any animal cell they access?…”

“…Clones were sequenced and plasmid in the forward orientation were selected and amplified for further use. Plen-tiCMVPuro plasmids (Addgene, #17452) encoding the FLAG tagged human, rodent, avian, toad and snake delta (SDAg-S and SDAg-L) viruses' antigens were generated by PCR amplification of the antigens coding sequences with primers listed in Table 1, from pCAGGS 1.2x plasmids of HDV, RDeV, SDeV, avian and toad deltaviruses described in Szirovicza et al 14 . The amplified fragments were then cloned into PlentiCMVPuro (Addgene, #17452) plasmids by Gibson assembly from NEB (#E2611L).…”

“…These discoveries indicated that this new viral family is far more diverse and widespread amongst the animal kingdom than originally thought. Evidence that HDVlike viruses found in snakes 13,14 , rodents 9,11 and birds 11 are able to replicate and the recent identification of thousands of sequences similar to deltaviruses in metatranscriptomes 12,15 led to the establishment of a novel realm, Ribozyviria with a single family, Kolmioviridae, that includes the genus Deltavirus as well as seven other novel genera of kolmiovirids 16 . Because other kolmiovirids were only recently discovered, most of our knowledge of the biology of these agents stems from research on HDV 17 .…”

Comparative analysis of human, rodent and snake deltavirus replication

“…These discoveries indicated that this new viral family is far more diverse and widespread amongst the animal kingdom than originally thought. Evidence that HDVlike viruses found in snakes [13,14], rodents [9,11] and birds [11] are able to replicate and the recent identification of thousands of sequences similar to deltaviruses in metatranscriptomes [12,15] led to the establishment of a novel realm, Ribozyviria, with a single family, Kolmioviridae, that includes the genus Deltavirus as well as seven other novel genera of kolmiovirids [16]. Because other kolmiovirids were only recently discovered, most of our knowledge of the biology of these agents stems from research on HDV [17].…”