Novel Insights into Endogenous RNA Viral Elements in and Other Arbovirus Vector Genomes

Many emerging arboviruses are not transmitted by traditional mosquito vectors, but by lesser-studied arthropods such as ticks, midges, and sand flies. Small RNA (sRNA) silencing pathways are the main antiviral defence mechanism for arthropods, which lack adaptive immunity. Non-retroviral integrated RNA virus sequences (NIRVS) are one potential source of sRNAs which comprise these pathways. NIRVS are remnants of past germline RNA viral infections, where viral cDNA integrates into the host genome and is vertically transmitted. In mosquitoes, NIRVS are widespread and produce PIWI-interacting RNAs (piRNAs). These are hypothesised to target incoming viral transcripts to modulate viral titre, perhaps rendering the organism a more efficient arbovirus vector. To explore the NIRVS landscape in alternative arbovirus vectors, we validated the NIRVS landscape in spp. and then identified novel NIRVS in six medically relevant arthropods and also in . We identified novel NIRVS in , , , , and . Due to their unexpected abundance, we further characterised NIRVS in the blacklegged tick ( = 143). Interestingly, NIRVS are not enriched in , another hard tick, suggesting this is an -specific adaptation. NIRVS are enriched in bunya- and orthomyxo-like sequences, reflecting that ticks are a dominant host for these virus groups. Unlike in mosquitoes, NIRVS are more commonly derived from the non-structural region (replicase) of negative-sense viruses, as opposed to structural regions (e.g. glycoprotein). Like other arthropods, NIRVS preferentially integrate into genomic piRNA clusters, and serve as a template for primary piRNA production in the commonly used embryonic ISE6 cell line. Interestingly, we identified a two-fold enrichment of non-long terminal repeat (non-LTR) retrotransposons, in genomic proximity to NIRVS, contrasting with studeis in , where LTR retrotransposons are instead associated with NIRVS formation. We characterised NIRVS phylogeny and integration patterns in the important vector, , revealing they are distinct from those in spp. Future studies will explore the possible antiviral mechanism conferred by NIRVS to ,which may help the transmission of pathogenic arboviruses. Finally, this study explored NIRVS as an untapped wealth of viral diversity in arthropods.