Known and novel nairoviruses in ticks (order Ixoidea) collected from Danish wild and domestic animals

Introduction: Climate change is perturbing ecological niches, contributing to animal distribution shifts. Disease vectors, like ticks, are expanding their habitat, contributing to arbovirus spread to new locations and hosts. It is, therefore, important to monitor arboviruses and identify them before they become a problem. Orthonairovirus (Nairoviridae) comprises significant tick-borne RNA viral pathogens (e.g., Crimean-Congo hemorrhagic fever andNairobi sheep disease viruses). Since orthonairovirus ecology is understudied, we investigated these viruses among ticks from Denmark, a previously unexplored territory for nairoviruses.

Methods: Viruses were detected using a pan-orthonairovirus hemi-nested RT-PCR (primers available on request), amplifying a conserved region (347bp) of the RNA-dependent RNA polymerase (RdRp) gene using RNA from 20 pools of ticks (n=228, mostly Ixodes ricinus) collected from Danish animals or the environment. Positives were Sanger-sequenced, Blast was used to identify the closest relatives of the detected viruses, and Nairoviridae-wide maximum-likelihood phylogenetic analysis was performed with partial RdRp protein alignments.

Results: Five samples were positive. A pool of ticks from European bison (n=45) and wild birds (n=16) contained a close relative (96.3-97.3% nt identity) of a Latvian Sulina virus (Orthonairovirus sulinaense) identified in Ixodes ricinus. Two pools of ticks from ungulates (horse, n=20; roe deer, n=15) contained viruses similar to members of Norwavirus grotenhoutense, previously identified in Ixodes ricinus across Europe (96.3- 98.6% nt identity). Finally, two pools of ticks from wild mink (n=39) contained a novel virus, whose closest relative was an unclassified virus from Spanish Ixodes simplex (nt=77.0%, aa=90.3%). Phylogenetic analysis placed it on a long branch in a clade with norwaviruses.

Conclusions: The method succeeded in identifying two genera of nairoviruses, discovering a probable new virus genus. Our results highlight that several nairoviruses circulate in Denmark, with more viruses likely awaiting discovery. Further analyses are required to fully characterize the identified viruses and assess their host tropism among vertebrates.