Potential spillover investigated by metagenome sequencing in Laikipia, Kenya reveals tick-borne pathogens and a novel bunyavirus

Tick-borne infections continue to present a global public health threat, and require a One Health approach for successful mitigation. We conducted cross-sectional tick screening utilizing an agnostic metagenomic screening strategy based on nanopore sequencing (NS), in an area spanning a range of habitats with intensified human-livestock-wildlife interactions in central Kenya. We further used targeted amplification by polymerase chain reaction (PCR) and sequence independent single primer amplification (SISPA) for confirmation and genome characterization, as necessary. We initially screened 44 ticks across pooled and individual samples belonging to seven species. Tick-associated bacteria—including spotted fever Rickettsia (13.3 %) and Coxiella -like endosymbionts, Francisella turcica and Francisella opportunistica , and tick-associated Borrelia —were detected in 86.6 % of samples . Viruses were detected in 93.3 % of samples, where Jingmen tick virus (JMTV) was observed as the most prevalent virus, detectable in 80 % of samples. A follow-up specific PCR confirmed JMTV virus detection in 75 %, associated with viral read abundance in NS. A complete JMTV genome was assembled from an Amblyomma sparsum tick, that displayed conserved motifs of putative structural and replication proteins. Maximum likelihood analyses placed the virus genome within a distinct clade in the proposed East African-Asian JMTV lineage. We further investigated a virus contig generated during the initial screening, with limited identities to Volzhskoe tick virus (VSTV). Subsequent NS and targeted PCR screening in an additional collection of 650 ticks from 11 species revealed a JMTV prevalence of 12.3 % in Amblyomma, Hyalomma and Rhipicephalus species. Follow-up NS and SISPA generated viral genomic segments, encoding a putative replicase and glycoprotein precursor. Pairwise comparisons and phylogeny indicated a novel virus—herein named as the Mpala tick virus—which is related to but distinct from VSTV and placed among unclassified members of Bunyaviricetes . In conclusion, our approach provides an effective strategy to detect a wide range of tick-borne bacteria and viruses, facilitating identification of opportunistic or endosymbiotic bacteria as well as novel viruses. • A tick pathogen screening strategy based on nanopore sequencing was utilized. • A wide range of tick-borne bacteria and viruses were detected, including opportunistic and endosymbiotic bacteria. • Complete genome of a potentially replication-competent JMTV was assembled. • A novel virus, distantly related to Volzhskoe tick virus, was described.