A Comprehensive Hybrid Capture Sequencing Platform for Tick-borne Disease Detection Reveals Zoonotic Emerging Pathogens in a Weather and Vector Transition Zone

Abstract

As weather systems quickly change, vector communities and their pathogens evolve faster than assay panels can be redesigned. Additionally, PCRs narrow target range makes it structurally unable to catch divergent or reassorted agents. We developed a hybrid capture next-generation sequencing enrichment platform that provides comprehensive detection and characterization of tick-borne agents alongside ecological vertebrate host identification with low-pass sequencing. Analytical validation demonstrated performance comparable to qPCR with superior variant tolerance and multiplexing capacity. Field deployment in subtropical, metropolitan New York detected Anaplasma phagocytophilum strains (n=3) linked to human granulocytic anaplasmosis and a Babesia microti-like species in urban raccoon (Procyon lotor) populations. Tick vector screening revealed a putatively novel chimeric Flavi-like virus in invasive Haemaphysalis longicornis ticks combining segmented and unsegmented genomic features, with codon adaptation analysis indicating strong human compatibility. Serology revealed high Flavivirus seropositivity in NYC raccoons, suggesting an unrecognized urban reservoir role. Blood meal analysis simultaneously revealed complex ecological pathogen transmission networks spanning multiple vertebrate hosts. This integrated surveillance system enables comprehensive pathogen discovery, real-time evolutionary monitoring, and ecological risk assessment, transforming our capacity to detect emerging tick-borne threats in rapidly changing environments and prevent spillovers.