Focal persistence and phylodynamics of Heartland virus in Georgia

Heartland virus (HRTV) is an emerging tick-borne virus associated with severe illness in the U.S. There are large gaps in knowledge of HRTV diversity, evolution, and transmission due to a paucity of HRTV-positive samples and genome sequences. We identified a focal site of HRTV-positive Amblyomma americanum ticks in central Georgia and developed a novel multiplex-amplicon sequencing assay to generate full HRTV genome sequences. By screening over 21,000 field-collected ticks from 2021-23, we identified six positive pools. Five were collected from the site in central Georgia where our group first detected HRTV-positive ticks in 2019, and one from a site in western Georgia approximately 175 km away. The HRTV genome sequences from Georgia were highly related, even across this distance and over five years. Reference HRTV genome sequences from across the U.S. were also geographically clustered. Time-scaled phylogenetic analysis suggested recent spread of HRTV in the U.S., with all available sequences sharing a common ancestor within the last seventy-five years, since the mid-1900s, and sequences from Georgia sharing a common ancestor within the last fifteen years, since 2010. Our observed spatial clustering of HRTV and the high degree of genetic conservation in our persistent focus suggest the importance of small spatial dynamics in HRTV transmission ecology.