Focal persistence and phylodynamics of Heartland virus in Georgia.

Heartland virus (HRTV) is an emerging tick-borne virus associated with severe illness in the USA. 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 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 to 2023, 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 ~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 USA were also geographically clustered. Time-scaled phylogenetic analysis suggested a recent spread of HRTV in the USA, with all available sequences sharing a common ancestor within the last 75 years, since the mid-1900s, and sequences from Georgia sharing a common ancestor within the last 15 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.