Gene duplication and phylogeography of North American members of the Hart Park serogroup of avian rhabdoviruses

•Flanders virus (FLAV) does not contain pseudogenes as previously reported.•The FLAV U1-U3 proteins arose by gene duplication.•The SH protein of FLAV is tentatively expressed by coupled translation.•Distinct lineages of FLAV circulate sympatrically in the United States.•Histone H4 and cyclophilin A are apparently incorporated into FLAV particles.

Flanders virus (FLAV) and Hart Park virus (HPV) are rhabdoviruses that circulate in mosquito-bird cycles in the eastern and western United States, respectively, and constitute the only two North American representatives of the Hart Park serogroup. Previously, it was suggested that FLAV is unique among the rhabdoviruses in that it contains two pseudogenes located between the P and M genes, while the cognate sequence for HPV has been lacking. Herein, we demonstrate that FLAV and HPV do not contain pseudogenes in this region, but encode three small functional proteins designated as U1-U3 that apparently arose by gene duplication. To further investigate the U1-U3 region, we conducted the first large-scale evolutionary analysis of a member of the Hart Park serogroup by analyzing over 100 spatially and temporally distinct FLAV isolates. Our phylogeographic analysis demonstrates that although FLAV appears to be slowly evolving, phylogenetically divergent lineages co-circulate sympatrically.