Identification of novel hepaciviruses in rock pigeon (Columba livia [Gmelin, 1789]), rusty-margined flycatcher (Myiozetetes cayanensis [Linnaeus, 1766]), and Hispaniolan amazon (Amazona ventralis [Statius Muller, 1776]).

Recent advances in sequencing technology and transcriptome mining have revealed highly divergent hepaciviruses in birds. However, only a limited number of avian hepaciviruses have been identified to date, leaving their diversity and evolutionary history poorly understood. Moreover, deep phylogenetic gaps among known avian hepaciviruses suggest that additional lineages remain undiscovered. Here, we screened publicly available RNA-seq data and identified three previously undescribed hepaciviruses from rock pigeon (Columba livia), rusty-margined flycatcher (Myiozetetes cayanensis), and Hispaniolan amazon (Amazona ventralis), named rock pigeon hepacivirus (RpHV), rusty-margined flycatcher hepacivirus (RfHV), and Hispaniolan amazon hepacivirus (HaHV). Although these three viruses meet the ICTV species demarcation criteria relative to their closest known relatives, the NS5B-based criterion was not satisfied between RfHV and HaHV. Notably, however, their genome sequence identity is low at 43.2%, and their hosts differ at the order level, suggesting that their classification warrants further consideration. Our phylogenetic analysis showed that avian hepaciviruses, including those found in this study, are monophyletic, but phylogenetic incongruence was observed between avian hepaciviruses and their hosts, suggesting past cross-species transmission among avian hepaciviruses. Overall, this study provides novel insights into the diversity and evolution of hepaciviruses.