Among the hepacivirus species recently described, the non-primate hepacivirus/hepacivirus A found in horses and donkeys is closely related to the human hepatitis C virus (HCV). Therefore, the equine is an attractive surrogate large animal model for the study of HCV therapy, pathogenesis and prophylaxis. Despite global efforts, epidemiological and genetic studies have not elucidated the risk factors, virus distribution or genetic variability of the hepacivirus A, which are also important issues for the equine welfare. Little information about this background scenery is available in Brazil. The aims of this study were to investigate potential risk factors associated with hepacivirus A infection among different horse cohorts throughout the state of Rio de Janeiro and to evaluate the diversity of the viral NS5B gene and protein. Hepacivirus A RNA was detected in horse cohorts from all geographical mesoregions, independent of horse activity or breed investigated. Statewide prevalence ranged from 4.0% to 27.5%. Potential risk factors such as geographical location and age of female horses were significantly associated with the presence of virus RNA. Phylogenetic analysis revealed the circulation of subtype 2 in all mesoregions. NS5B gene sequences clustered according to geographical origin, while the NS5B fragments did not allow discriminant analysis. The predicted NS5B protein showed marked conservation, especially in the thumb domain. In conclusion, the higher frequency of hepacivirus A RNA detection in horses bred for reproduction purposes as well as in young females suggests a direct link between reproduction practices and the virus's spread. Additional studies are necessary to understand the distribution of this genetically conserved hepacivirus.2030-01-01