peer reviewed ; Fruit trees, like sweet cherry (Prunus avium) are known to be infected by numerous plant viruses, predominantly as a consequence of their vegetative mode of propagation and extended cropping. Prunus virus F (PrVF) is a recently described member of the genus Fabavirus (Secoviridae) which has a poorly understood pathology (Villamor et al., 2017). During a sweet cherry resistance screening greenhouse experiment (July 2018) against Little cherry virus 1 (LChV-1) and Little cherry virus 2 (LChV-2), branches and leaves from 10 non symptomatic trees, representing propagation material from different cultivars grafted on Gisela 5 rootstocks, were collected and pooled as a single composite sample. Total RNA was extracted from 100 mg fresh tissue using the Spectrum Total Plant RNA kit (Sigma-Aldrich, Belgium) and after passing quality control, the sample was sent for library preparation (NEBNext Ultra RNA library kit; New England BioLabs, MA, USA) and high-throughput sequencing (Illumina NextSeq v2, totRNA sequencing; 40M single reads (20M each direction (2x 150bp)) by Admera Health (USA). The reads were quality filtered and submitted to the automated VirusDetect pipeline (Zheng et al., 2017). In total, 48 viral contigs mapped to specific PrVF accessions. A reference mapping was then conducted against the closest accessions of RNA1 (NC_039077), and RNA2 (NC_039078), in both cases revealing a 100% coverage (CLC Genomics Workbench 12, Qiagen, Denmark). The obtained RNA1 (GenBank Accession MK834285) and RNA2 (MK834286) genomic fragments were 6150 bp and 3612 bp in length, respectively. The presence of PrVF was confirmed both in the composite sample and individual trees by RT-PCR, using primers PVF1-CAF/PVF1-CAR (James et al., 2018), targeting RNA1. RNA2 was detected using primer pair Fab-R2 1808F/Fab-R2 2546R (Villamor et al., 2017). RT-PCR products were bidirectionally sequenced (Genewiz, Leipzig, Germany). Phylogenetic analysis revealed the genetic relationship between the Belgian PrVF strain with other ...