Molecular Characterization of Tomato Brown Rugose Fruit Virus in Portugal and Its Global Phylogenetic Context

(ToBRFV) has emerged as one of the most damaging tobamovirus affecting tomato, a crop of major economic importance worldwide. ToBRFV has been reported in more than 45 countries, including Portugal. However, to date, no peer-reviewed molecular characterization of local isolates has been published, and official records classify its presence in Portugal as transient. This study confirms the occurrence of ToBRFV and provides the first comprehensive genomic and phylogenetic characterization of local virus isolates in Portugal. RNA-seq generated 192,852,438 reads, of which 103,882,115 (58.9%) mapped to ToBRFV, allowing reconstruction of a complete 6393 nt viral genome. A second full-length consensus sequence was independently obtained from the same composite sample using an overlapping Sanger sequencing strategy, differing by only two SNPs. Comparative genomic, functional, structural, and phylogenetic analysis revealed low diversity, with most variation located in replicase-coding regions, while movement and coat protein genes remained highly conserved. Nucleotide-based phylogenies resolved geographically structured clades, although the Portuguese sequences formed a strongly supported subclade with a Chinese isolate. These findings support recent global dissemination of ToBRFV and reinforce the importance of integrated surveillance and genomic monitoring for effective virus management.