Discovery of tobacco necrosis virus A in a diseased Colombian Cannabis sativa plant

Plant viral infections pose a significant threat to global crop productivity. Despite their profound impact on agriculture, plant viruses have been relatively understudied, primarily due to technological limitations associated with classical molecular methods. However, the advent of RNA Next-Generation Sequencing (NGS) RNA-seq analysis has revolutionized virus characterization in environmental settings, overcoming previous limitations and providing a powerful tool for studying plant viruses. In an RNA-seq experiment conducted on a diseased Colombian Cannabis sativa hemp plant, we identified a linear positive-sense single-stranded RNA genome belonging to Tobacco Necrosis Virus A (TNV-A), a common cause of necrotic lesions in plants such as tobacco and tulipa. The affected Cannabis sativa hemp plant exhibited severe symptoms, including alterations in pigmentation, leaf morphology such as chlorosis, necrotic tissue formation, and surface wear on the leaves. The complete genome sequence of the Cannabis sativa TNV-A was 3656 nucleotides long, containing five putative ORFs, and was classified in the family Tombusviridae, genus Alphanecrovirus, and belonging to the Necro-like clade based on RdRp protein phylogenetic analysis. Our analysis revealed a well-conserved RdRp protein among the Alphanecroviruses, with 89 % of the amino acid residues in the peptide being entirely conserved. In contrast, the coat protein exhibited significantly higher variability, with only 49.3 % of the residues being 100 % conserved. Regarding the viral genome expression of Cannabis sativa TNV-A, we observed that the virus was highly abundant in the leaves of the diseased plant, ranking among the topmost abundant transcripts, occupying the percentile position of 3 %. Overall, our study generated the first reference genome of TNV-A virus in the tropical region and reported the first case of this virus infecting a Cannabis sativa plant.