Discovery of novel members of the genera Carlavirus and Potexvirus, and detection of Nepovirus, Cilevirus, Betacarmovirus and Tobamovirus members in a single Hibiscus rosa-sinensis plant in Colombia using Metagenomic Sequencing

Hibiscus is a well-known host for Brevipalpus transmitted viruses (BTVs). During surveys of BTV reservoir hosts in the citrus growing regions in Colombia, several hibiscus samples were collected for virus testing. Although hibiscus is not native to Colombia, it is well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of leaves, was selected for virome analysis using metatranscriptomic next generation sequencing technology. Bioinformatic analysis detected 12.5% of total reads in the library which mapped to viral genomes. BLAST searches of assembled contigs revealed three complete and one partial novel sequence resembling carlaviruses and a full genome sequence of a presumed potexvirus. The presence of novel carlaviruses, a potexvirus and of known members of genera nepovirus, cilevirus, betacarmovirus and tobamovirus in the same sample was confirmed by virus-specific primers RT-PCR assays followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the same plant in mixed infection. Overall, RdRp and coat protein gene sequences of potex - and carla- viruses shared less than 72% nt and 80% aa identities with any alpha- and beta- flexivirus sequences available in the GenBank, revealing the existence of three novel carlavirus and one potexvirus species in nature. In addition to previously known hibiscus viruses, here we report the first incidence of physalis vein necrosis nepovirus and passionfruit green spot cilevirus in hibiscus in Colombia.