Detection and Differentiation of Comoviridae Species using a Semi-nested RT-PCR and a Phylogenetic Analysis based on the Polymerase Protein

nested reverse transcription polymerase chain reaction (RT-PCR) method is described that allows detection and identification of Comoviridae species and possibly other bipartite plant picorna-like viruses. It involves an RT-PCR, in which a combination of degenerate deoxyinosine (dI)-substituted primers amplified part of the RNA-dependent RNA polymerase (RdRp) domain, followed by a semi-nested PCR amplification that increased detection sensitivity. The PCR amplicons can be easily cloned and sequenced to obtain initial sequence information of Comoviridae genomes for virus species differentiation and characterization. The method can be used on total RNA extracts from infected plants, when viral genomic double- stranded RNA (dsRNA) or the viral RNA is unavailable. Previously uncharacterized RdRp domains of Potato black ringspot virus (PBRSV), Cherry leafroll virus (CLRV) and Arracacha virus B (AVB) were amplified and sequenced. Neighbour-joining trees calculated from the homologous partial RdRp amino acid sequences were used to establish the taxonomic relationships between members of plant picorna-like viruses, revealing lineages that classified all species within their respective genera and clearly distinguished each genus from the other genera. Phylogenetic analysis confirmed the classification of PBRSV and CLRV within the genus, Nepovirus whereas AVB was placed into a cluster different from the Nepovirus, Comovirus and Fabavirus lineages