Analysis of Raspberry ringspot virus in raspberry by amplification of RNA-2

Abstract: So far, only limited research has been carried out on viruses infecting raspberry, even though the diseases that these viruses cause result in economical losses for raspberry growers. Raspberry ringspot virus (RpRSV) belongs to the family Comoviridae and the genus Nepovirus. Nepoviruses are characterized by their icosahedral virus particles and bipartite genome of single-stranded (+) RNA (RNA-1 and RNA-2). These viruses are transmitted mainly by nematodes of the genus Longidorus, which feed on the roots of plants. RpRSV causes disease in raspberry, grapevine, strawberry and many other plants. The aim of this project was to confirm infection of RpRSV in raspberry samples from Belarus by amplification and sequencing of the coat protein gene. The aim was also to characterize the detected viruses in the samples, since there is little knowledge about nepoviruses in raspberry. RNA was extracted from raspberry stem tissue and cDNA was synthesized by reverse transcription with random primers. The cDNA samples were then used for PCR with different RpRSV-specific primers, in order to amplify parts of the RNA-2 component. However, there was no success in amplification of RpRSV RNA-2 for any of the primer pairs tested. Changing annealing temperature and reagent mixture composition did not improve the results. It is hypothesized that this can be due to sequence differences between these RpRSV isolates and previously studied isolates that prevent the primers from binding. However, more extensive analyses are needed to be able to make any reliable conclusions