A Tobacco necrosis virus D isolate from Olea europaea L.: viral characterization and coat protein sequence analysis

A virus isolated from Olea europaea L. grown in Portugal, was identified as a member of the species Tobacco necrosis virus D (TNV-D, genus Necrovirus, family Tombusviridae), based on the molecular and serological properties of the purified virus particles. The genomic region encoding the coat protein (CP) of this isolate (named GP isolate) was amplified by RT-PCR and the cDNA was cloned and sequenced. The CP gene encodes a predicted protein of 269 amino acids showing high identity (86.2%) to TNV-D coat protein sequence. Phylogenetic analysis based on necroviruses CP sequences, confirmed GP as a TNV-D isolate. The alignment with homologous TNV-D CP sequences revealed four conserved amino acids involved in Ca(2+) binding as well as the plant virus icosahedral capsid protein "S' signature. Based on the determined nucleotide sequence, specific primers were designed and successfully used in RT-PCR for virus diagnosis in naturally infected olive trees.