Arch Virol (2004) 149: 1129–1138 DOI 10.1007/s00705-003-0258-7 A Tobacco necrosis virus D isolate from Olea europaea L.: viral characterization and coat protein sequence analysis J. M. S. Cardoso 1 , M. R. F´ elix 1,2 , S. Oliveira 1,3 , and M. I. E. Clara 1,2 1 I.C.A.M. Instituto de Ciˆ encias Agr´ arias Mediterrˆ anicas, ´ Evora, Portugal 2 Departamento de Sanidade Animal e Vegetal, Universidade de ´ Evora, ´ Evora, Portugal 3 Departamento de Biologia, Universidade de ´ Evora, ´ Evora, Portugal Received May 12, 2003; accepted December 1, 2003 Published online February 16, 2004 c  Springer-Verlag 2004 Summary. A virus isolated from Olea europaea L. grown in Portugal, was iden- tified as a member of the species Tobacco necrosis virus D (TNV-D, genus Necrovirus, family Tombusviridae), based on the molecular and serological prop- erties 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. Introduction Tobacco necrosis virus D (TNV-D) is a small icosahedral virus ca. 28nm in diameter with a single stranded positive-sense RNA genome approximately 3.8 kb long [6, 34]. It belongs to the genus Necrovirus [27] and is naturally transmitted by the soil inhabiting fungus Olpidium brassicae (Wor.) Dang [32]. Strains of TNV have been grouped into two distinct species, TNV-A and TNV- D [2, 34]. The complete nucleotide sequences of TNV-A and TNV-D were reported by Meulewaeter et al. (1990) and Coutts et al. (1991) respectively. A similar genome organization of these two species has been deduced from the determined Note: Nucleotide sequence data reported is available in the GenBank database under the accession number AY263376.