First molecular characterization of grapevine virus B (GVB) in portuguese grapevine cultivars and improvement of the RT-PCR detection assay

Fonseca, Filomena; Fernandes Duarte, Vilma Vanessa; Santos, Margarida Teixeira; Brazão, João; Eiras-Dias, Eduardo

http://hdl.handle.net/10400.1/9201

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Autores

Fonseca, Filomena

Fernandes Duarte, Vilma Vanessa

Santos, Margarida Teixeira

Brazão, João

Eiras-Dias, Eduardo

Resumo(s)

This work describes the first molecular characterization of grapevine virus B (GVB) in Portuguese grapevine cultivars. During a routine screening of 44 accessions in the National Collection of Grapevine Varieties (CAN PRT051), 17 were found infected with GVB in DAS-ELISA assays with commercial antibodies. However, only six of the corresponding isolates were successfully amplified using primer pairs described in the literature. The sequence variants (ORF4-3'UTR, 1147 nt) retrieved from these isolates segregated into two phylogenetic groups, which included sequences from complete genomes available in GenBank. The highly discrepant results obtained using serological and RT-PCR-based diagnostic tools led to the design of a primer pair for detection of GVB, which allowed the amplification of a 606-bp GVB-specific fragment from all DAS-ELISA-positive isolates and also revealed the existence of false negatives in the serological testing.