First molecular characterization of grapevine virus B (GVB) in Portuguese grapevine cultivars and improvement of the RT-PCR detection assay.
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.
CIMA, Universidade do Algarve, Campus de Gambelas, edifício 7, 8005-139, Faro, Portugal. ffonseca@ualg.pt.
Filomena
http://orcid.org/0000-0003-3824-0459
F
Fonseca
CIMA, Universidade do Algarve, Campus de Gambelas, edifício 7, 8005-139, Faro, Portugal.
Vilma
V
Duarte
INIAV, Quinta do Marquês, 2780-159, Oeiras, Portugal.
Margarida
M
Teixeira Santos
INIAV, Quinta da Almoínha, 2565-191, Dois Portos, Portugal.
João
J
Brazão
INIAV, Quinta da Almoínha, 2565-191, Dois Portos, Portugal.
Eduardo
E
Eiras-Dias
D017931:DNA Primers / Q000235:genetics; D052677:Flexiviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014644:Genetic Variation; D016366:Open Reading Frames; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology; D011174:Portugal; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods*; D027843:Vitis / Q000821:virology*
D017931:DNA Primers
Corky bark; Rugose wood complex; Vitivirus