Detecção do Grapevine leafroll-associated virus 5 no Estado de São Paulo

Kuniyuki, Hugo; Rezende, J. A. M.; Gaspar, José Carlos; Yuki, Valdir Atsushi

doi:10.1590/s0100-54052008000400014

Cited by 3 publications

(3 citation statements)

References 4 publications

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“…GLRaV-2 is one among twelve (GLRaV-1 to -11 and WC-HSP-10) species involved in the grapevine leafroll disease complex. Detection of GLRaV-2 has been previously reported in Brazil (Kuniyuki et al, 2002).…”

Section: Introductionmentioning

confidence: 87%

“…Antisera and several variants of enzyme-linked immunosorbent assay (ELISA) (Clark, 1981;Koenig, 1981;Barbara & Clark, 1982) are widely used tools for the routine detection of grapevine viruses (Kuniyuki et al, 2002;Fajardo et al, 2003). However, the production of high-quality virus-specifi c antisera to GLRaV-2 and GVB, suitable for their large scale detection and based on virus purifi cation procedures, faces substantial drawbacks, including complex virus infections, low yields of virus particles, presence of inhibitory compounds -such as polyphenols, tannins and polysaccharides -, narrow range and poor mechanical transmission to herbaceous hosts, contamination of antigens with host proteins (Uyemoto et al, 1997;Xu et al, 2006;Beuve et al, 2007;Fajardo et al, 2007;Ling et al, 2007), and, especially in the case of GVB, low immunogenicity (Boscia et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

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Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Radaelli

Fajardo

Nickel

et al. 2008

Pesq. agropec. bras.

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-The objective of this work was to produce and characterize specifi c antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplifi ed, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purifi ed, and their identities were confi rmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A 405 ) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.Index terms: Vitis, GLRaV-2, GVB, indirect ELISA, recombinant protein, Western blot. Produção de anti-soros policlonais a partir de proteínas capsidiais recombinantes de Grapevine leafroll-associated virus 2 e Grapevine virus BResumo -O objetivo deste trabalho foi produzir e caracterizar anti-soros específi cos contra isolados brasileiros do Vírus do enrolamento-da-folha da videira 2 (GLRaV-2) e do Vírus B da videira (GVB), desenvolvidos a partir das proteínas capsidiais expressas em Escherichia coli, e testar seu possível uso para a detecção destes dois vírus em videiras infectadas. Os genes da proteína capsidial (CP) foram amplifi cados via RT-PCR, clonados e seqüenciados. Foram, subseqüentemente, subclonados, e os plasmídeos recombinantes foram empregados na transformação das células de E. coli e na expressão das proteínas capsidiais. As proteínas capsidiais recombinantes foram purifi cadas, e suas identidades foram confi rmadas em SDS-PAGE e "Western blot" e utilizadas para imunizar coelhos. Os anti-soros produzidos contra essas proteínas foram capazes de reconhecer as proteínas recombinantes correspondentes em "Western blot", de detectar GLRaV-2 e GVB em tecidos infectados de videiras pelo ELISA indireto, e de discriminar videiras sadias e infectadas, com absorbâncias (A 405 ) de 0,08/1,15 e 0,12/1,30, respectivamente. A expressão dos genes CP pode produzir grandes quantidades de proteínas virais, com elevada antigenicidade, e os anti-soros de GLRaV-2 e GVB obtidos neste trabalho possibilitam a diagnose confi ável desses vírus.Termos para indexação: Vitis, GLRaV-2, GVB, ELISA indireto, proteína recombinante, Western blot.

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Section: Introductionmentioning

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Radaelli

Fajardo

Nickel

et al. 2008

Pesq. agropec. bras.

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show abstract

“…In Brazil, leafroll is one of the most important viral diseases of grapevine. So far, GLRaV-5 and -6 were serologically detected in leafroll diseased grapevines in Brazil (KUNIYUKI et al, 2003;KUNIYUKI et al, 2008). The objective of this work was to perform the partial molecular characterization of Brazilian isolates of GFkV, GVD, GLRaV-5 and -6.…”

Section: Mancha Das Nervuras Lenho Rugoso E Enrolamento Das Folhas Smentioning

confidence: 99%

Detection and partial molecular characterization of Grapevine fleck virus, Grapevine virus D, Grapevine leafroll-associated virus -5 and -6 infecting grapevines in Brazil

et al. 2012

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Survey of potential sharpshooter and spittlebug vectors of Xylella fastidiosa to grapevines at the São Francisco River Valley, Brazil

Ringenberg¹,

Lopes²,

Müller³

et al. 2014

Rev. Bras. entomol.

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Detecção do Grapevine leafroll-associated virus 5 no Estado de São Paulo

Cited by 3 publications

References 4 publications

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Detection and partial molecular characterization of Grapevine fleck virus, Grapevine virus D, Grapevine leafroll-associated virus -5 and -6 infecting grapevines in Brazil

Survey of potential sharpshooter and spittlebug vectors of Xylella fastidiosa to grapevines at the São Francisco River Valley, Brazil

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