Produção de anti‑soros policlonais a partir de proteínas capsidiais recombinantes de Grapevine leafroll‑associated virus 2 e Grapevine virus B

The objective of this work was to produce and characterize specific 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-amplified, 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 purified, and their identities were confirmed 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 (A405) 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.O objetivo deste trabalho foi produzir e caracterizar anti-soros específicos 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 amplificados 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 purificadas, e suas identidades foram confirmadas 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 (A405) 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 confiável desses vírus

Desempenho agronômico de videiras com e sem sintomas de viroses, e comparação molecular de isolados virais

The objective of this work was to evaluate the effect of viroses in symptomatic and asymptomatic grapevines on the agronomic variables related to the plant vigor and the enological quality of grapes, and to compare viral isolates obtained from these two conditions. Two experiments were performed with four cultivars. All plants were indexed by real time, reverse transcription‑polymerase chain reaction (RT‑PCR) for the probable occurrence of the following viruses: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine leafroll‑associated virus (GLRaV‑1 to ‑4, GLRaV‑4 strain 5), Grapevine rupestris stem pitting‑associated virus (GRSPaV), and Grapevine fleck virus (GFkV). The evaluated variables were: number of buds burst and not burst, number of branches with or without bunches, total number of buds, number of bunches, fresh bunch weight, total berry weight, rachis weight, number of berries per bunch, average berry weight, total soluble solids, total titratable acidity, pH, pruned branch weight, or rootstock and scion trunk diameters. The negative effects were more pronounced on the plants with virus symptoms; however, it was observed that plants without symptoms were also often infected. The molecular analysis of GRSPaV, GVA, and GLRaV‑2, isolates from symptomatic and asymptomatic plants, resulted in a high percentage of nucleotide identities between homologous isolates.O objetivo deste trabalho foi avaliar os efeitos de viroses em videiras sintomáticas e assintomáticas sobre as variáveis agronômicas relacionadas ao vigor das plantas e à qualidade enológica da uva, e comparar os isolados virais obtidos nessas duas condições. Realizaram-se dois experimentos com quatro cultivares. Todas as plantas foram indexadas, por meio da reação em cadeia da polimerase via transcrição reversa (RT‑PCR) em tempo real, quanto à provável ocorrência dos seguintes vírus: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine leafroll‑associated virus (GLRaV‑1 ao ‑4, GLRaV‑4 estirpe 5), Grapevine rupestris stem pitting‑associated virus (GRSPaV) e Grapevine fleck virus (GFkV). As variáveis avaliadas foram: número de gemas brotadas e não brotadas, número de ramos com ou sem cachos, número total de gemas, número de cachos, massa de cachos frescos, massa total de bagas, massa do engaço, número de bagas por cacho, massa média de baga, sólidos solúveis totais, acidez total titulável, pH, massa de ramos podados ou diâmetros do tronco do porta‑enxerto e da copa. Os efeitos negativos foram mais pronunciados nas plantas com sintomas de viroses; no entanto, constatou-se frequentemente que plantas sem sintomas também estavam infectadas. A análise molecular de GRSPaV, GVA e GLRaV‑2, isolados de plantas sintomáticas e assintomáticas, resultou em alta percentagem de identidade de nucleotídeos entre isolados homólogos

Real-time RT-PCR high-resolution melting curve analysis to detect and differentiate Brazilian variants of grapevine viruses

Detecting and identifying viral infections in perennial plants, such as grapevines, can be challenging. Therefore, the aim of this study was to perform a real-time RT-PCR (RT-qPCR) high-resolution melting (HRM) curve analysis to detect and differentiate Brazilian variants of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine fanleaf virus (GFLV) in 74 and 10 infected plants, respectively, maintained in a collection block of grapevines. A single amplification curve was generated for each sample by RT-qPCR. Considering the amplified region of genomes of these two viruses, it was possible to identify and distinguish different variants of GLRaV-3 and of GFLV, which showed significantly different melting temperature (Tm) values between themselves, reflecting differences in the nucleotide sequences of the respective amplicons, and allowing discriminating variants and assess the viral diversity in grapevine accessions. The HRM analysis was validated by sequencing and nucleotide comparisons among Brazilian isolates of GLRaV-3 and GFLV

Molecular variants of Grapevine rupestris stem pitting-associated virus infecting grapevines (Vitis spp.) in Brazil

<div><p>ABSTRACT: Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most common viruses of grapevine. It is involved in the graft-transmissible disease rupestris stem pitting of the rugose wood complex. The objective of the research was to perform the molecular characterization of the coat protein (CP) gene of sixteen Brazilian GRSPaV isolates aiming to determine the occurrence of molecular variants (strains) of this virus. Nine grapevine samples were evaluated, from which dsRNA was extracted. Nucleotide sequences were generated by Next generation sequencing (NGS). Fifteen complete sequences of the GRSPaV CP gene were obtained and phylogenetically analyzed. Multiple alignments of the sequences showed identities of nucleotides ranging from 82% to 99%, suggesting high variability among the CPs of Brazilian isolates. The study revealed that genetic variability of GRSPaV comprising three molecular variants is also present in Brazilian grapevine genotypes.</p></div

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B Produção de anti-soros policlonais a partir de proteínas capsidiais recombinantes de Grapevine leafroll-associated virus 2 e Grapevine virus B

The objective of this work was to produce and characterize specific 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-amplified, 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 purified, and their identities were confirmed 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 (A405) 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.O objetivo deste trabalho foi produzir e caracterizar anti-soros específicos 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 amplificados 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 purificadas, e suas identidades foram confirmadas 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 (A405) 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 confiável desses vírus