Thor Vinícius Martins Fajardo

Sequence analysis of the capsid protein gene of an isolate of Apple stem grooving virus, and its survey in Southern Brazil

Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).

Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus

Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties

ABSTRACT Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.

Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the Sao Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong No2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Systemic transport of Alfalfa mosaic virus can be mediated by the movement proteins of several viruses assigned to five genera of the 30K family.

We previously showed that the movement protein (MP) gene of Alfalfa mosaic virus (AMV) is functionally exchangeable for the cell-to-cell transport of the corresponding genes of Tobacco mosaic virus (TMV), Brome mosaic virus, Prunus necrotic ringspot virus, Cucumber mosaic virus and Cowpea mosaic virus. We have analysed the capacity of the heterologous MPs to systemically transport the corresponding chimeric AMV genome. All MPs were competent in systemic transport but required the fusion at their C terminus of the coat protein-interacting C-terminal 44 aa (A44) of the AMV MP. Except for the TMV MP, the presence of the hybrid virus in upper leaves correlated with the capacity to move locally. These results suggest that all the MPs assigned to the 30K superfamily should be exchangeable not only for local virus movement but also for systemic transport when the A44 fragment is present.

Citrus exocortis viroid and Hop Stunt viroid Doubly infecting grapevines in Brazil

Part of the Doctoral Thesis of the first author. ESALQ, Universidade de Sao Paulo, Piracicaba, SP. 2006.

Fisiologia foliar e qualidade enológica da uva em videiras infectadas por vírus

Leaf physiology and enologic grape quality of virus-infected plants Viruses may induce metabolic and structural disarray in plant cells to varying degrees depending on viral species and plant susceptibility. With this focus, grapevine plants (Vitis vinifera) cvs. Cabernet Franc and Cabernet Sauvignon, symptomless and showing symptoms of virus-infection, in two commercial vineyards were comparatively analyzed. The parameters were: 1. photosynthetic potential (light-saturated rate of photosynthesis, saturating light intensities, light compensation point, dark respiration rate, apparent quantum yield, chlorophyll a and b); 2. foliar carbon metabolism (total soluble sugars and starch), and 3. enologic quality of the produced grapes (total soluble solids - °Brix, density, pH and titerable total acidity in the must; total color intensity and total polyphenols in berry skins) were recorded. In symptomatic plants Grapevine leafroll-associated virus 2 (GLRaV-2) and Rupestris stem pitting-associated virus (RSPaV) have been detected by ELISA carried out for six viruses. The virus infections induced significant reductions in chlorophyll content and in photosynthetic potential of both cultivars. Leaves of infected plants also showed significant accumulation of carbohydrates, suggesting a blockage of carbon transport out of these tissues. Concerning the enologic quality and based on technological parameters of the must (°Brix, density, titerable total acidity and polyphenols), grapes of infected plants showed at harvest a significantly lower level of maturation. These viruses directly affect the productive capacity as well as the quality of the grapes produced by these cultivars.

Detecção e identificação molecular de vírus associados a videiras sintomáticas e assintomáticas

The vegetative propagation of grapevine facilitates multiple viral infections, with different symptoms which vary according to combinations of cultivar or host species with viral species. The aims of this research were to detect and identify the viral species infecting two grapevine species/cultivars: one symptomatic and one symptomless. DsRNA from both samples was assayed by RT-PCR using 17 pairs of specific primers for detection of the Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV) and Grapevine leafroll-associated virus 1-4 (GLRaV-1 to -4), besides three degenerate primer pairs. For each primer pair at least one amplicon was cloned and sequenced. Symtomatic and symptomless plants were multiple infected by RSPaV, GLRaV-2 and/or GLRaV-3. The nucleotide sequences of seven isolates of RSPaV, three of GLRaV-2 and two of GLRaV-3 showed identities higher than 90% with the homologous viral species and allowed to identify possible viral strains in infected samples. These results highlight the necessity of viral diagnosis based on specific assays to determine grapevine sanitary status.

Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil

ABSTRACT An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca andV. vinifera grapevines on the indicator LN33,was transmitted mechanically to several Nicotiana species. The viruswas partially purified from N. cavicola and the coat protein estimatedat 23 kDa by SDS-PAGE. In negatively stained leaf extracts ofexperimentally inoculated N. cavicola and N. occidentalis , flexuousparticles with cross banding were observed, predominantlymeasuring 750-770 x 12 nm, with a modal length of 760 nm.Decoration indicated a clear, positive reaction against AS-GVB. InDAS-ELISA, GVB was detected in N. cavicola and grapevineextracts, and Western blots showed homologous and cross reactionof GVB and GVA antisera with GVB coat protein. Using specificprimers for GVB, a fragment of 594 bp, comprising the coat proteingene coding for 197 amino acids, was amplified by RT-PCR withviral RNA extracted from GVB-infected N. occidentalis . Thenucleotide and the deduced amino acid sequences of the coat proteingene showed high identities with Italian and Japanese isolates ofGVB.