Analysis of the nucleotide sequence of the coat protein and 3'-untranslated region of two Brazilian Potato virus Y isolates

Two Brazilian Potato virus Y (PVY) isolates were biologically characterized as necrotic (PVY-NBR) and common (PVY-OBR) based upon symptoms on test plants. Additional characterization was performed by sequencing a cDNA corresponding to the 3' terminal region of the viral genome. The sequence consisted of 195 nucleotides (nt) coding part of the nuclear inclusion body b (NIb) gene, 804 nt of the coat protein (CP) gene, and 328 nt (PVY-OBR) or 326 nt (PVY-NBR) of the 3'-untranslated region (UTR). Translation of the sequence resulted in one single open reading frame with part of the NIb and a CP of 267 amino acids. The two isolates shared 95.1% similarity in the CP amino acid sequence. The CP and the 3'-UTR sequence of the Brazilian isolates were compared to those of other PVY isolates previously reported and unrooted phylogenetic trees were constructed. The trees revealed a separation of two distinct clusters, one comprising most of the common strains and the other comprising the necrotic strains. PVY-OBR was clustered in the common group and PVY-NBR in the necrotic one.

Sequences of the coat protein gene from brazilian isolates of Papaya ringspot virus

Papaya ringspot virus (PRSV) is the causal agent of the main papaya (Carica papaya) disease in the world. Brazil is currently the world's main papaya grower, responsible for about 40% of the worldwide production. Resistance to PRSV on transgenic plants expressing the PRSV coat protein (cp) gene was shown to be dependent on the sequence homology between the cp transgene expressed in the plant genome and the cp gene from the incoming virus, in an isolate-specific fashion. Therefore, knowledge of the degree of homology among the cp genes from distinct PRSV isolates which are present in a given area is important to guide the development of transgenic papaya for the control of PRSV in that area. The objective of the present study was to assess the degree of homology among the PRSV cp genes of several Brazilian isolates of this virus. Papaya and PRSV are present in many different ecosystems within Brazil. Twelve PRSV isolates, collected in eight different states from four different geographic regions, were used in this study. The sequences of the cp gene from these isolates were compared among themselves and to the gene used to generate transgenic papaya for Brazil. An average degree of homology of 97.3% at the nucleotide sequence was found among the Brazilian isolates. When compared to 27 isolates from outside Brazil in a homology tree, the Brazilian isolates were clustered with Australian, Hawaiian, and Central and North American isolates, with an average degree of homology of 90.7% among them.

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Development of virus resistant transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus

Translatable and nontranslatable versions of the coat protein (cp) gene of a Papaya ringspot virus (PRSV) isolate collected in the state of Bahia, Brazil, were engineered for expression in Sunrise and Sunset Solo varieties of papaya (Carica papaya). The biolistic system was used to transform secondary somatic embryo cultures derived from immature zygotic embryos. Fifty-four transgenic lines, 26 translatable and 28 nontranslatable gene versions, were regenerated, with a transformation efficiency of 2.7%. Inoculation of cloned R0 plants with PRSV BR, PRSV HA or PRSV TH, Brazilian, Hawaiian and Thai isolates, respectively, revealed lines with mono-, double-, and triple-resistance. After molecular analysis and a preliminary agronomic evaluation, 13 R1 and R2 populations were incorporated into the papaya-breeding program at Embrapa Cassava and Tropical Fruits, in Cruz das Almas, Bahia, Brazil.

Sequence diversity in the coat protein gene of Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus infecting lettuce in Brazil

Lettuce big vein associated virus (LBVaV) and Mirafiori lettuce big vein virus (MLBVV) have been found in mixed infection in Brazil causing the lettuce big vein disease. Analysis of part of the coat protein (CP) gene of Brazilian isolates of LBVaV collected from lettuce, showed at least 93% amino acid sequence identity with other LBVaV isolates. Genetic diversity among MLBVV CP sequences was higher when compared to LBVaV CP sequences, with amino acid sequence identity ranging between 91% to 100%. Brazilian isolates of MLBVV belong to subgroup A, with one RsaI restriction site on the coat protein gene. There is no indication for a possible geografical origin for the Brazilian isolates of LBVaV and MLBVV.

High-affinity RNA-binding domains of alfalfa mosaic virus coat protein are not required for coat protein-mediated resistance.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.

Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

Naturally acquired antibodies against the major merozoite surface coat protein (MSP-1) of Plasmodium falciparum acquired by residents in an endemic area of Colombia

A preliminary baseline epidemiological malaria survey was conducted in the village of Punta Soldado, Colombia. Parasite prevalence and density as well as serological data were obtained from 151 asymptomatic children and adults. Fifty individuals were infected with Plasmodium falciparum. The mean parasite density was 184 parasites/mm3. Greater than 90 of the sample population were P. falciparum antibody positive as detected by the indirect immunofluorescent antibody test (IFAT). The enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against the major merozoite surface protein (MSP-1) of P. falciparum. In this population, anti-MSP-1 antibody concentration is acquired in an age dependent manner with equal immunogenicity to both the N- and C-terminal regions of the molecule. Infection at the time of sampling was associated with a higher anti-MSP-1 antibody concentration than that found in non-infected individuals. Further studies are planned to assess the role of immune and non-immune factors in limiting the number of cases of severe malaria seen in this population.

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

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

An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca and V. vinifera grapevines on the indicator LN33, was transmitted mechanically to several Nicotiana species. The virus was partially purified from N. cavicola and the coat protein estimated at 23 kDa by SDS-PAGE. In negatively stained leaf extracts of experimentally inoculated N. cavicola and N. occidentalis, flexuous particles with cross banding were observed, predominantly measuring 750-770 x 12 nm, with a modal length of 760 nm. Decoration indicated a clear, positive reaction against AS-GVB. In DAS-ELISA, GVB was detected in N. cavicola and grapevine extracts, and Western blots showed homologous and cross reaction of GVB and GVA antisera with GVB coat protein. Using specific primers for GVB, a fragment of 594 bp, comprising the coat protein gene coding for 197 amino acids, was amplified by RT-PCR with viral RNA extracted from GVB-infected N. occidentalis. The nucleotide and the deduced amino acid sequences of the coat protein gene showed high identities with Italian and Japanese isolates of GVB.

Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

Cole latent virus (CoLV), genus Carlavirus, was studied by electron microscopy and biochemical approaches with respect both to the ultrastructure of the Chenopodium quinoa infected cells and to its association with chloroplasts. The CoLV was observed to be present as scattered particles interspersed with membranous vesicles and ribosomes or as dense masses of virus particles. These virus particles reacted by immunolabelling with a polyclonal antibody to CoLV. Morphologically, chloroplasts, mitochondria and nuclei appeared to be unaltered by virus infection and virus particles were not detected in these organelles. However, virus particle aggregates were frequently associated with the outer membrane of chloroplasts and occasionally with peroxisomes. Chloroplasts were purified by Percoll gradient, and the coat protein and virus-associated RNAs were extracted and analyzed by Western and Northern blots respectively. Coat protein and CoLV-associated RNAs were not detected within this organelle. The results presented in this work indicate that the association CoLV/chloroplasts, observed in the ultrastructural studies, might be a casual event in the host cell, and that the virus does not replicate inside the organelle.

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 São 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.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.