PREDICTING THE BOILING POINT OF PCDD/Fs BY THE QSPR METHOD BASED ON THE MOLECULAR DISTANCE-EDGE VECTOR INDEX

The quantitative structure property relationship (QSPR) for the boiling point (Tb) of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) was investigated. The molecular distance-edge vector (MDEV) index was used as the structural descriptor. The quantitative relationship between the MDEV index and Tb was modeled by using multivariate linear regression (MLR) and artificial neural network (ANN), respectively. Leave-one-out cross validation and external validation were carried out to assess the prediction performance of the models developed. For the MLR method, the prediction root mean square relative error (RMSRE) of leave-one-out cross validation and external validation was 1.77 and 1.23, respectively. For the ANN method, the prediction RMSRE of leave-one-out cross validation and external validation was 1.65 and 1.16, respectively. A quantitative relationship between the MDEV index and Tb of PCDD/Fs was demonstrated. Both MLR and ANN are practicable for modeling this relationship. The MLR model and ANN model developed can be used to predict the Tb of PCDD/Fs. Thus, the Tb of each PCDD/F was predicted by the developed models.

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.

Etiologia e estratégias de controle de viroses do mamoeiro no brasil

As viroses constituem o principal grupo de doenças do mamoeiro (Carica papaya), ocasionando grandes perdas na produção, podendo chegar à destruição total das plantações afetadas. Embora mais de dez vírus tenham sido constatados infetando naturalmente o mamoeiro, em todo o mundo, no Brasil, até o presente, foram assinaladas apenas as ocorrências do vírus da mancha anelar do mamoeiro (Papaya ringspot virus, PRSV), do vírus do amarelo letal do mamoeiro (Papaya lethal yellowing virus, PLYV) e do vírus da meleira que se encontra em fase de caracterização. A mancha anelar causada pelo PRSV é, inquestionavelmente, o mais importante problema sanitário do mamoeiro. O controle do PRSV mostra-se imprescindível, apesar de bastante difícil, em razão da sua forma de disseminação rápida e eficiente por diversas espécies de afídeos e ausência de resistência genética em C. papaya. Na tentativa de controlar o PRSV, várias medidas já foram testadas, não existindo, até o momento, nenhuma estratégia eficiente e duradoura para seu controle no Brasil. O desenvolvimento de plantas transgênicas de mamoeiro expressando o gene da capa protéica (cp) do PRSV, imunes ao mesmo, abriu nova possibilidade para solução do problema.

Mosaic in Senna occidentalis in southern Brazil induced by a new strain of Soybean mosaic virus

Plants of Senna occidentalis (sin. Cassia occidentalis) with mosaic symptoms were collected near a soybean (Glycine max) field where some plants exhibited symptoms of mosaic and blistering. A preliminary examination of leaf tissue from diseased S. occidentalis by electron microscopy revealed the presence of pinwheel inclusions as well as long flexuous particles, indicating the presence of a potyvirus. Host range, serology, and amino acid sequence from this potyvirus were similar to those from other Brazilian isolates of Soybean mosaic virus (SMV). The 3'- terminal region of the genomic RNA was cloned and a cDNA sequence of 1.9 kb upstream of the poly (A) tract was determined. The sequence contains a single open reading frame and a 3'- non-translated region (NTR) of 259 bp. The nucleotide sequence of the CP gene of SMV-Soc was 98% identical to that of Brazilian isolates SMV-B, SMV-L, and SMV-FT10. The percentage of nucleotide identity of their 3'-NTR's was 91, 98, and 99% in relation to SMV-L, SMV-B, and SMV-FT10, respectively. In contrast to other Brazilian SMV isolates studied, SMV-Soc was able to infect sunflower (Helianthus annuus). Based on these results, the S. occidentalis isolate was identified as a new strain of SMV belonging to the SMV strain, group G5 and was named SMV-Soc. This is the first report of naturaly occurring SMV infecting plants of S. occidentalis in Brazil, adding this weed as a new source of SMV in the field.

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.

Caracterização do Tomato chlorotic spot virus isolado de jiló no Vale do Paraíba, Estado de São Paulo

Os tospovírus são responsáveis por perdas significativas em diversas culturas, principalmente solanáceas. No município de São José dos Campos (SP), plantas de jiló (Solanum gilo) apresentando sintomas de mosaico, bolhosidades, nanismo e queda acentuada da produção foram coletadas para análise. Visando a caracterização do agente causador dos sintomas, testes biológicos, elétrono microscópicos, sorológicos e moleculares foram realizados. Através de inoculação mecânica em plantas indicadoras das famílias Amaranthaceae, Chenopodiaceae e Solanaceae obtiveram-se resultados típicos aos esperados para tospovírus. Ao microscópio eletrônico de transmissão, observaram-se, em contrastação negativa, partículas pleomórficas com diâmetro entre 80 e 110 nm e em cortes ultra-finos partículas presentes em vesículas do retículo endoplasmático. Através de DAS-ELISA, identificou-se o Tomato chlorotic spot virus (TCSV). A partir de RNA total extraído de folhas infetadas, amplificaram-se, via RT-PCR, fragmentos correspondentes ao gene da proteína do capsídeo (cp) os quais foram seqüenciados e comparados com outros depositados no "GenBank". A homologia de nucleotídeos e aminoácidos deduzidos foi respectivamente de 99 e 95% quando comparada com seqüências de isolados de TCSV. A comparação com as outras espécies do gênero Tospovirus apresentou valores de homologia entre 72 e 84%. Estes resultados confirmam a identidade deste vírus como pertencente à espécie TCSV, que é predominante no Estado de São Paulo e importante patógeno de outras plantas cultivadas. Além disso, variedades de jiló quando inoculadas foram susceptíveis tanto ao TCSV como às espécies Tomato spotted wilt virus (TSWV) e Groundnut ringspot virus (GRSV).

Comparação da seqüência de nucleotídeos do gene da proteína capsidial de estirpes severas e premunizantes do Papaya ringspot virus

This study compared three mild and three severe strains of Papaya ringspot virus - type W (PRSV-W), based on nucleotide and amino acid sequences of the capsid protein (CP) gene. The CP nucleotide sequences of the mild strains shared 98% to 100% identity. When compared to the severe strains the identity ranged from 93% to 95%, except in the case of PRSV-W-2R, which resulted from reversion of the mild strains PRSV-W-2. The CP sequence of the reverting strain showed 100% identity with the sequence of its parental strain. An insertion of six nucleotides in the core region of the CP gene, which reflected the addition of two amino acids (Asn and Asp) in the deduced sequence of the protein, was found in all mild strains. These sequence comparisons were used to design strain-specific primers that were used to specifically amplify regions for either the mild or severe strains.

Identificação e caracterização de um potyvírus isolado de Zinnia elegans

O presente trabalho teve como objetivo a identificação e caracterização de um potyvírus isolado de Zinnia elegans, na Região Noroeste do Estado de São Paulo. O potyvírus foi transmitido por inoculação mecânica e apresentou uma gama restrita de hospedeiras sendo que as espécies mais afetadas pertencem à família Asteraceae. Em SDS-PAGE, a massa molecular da proteína capsidial (CP) foi estimada em 33 kDa e, em "Western-blot", reagiu com anti-soro para o Bidens mosaic virus (BiMV). Um fragmento de aproximadamente 820 pb foi amplificado por RT/PCR, clonado e seqüenciado. O fragmento, que inclui o gene da proteína capsidial, mostrou similaridade de aminoácidos do "core" da CP variando de 55% (Tobacco vein mottling virus, TVMV) a 95% (Sunflower chlorotic mottle virus, SuCMoV) e da CP completa de 55% (TVMV) a 91% (SuCMoV). Na região N-terminal, o potyvírus de Zinnia tem uma deleção de quatro aminoácidos (posições 9 a 12 após o sítio de clivagem entre a proteína NIb e a CP) quando comparada com a seqüência do SuCMoV. A análise filogenética agrupou o potyvírus de Zinnia e o SuCMoV em um mesmo ramo em 100% das réplicas, mostrando uma relação de parentesco muito próxima entre esses dois vírus. Os resultados obtidos no presente trabalho demonstraram que o potyvírus de Zinnia e o SuCMoV são estirpes do mesmo vírus. Sugere-se o nome Sunflower chlorotic mottle virus, isolado Zinnia (SuCMoV-Zi), ao potyvírus encontrado em Z. elegans no Brasil.

Identidade e propriedades de isolados de potyvírus provenientes de Capsicum spp.

Vinte isolados virais provenientes de Capsicum spp. foram coletados em Minas Gerais, São Paulo, Espírito Santo e Rio de Janeiro visando definir a etiologia dos mosaicos. Para a caracterização biológica realizou-se teste de gama de hospedeiros e inoculação em cultivares diferenciadoras de pimentão (Capsicum annuum). Dois isolados provenientes de batata (Solanum tuberosum) (PVY N-BR e PVY O-BR) foram utilizados como controles. Os resultados indicaram considerável grau de variabilidade biológica entre os isolados, embora todos tenham sido identificados preliminarmente como Potato virus Y (PVY). A reação das cultivares diferenciadoras classificou os isolados como patótipo 1 ou 1.2 de PVY. Anti-soros foram produzidos a partir de partículas virais purificadas de um isolado fraco e um forte. O uso desses anti-soros em ELISA indireto levou a resultados positivos contra os isolados testados. Os anti-soros reagiram também contra PVY N-BR e PVY O-BR, embora este último tenha apresentado reação mais fraca. Para caracterização molecular, seqüenciaram-se os genes da polimerase (NIb) e da proteína capsidial (cp), e da região 3' não-traduzida (3'NTR) de isolados biologicamente distintos. A análise filogenética confirmou a identidade de seis isolados como Pepper yellow mosaic virus (PepYMV), um potyvírus descrito recentemente infetando pimentão no Brasil. Esse resultado sugere que o PepYMV pode ser a espécie de potyvírus predominante em Capsicum spp. no Brasil. O fato de isolados de PepYMV apresentarem gama de hospedeiros semelhante à do PVY, e de os dois vírus apresentarem relacionamento sorológico, ressalta a utilidade da análise molecular para a classificação de potyvírus provenientes de Capsicum spp.

Expressão em Escherichia coli da proteína capsidial do Watermelon mosaic virus e produção de anti-soro

Um anti-soro policlonal específico para Watermelon mosaic virus (WMV) foi produzido por meio da imunização de coelhos com proteína capsidial purificada, expressa in vitro em células de Escherichia coli. O gene cp foi amplificado via RT-PCR utilizando oligonucleotídeos específicos, a partir de RNA viral extraído de preparações virais concentradas. O fragmento amplificado foi clonado em pBLUESCRIPT KS+ e completamente seqüenciado para confirmação de sua identidade e integridade. Em seguida, o fragmento foi subclonado no vetor de expressão pRSET-A. Plasmídeos recombinantes foram utilizados para a expressão da proteína capsidial em E. coli BL21::DE3. A proteína foi purificada por meio de cromatografia de afinidade em coluna de Ni+-NTA, a partir de proteínas totais extraídas de E. coli. Uma vez purificada, a proteína foi quantificada e utilizada para imunização dos coelhos. O anti-soro foi testado quanto a sua especificidade e sensibilidade em testes de Western blot, DAS-ELISA, imunodifusão e ELISA indireto. Todos os testes demonstraram que o anti-soro produzido a partir da expressão in vitro da proteína capsidial é altamente específico para a detecção do WMV em extratos foliares, não tendo sido observada nenhuma reação heteróloga interespecífica.

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.

Transgenic passionfruit expressing RNA derived from Cowpea aphid-borne mosaic virus is resistant to passionfruit woodiness disease

Sixteen transgenic yellow passionfruit (Passiflora spp.) plants (R0) were obtained which express a non-translatable transgenic RNA corresponding to the 3' region of the NIb gene and the 5' region of the CP gene, derived from the genome of a Brazilian isolate of Cowpea aphid-borne mosaic virus (CABMV). The transgenic plants were propagated by stem cuttings and challenged by sap inoculation with isolates CABMV-MG1 and CABMV-PE1. One transgenic plant (TE5-10) was resistant to the isolate CABMV-MG1, but susceptible to CABMV-PE1. The remaining transgenic plants developed systemic symptoms, equal to non-transformed plants, when inoculated with either isolate. The absence of virus in TE5-10 plants was confirmed by indirect ELISA. Transcription analysis of the transgene demonstrated that the TE5-10 plant did not accumulate transgenic mRNA, even before inoculation. After inoculation, viral RNA was only detected in plants inoculated with CABMV-PE1. These results confirm that the transgenic plant TE5-10 is resistant to isolate CABMV-MG1, and suggest that the resistance mechanism is post-transcriptional gene silencing, which is already activated in the transgenic plants before virus inoculation.

Transmissão por afídeos e afinidade a Buchnera sp. GroEL de um mutante deletério da proteína de RTD do Potato leafroll virus

Potato leafroll virus (PLRV), gênero Polerovirus, família Luteoviridae, é transmitido por afídeos de um modo persistente e circulativo. Membros da família Luteoviridae associam-se a um homólogo de GroEL produzido pelo endosimbionte primário (Buchnera sp.) de afídeos para evitar a degradação na hemolinfa. Partículas purificadas de luteovirus contêm dois tipos de proteínas: a capa protéica (CP) de ~22 kDa e um componente "capsidial" de 54 kDa, o qual é uma forma truncada de uma proteína de "transleitura" a partir do códon de terminação do gene da CP. O domínio de transleitura (RTD) contém determinantes responsáveis pela transmissão do vírus. Um clone de cDNA infeccioso do PLRV e um mutante deletério da RTD foram usados para analisar as interações entre esse luteovirus e seu afídeo vetor Myzus persicae. As partículas mutantes do PLRV, deficientes da proteína RTD inteira, não foram transmissíveis por M. persicae e não se ligaram a Buchnera GroEL. Adicionalmente, esse mutante foi menos persistente na hemolinfa do afídeo do que o vírus selvagem.

Protection between strains of Passion fruit woodiness virus in sunnhemp

The main objective of the present study was to evaluate the effect of the sunhemp (Crotalaria juncea) host species on the protective ability of two mild strains of Passion fruit woodiness virus (PWV), named F-101 and F-144, which had failed to protect passion flowers (Passiflora edulis f. flavicarpa) in previous experiments. The nucleotide sequences of the capsid protein (CP) gene and the 3'-non-translated region (3'-NTR) of these mild strains and the severe strain of PWV-SP were compared to confirm their relationship. The results of two protective tests with sunhemp plants in the greenhouse and one test under field conditions showed that all plants infected with either mild strain were protected against infection and/or symptom expression of the severe strain of PWV-SP. Evaluation of the relative concentration of the mild strains in sun hemp leaves showed an apparent uniformity in virus distribution in the leaf tissues, different than that which was previously reported for these mild strains in passion flower leaves. These results agree with previous studies that showed the effect of the concentration of the protective strains and the host species in the protection process.

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.