Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Passion fruit (Passiflora edulis) and cowpea (Vigna unguiculata) are important crops of economical impact for the Northeast of Brazil. Fruit woodiness is an important virus disease of passion fruit, caused by Passionfruit woodiness virus (PWV) and Cowpea aphid-borne mosaic virus (CABMV), both from the genus Potyvirus. CABMV is also responsible for important disease on cowpea. The present research had the objective to study and compare the biological, serological and molecular properties of isolates of CABMV obtained from passion fruit (CABMV-P Pet, CABMV-P Gua, CABMV-P Sb and CABMV-P Uba) and from cowpea (CABMV-C Fort and CABMV-C Bv). CABMV-C Fort and CABMV-C Bv were purified from infected cowpea cv. Pitiuba systemically infected. The obtained purified preparations presented concentrations of 16.4 mg of virus per mL (CABMV-C Bv) and 15.9 mg of virus per mL (CABMV-C Fort). Both purified virus preparations were used for rabbit immunizations to produce polyclonal antisera with reactive titers of 1:128.000 in PTA-ELISA. Electrophoresis analyses of the virus purified preparations revealed the presence of only one capsidial protein with molecular weight of 34 kDa for both virus isolates. Parts of the genomes, corresponding to the coat protein gene (cp), from the virus isolates obtained from cowpea and from passion fruit were amplified by RT-PCR. The philogenetic analyses of the amplified cDNA fragments grouped with the CABMV isolates sequences deposited in the GenBank, according to their original host. Based on the biological, serological and molecular results, the virus isolates studied were classified into two biotypes: Biotype Cowpea (CABMV-C) including CABMV-C Bv and CABMV-C Fort obtained from cowpea, and biotype Passion fruit (CABMV-P) including CABMV-P Pet, CABMV-P Gua, CABMV-P Sb and CABMV-P Uba obtained from passion fruit. Source of immunity to CABMV-C Bv and CABMV-C Fort was identified in cowpea and the genotype was designate Lab-Poty. Studies of virus interaction in cowpea demonstrated strong synergistic effect among CABMV-C, Cucumber mosaic virus (CMV) and Cowpea severe mosaic virus (CPSMV). / O maracujazeiro (Passiflora edulis) e o feijoeiro caupi (Vigna unguiculata) sÃo culturas de elevada importÃncia econÃmica para o Nordeste brasileiro. Entre as viroses que se manifestam no maracujazeiro, destaca-se o endurecimento dos frutos causado por Passionfruit woodiness virus (PWV) e Cowpea aphid-borne mosaic virus (CABMV), ambos pertencentes ao gÃnero Potyvirus, sendo o CABMV responsÃvel por importante doenÃa do feijoeiro caupi. A presente pesquisa teve como objetivo estudar e comparar as propriedades biolÃgicas, sorolÃgicas e moleculares de isolados de CABMV obtidos de maracujazeiro (CABMV-P Pet, CABMV-P Gua, CABMV-P Sb e CABMV-P Uba) e isolados obtidos de feijoeiro caupi (CABMV-C Fort e CABMV-C Bv). Os isolados CABMV-C Fort e CABMV-C Bv foram purificados a partir de plantas de feijoeiro caupi cv. Pitiuba sistemicamente infetadas. As preparaÃÃes purificadas obtidas apresentaram concentraÃÃes de 16,4 mg de vÃrus por mL (CABMV-C Bv) e 15, 9 mg de vÃrus por mL (CABMV-C Fort), as quais foram usadas na imunizaÃÃo de coelhos, para a produÃÃo de antissoros policlonais, com tÃtulo de 1:128.000 em PTA-ELISA. AnÃlises eletroforÃtica das preparaÃÃes virais revelaram uma Ãnica proteÃna capisidial com peso molecular de 34 kDa. Partes do genoma correspondente ao gene da capa protÃica (cp) dos isolados virais obtidos de feijoeiro caupi e de maracujazeiro foram amplificadas por RT-PCR. AnÃlises filogenÃticas dos fragmentos dos cDNA amplificados se agruparam com sequencias de isolados do CABMV depositadas no GenBank, de acordo com os hospedeiros originais. Com base nos resultados de estudos biolÃgicos, sorolÃgicos e moleculares, os isolados virais estudados foram classificados em dois biÃtipos: BiÃtipo Cowpea (CABMV-C) incluindo CABMV-C Bv and CABMV-C Fort obtidos de feijoeiro caupi e biÃtipo Passion fruit (CABMV-P) incluindo CABMV-P Pet, CABMV-P Gua, CABMV-P Sb e CABMV-P Uba obtidos de maracujazeiro. Fontes de imunidade a CABMV-C Bv e CABMV-C Fort foi identificada em feijoeiro caupi e o genÃtipo foi designado de Lab-Poty. Estudos de interaÃÃo viral em feijoeiro caupi, mostraram forte sinergismo entre CABMV-C, Cucumber mosaic virus (CMV) e Cowpea severe mosaic virus (CPSMV).

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Plant virus identification and characterization can be achived by several methods based in the biological, morphological, cytological, serological and molecular virus properties. The molecular properties have been used with frequency for vÃrus identification and characterization and the reverse transcription polymerase chain reaction (RT-PCR) has constituted an efficient and precise method for researches with RNA plant viruses. On the other hand, the enzyme-linked immunosorbent assay (ELISA) is the most used serological method for virus detection in plant tissues. A techncal innovation developed for plant virus identification represents a great technological development and support for plant virus research. A new approach involving virus particle immune precipitation to be used for RNA amplification by RT-PCR named IP-RT-PCR was sucessefuly used for amplification of RNA fragments from five virus species from the genera Comovirus, Cucumovirus, Potyvirus and Sobemovirus. Considering that the immune biological Companies have developed several DAS-ELISA kits, but neither of them produce and commercialize PTA-ELISA kits, a simple and practical PTA-ELISA kit was developed and validated for plant virus detection. The second part of the research had the objective to study, comparatively, the biological, serological and molecular properties of four plant virus isolates obtained from naturally infected Passiflora edulis (PWV-PET and PWV-GUA) and from naturally infected Vigna unguiculata (CABMV-BV and CABMV-FOR) with the objective to elucidate the identity of the causal agent of passion fruit woodiness in Brazil. In host range studies onle Canavalia ensiformes and Macroptilium lathyroides were infected by virus isolates obtained from cowpea and from passion fruit. The isolate PWV-GUA was purified from systemically infected M. lathyroides plants and the virus purified preparation (18.24 mg of virus.ml-1) was used for rabitt immunization for polyclonal antiserum production, which showed a title of 1:128,000 in PTA-ELISA. The electrophoresis analysis of the purified virus showed a unique capsidial protein with 34 kDA. Plant virus interaction studies in C. ensiformis indicated unilateral cross protection between PWV-GUA and CABMV-FOR. On the other hand, the isolate PWV-PET did not cross protect passion fruit plants against PWV-GUA. Filogenetic analysis of nucleotiode sequencies from cDNA fragments corresponding to coat protein (CP) genes amplified by IP-RT-PCR from the genomic virus isolates compared with virus sequencies from the Genbank grouped according to the host specifities. Based on the biological, serological and mainly molecular results, the virus isolates studied were classified into two biotypes: Biotype CABMC-C (Cowpea) to include isolates obtained from cowpea that do not infect passion fruit, and biotype CABMV-P (Passion fruit) to include the virus isolates responsible for the passion fruit woodiness in Brazil. / A identificaÃÃo e a caracterizaÃÃo de vÃrus de planta podem ser realizadas por vÃrios mÃtodos envolvendo propriedades morfolÃgicas, biolÃgicas, citolÃgicas, moleculares e sorolÃgicas. As tÃcnicas moleculares tÃm sido usadas com frequencia para identificaÃÃo e caracterizaÃÃo de vÃrus, e a tÃcnica de âreverse transcription polymerase chain reactionâ (RT-PCR) tem se constituÃdo em mÃtodo eficiente e preciso para pesquisas com vÃrus de planta com genoma de RNA. De outra parte, a tÃcnica de enzyme-linked immunosorbent assay (ELISA) constitui o mÃtodo sorolÃgico mais usado para detecÃÃo de vÃrus em tecidos vegetais. Uma inovaÃÃo tecnolÃgica desenvolvida nesta pesquisa para diagnose de vÃrus de planta representa grande avanÃo tecnolÃgico e suporte para pesquisa em virologia vegetal. A inovaÃÃo envolvendo a imunoprecipitaÃÃo (IP) de partÃculas de vÃrus para uso na RT-PCR denominada de IP-RT-PCR foi usada com sucesso para amplificaÃÃo de fragmentos de RNA de cinco espÃcies de vÃrus dos gÃneros Comovirus, Cucumovirus, Potyvirus e Sobemovirus. Considerando que kits de DAS-ELISA tÃm sido produzidos e comercializados por companhias de imunobiologicos, mas nenhuma companhia produz kits de PTA-ELISA, um kit simples e prÃtico de PTA-ELISA foi desenvolvido e validado para detecÃÃo de vÃrus de planta. A segunda etapa da pesquisa teve como objetivo estudar as propriedades biolÃgicas, sorolÃgicas e moleculares de isolados de vÃrus do gÃnero Potyvirus obtidos de maracujazeiro (Passiflora edulis) (PWV-PET e PWV-GUA) e isolados de Cowpea aphid-borne mosaic virus (CABMV-FOR e CABMV-BV) obtidos de feijoeiro caupi (Vigna unguiculata), visando elucidar a identidade do agente causal do endurecimento dos frutos do maracujazeiro no Brasil. Em estudos de gama de plantas hospedeiras, somente Canavalia ensiformis e Macroptilium lathyroides foram infetadas por isolados obtidos de maracujazeiro e de feijoeiro caupi. O PWV-GUA foi purificado a partir de plantas de M. lathyroides sistemicamente infetadas e a preparaÃÃo viral purificada (18,24 mg de vÃrus.ml-1) foi usada para imunizaÃÃo de coelho com a produÃÃo de antissoro policlonal com tÃtulo de 1:128.000 em PTA-ELISA. AnÃlise eletroforÃtica da preparaÃÃo viral purificada revelou uma Ãnica proteÃna capisidial com peso molecular de 34 kDa. Experimentos de interaÃÃo entre os isolados virais em C. ensiformis indicaram proteÃÃo unilateral entre PWV-GUA e CABMV-FOR. De outra parte, o isolado PWV-PET nÃo protegeu plantas de maracujazeiro contra a super infecÃÃo de PWV-GUA. AnÃlises filogenÃticas das seqÃÃncias dos fragmentos de cDNA correspondentes Ãs capas protÃicas (CP), amplificados a partir dos genomas dos isolados virais de maracujazeiro e de feijoeiro caupi por IP-RT-PCR, agruparam-se com as seqÃÃncias de isolados virais de referidas culturas depositadas no GenBank, apresentando um agrupamento em funÃÃo da especificidade de hospedeiros. Com base nos resultados dos estudos biolÃgicos, sorolÃgicos e, sobretudo moleculares, os isolados virais estudados foram classificados em dois biÃtipos: BiÃtipo CABMV-C (Cowpea) incluindo os isolados obtidos de feijoeiro caupi e biÃtipo CABMV-P (Passion fruit) para incluir os isolados responsÃveis pelo endurecimento dos frutos do maracujazeiro no Brasil.