The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species

[EN] The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the `30K family¿. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.We thank L. Corachan for her excellent technical assistance. This work was supported by grant BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT), the Prometeo Program GV2014/010 from the Generalitat Valenciana, CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Capes (Conselho de Aperfeicoamento de Pessoal de Nivel Superior) and FAP-DF (Fundacao de Apoio a Pesquisa do Distrito Federal)Leastro, MO.; Pallás Benet, V.; Resende, RO.; Sanchez Navarro, JA. (2017). The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species. Virus Research. 227:57-68. https://doi.org/10.1016/j.virusres.2016.09.023S576822

Factors related to incidence and dissemination of Maize common mosaic virus

O mosaico comum do milho (Zea mays) destaca-se, atualmente, entre as doenças mais importantes dessa cultura, sendo causada por um complexo de potyvirus transmitido por afídeos. Esse trabalho teve por objetivo a identificação de fatores que podem contribuir para a incidência dessa virose e disseminação do seu agente causal. Plântulas de 115 cultivares dos Ensaios Nacionais de Milho-Centro foram submetidas a quatro inoculações com o complexo viral, utilizando-se, em cada uma, parcelas com cinco plantas de cada cultivar. A maioria das cultivares mostrou-se suscetível, apresentando sintomas da virose, aos 15 dias após a inoculação. Espécies de gramíneas também foram inoculadas e mostraram-se hospedeiras desses vírus. Através do teste dot-ELISA, a presença dos vírus do mosaico comum foi detectada em folhas de milho provenientes de vários municípios dos estados de São Paulo, Minas Gerais e Goiás. No período de mar/97 a fev/98, dois híbridos de milho foram plantados mensalmente, e semanalmente avaliados quanto à incidência do mosaico comum. Observou-se tendência de maior incidência nos plantios de novembro, dezembro e janeiro, coincidindo com as elevadas temperaturas e precipitações pluviométricas do verão. Os resultados obtidos contribuem para a recomendação de medidas para o controle dessa virose.In recent years, mosaic became one of the most important diseases affecting maize (Zea mays) crops. It is caused by a potyvirus complex transmitted by aphid species. The aim of this work was to identify factors that may contribute to increase the incidence and dissemination of this disease. Seedlings of 115 cultivars from three different National Maize Cultivars Trials were tested for their susceptibility to the virus complex via mechanical inoculation of five plants of each cultivar, between October/97 and February/98. Most cultivars were susceptible and showed mosaic symptoms 15 days after inoculation. Several graminaceous species were also inoculated and showed to be hosts of the potyvirus complex. Using the dot-ELISA test, the virus complex could be detected in maize plants collected from different regions of São Paulo, Minas Gerais and Goias states. Between March/97 and February/98 two maize inbred lines were planted monthly and incidence of mosaic was evaluated weekly. The highest incidence was observed in the summer, coinciding with the increase of temperature and the levels of rainfall. These results may contribute to recommending control measures

High incidence of Pepper yellow mosaic virus in tomatoes in productive areas of Brazil s Federal Distric

Várias doenças ameaçam a produção de tomate, especialmente aquelas causadas por vírus dos gêneros Begomovirus, Tospovirus e Potyvirus. Uma análise de amostras foliares de tomateiro para detecção dos vírus de maior importância no Distrito Federal foi realizada utilizando-se as técnicas de PCR e DAS-ELISA. Em um total de 54 amostras avaliadas, o vírus mais prevalente foi o Pepper yellow mosaic virus, um potyvírus responsável por sérias perdas em áreas de produção de pimentão no Brasil. Este resultado reforça a crescente importância deste vírus na produção de tomate. Os sintomas causados por PepYMV são muito similares àqueles causados por begomovírus, portanto é recomendada que uma técnica de detecção apropriada seja utilizada em adição à avaliação visual de sintomas.Various diseases threaten tomato production; especially those caused by viruses of the genera Begomovirus, Tospovirus and Potyvirus. A survey on the major viral diseases occurring on tomatoes in the Federal District was carried out using PCR and DAS-ELISA. Out of 54 evaluated samples, the most prevalent virus was Pepper yellow mosaic virus, a potyvirus responsible for heavy losses in sweetpepper production in Brazil. This result highlights the increasing importance of this virus on the tomato crop. Since the symptoms caused by PepYMV infection are similar to those caused by begomoviruses, the use of a proper detection technique is recommended in addition to visual evaluation of symptoms

Métodos de RT-PCR e de hibridização Dot Blot para detecção universal de tospovirus

Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.Visando a um método para a detecção universal de tospovírus, utilizaram-se as técnicas de "Transcriptase reverse - polymerase chain reaction" (RT-PCR) e hibridização com sondas marcadas com digoxigenina. As espécies de tospovirus testadas foram: Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Os oligonucleotídeos foram sintetizados para anelar em regiões conservadas do genoma viral, sendo os produtos de PCR utilizados como sondas para hibridização através de "dot blot". Através de RT-PCR, utilizando-se diferentes combinações de oligonucleotídeos, somente foi possível a amplificação de todas as espécies quando se utilizou RNA de vírus purificado, sendo que, para a detecção a partir de RNA total, a RT-PCR apresentou problemas para a detecção das espécies ZLCV e IYSV. Sob condições de baixa adstringência, os testes de hibridização por "dot blot" com a sonda M (correspondente ao gene G1/G2) detectaram todas as espécies testadas, exceto IYSV. Com a sonda L, também sob condições de baixa adstringência, pôde-se detectar todas as espécies de tospovirus testadas simultaneamente em um único ensaio. Este método de detecção pode ser utilizado em serviços de quarentena e para indexação de germoplasma in vitro

Complexo viral do alho: identificação de Potyvirus e Carlavirus na região central do Brasil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virus-free garlic was evaluated.Infecções virais em alho são normalmente causadas por um complexo viral. Neste estudo, um complexo viral de alho, coletado em campo, foi purificado. Procedeu-se à amplificação por RT-PCR usando oligonucleotídeos desenhados para regiões-consenso dos genes das proteínas capsidiais de Onion yellow dwarf virus, estirpe do alho e de Leek yellow stripe virus. cDNA de Garlic common latent virus foi sintetizado usando oligo-dT e oligonucleotídeos aleatórios. Por estes procedimentos clones de diferentes espécies virais foram isolados e sequenciados. A análise das sequências nucleotídicas e os resultados sorológicos revelaram a presença dos Potyvirus OYDV-G e LYSV e do Carlavirus GCLV, simultaneamente infetando plantas de alho. As seqüências de aminoácidos deduzidos dos isolados brasileiros foram comparadas com aquelas de vírus relacionados, relatados em diferentes regiões do mundo. A análise mostrou pequena variabilidade em relação aos isolados brasileiros de OYDV-G e GCLV, e maior divergência em relação ao isolado de LYSV. A detecção destas espécies virais também foi obtida por reações específicas observadas quando o gene da proteína capsídica dos isolados brasileiros foi usado como sonda em ensaios de hibridização do tipo dot-blot e Southern blot. Em campo, a re-infecção natural de alho livre de vírus foi avaliada

Molecular characterization reveals Brazilian Tomato chlorosis virus to be closely related to a Greek isolate

Tomato chlorosis virus (ToCV, genus Crinivirus, family Closteroviridae) is a whitefly-transmitted crinivirus with a bipartite RNA genome. This virus is emerging as a serious threat to tomato crops worldwide. To date, only three complete genomic sequences of ToCV have been described from North America, Spain, and Greece isolates. In this study, we present the fourth complete nucleotide sequence of the RNA 1 (8594 nt) and RNA 2 (8242 nt) components of a Brazilian ToCV isolate (ToCV-BR). The complete genome sequences of RNA 1 and RNA 2 have been deposited in the GenBank database under the accession numbers JQ952600 and JQ952601, respectively. The sequences of RNA 1 and RNA 2 shares the highest nucleotide identity of 99.6% and 99.5%, respectively, with the Greek isolate sequences. Phylogenetic analysis confirmed that both RNA 1 and RNA 2 of the Brazilian isolate are most closely related to the Greek isolate of that virus. These results suggest that ToCV may have been recently introduced to Brazil from Europe

Occurrence of corn stunt diseases and maize viruses in the Provinces of Tucumán and Córdoba in Argentina

A incidência de doenças causadas por molicutes e por vírus foi avaliada em lavouras de milho (Zea mays) nas Províncias de Tucumán e de Córdoba, na Argentina, em fevereiro de 2000. Na Província de Tucumán verificou-se que 44% das lavouras apresentaram altos níveis de incidência de plantas com sintomas de enfezamentos causados por molicutes (50 a 100%), em altitudes variando de 300 a 2.000 m. A presença de fitoplasma e de espiroplasma foi confirmada em amostras de folhas de plantas com sintomas de enfezamentos, através dos testes de PCR e de "Western blotting". Constatou-se, porém, que a eficiência desses testes para detecção destes patógenos, quando os sintomas apresentados pelas plantas eram muito acentuados, foi da ordem de 70%, e de apenas 30% quando os sintomas eram menos acentuados. Na localidade Jesus Maria, foram encontradas plantas apresentando acentuado nanismo, folhas estreitas e com deformações. Dentre quatro amostras destas plantas, submetidas a testes de PCR, em duas foi detectada a presença de fitoplasma, possivelmente d istinto do "Maize Bushy Stunt Phytoplasma". A cigarrinha Dalbulus maidis, inseto vetor dos molicutes, foi encontrada apenas em Tucumán, estando ausente em Córdoba. O Mal de Rio Cuarto virus foi detectado em seis lavouras em Córdoba, e em três em Tucumán. A cigarrinha Delphacodes kuscheli foi detectada em todas as lavouras em Córdoba, e em apenas três lavouras em Tucumán. O Maize dwarf mosaic virus foi detectado em cerca de 60% das lavouras amostradas nas duas Províncias e o Maize rayado fino virus em apenas uma localidade em Tucumán.The incidence of "corn stunt diseases" and maize (Zea mays) viruses was evaluated in maize fields located at the Provinces of Tucumán and Córdoba in Argentina in February 2000. A high number of plants infected with "corn stunt disease" were observed in Tucumán (up 50 to 100%) in 44% of maize crops surveyed in areas varying from up 300 to 2000 m high. The presence of Maize Bushy Stunt Phytoplasma (MBSP) and Corn Stunt Spiroplasma (CSS) was confirmed by PCR and Western blotting tests. In plants showing typical symptoms of MBSP or CSS, the pathogens were detected in 70% of the samples. However, when symptoms were weak, the efficiency of detection dropped to approximately 30%. In Rio Cuarto, Province of Córdoba, the presence of phytoplasma was detected only in three plants showing red leaf symptoms. In Jesus Maria locality, plants showing symptoms different from those caused by MBSP, were demonstrated to be infected by phytoplasma when analyzed by PCR using universal primers. The leafhopper Dalbulus maidis, vector of MBSP and CSS, was found only in Tucumán. The Mal de Rio Cuarto virus (MRCV) was found in six maize fields in Córdoba and in three maize fields located at 1970 to 2.000 m high in Tucumán. The plant hopper Delphacodes kuscheli, vector of MRCV, was found in both provinces sampled. The Maize dwarf mosaic virus (MDMV) was found in 60% of maize crops in both Provinces and the Maize rayado fino virus (MRFV)was found only in one location in Tucumán