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%).

An isolate of Apple stem grooving virus associated with Cleopatra mandarin fruit intumescence

A citrus tatter leaf isolate (CTLV-Cl) of Apple stem grooving virus (ASGV) has been found to be associated with a fruit rind intumescence in Cleopatra mandarin (Citrus reshni) in Limeira (SP). The CTLV-Cl was mechanically transmitted to the main experimental herbaceous hosts of CTLV. Chenopodium quinoa and C. amaranticolor reacted with local lesions and systemic symptoms while other test plants reacted somewhat differently than what is reported for CTLV. A pair of primers designed for specific detection of ASGV and CTLV amplified the expected 801 bp fragment from the CTLV-Cl-infected plants. Typical capillovirus-like particles were observed by the electron microscope in experimentally infected C. quinoa and C. amaranticolor leaves.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Caracterização do gene da proteína capsidial do Grapevine virus A em videiras afetadas pela acanaladura do lenho de Kober no Estado de São Paulo

O presente trabalho caracteriza o gene codificador da proteína capsidial do isolado do Grapevine virus A (GVA) encontrado no Estado de São Paulo (GVA-SP). RNA total foi extraído de folhas e pecíolos de plantas de videira (Vitis spp.) da variedade 'Kober 5BB' e submetido a RT-PCR usando oligonucleotídeos desenhados para amplificar um fragmento entre as posições 6409 e 7175 do RNA do GVA ("GenBank", acesso X75433). Foi obtido um fragmento de tamanho esperado (767 nt) que inclui o gene da proteína capsidial, codificando 198 aminoácidos. A seqüência do GVA-SP apresentou similaridade de nucleotídeos e aminoácidos de, respectivamente, 86-92,3% e 94,5-98% com isolados do GVA da Europa, África e Japão (Acessos X75433, AF441234, AF007415, AB039841) e da região Sul do Brasil (Acesso AF494187), sendo, entretanto, mais similar aos isolados africano e italiano.

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.

Caracterização do gene da proteína capsidial do Grapevine virus A em videiras afetadas pela acanaladura do lenho de Kober no Estado de São Paulo

O presente trabalho caracteriza o gene codificador da proteína capsidial do isolado do Grapevine virus A (GVA) encontrado no Estado de São Paulo (GVA-SP). RNA total foi extraído de folhas e pecíolos de plantas de videira (Vitis spp.) da variedade 'Kober 5BB' e submetido a RT-PCR usando oligonucleotídeos desenhados para amplificar um fragmento entre as posições 6409 e 7175 do RNA do GVA (GenBank, acesso X75433). Foi obtido um fragmento de tamanho esperado (767 nt) que inclui o gene da proteína capsidial, codificando 198 aminoácidos. A seqüência do GVA-SP apresentou similaridade de nucleotídeos e aminoácidos de, respectivamente, 86-92,3% e 94,5-98% com isolados do GVA da Europa, África e Japão (Acessos X75433, AF441234, AF007415, AB039841) e da região Sul do Brasil (Acesso AF494187), sendo, entretanto, mais similar aos isolados africano e italiano.

Occurrence of apple stem grooving capillovirus in Santa Catarina, Brazil, detected by RT-PCR.

1999

Recombination events in full genome sequences of apple stem grooving virus.

2016

Detecção do Grapevine virus A e Grapevine virus B por hibridização "dot-blot" com sondas moleculares não radioativas

O vírus A da videira (Grapevine virus A, GVA) e o vírus B da videira (Grapevirus virus B, GVB) estão associados à acanaladura do lenho de Kober ("Kober stem grooving") e ao fendilhamento cortical da videira ("grapevine corky bark"), respectivamente. Este trabalho descreve o uso de sondas moleculares de cDNA na detecção de isolados do GVA (GVA-SP) e do GVB (GVB-C-SP e GVB-I-SP) em videiras (Vitis spp.) e fumo (Nicotiana occidentalis). As sondas marcadas com digoxigenina foram produzidas por RT-PCR utilizando oligonucleotídeos específicos para os genes da proteína capsidial. Os RNA totais foram extraídos de 45 plantas de diversas variedades de videira e de 13 plantas de fumo inoculadas mecanicamente com o GVB. Os RNA extraídos das plantas infetadas, indexadas biologicamente, hibridizaram com as sondas, não se verificando reação com plantas sadias. Para confirmar os resultados de hibridização, foram também feitos testes de RT-PCR. A utilização de hibridização "dot-blot" com sondas de cDNA mostrou-se eficaz na detecção dos vírus com especificidade e sensibilidade, ressaltando-se que, preferencialmente, folhas maduras e ramos dormentes devem ser utilizados nos testes diagnósticos para o GVB e GVA, respectivamente.

Detecção do Grapevine virus A e Grapevine virus B por hibridização dot-blot com sondas moleculares não radioativas

O vírus A da videira (Grapevine virus A, GVA) e o vírus B da videira (Grapevirus virus B, GVB) estão associados à acanaladura do lenho de Kober (Kober stem grooving) e ao fendilhamento cortical da videira (grapevine corky bark), respectivamente. Este trabalho descreve o uso de sondas moleculares de cDNA na detecção de isolados do GVA (GVA-SP) e do GVB (GVB-C-SP e GVB-I-SP) em videiras (Vitis spp.) e fumo (Nicotiana occidentalis). As sondas marcadas com digoxigenina foram produzidas por RT-PCR utilizando oligonucleotídeos específicos para os genes da proteína capsidial. Os RNA totais foram extraídos de 45 plantas de diversas variedades de videira e de 13 plantas de fumo inoculadas mecanicamente com o GVB. Os RNA extraídos das plantas infetadas, indexadas biologicamente, hibridizaram com as sondas, não se verificando reação com plantas sadias. Para confirmar os resultados de hibridização, foram também feitos testes de RT-PCR. A utilização de hibridização dot-blot com sondas de cDNA mostrou-se eficaz na detecção dos vírus com especificidade e sensibilidade, ressaltando-se que, preferencialmente, folhas maduras e ramos dormentes devem ser utilizados nos testes diagnósticos para o GVB e GVA, respectivamente.

Evaluation of detection methods for Virus, Viroids and Phytoplasmas affecting pear and apple

The RT-PCR technique for the detection of apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV) and pear blister canker viroid (PBCV) was evaluated for health control of fruit plants from nurseries. The technique was evaluated in purified RNA and crude extracts and also in phloem collected in autumn and from young spring shoots. The results obtained for phytoplasma detection with ribosomal and non-ribosomal primers are also presented.

Estudo in silico de sítios de restrição enzimática de genes da proteína capsidial e análise de diversidade genética de Apple chlorotic leaf spot virus em fruteiras de semente e caroço.

Cultivares comerciais de macieiras são infectadas por 3 espécies principais de vírus: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) e Apple stem pitting virus (ASPV), geralmente em infecções complexas. O objetivo do estudo foi caracterizar a diversidade genética de genes da proteína capsidial (CP) de isolados de ACLSV.

Clonagem de construto de RNA interferente para produção de Maruba-Kaido resistente a vírus de macieira.

2012

Development of Glomerella leaf spot is enhanced in virus-infected Maxi Gala apples.

Apples are commercially grown in Brazil in a subtropical environment that favors the development of fungal diseases such as Glomerella leaf spot (GLS) caused mainly by Glomerella cingulata (anamorph Colletotrichum gloeosporioides). The main objective of this work was to evaluate the effect of mixed infections by Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) on the infection and the colonization processes of C. gloeosporiodes in cv. Maxi Gala plants. Leaves of 16-month-old potted plants were spray-inoculated and both the disease incidence and lesion count were monitored over time and leaf severity was assessed in the final evaluation using an image analysis tool. Results showed that initial infection estimated from a monomolecular model fitted to progress of lesion count was higher and the incubation period (time to reach 50% incidence) was on average 10 h shorter in virus-infected plants compared to non-infected plants. It is hypothesized that initial events such as conidial germination and fungal penetration into plant cells were facilitated by the presence of viral infection. Also, final GLS severity was significantly higher in the virus-infected plants. Mixed infections by ASGV/ASPV seemed to make apple leaves more susceptible to the initial infection and colonization by C. gloeosporioides.

Sinonasal Disorders In Hematopoietic Stem Cell Transplantation.

hematopoietic stem cell transplantation (HSCT) is associated with more respiratory infections due to immunosuppression. this study aimed to verify the frequency of rhinosinusitis after HSCT, and the association between rhinosinusitis and chronic graft vs. host disease (GVHD) and type of transplantation, clinical treatment, surgical treatment, and survival. this was a retrospective study in a tertiary university hospital. A total of 95 patients with hematological diseases undergoing HSCT between 1996 and 2011 were selected. chronic myeloid leukemia was the most prevalent disease. The type of transplant most often performed was the allogenic type (85.26%). The frequency of rhinosinusitis was 36%, with no difference between the autologous and the allogenic types. Chronic GVHD occurred in 30% of patients. Patients with GVHD had a higher frequency and recurrence of rhinosinusitis, in addition to more frequent need for endoscopic sinusectomy and decreased overall survival. there was a higher frequency of rhinosinusitis in HSCT and GVHD. The type of transplant does not appear to predispose to the occurrence of rhinosinusitis. GVHD seems to be an aggravating factor and requires a more stringent treatment.