Characterization and Experimental Host Range of a Brazilian Tomato Isolate of Tomato severe rugose virus

We report the complete molecular characterization of the DNA-A and DNA-B of a Brazilian tomato isolate of Tomato severe rugose virus (ToSRV) and the experimental host range of the virus determined using white-fly transmission tests. Genome analysis showed that ToSRV has a close evolutionary relationship with Tomato rugose mosaic virus. Of 33 plants species inoculated with viruliferous Bemisia tabaci biotype B, 13 species were susceptible to ToSRV, nine asymptomatically. Therefore, ToSRV disease management strategy should include the control of infected weeds close to tomato fields.

Natural infection of Nicandra physaloides by Tomato severe rugose virus in Brazil

Nicandra physaloides, a common weed in South America, was found to be infected by an isolate of Tomato severe rugose virus (ToSRV), a bipartite begomovirus. The plants developed severe yellow rugose mosaic and were collected in So Paulo State, Brazil. This isolate of ToSRV was transmitted by Bemisia tabaci B biotype from infected plants of N. physaloides to healthy plants of N. physaloides and tomato in a glasshouse. This is the first report of natural infection of N. physaloides by ToSRV in Brazil.

Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Detection of human cytomegalovirus and Epstein-Barr virus in coronary atherosclerotic tissue

Previous studies indicated that patients with atherosclerosis are predominantly infected by human cytomegalovirus (HCMV), but rarely infected by type 1 Epstein-Barr virus (EBV-1). In this study, atheromas of 30 patients who underwent aortocoronary bypass surgery with coronary endartherectomy were tested for the presence of these two viruses. HCMV occurred in 93.3% of the samples and EBV-1 was present in 50% of them. Concurrent presence of both pathogens was detected in 43.3% of the samples.

Effect of Potato virus X on total phenol and alkaloid contents in Datura stramonium leaves

The present paper reports results of the effect of Potato virus X (PVX) on the contents of total phenols and alkaloids in leaves of Datura stramonium. A significant decrease in the contents of phenols and alkaloids was observed in leaves inoculated with PVX (X-I). However, there was an increase in the percentage of phenols in leaves rubbed with phosphate buffer (C1-I) and in leaves from the nodes immediately above, possibly induced by mechanical injury. Gas chromatography/mass spectroscopy revealed amounts of scopolamine in samples submitted to all treatments, except X-I, in which the amount of this alkaloid was low. High amounts of an unidentified compound (molecular ion m/z 302 and a prominent peak at m/z 129) were noted in extracts from leaves X-I, C1-I and leaves from the nodes immediately above the leaves inoculated with PVX. It is suggested that the synthesis and accumulation of the unidentified compound is a result of stress from mechanical injury and virus inoculation.

Comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants

A total of 316 samples of nasopharyngeal aspirate from infants up to two years of age with acute respiratory-tract illnesses were processed for detection of respiratory syncytial virus (RSV) using three different techniques: viral isolation, direct immunofluorescence, and PCR. Of the samples, 36 (11.4%) were positive for RSV, considering the three techniques. PCR was the most sensitive technique, providing positive findings in 35/316 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). A sample was positive by immunofluorescence and negative by PCR, and 11 (31.4%) were positive only by RT-PCR. We conclude that RT-PCR is more sensitive than IF and viral isolation to detect RSV in nasopharyngeal aspirate specimens in newborn and infants.

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera Hydroelectric Plant, São Paulo, Brazil

Yellow fever (YF) is an acute viral infectious disease transmitted by mosquitoes which occurs in two distinct epidemiological cycles: sylvatic and urban. In the sylvatic cycle, the virus is maintained by monkey's infection and transovarian transmission in vectors. Surveillance of non-human primates is required for the detection of viral circulation during epizootics, and for the identification of unaffected or transition areas. An ELISA (enzyme-linked immunosorbent assay) was standardized for estimation of the prevalence of IgG antibodies against yellow fever virus in monkey sera (Alouatta caraya) from the reservoir area of Porto Primavera Hydroelectric Plant, in the state of São Paulo, Brazil. A total of 570 monkey sera samples were tested and none was reactive to antibodies against yellow fever virus. The results corroborate the epidemiology of yellow fever in the area. Even though it is considered a transition area, there were no reports to date of epizootics or yellow fever outbreaks in humans. Also, entomological investigations did not detect the presence of vectors of this arbovirus infection. ELISA proved to be fast, sensitive, an adequate assay, and an instrument for active search in the epidemiological surveillance of yellow fever allowing the implementation of prevention actions, even before the occurrence of epizootics.

Evaluation of a recombinant p24 antigen for the detection of Feline Immunodeficiency Virus-specific antibodies

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

Pathogenicity of different rabies virus isolates and protection test in vaccinated mice

This study was aimed to evaluate and compare the pathogenicity of rabies virus isolated from bats and dogs, and to verify the efficacy of a commercial rabies vaccine against these isolates. For evaluation of pathogenicity, mice were inoculated by the intramuscular route (IM) with 500MICLD50/0.03mL of the viruses. The cross-protection test was performed by vaccinating groups of mice by the subcutaneous route and challenged through the intracerebral (IC) route. Isolates were fully pathogenic when inoculated by the IC route. When inoculated intramuscularly, the pathogenicity observed showed different death rates: 60.0% for the Desmodus rotundus isolate; 50.0% for dog and Nyctinomops laticaudatus isolates; 40.0% for Artibeus lituratus isolate; 9.5% Molossus molossus isolate; and 5.2% for the Eptesicus furinalis isolate. Mice receiving two doses of the vaccine and challenged by the IC route with the isolates were fully protected. Mice receiving only one dose of vaccine were partially protected against the dog isolate. The isolates from bats were pathogenic by the IC route in mice. However, when inoculated through the intramuscular route, the same isolates were found with different degrees of pathogenicity. The results of this work suggest that a commercial vaccine protects mice from infection with bat rabies virus isolates, in addition to a canine rabies virus isolate.

Isolation of rabies virus from the parotid salivary glands of foxes (Pseudalopex vetulus) from Paraíba State, Northeastern Brazil

To determine the presence of rabies virus in the parotid salivary glands, 12 road-killed rabies-positive hoary foxes (Pseudoalopex vetulus) were tested by using the fluorescent antibody test (FAT) and mouse inoculation test (MIT). All 12 parotid salivary glands were positive for both tests, although in some cases several passages were required. The findings of this study support the importance of the hoary fox as rabies reservoir in the semi-arid region of Paraíba State, Northeastern Brazil.

Detection of a new mumps virus genotype during parotitis epidemic of 2006-2007 in the State of São Paulo, Brazil

Nucleotide sequence analyses of the SH gene of 18 mumps virus isolates collected in the 2006-2007 parotitis epidemic in the state of São Paulo identified a new genotype, designated genotype M. This new designation fulfills all the parameters required to define a new mumps virus genotype. The parameters were established by an expert panel in collaboration with the World Health Organization (WHO) in 2005. This information will enhance the mumps virus surveillance program both at the national and global levels

Investigation of measles IgM-seropositive cases of febrile rash illnesses in the absence of documented measles virus transmission, State of São Paulo, Brazil, 2000-2004

Introdução: Revisar os casos de doenças febris exantemáticas com IgM reagente contra o sarampo, no estado de São Paulo, Brasil, durante os cinco anos seguidos a interrupção da transmissão do vírus do sarampo. Métodos: Nós revisamos 463 casos de doenças febris exantemáticas com IgM reagente contra o sarampo, no estado de São Paulo, Brasil, de 2000 a 2004. Indivíduos vacinados contra o sarampo 56 dias antes da coleta de amostra foram considerados expostos à vacina. Soros da fase aguda e de convalescença foram testados para a evidência de infecção de sarampo, rubéola, parvovírus B19 e herpes vírus 6. Na ausência de soroconversão para imunoglobulina G contra o sarampo, casos com IgM reagente contra o sarampo foram considerados falsos positivos em pessoas com evidência de outras infecções virais. Resultados: Entre as 463 pessoas com doenças febris exantemáticas que testaram positivo para anticorpos IgM contra o sarampo durante o período, 297 (64 por cento) pessoas foram classificadas como expostas à vacina. Entre os 166 casos não expostos à vacina, 109 (66 por cento) foram considerados falsos positivos baseado na ausência de soroconversão, dos quais 21 (13 por cento) tiveram evidência de infecção por vírus da rubéola, 49 (30 por cento) parvovírus B19 e 28 (17 por cento) infecção por herpes vírus humano 6. Conclusões: Após a interrupção da transmissão do vírus do sarampo é necessária exaustiva investigação dos casos com IgM reagente contra o sarampo, especialmente dos casos não expostos à vacina. Testes laboratoriais para etiologias das doenças febris exantemáticas ajudam na interpretação destes casos

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera hydroeletric plant, São Paulo, Brazil

A febre amarela (FA) é doença infecciosa aguda de origem viral transmitida por mosquitos. No ciclo silvestre, o vírus é mantido por meio da infecção de macacos e da transmissão transovariana nos vetores. A vigilância sobre populações de primatas não humanos torna-se necessária para detectar a circulação viral, quando ainda está restrito a epizootias, e para determinar sua presença em regiões indenes ou de transição para a doença. Padronizou-se a técnica ELISA (Enzyme Linked Immunosorbent Assay) para determinar a prevalência de anticorpos da classe IgG contra o vírus da FA em soros de bugios (Alouatta caraya) da região do reservatório da Usina Hidrelétrica de Porto Primavera, SP. Foram testados soros de 570 macacos sendo que nenhuma amostra mostrou-se reativa para a presença de anticorpos contra o vírus da FA. Os resultados são coerentes com a epidemiologia da FA na região. Mesmo sendo área de transição, não se conhece, até o momento, ocorrência de epizootia ou surto de FA em humanos e investigações entomológicas não apontaram a presença de vetores para esta arbovirose. A técnica mostrou-se sensível, rápida e útil à vigilância epidemiológica como instrumento de busca ativa permitindo desencadear ações preventivas, como vacinação, antes mesmo do surgimento de epizootias

The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system

Background: The cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps. Results: Here we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 mu M 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 mu M 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization. Conclusions: The genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research

Background: The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. Results: We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. Conclusions: The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.