Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

Epizootic activity of Murray Valley encephalitis and Kunjin viruses in an aboriginal community in the Southeast Kimberley region of Western Australia: Results of mosquito fauna and virus isolation studies

We undertook annual surveys of flavivirus virus activity in the community of Billiluna of Western Australia in the southeast Kimberley region between 1989 and 2001. Culex annulirostris was the dominant mosquito species, particularly in years of above average rains and flooding. Murray Valley encephalitis (MVE) virus was isolated in 8 of the 13 years of the study from seven mosquito species, but more than 90% of the isolates were from Cx. annulirostris. The results suggest that MVE virus is epizootic in the region, with activity only apparent in years with average or above average rainfall and increased numbers of Cx. annulirostris. High levels of MVE virus activity and associated human cases were detected only once (in 1993) during the survey period. Activity of MVE virus could only be partially correlated with wet season rainfall and flooding, suggesting that a number of other factors must also be considered to accurately predict MVE virus activity at such communities.

Distribution of Sugarcane mosaic virus in sugarcane plants

Variation in the concentration of virus in different parts of the plant has implications for virus-indexing programs. To allow more reliable detection of Sugarcane mosaic virus (SCMV), the distribution of the virus in sugarcane plants after artificial inoculation was studied using a reverse transcription polymerase chain reaction (RT-PCR) assay. Leaves of susceptible and moderately resistant sugarcane were mechanically inoculated with SCMV 6 weeks after planting. Weekly for 8 weeks after inoculation, plants were examined for mosaic symptoms and samples of leaves, roots and tillers were tested by RT-PCR to detect virus. SCMV moved from the point of inoculation to younger leaves, roots and tillers and eventually to leaves that emerged prior to inoculation. The pattern of SCMV distribution in moderately resistant and susceptible cultivars was not substantially different. However, the virus moved more slowly in the moderately resistant than in the susceptible cultivar. Young leaves proved to be the most suitable tissue for testing.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of tine of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can he reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the V formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Análise transcriptômica da interação mamoeiro-Papaya Meleira Virus

O mamoeiro (Carica papaya L.) é uma das fruteiras mais cultivadas nas regiões tropicais e subtropicais do mundo. O Brasil faz parte do grupo dos países que mais produzem e exportam mamão no mundo. O Espírito Santo e a Bahia são responsáveis por mais de 70% da área brasileira produtora deste fruto. Porém, doenças causadas por microrganismos infecciosos afetam de modo considerável sua produção. Entre as principais doenças, destaca-se a meleira do mamoeiro, causada pelo Papaya meleira virus (PMeV), que ainda não possui uma cultivar resistente. Interessantemente os sintomas somente são desencadeados após a frutificação. Os mecanismos moleculares envolvidos no desenvolvimento dos sintomas e na resposta de defesa da planta ao PMeV ainda não foram esclarecidos. Para entender os pontos chaves desta interação, que permitam o desenvolvimento de metodologias de melhoramento genético, um estudo transcriptômico foi abordado. A tecnologia RNA-seq foi usada para o sequenciamento do transcriptoma a partir de plantas com 3, 6 e 8 meses de idade após plantio, inoculadas e não inoculadas com o PMeV. Os genes diferencialmente expressos nos 3 tempos e nas duas condições foram preditos e analisados. Estas análises revelaram um padrão de expressão geral dos genes envolvidos nesta interação. Foram encontrados 21 genes com o perfil de expressão alterado nas plantas inoculadas exclusivamente nos seis meses de idade. Destes, 8 genes envolvidos em processos de respostas de defesa e morte celular, resposta ao estresse e resposta ao estímulo biótico e abiótico foram reprimidos; enquanto os demais (13 genes), envolvidos principalmente em processos metabólicos primários, biogêneses, diferenciação e ciclo celular, comunicação e crescimento celular, bem como processos envolvidos em reprodução, e desenvolvimento da floração, foram superexpressos. Estes resultados sugerem que, aos seis meses de idade, a planta é obrigada a alterar seu programa de expressão gênica, direcionando a resposta para os processos próprios do desenvolvimento, requeridos nesse estádio fisiológico, que primam sob a resposta ao estresse, fato que finalmente leva ao desenvolvimento dos sintomas.

Effect of cattle manure on sunflower production and water use in two types of soil

The aim of the present study was to evaluate water consumption, use efficiency and yield components of sunflower variety Embrapa 122 V/2000 cultivated in two types of soil (Fluvissol and Haplic Luvisol) subjected to increasing doses of cattle manure. The experiment was carried out in a greenhouse at Universidade Estadual da Paraíba. The experimental design was completely randomized in a factorial scheme. The irrigation was performed every other day, replacing the water absorbed by the plants. The water consumption and the use efficiency were evaluated, being the use efficiency determined by the ratio of the total dry mass of sunflower and the amount of water used to produce it in each treatment. Plants were harvested at 95 days after sowing when the following parameters were evaluated: number of seeds per plant, weight of seeds per plant, weight of 1000 seeds and the outer diameter of the capitulum (head). The results showed that the sunflower was positively affected by cattle manure application, increasing the production components and the water use efficiency, regardless of the type of soil. Excepting for the 1000 seeds weight and the water use efficiency, the type of soil affected significantly the water use, the number and weight of seeds per plant. The plants cultivated in Haplic Luvisol had a better performance.

Yield and nutrition of sunflower fertilized with sewage sludge stabilized by different processes

The different methods of sewage sludge stabilization modify their physical chemical and biological properties, altering its efficiency when applied in agriculture. The objective of this study was to evaluate the nutrient levels in soil and the yield of sunflower fertilized with sewage sludge stabilized by different processes. The experiment was conducted in Cambisol, with the treatments: control (without fertilization), fertilization with sewage sludge solarized, composted, vermicomposted, limed and chemical fertilizer recommended for sunflower crop. The experimental design a randomized block with four replications. The different methods of sewage sludge treatment did not affect the yield; however, the application of sewage sludge, regardless the stabilization process adopted, was more effective than chemical fertilizer and the control treatment. Overall, fertilization with limed sewage sludge provided higher soil nutrients concentrations, while treatments with composted and vermicomposted sewage sludge showed higher levels of nutrients in the plant.

Herança da resistência a Watermelon mosaic virus em melancia

Entre as doenças que ocorrem na cultura da melancia (Citrullus lanatus), a virose ocasionada por Watermelon mosaic virus (WMV) se destaca entre as principais, sendo a resistência genética a forma mais indicada de controle. Dessa forma, é importante o conhecimento do controle genético da resistência que se pretende trabalhar. Objetivando estudar a herança da resistência ao WMV em melancia, foram realizados cruzamentos entre o cultivar Crimson Sweet (CS) suscetível e a linha L26 resistente. Populações segregantes e não segregantes obtidas dos cruzamentos foram inoculadas com um isolado de WMV e avaliadas quanto ao aparecimento de sintomas e à presença do vírus por testes de ELISA indireto contra antissoro específico para WMV. A hipótese de herança monogênica foi avaliada em diferentes graus médios de dominância e pelo método da máxima verossimilhança. Foram obtidas variâncias genética (σ²G), ambiental (σ²E), fenotípica (σ²F2), aditiva (σ²A) e de dominância (σ²D), herdabilidades nos sentidos amplo (h²a) e restrito (h²r). A herança monogênica foi rejeitada. O grau médio de dominância indicou efeito de dominância completa. As herdabilidades no sentido amplo foram baixas; portanto, constatou-se que o controle da resistência a WMV nas populações de melancia estudadas é do tipo oligogênica, com presença de efeitos aditivos e não aditivos e presença de genes maiores e poligenes.

Effect of spatial arrangement on the production components and yield of sunflower

ABSTRACT The sunflower plant is an oilseed crop that has aroused a great interest in the Brazilian and international scenery especillay because of the possibility of using its oil for biodiesel production. The objective of this study was to evaluate productivity and yield components of Embrapa 122 sunflower according to the spatial arrangement. Treatments were arranged in 4 x 4 factorial arrangement, which are the four spacings between rows (0.30; 0.50; 0.70 and 0.90 m) and four sowing densities (30,000; 45,000; 60,000 and 75,000 plants ha-1). The experiment was carried out in a complete randomized block design with four replications. The experiments were conducted in the experimental area of the Plant Science Department in Fortaleza, State of Ceará-Brazil and on the Curu Vale Experimental Farm in Pentecoste, State of Ceará-Brazil. Productivity and the following production components were analyzed in the end of the crop cycle: harvested capitula, capitulum diameter, capitulum mass, achene mass per capitulum, mass of 100 achenes, achenes per capitulum, harvest index and oil content in the achenes. The experiments were analyzed jointly in relation to the cropping area and the data submitted to analysis of variance and quantitative factors tested by polynomial regression. The variables, spacing, density and cropping area did not interact with these variables and the spatial arrangement of the plants affected only the components. The cropping area influences the productive behavior of sunflower Embrapa 122. The spatial arrangement of the plants of sunflower of variety Embrapa 122 influences yield components but does not affect productivity.

Surface plasmon resonance as a tool in the functional analisys of an immunodominant site in foot-and-mouth disease virus

A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates, medium antibody surface densities and high peptide concentrations. The method was applied to an extensive analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total agreement with previous competition ELISA experiments. Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21 residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features previously correlated with antigenicity were more pronounced in the cyclic peptide. Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicity-enhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide ? mAb 4C4 complex showed paratope ? epitope interactions identical to all FMDV peptide ? mAb complexes studied so far. Key residues for these interactions are those directly involved in epitope ? paratope contacts (141Arg, 143Asp, 146His) as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds, delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif).

Virus-Resistance Genes: The Mouse Model

Os Homens e os animais diferem grandemente nas suas respostas às infecções virais. Os vírus podem induzir, em alguns, sintomas ligeiros, enquanto que em outros podem provocar patologias graves mesmo mortais. Acumulam-se evidências que o património genético é um dos factores primordiais a condicionar e contribuir para a complexidade das interações vírus-hospedeiro. A identificação de genes com papel na resposta à infecção viral tornouse pois o tema de investigação de muitos laboratórios, com o objectivo de elucidar os processos fisiopatológicos que regem e determinam esse tipo de resposta. Neste artigo de revisão pretende-se ilustrar como o modelo murino têm sido utilizado para a identificação de genes de resistência viral, e como estes podem funcionar como base para a descoberta de genes homólogos em outras espécies. na elucidação dos mecanismos de resistência, e em novos componentes da reunir todos os genes de resistência viral descobertos em murganhos.

The measurement by serological means of the impact of the Hong Kong/68 influenza virus on a population

In order to obtain evidence on the size of the impact of the Hong Kong/68 variant of influenza A2 virus on the population of São Paulo, Brazil, serum samples taken in 1967 before this variant appeared and during successive years after it appeared were examined for their antibody content. Haemagglutination-inhibition tests performed on a total of 2726 serum samples from adults showed a sharp decrease in 1969 of the proportion of sera without antibody to the Hong Kong/68 variant and a corresponding mercase in the proportion with high titres. It was concluded that about three-quarters of the adult population became infected at some time after the variant appeared, the majority in the first year of prevalence.

Dados preliminares sobre partículas semelhantes a virus em células de triatomíneos

Nos tubos de Malpighi de Triatoma infestans Klug e de Panstrongylus megistus Burmeister foram encontradas estruturas víricas distribuídas no citoplasma, no interior de citolissomos ou outros tipos de glóbulos, formando às vezes arranjos para cristalinos envolvidos por membrana, e entre plasmalemas de células contíguas. Outros tecidos dos mesmos insetos apresentam arranjos paracristalinos desses virus. A análise citoquímica ao nível da microscopia eletrônica indica serem estes virus compostos de RNP. Não são encontradas alterações morfológicas drásticas nos tecidos infectados. Admite-se que tais virus tenham sido ingeridos durante uma alimentação de sangue infectado de ave.

Soro-epidemiologia do virus de Epstein-Barr (VEB)

É feita revisão dos estudos soro-epidemiológicos do vírus de Epstein-Barr (vírus EB), mostrando a ubicuidade deste vírus, bem como sua relação com doenças malígnas e não malígnas do homem. O desenvolvimento de novas técnicas laboratoriais e o acúmulo, nos últimos 10 anos, de grande número de estudos soro-epidemiológicos, levaram à conclusão de que, além da associação etiológica com a mononucleose infecciosa, torna-se cada vez mais evidente a ligação do vírus EB com o linfoma de Burkitt e com o carcinoma do nasofaringe. Estas observações, além de sua extraordinária importância intrínseca, tem sido de grande utilidade para a melhor compreensão dos possíveis mecanismos e efeitos das infecções virais no homem.