A rainbow trout (Slamo gairdneri Richardson) population of IPN virus carriers was studied over a one-year period using both homogenisatian and co-cultivation for virus isolation. The percentage of virus-yielding fish was high between March and June but then declined. This was diametrically opposite to the trend in the serum antibody levels indicating that the marked humoral immune response resulted in a very significant reduction in the virus titres. The highest isolation rate was obtained from the kidneys after co-cultivation (from seventeen of the twenty-three virus-positive fish) underlining the very high sensitivity of this recently developed method for virus detection. Twelve of the twenty-three virus-positive fish yielded virus from the pyloric caeca after homogenisation. Virus was occasionally isolated from the faeces indicating that this may well be a possible avenue for horizontal transmission of the virus. No virus was ever detected in gonadal tissue. The virulence of the rainbow trout virus was enhanced in various ways and used to infect tilapia Oreochromis spilurus Gunther of different ages through a variety of routes. Fry infected by direct immersion, orally and by force feeding showed little or no signs of infection. Intraperitoneally and intramuscularly injected fingerling and adult fish developed marked haemorrhaging, severe loss of skin mucus and up to 50% mortalities were recorded. Gross pathology included enlarged and liquifying liver, gastroenteritis and mild brain haemorrhaging. Histopathologically there was extensive cellular vacuolation and degeneration as well as marked leucocytic infiltration in the liver, intestine and swimbladder. Eosinophilic granule cell infiltration of the intestinal wall was also very prominent. Virus was recovered from several organs and determination of virus titres revealed that active viral replication had occurred in the tilapia tissues, a finding further supported by electron microscopical evidence. The fish showed a clear humoral antibody response.

No description available.

There is an urgent need to discover new antimicrobial and antiviral compounds owing to increasing problems of resistance to drugs encountered in many pathogenic organisms. There are also problems with currently used drugs in terms of side effects and expense. Plants have been used for many generations for healing purposes, and screening of extracts of these plants has often yielded positive results. In particular, plants with antimicrobial properties are the subject of much investigation. This study focuses on isolating the compounds responsible for biological activity in one such medicinal plant, Combretum paniculatum, extracts of which have been shown to possess antimicrobial activity. Members of the genus Combretum are widely used for medicinal purposes by many groups in Africa, to treat various conditions. Other researchers have discovered antifungal, antibacterial, anti-inflammatory and molluscicidal effects of these plants. One species of this genus, C. paniculatum, has been reported in the literature to have antiviral activity against HIV-2 with a promising selectivity index. It is important to exclude highly toxic effects of potential antimicrobial preparations. C. paniculatum extracts also displayed good antibacterial activity and some anti-inflammatory activity in other studies. Although many active compounds, especially antibacterial and antifungal, have been isolated from other Combretum species, little is known about the identity of compounds responsible for activity in C. paniculatum. In the initial stages of this project, the crude extracts of leaves of C. paniculatum were investigated for antiviral and cytotoxic activity. It was found that the acetone and water extracts of C. paniculatum leaves reduced the cytopathic effect of feline herpesvirus type 1 by 3.0 log10, a very promising result. Investigations were carried out to determine the best solvent to use for extracting the active components. It was found that acetone was the best extractant in terms of the number of compounds extracted from the plant after analysis using thin layer chromatography (TLC) and the number of bioactive compounds using bioautography against bacteria. Water extracted a large quantity of material. Different plant parts, namely stem bark, root bark and leaves, were screened for antiviral and antibacterial activity and the leaves and stem bark showed good activity. The test organisms were feline herpesvirus type 1 (FHV-1) for antiviral testing, and a range of Gram-positive and Gram-negative bacteria for antibacterial activity. Cytotoxicity against African green monkey kidney (Vero) cells was observed only at a relatively high concentration of 0.28 mg/ml. Based on availability and sustainability, the leaves were chosen for further work especially since leaves were used in the published data. Isolation of active compounds from a 70% acetone extract of a large quantity of C. paniculatum leaf material was carried out using bioassay-guided fractionation. The bioassay used to select the active fractions for further fractionation was an antibacterial assay since it is easier and more rapid to detect antibacterial activity than antiviral activity. Various techniques including column chromatography and high performance liquid chromatography (HPLC) were used to fractionate the extract to result in pure compounds. The isolated compounds were structurally elucidated by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) analysis. Nine compounds were identified as cholest-5-en-3-ol, 2-phyten-1-ol, isoquercitrin, p-coumaric acid, 2, 3, 8-tri-O-methylellagic acid, beta-sitosterol, gallocatechin, apigenin and apigenin-7-glucoside. The compounds were subjected to various bioassays to evaluate their biological activity. The isolated compounds had a broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens, as well as some antifungal and antimycobacterial activity. Cholest-5-en-3-ol, 2-phyten-1-ol, gallocatechin and apigenin were active against Escherichia coli (Gram-negative) and Mycobacterium vaccae, and against the fungi Sporobolomyces salmonicolor and Penicillium notatum. Cholest-5-en-3-ol and 2-phyten-1-ol were also active against Bacillus subtilis (Gram-positive). None of the compounds showed substantial antiviral activity against coxsackievirus strain B3 Nancy, influenzavirus type A strain Hong Kong and herpes simplex virus type 1 strain K1. The compounds were generally moderately cytotoxic to the HeLa cell line but were less toxic to the Madin-Darby Canine Kidney (MDCK) and Vero cell lines. The results obtained confirm the ethnobotanical use of C. paniculatum. Nine compounds with various biological activities were isolated from the leaf extract. The constituents responsible for antiviral activity still remain to be isolated and further work should be carried out making use of antiviral assay-guided isolation. These compounds may be present in low concentrations in C. paniculatum. Synergistic effects of isolated compounds on biological activity, particularly antiviral activity, could be investigated. The results reported here confirm that the presence of antibacterial activity in plant extracts is not an indicator of antiviral activity. Although the crude extracts of C. paniculatum had both antibacterial and antiviral activity, different compounds are responsible for antibacterial and antiviral activity respectively. / Thesis (PhD (Paraclinical Science))--University of Pretoria, 2007. / Paraclinical Sciences / PhD / unrestricted

Ross River virus (RRV) is an Alphavirus distributed throughout Australia. It is transmitted by mosquitoes and is known to cause moderate to severe disease symptoms in humans. Along with other alphaviruses such as Sindbis virus and Chikungunya virus, RRV is known to cause arthritic symptoms, characterised by muscle and joint inflammation. Several investigations have established the role of macrophage cells and pro-inflammatory host factors in the development of RRV-induced disease. In this study, we attempted to determine differences between RRV passaged in mammalian and mosquito cells. There is strong evidence that arthropod-borne viruses are able to display enhanced infectivity when passaged into arthropod cell line. We showed that mosquito cell-derived RRV (mos-RRV) was able to replicate to higher titres than mammalian cell-derived RRV. We also showed that mos-RRV failed to induce Type I IFN-associated antiviral responses. The second aim of this study was to investigate the role of TNF-ᬠa pro-inflammatory cytokine implicated in arthritic diseases, in the development of RRV disease. We treated RRV-infected C57BL/6J mice with a commercially available TNF-ᠩnhibitor drug and monitored disease signs. We found that the TNF-ᠩnhibitor does not ameliorate RRV disease (RRVD) symptoms, and that it does not prevent muscle and joint inflammation. We analysed histological sections of muscle and joint tissue of Enbrel-treated and untreated, RRV-infected cells. We also determined and compared host cytokine expression profiles. Finally, we sought to determine the requirement for natural killer (NK) cells in RRV disease. NK cells have been detected in the synovium of RRV-infected patients since early studies, but their role in disease pathogenesis remains unclear. Using a NK-dysfunctional mouse (C57BL/6J-Lystbg), we showed that mice lacking a functional NK system are more susceptible to RRV disease than wildtype, C57BL/6J mice. We monitored disease symptoms following RRV infection and assessed muscle and joint inflammation in Lystbg and C57BL/6J mice. This thesis examines mechanisms of viral infection and immune evasion employed by RRV, as well as into the role of host cells and cytokines in RRVD pathogenesis disease mechanisms. We showed that a functional NK cell system is required for the regulation of RRV-induced muscle and joint inflammation. Our characterisation of the use of a commercial TNF-ᠩnhibitor in RRV-induced disease in mice may provide information on the role of TNF-ᠩn viral arthritis, and may help towards developing safe and effective treatment.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / PapilomavÃrus Humano (HPV) à um agente viral que causa doenÃa infecciosa, de transmissÃo freqÃentemente sexual, conhecida usualmente como condiloma acuminado, verruga genital ou crista de galo, à o principal agente causal do cÃncer de colo do Ãtero, podendo repercutir na vida do indivÃduo infectado e na saÃde da famÃlia. Os objetivos foram avaliar a estrutura, o desenvolvimento e o funcionamento da famÃlia composta por mulher acometida pelo PapilomavÃrus Humano, com base no Modelo Calgary; identificar possÃveis influÃncias causadas na estrutura familiar, em decorrÃncia da doenÃa; reconhecer influÃncias no desenvolvimento e funcionamento da famÃlia, em decorrÃncia da doenÃa; identificar fatores os quais a mulher relaciona com a causa da doenÃa. A pesquisa foi do tipo descritiva com abordagem qualitativa, tendo sido realizado um estudo de caso, no domicÃlio da participante, utilizando como referencial o Modelo Calgary de AvaliaÃÃo de FamÃlia. A coleta de dados foi realizada em janeiro e fevereiro de 2007 atravÃs de entrevista semi-estruturada com o casal, genograma e ecomapa da famÃlia. A famÃlia era composta por quatro membros, o casal: Josuà e Rosa; e seus dois filhos: Gabriel e Bela. Em exame ginecolÃgico de rotina foi diagnosticado em Rosa uma verruga externa e uma pequena lesÃo na junÃÃo escamo-colunar do colo uterino. Todos os membros da famÃlia mantÃm relacionamento saudÃvel entre si, hà um pouco de negatividade no vÃnculo entre Rosa e Gabriel, em decorrÃncia das cobranÃas com relaÃÃo aos estudos. O casal nÃo se distanciou da sua rede social em decorrÃncia do acometimento com o vÃrus. A esposa compartilhou o caso com o marido, culpando-o, mas posteriormente conseguiu conscientizÃ-lo da importÃncia dos hÃbitos sexuais seguros. O casal conseguiu superar a fase crÃtica vivenciada, deixando como melhor repercussÃo a mudanÃa de hÃbitos e maior afeiÃÃo entre a famÃlia. Com relaÃÃo ao tema HPV, verifica-se que muitas pessoas ainda o desconhecem, ficando a cargo dos profissionais difundirem o assunto com maior Ãnfase tanto entre as mulheres como os homens. Assim como tambÃm promover a educaÃÃo em saÃde, para que as pessoas reflitam a respeito da importÃncia de utilizar o preservativo. / Human Papillomavirus (HPV) is one viral agent that causes infective disease, of transmission frequently sexual, known usually as acuminated conduloma, genital wart or cockscomb, it is the main agent that causes the cancer of the uterus colon as sure as can be with repercussion in the life of the infected individual and in the health of the family. The aims were to evaluate the structure, the development and the function of the family composed by attacked woman by Human Papillomavirus, based in the Calgary Model; to identify possible influences caused within structural family due to disease; to identify factors that the woman relates with the cause of the disease. The search was the descriptive type with qualitative boarding, having been realized one case study, at the home of the informer making use of the Calgary Model as one touched topic of family valuation. Datum collection was realized in January and February from 2007, through interview semi-structured with the couple, genogram and echomap from family. The family was composed of four members such as: the couple Josuà and Rosa and their two children: Gabriel and Bela. By gynecological routine exam it was diagnosed in Rosa, one external wart and one small lesion in the scale-columnar junction of the uterus colon. All the members of the family maintain healthy relationship among them; there is a little bit of negativity in the entail between Rosa and Gabriel, due to charges with relation to studies. The couple didnât keep away from their social net, due to undertaking with the virus. The housewife shared the case with her husband, accusing him, but lately she got acquired him of the importance from safe sexual habits. The couple got over comes the grasped critical phases, overlooking as better repercussion, the changing of their habits and bigger affection among the family. With regard to the theme HPV, it had seen that many people unknown still, staying to the load from professional spread the subject with more emphasis as among women well as men. As well as to promote the education within health, in order to the people reflect to respect of the great importance in utilizing the prophylactic.

Integrase catalyzes insertion of a retroviral genome into the host chromosome. Following reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. Additionally, it has been observed that integrase can catalyze the removal of inserted viral ends (the disintegration activity) in vitro. Presteady-state experiments were performed using synapsed substrates to probe the processing reaction and a disintegration substrate to determine the number of protomers in a functional multimeric complex. In single-turnover studies, a novel "splicing" reaction was observed that revealed complications with accurate quantification of enzymatic activity using the synapsed substrates. The splicing reaction was further used to gain insight into the selection of nucleophiles and electrophiles at the binding site. To reduce the complexity introduced by the integrase-catalyzed splicing reaction, 5'-5' reverse-polarity synapsed substrates were designed that were not susceptible to the splicing reaction and that allowed direct comparison of LTR ends simultaneously bound at the active site. Analysis of the presteady-state assays using these reverse-polarity substrates revealed that the concurrent binding of the biologically relevant U3/U5 combination of viral ends facilitates maximal activity of the processing reaction. A disintegration substrate was used in presteady-state active site titrations to determine a reaction stoichiometry of four integrase protomers per one substrate molecule for the disintegration reaction. A tetrameric active complex was then confirmed using atomic force microscopy to image integrase-DNA complexes during the first catalytic turnover. The observed increase of the tetramer population in the presence of substrate DNA demonstrates that the binding of the disintegration substrate induces assembly of the active tetramer and suggests that tetramer assembly may be an integral and dynamic component of the catalytic pathway. / Graduation date: 2003

Made available in DSpace on 2014-10-09T12:52:47Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:54Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:52:47Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:54Z (GMT). No. of bitstreams: 0 / Radiofármacos marcados com diferentes radionuclídeos são utilizados em aplicações diagnósticas e terapêuticas em Medicina Nuclear. Nos últimos anos houve um aumento no interesse pela terapia radionuclídica, com a introdução de novos radiofármacos aplicados para destruir especificamente determinada célula ou impedir sua proliferação indesejável. Uma modalidade terapêutica que emprega radiofármacos é a radiosinovectomia (RSV), na qual o radiofármaco é administrado na cavidade articular, sendo uma alternativa de tratamento existente para artropatia de várias etiologias e, em particular, àquelas associadas à artrite reumatóide e à hemofilia. O presente trabalho objetivou estudar a marcação de compostos com 90Y e 177Lu visando otimizar as condições de produção e controle de qualidade de pureza radioquímica, avaliar a estabilidade dos produtos gerados e realizar estudos preliminares de biodistribuição animal de radiofármacos com potencial para aplicação em radiosinovectomia. O estudo da produção do citrato coloidal de 90Y (Cit-90Y) foi baseado em procedimento de marcação utilizando solução de 90YCl3 (37 - 54 MBq) levada previamente à secura, seguida da adição de solução de nitrato de ítrio, e citrato de sódio em pH 7,0 com aquecimento à 37º C por 30 minutos. A produção de hidroxiapatita (HA) marcada com 90Y foi estudada tendo como base procedimento de marcação utilizando ácido cítrico monohidratado, nitrato de ítrio e a solução de 90YCl3 (37 - 370MBq). Incubou-se a mistura durante 30 minutos à temperatura ambiente e adicionou-se a hidroxiapatita em meio aquoso e incubou-se à temperatura ambiente durante 30 minutos sob forte agitação. Na produção de HA-177Lu, utilizou-se solução de 177LuCl3 (296 MBq), em presença de óxido de lutécio em meio de NaCl 0,9% pH 7, sob agitação contínua à temperatura ambiente durante 30 minutos. Diversos parâmetros de reação foram estudados para os três radiofármacos. O rendimento das marcações foi determinado por meio de centrifugação após lavagem das partículas com NaCl 0,9%. A análise de pureza radioquímica das marcações foi realizada por meio de cromatografia líquida ascendente utilizando diferentes sistemas cromatográficos. A análise do tamanho das partículas radiomarcadas foi realizada utilizando-se membranas filtrantes de poros de diferentes tamanhos. O comportamento biológico da HA-90Y e HA-177Lu foi estudado a partir da administração intra-articular (joelho) de 18,5 22,2 MBq /0,1 mL do respectivo radiofármaco. Foram adquiridas imagens cintilográficas em gama-câmara em diferentes tempos para determinar a retenção e o extravasamento da atividade da articulação. O método utilizado para produção do Cit-90Y resultou em baixo rendimento de marcação (cerca de 20%), com baixa porcentagem de atividade ligada às partículas com tamanho apropriado para aplicação em RSV. Apesar do baixo rendimento de marcação, as partículas radiomarcadas separadas por centrifugação apresentaram estabilidade relativa de cerca de 70% após 5 dias. A marcação da HA-90Y resultou em excelentes rendimentos de marcação (> 95%). A reação foi otimizada para aplicação rotineira com a redução do tempo de reação para 15 minutos e utilização de apenas um procedimento de centrifugação e lavagem. A marcação da HA com 177Lu resultou em excelente rendimento (> 95%), com otimização da % de ligação às partículas >12m, sendo que os melhores resultados foram obtidos nas marcações realizadas na ausência de óxido de lutécio. A HA marcada com 90Y e 177Lu apresentaram estabilidade in vitro, armazenado à temperatura ambiente dentro do período avaliado de 5 e 7 dias, respectivamente. Foram definidos sistemas cromatográficos em papel e em camada delgada para determinação da pureza radioquímica das preparações. Os estudos de biodistribuição realizados com a hidroxiapatita marcada com 90Y e 177Lu mostraram a estabilidade in vivo dos compostos, não tendo ocorrido extravasamento articular nem liberação do radionuclídeo livre para a circulação, confirmando o potencial de ambos para aplicação em radiosinovectomia. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

No description available.

Following rabies virus (RABV) exposure, a combination of thorough wound washing, multiple-dose vaccine administration and the local infiltration of rabies immune globulin (RIG) are essential components of modern post-exposure prophylaxis (PEP). Although modern cell-culture-based rabies vaccines are increasingly used in many countries, RIG is much less available. The prohibitive cost of polyclonal serum RIG products has prompted a search for alternatives and design of anti-RABV monoclonal antibodies (MAbs) that can be manufactured on a large scale with a consistent potency and lower production costs. Robust in vitro neutralization activity has been demonstrated for the CL184 MAb cocktail, a 1:1 protein mixture of two human anti-RABV MAbs (CR57/CR4098), against a large panel of RABV isolates. In this study, we used a hamster model to evaluate the efficacy of experimental PEP against a lethal challenge. Various doses of CL184 and commercial rabies vaccine were assessed for the ability to protect against lethal infection with representatives of four distinct bat RABV lineages of public health relevance: silver-haired bat (Ln RABV); western canyon bat (Ph RABV); big brown bat (Ef-w1 RABV) and Mexican free-tailed bat RABV (Tb RABV). 42–100% of animals survived bat RABV infection when CL184 (in combination with the vaccine) was administered. A dose-response relationship was observed with decreasing doses of CL184 resulting in increasing mortality. Importantly, CL184 was highly effective in neutralizing and clearing Ph RABV in vivo, even though CR4098 does not neutralize this virus in vitro. By comparison, 19–95% survivorship was observed if human RIG (20 IU/kg) and vaccine were used following challenge with different bat viruses. Based on our results, CL184 represents an efficacious alternative for RIG. Both large-scale and lower cost production could ensure better availability and affordability of this critical life-saving biologic in rabies enzootic countries and as such, significantly contribute to the reduction of human rabies deaths globally.