Thesis (PhD.(Medical Virology)--Faculty of Health Sciences)-University of Pretoria, 2004. / Summary in English and Afrikaans. Includes bibliographical references.

This diploma thesis deals with the issue of the eradication of infectious polio and its current problems. Polio is a highly infectious disease of viral origin. The most effective form of protection from the disease is vaccination. The main aim of this work was to determine the major issues in polio surveillance in the Pilsen region at present. Intermediate goals were determined in relation to the main aim: to ascertain the awareness of practicing paediatricians about the principles of poliomyelitis surveillance with an emphasis on diagnosing and reporting cases of acute palsy and further to ascertain the quality of the cooperation between the locally competent public health authority and practicing paediatricians in the field of poliomyelitis surveillance. The work is divided into two parts, theoretical and practical. The practical part was elaborated using a qualitative method based on semi-structured interviews with practicing paediatricians and with the locally competent public health authority. The research was participated in by 9 practicing paediatricians and one public health authority worker from the Pilsen region, with an average length of practice of 20 years. The resulting data was then evaluated by a coding method and divided into schemes according to Švaříček and Šeďová (2007). Three research questions were defined based on the set aims: RQ1: What do practising paediatricians see as the prime problems in polio surveillance? RQ2: How many cases of acute palsy do practising paediatricians record and subsequently report to the competent public health authority? RQ3: What is the quality of the cooperation between practising paediatricians and the locally competent public health authority? The research revealed that, according to the practising paediatricians, the problem in poliomyelitis surveillance is refusal of the vaccination and the associated decreasing immunisation coverage of the population. The next most commonly reported problem was population migration. Furthermore, it was found that not one practising paediatrician recorded or reported cases of acute palsy in their surgery. Only one respondent encountered acute palsy 10 years ago. The answer to the last research question is also apparent from the information obtained. More than half of the respondents agreed that so far there is no cooperation on this issue. Although four respondents stated that the cooperation is at a high level and is of very good quality.

The Picornaviridae family contains a number of pathogens which are economically important including Poliovirus, Coxsakievirus, Hepatitis A Virus, and Foot-and-Mouth-Disease-Virus. Recently the emergence of novel picornaviruses associated with gastrointestinal, neurological and respiratory diseases in humans has been reported. Although effective vaccines for viruses such as FMDV, PV and HAV have been developed there are currently no antivirals available for the treatment of picornavirus infections. Picornaviruses proteins are classified as: the structural proteins VP1, VP2, VP3 and VP4 which form the subunits of the viral capsid and the replication proteins which function as proteases, RNA-polymerases, primers and membrane binding proteins. Although the host specificity and viral pathogenicity varies across members of the family, the icosahedral capsid is highly conserved. The capsid consists of 60 protomers, each containing a single copy of VP1, VP2 and VP3. A fourth capsid protein, VP4, resides on the internal side of the capsid. Capsid assembly is integral to life-cycle of picornaviruses; however the process is complex and not fully-understood. The overall aim of the study was to broaden the understanding of the evolution and function of the structural proteins across the Picornaviridae family. Firstly a comprehensive analysis of the phylogenetic relationships amongst the individual structural proteins was performed. The functions of the structural proteins were further investigated by an exhaustive motif analysis. A subsequent structural analysis of highly conserved motifs was performed with respect to representative enteroviruses, Foot-and-Mouth-Disease-Virus and Theiler’s Virus. This was supplemented by the in silico prediction of interacting residues within the crystal structures of these protomers. Findings in this study suggest that the capsid proteins may be evolving independently from the replication proteins through possible inter-typic recombination of functional protein regions. Moreover the study predicts that protomer assembly may be facilitated through a network of multiple subunit-subunit interactions. Multiple conserved motifs and principle residues predicted to facilitate capsid subunit-subunit interactions were identified. It was also concluded that motif conservation may support the theory of inter-typic recombination between closely related virus sub-types. As capsid assembly is critical to the viral life-cycle, the principle interacting motifs may serve as novel drug targets for the antiviral treatment of picornavirus infections. Thus the findings in the study may be fundamental to the development of treatments which are more economically feasible or clinically effective than current vaccinations.

Diversos trabalhos têm demonstrado a presença de peptídeos bioativos em hemolinfa de insetos e seu potencial uso como agentes terapêuticos. Este trabalho buscou identificar e isolar proteínas da hemolinfa de Lonomia obliqua com ação antiviral. A adição de hemolinfa antes da infecção reduziu o título de poliovírus de 1,5x107 TCID50/mL no cultivo controle para 4x105 TCID50/mL e em aproximadamente 100 vezes o título do sarampo. Após cromatografia de gel filtração, foram obtidos 3 pools de proteína. O pool responsável pela ação antiviral foi identificado (Pool 2), uma vez que reduziu em 87 vezes o título de poliovírus, e de 1,6x106 TCID50/mL para 2,7x105 TCID50/mL o título do sarampo. O Pool 2 foi fracionado por cromatografia de troca iônica. A fração responsável pela ação antiviral da hemolinfa de Lonomia obliqua frente ao poliovírus e sarampo foi identificada (RQ 3-4), esta proteína, de aproximadamente 20 kDa, tem ação sobre vírus envelopado (sarampo) e vírus desprovido de envelope viral (poliovírus). / Several works have demonstrated the presence of bioactive peptides in insect hemolymph and their potential use as therapeutic agents. This work sought to identify and purify proteins from Lonomia obliqua hemolymph with antiviral activity. The analyses demonstrated that the best time for hemolymph addition in the cell culture is before the infection, the poliovirus titer had reduced from 1.5x107 TCID50/mL to 4x105 TCID50/mL and for measles, the virus titer was about 100 times lower than the control. After gel filtration chromatography, the hemolymph was divided into 3 pools of protein. The pool responsible for the antiviral activity was identified (Pool 2), once it reduced to 87 times the poliovirus titer, and the measles titer from 1.6x106 TCID50/mL to 2.7x105 TCID50/mL. The Pool 2 was fractionated by ion-exchange chromatography. The fraction from Lonomia obliqua hemolymph responsible for the antiviral activity was identified - RQ 3-4, this protein of approximately 20 kDa, has antiviral effect on enveloped virus (measles) and on non enveloped virus (poliovirus).

No description available.

The aim of this study is the three-dimensional structural characterization of the genome packaging inside viral capsids via cryo-electron microscopy and three-dimensional reconstruction. The genome of some single-stranded viruses can be densely packaged within their capsid shells. Several stretches of the genome are known to adopt stable secondary structures, however, to date, little is known about the three-dimensional organization of the genome inside their capsid shells. Two techniques have been developed to facilitate the structural elucidation of genome packaging: the asymmetric random-model method, and the symmetry-mismatch, random model method. Both techniques were successfully tested with model and experimental data. The new algorithms were applied to study the genome structure of poliovirus and satellite tobacco mosaic virus. We have not yet found a consistent structure for the two genomes. Nevertheless, we have found that the genome of satellite tobacco mosaic genome is very stable, supporting a model where the RNA acts as a scaffold, with potential implications in capsid stability and assembly.

No description available.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-11-28T12:37:39Z No. of bitstreams: 1 eliane_v_costa_ioc_bcm_0043_2011.pdf: 4529718 bytes, checksum: fa63bf5abfb95e39c6504892dd9a4e9e (MD5) / Made available in DSpace on 2012-11-28T12:37:39Z (GMT). No. of bitstreams: 1 eliane_v_costa_ioc_bcm_0043_2011.pdf: 4529718 bytes, checksum: fa63bf5abfb95e39c6504892dd9a4e9e (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / A poliomielite (poliomielite anterior aguda, paralisia infantil) é uma doença infecciosa de caráter agudo que ocorre seguida a uma infecção causada por um dos três sorotipos de poliovírus, denominados poliovírus tipos 1, 2 e 3. Em 1988, quando os poliovírus selvagens eram endêmicos em 125 países ficou estabelecida durante a 41a Assembléia Mundial da Saúde, em Genebra, a meta da erradicação da poliomielite até o ano 2000. Os poliovírus selvagens estão hoje restritos a apenas quatro países (Nigéria, Afeganistão, Paquistão e Índia). Este grande sucesso é atribuído à utilização sistemática da vacina oral contra a poliomielite (VOP), desenvolvida pelo Dr. Albert Sabin. Esta vacina, licenciada nos anos de 1960s, e que vem sendo utilizada por mais de quatro décadas, consiste de variantes atenuadas de cada um dos três sorotipos de poliovírus. Por ser uma vacina constituída por vírus vivos atenuados, os problemas a ela associados estão principalmente ligados à sua instabilidade genética e a possibilidade do aparecimento de mutações e recombinações nas subpopulações virais excretadas. Durante a replicação do vírus vacinal em humanos, frequentemente ocorre reversão de algumas substituições nucleotídicas que conferem o fenótipo atenuado. Essa é a principal causa do aparecimento de raros casos de poliomielite associados à vacina (VAPP), relatados em diferentes países, assim como surtos de poliomielite paralítica devidos à infecção por estes vírus vacinais derivados (PVDV) que possuem propriedades biológicas semelhantes aos poliovírus selvagens. A análise e caracterização de quatro regiões distintas do genoma viral (5’NC, VP1, 2C e 3D), dos poliovírus vacinais isolados no Laboratório de Enterovírus do Instituto Oswaldo Cruz, a partir de amostras clínicas de casos de paralisia flácida aguda que ocorreram no Brasil, no período de 1995 a 2007, constituíram no principal objetivo do presente trabalho. Um total de 177 amostras dos três sorotipos foi analisado por sequenciamento nucleotídico em todo o gene que codifica a VP1; 222 amostras foram analisadas por duplex RT-PCR nas regiões 2C e 3D; 68 amostras foram analisadas na região 5´- NC e 44 amostras foram analisadas nas 4 regiões (5´NC, VP1, 2C e 3D). Dois isolados de PV2, apresentando mais de 1% de mutação em VP1, foram identificados e classificados como PVDV2. Existem aproximadamente 40 relatos de PVDV relacionados a pacientes imunodeficientes de 1962 a 2010, globalmente distribuídos, porém nenhum identificado no Brasil. Das 68 amostras sequenciadas na região 5’NC visando à avaliação de uma importante posição nucleotidica envolvida na atenuação da neurovirulência em cada um dos 3 sorotipos (PV1480, PV2481 e PV3472), os seguintes resultados foram encontrados: PV1=18.5% mutadas na posição 480, PV2=65% mutadas em 481 e PV3=81% mutadas em 472. Recombinação (em 2C/3D) foi encontrada em 15 amostras: 1 PV1 (1,3%), 5 PV2 (6,6%) e 9 PV3 (13,2%). Os três sorotipos de poliovírus apresentaram comportamento distinto em relação às análises realizadas, sendo o sorotipo 3 o menos estável em relação ao padrão de recombinação e reversão à neurovirulência seguido pelo sorotipo 2 e o sorotipo 1 foi o mais estável. A monitorização do perfil genômico dos poliovírus vacinais em circulação é essencial no estágio final de erradicação global da poliomielite. / Poliomyelitis (acute anterior poliomyelitis, infantile paralysis) is an acute infectious disease which occurs subsequent to an infection caused by any of the three serotypes of polioviruses, designated poliovirus types 1, 2 and 3. In 1988, when wild polioviruses were endemic in 125 countries, the 41st World Health Assembly in Geneva set a goal for poliomyelitis eradication by the year 2000. Nowadays, poliomyelitis in its endemic form is restricted to only four countries: Nigeria, Afghanistan, Pakistan, and India. This remarkable success is attributed to the systematic use of oral poliomyelitis vaccine (OPV), developed by Dr. Albert Sabin. This vaccine, which was licensed in the 1960´s and is in use for over four decades, consists of attenuated variants of each of the three poliovirus serotypes. Because OPV consists of live attenuated viruses, the problems associated with this vaccine are mainly related to its genetic instability, which favor the emergence of mutations and recombinations among the excreted viral sub-populations. Also, during the process of virus replication in humans, some nucleotide substitutions responsible for the attenuated phenotype are often reverted. That is the main cause of the appearance of rare cases of vaccine-associated paralytic poliomyelitis (VAPP) reported in several countries as well as paralytic poliomyelitis outbreaks caused by vaccine-derived polioviruses which exhibit biological properties similar to wild polioviruses. The genomic characterization in four distinct regions of the genome (5’NC, VP1, 2C, and 3D) of vaccine-related polioviruses isolated in our laboratory from clinical samples of acute flaccid paralysis cases which occurred in Brazil, from 1995 to 2007 was the main purpose of the present work. A total of 177 isolates of the three serotypes of polioviruses were analyzed by nucleotide sequencing on the entire gene that codifies VP1; 222 isolates were analyzed by duplex PCR in 2C and 3D regions; 68 isolates were analyzed at the 5´- NCR while 44 isolates were analyzed in the four regions (5´- NCR, VP1, 2C and 3D)., Two PV2 isolates presenting over 1% of mutations in VP1 were identified and classified as VDPV2. There are approximately 40 reports of VDPV related to immunodefficient patients around the world from 1962 to 2010, but none of those occurred in Brazil. From the 68 isolates which were analyzed on the 5’NC region, in order to evaluate an important nucleotide position involved in attenuation of neurovirulence in each of the 3 serotypes (PV1480, PV2481 e PV3472). The following results were found: PV1 = 18.5% mutated at position 480, PV2 = 65% mutated at position 481 and PV3 = 81% mutated at 472. Recombination was found in 15 isolates: 1 PV1 (1.3%), 5 PV2 (6.6%) e 9 PV3 (13.2%). The three serotypes of polioviruses behavior distinctly to the analyses: type 3 showed to be the the least stable regarding the recombination and neurovirulence reversion patterns followed by serotype 2 while serotype 1 was the most stable. The knowledge regarding the genomic pattern of circulating polioviruses is essential at the final stages of poliomyelitis eradication

BACKGROUND:In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.RESULTS:Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected.CONCLUSIONS:This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.

Arrabidaea chica Verlot (Bignoniaceae), conhecida como crajiru, fornece pigmentos vermelhos utilizados pelos índios no Brasil como corante e agente cicatrizante. Este estudo visou otimizar a extração de compostos fenólicos de A. chica e avaliar sua atividade farmacológica. Extratos de A. chica foram obtidos através de tratamento com xilanases de Bacillus pumilus previamente à extração. Os ensaios foram monitorados por CLAE e ESI-MS. O tratamento enzimático forneceu extratos enriquecidos em antocianidinas. Extratos sem tratamento enzimático apresentaram maior teor de antocianosídeos. O estudo farmacológico demonstrou que as atividades anticâncer e antioxidante in vitro estão diretamente relacionadas ao maior teor de agliconas. O ensaio in vitro de indução de crescimento de fibroblastos indicou que o maior teor da aglicona carajurina é inversamente proporcional à ação cicatrizante. Portanto, foi desenvolvida uma metodologia inovadora, através de processos biotecnológicos, para extração de antocianidinas que apresentam propriedades corantes e terapêuticas. / Arrabidaea chica Verlot (Bignoniaceae), known as crajiru, produces red pigments used by Brazilian indians as dye and as healing agent. This study has aimed the optimization of phenolic compounds extraction from A. chica and to evaluate its pharmacological activities. Extracts from A. chica were obtained through treatment with xylanases from Bacillus pumilus before the extraction. The assays were monitored by HPLC and ESI/MS-MS. The enzymatic treatment has produced more concentrated in anthocyanidins extracts. Those obtained without enzymatic treatment have presented higher glycosilated anthocyanins content. The pharmacologic study has demonstrated that the antitumoral and the antioxydant in vitro properties for A. chica are directly related to the higher contents of aglycones. In vitro assay for fibroblasts growth induction has demonstrated that a higher content of carajurin is inversely proportional to the healing action. In conclusion, a novel approach has been developed, through biotechnological process, aiming the extraction of anthocyanidins presenting dye and therapeutic properties.