Made available in DSpace on 2014-08-26T17:15:01Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 137.pdf: 791646 bytes, checksum: 39acf8b8b6ba9b68e5a239ea71e51227 (MD5) Previous issue date: 2006 / A raiva é uma das doenças mais temidas entre as diferentes zoonoses que ameaçam o Homem, pois evolui sempre para o êxito letal, sendo, na prática, sua morbidade igual à mortalidade. Acrescem-se ainda os prejuízos econômicos, causados ao rebanho bovino nos focos de raiva epizoótica, seu impacto geral na economia pecuária, reduzindo a produção de leite e carne, e suas implicações na área de Saúde Pública. Através da utilização de técnicas clássicas e moleculares, o presente estudo, visou estudar a diversidade molecular das amostras de vírus da raiva provenientes de animais domésticos e silvestres que atualmente são identificadas nas diferentes regiões do Brasil (Norte, Nordeste, Sudeste, Sul e Centro-Oeste), comparando-as entre si e entre amostras originárias de um município do Estado do Rio de Janeiro (Porciúncula), com base em seqüenciamento do gene codificador da nucleoproteína. A técnica de RT-PCR foi aplicada em 32 amostras de tecido nervoso oriundo de animais suspeitos de estarem acometidos pela raiva, demonstrando 100% de concordância com os resultados apresentados pelas provas clássicas, permitindo diagnóstico positivo inclusive em amostras que se apresentam em estado de putrefação. Treze das amostras de vírus isoladas foram parcialmente seqüenciadas, tendo sido encontrado formação dos principais grupos esperados de amostras de vírus da raiva, ou seja, variante antigênica 2,3, amostras fixas e variante de vírus de raiva de sagüi. Foi evidenciado, ainda, um padrão regional de distribuição de vírus da raiva associado à variante antigênica 3. A obtenção de dados derivados do seqüenciamento vem a permitir um melhor entendimento da diversidade molecular das amostras de vírus da raiva circulantes nas regiões em estudo, representando um passo fundamental para a geração de informações a serem utilizadas na epidemiologia molecular da raiva, determinando fontes de infecção, origens de surtos e relações genéticas geográficas entre os vírus da raiva detectados a partir de diferentes espécies animais. / Rabies is one of the most feared zoonosis, once it always results in the death of the affected patient, what makes rabies morbidity equal to its mortality. Furthermore, economic losses due to rabies epidemics in cattle, the economic impact in agrobusiness derived from decreased milk and meat production and implications in public health must also to be taken into account. Using classic and traditional techniques, the present research aimed to study the molecular diversity of rabies virus strains from domestic and wild animals that circulate in different Brazilian regions (North, Northeastern, Southeastern, South and Center-Western) comparing the strains amongst them and with strains from a municipality from Rio de Janeiro State (Porciúncula) based on the gene coding for the nucleoprotein. The RT-PCR was applied to 32 samples of central nervous system tissue of rabies-suspected animals, showing an agreement of 100% with the classic tests, allowing a positive diagnosis even in decomposed samples. Thirteen out of these 32 samples were submitted to partial sequencing resulting in the expected groups of rabies virus variants, i. e., antigenic variants 2, 3, fixed strains and marmoset strains. A regional pattern was found regarding the variant 3 of rabies virus. Data obtained from DNA sequencing allow a better understanding of the molecular diversity of the rabies virus strains circulating in the regions under study, a fundamental step for the generation of information to be used in the molecular epidemiology of rabies, as the determination of sources of infection, origins of outbreaks and phylogeographic relationships among rabies virus strains from different species.

Made available in DSpace on 2014-08-26T17:15:07Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 106.pdf: 11011427 bytes, checksum: 47de23ce5361c89a3fe418a1ea675e43 (MD5) Previous issue date: 2006 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Cinco cepas P1/Sabin, 20 cepas P2/Sabin e 2 cepas P3/Sabin isolados a partir de amostras do meio ambiente no Brasil foram analisadas. Neste estudo todos os isolados foram caracterizadas pelo seqüenciamento parcial da região 5 não codificante (NCR) e pelo seqüenciamento completo do gene da proteína VP1, com o objetivo de demonstrar mutações, as quais são importantes para a reversão à neurovirulência. O seqüenciamento parcial da região 5'NCR revelou que todos os poliovírus isolados do sorotipo 1 apresentaram populações de revertentes G480 ->A e os 2 isolados do sorotipo 3 apresentaram a reversão U472 ->C. Nenhum isolado do sorotipo 2 apresentou mutação A481 ->G. O completo seqüenciamento da região de VP1 demonstrou que nenhum isolado P1/Sabin apresentou substituição nucleotídica, as 2 cepas P3/Sabin apresentaram VP1-6 (Thr ->Iso) e 16 cepas P2/Sabin apresentaram VP1-109 (Lys ->Arg). O isolado P2/26183 também apresentou mutações VP1-103 (Ser ->Lys) e VP1-116 (Val ->Gly), enquanto P2/26184 apresentou VP1-155 (Cys ->Tyr). As regiões 2C e 3D do genoma foram investigadas pelo uso de primers que hibridizam nestas áreas com o objetivo de verificar a presença de recombinação. Nenhuma cepa envolvida no estudo foi identificada como recombinante. Nenhum poliovírus derivado da vacina foi detectado. / Five strains of P1/Sabin, 20 strains of P2/Sabin and 2 strains of P3/Sabin isolated from environmental samples in Brazil were analyzed. In this study all isolates were characterized by partial genomic sequencing of the 5’ noncoding region (NCR) and the complete VP1 protein gene VP1, with the objective of finding mutations, which are important for neurovirulence reversion. Partial sequencing of 5’NCR revealed that all type 1 poliovirus isolates had the G→A substitution at the nt 480 and the 2 type 3 poliovirus isolates had the U→C substitution at the nt 472. None type 2 isolate had A→G substitution 481. The complete sequencing of the VP1 region showed that none P1/Sabin strain had nucleotide substitution, the 2 P3/Sabin strain had VP1"6 (Thr→Iso) and 16 P2/Sabin strain had VP1"109 (Lys→Arg). The isolate P2/26183 also presented mutations VP1"103 (Ser→Lys) and VP1"116 (Val→Gly), while P2/26184 showed VP1"155 (Cys→Tyr). The 2C and 3D regions of the viral genome were investigated by the use of primers that hybridize in these areas with the objective to verify the presence of recombination. None of the strain involved in this study was identified as recombinant. None vaccine derived poliovirus was detected.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-13T15:29:17Z No. of bitstreams: 2 TESE Bethânia de Araújo Amaral.pdf: 4449052 bytes, checksum: f014da1c40afd7489d798e17efed3d13 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T15:29:17Z (GMT). No. of bitstreams: 2 TESE Bethânia de Araújo Amaral.pdf: 4449052 bytes, checksum: f014da1c40afd7489d798e17efed3d13 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / UFPE; CAPES; FACEPE / A leucemia promielocítica aguda (LPA) requer uma atenção especial dentre as leucemias mielóides agudas devido às suas implicações prognósticas e terapêuticas. A taxa de sobrevida de no mínimo cinco anos na LPA chega a 80% dos pacientes com a terapia atual e a taxa de cura excede os 70%. No entanto, a real situação dos resultados do tratamento da LPA em países em desenvolvimento, como o Brasil, é desconhecida. Este trabalho visou contribuir na investigação dos casos de LPA infantil em pacientes na nossa população objetivando a identificação de marcadores que possam redirecionar o acompanhamento e tratamento destes pacientes. A investigação da ocorrência de mutações dos genes FLT3 e NPM1 é importante na determinação prognostica das LMAs, contribuindo para a estratificação de grupos de maior ou menor risco e auxiliando o direcionamento do tratamento. Dezesseis pacientes pediátricos com LPA, representando 29,09% dos casos de LMA infantil, foram atendidos no CEONHPE/HUOC/UPE de 2004 a 2013. A idade variou de 5 a 17 anos (média de 11,81 anos). Análises citogenética e molecular por FISH e RT-PCR foram realizadas para confirmação do diagnóstico genético, na identificação do rearranjo PML-RARα decorrente da t(15;17), além da pesquisa das mutações dos genes FLT3 e NPM1. A análise cariotípica com o bandeamento G revelou alterações cromossômicas em 10 pacientes. Três apresentaram cariótipos complexos com presença de cromossomos marcadores. A técnica de FISH para a t(15;17) confirmou o rearranjo em 14 pacientes. Divergência entre as análises moleculares foram observadas em cinco pacientes, sendo positiva a detecção da t(15;17) pela FISH e negativa pela RT-PCR. A mutação do FLT3/ITD foi detectada em dois pacientes (12,5%), enquanto a mutação FLT3/TKD foi detectada em apenas um paciente (6,25%). A pesquisa para a mutação no gene NPM1 foi negativa para todos os casos. Quanto ao status oito pacientes encontramse vivos ou na fase de manutenção do tratamento e oito foram a óbito, sendo que destes sete tiveram óbitos precoces ainda na fase de indução. Os motivos primários dos óbitos foram hemorragias, septicemia e manifestação da síndrome do ATRA. Este dado é alarmante uma vez que estas mortes são difíceis de prevenir e assinalam o alto risco de ocorrência destes eventos em nossa população. Destacamos a importância do uso de técnicas citogenéticas moleculares na confirmação genética do diagnóstico da LPA e a necessidade de uma melhor adaptação do regime terapêutico aos casos de LPA na infância em nossa população.

A vinca (Catharanthus roseus) é uma planta perene, arbustiva, pertencente à família Apocinaceae, cujas folhas e raízes possuem propriedades medicinais. A presença de sintoma de mosaico e deformação foliar em plantas dessa espécie, associados com a presença de partículas alongadas e flexuosas, característica de vírus pertencentes ao gênero Potyvirus, conduziu a estudos complementares para a identificação e caracterização desse vírus. No estudo da gama parcial de hospedeiras foram testadas 28 espécies, envolvendo oito famílias botânicas. Catharanthus roseus e Nicotiana benthamiana apresentaram sintomas de mosaico foliar e Chenopodium amaranticolor e C. quinoa apresentaram lesões locais cloróticas nas folhas inoculadas. A transmissão do vírus com afídeos foi avaliada com as espécies Aphis gossypii, Myzus nicotianae e Toxoptera citricidus. Apenas Aphis gossypii e Myzus nicotianae transmitiram o vírus. O antissoro policlonal produzido contra este potyvirus reagiu com o vírus homólogo e com o Passionfruit woodiness virus (PWV) e Cowpea aphid-borne mosaic virus (CABMV), mas não com o Lettuce mosaic virus (LMV), Papaya ringspot virus - type W (PRSV-W), Potato virus Y (PVY) e Zucchini yellow mosaic virus (ZYMV). O peso molecular da proteína capsidial (CP) foi de aproximadamente 34 kDa. A reação de PCR realizada com os oligonucleotídeos universais de potyvirus e oligonucleotídeos específicos posteriomente confeccionados amplificaram três fragmentos de aproximadamente 0,8, 1,0 e 1,4 Kb, os quais após o seqüênciamento geraram um fragmento de 1654 nucleotídeos (nt) da região 3' terminal do genoma, que inclui parte do gene da replicase viral (Nib), a região codificadora completa do gene da proteína capsidial (CP), seguida de 286 nt da região 3' não traduzida (3'NTR). A identidade da seqüência de nucleotídeos do gene da CP variou de 67,0 a 76,0%, quando comparada com as de outros membros da família Potyviridae. A maior identidade foi com o Omphalodes virus Y (76,0%). A identidade dos aminoácidos deduzidos da proteína capsidial variou de 62,0 a 71,0%, sendo a maior com East Asian Passiflora virus (71%). Para a região não traduzida (3'NTR) a identidade variou de 16,8 a 28,6%. Em conjunto esses dados indicam que este vírus é uma nova espécie dentro do gênero Potyvirus, para o qual se propõe o nome de Vírus do mosaico do Catharanthus (Catharanthus mosaic virus - CatMV). / Catharanthus roseus is known as the common periwinkle or Madagascar periwinkle. It is a perennial, evergreen herb in the family Apocynaceae, which was originally native to the island of Madagascar, although both name and classification are contradictory in some literature. The plants grow up to 80 cm high; have glossy, dark green leaves and bloom during summer. The flowers range from white to hot pink to purple. The species has historically been used in popular medicine to treat a wide assortment of human diseases, as it contains more than 150 useful alkaloids. Plants of C. roseus exhibiting mosaic symptoms followed by malformation of the leaf blades and flower variegation were collected from a garden at the University of São Paulo, School of Agriculture (Piracicaba, State of São Paulo, Brazil). Preliminary electron microscopy exams of negatively stained leaf sap revealed that the symptoms were associated with potyvirus-like particles. The objective of the present work was to obtain further biological, immunological and molecular data to better characterize this species of the genus Potyvirus, family Potyviridae. Of 28 plant species from eight botanical families inoculated mechanically with this potyvirus, only C. roseus and Nicotiana benthamiana developed systemic mosaic, whereas Chenopodium amaranticolor and C. quinoa exhibited only chlorotic local lesions. The virus was transmitted by Aphis gossypii and Myzus nicotianae, but not by Toxoptera citricidus. Polyclonal antiserum raised against this potyvirus reacted with the homologous virus, Passion fruit woodiness virus (PWV) and Cowpea aphid borne mosaic virus (CABMV) in PTA-ELISA. The molecular mass of the coat protein (CP) was approximately 34 kDa. RT-PCR from viral RNA amplified a fragment of approximately 1654 nucleotides (nt) at the 3'-terminal of the viral genome, containing portion of the replicase gene (Nib), the entire CP gene and the 3' untranslated region (3'UTR) (286 nt). When the nucleotide sequence of the CP gene was compared with other members of the Potyviridae family, identities varied from 67.0 to 76.0%. The highest identity was with Omphalodes virus Y. Identity of the deduced amino acid of the CP varied from 62.0 to 71.0%, with the highest for East Asian Passiflora virus. For the 3' UTR, identities varied from 16.8 to 28.6%. The name Catharanthus mosaic virus (CatMV) is proposed for this new potyvirus.

O câncer de mama em mulheres jovens (idade igual ou abaixo de 35 anos) apresenta-se de forma mais avançada ao diagnóstico, possuindo grau histopatológico menos diferenciado. Além disso, as pacientes apresentam maior taxa de mortalidade e menor sobrevida livre de doença quando comparadas às pacientes menopausadas. Dentre os cânceres de mama em mulheres jovens, apenas 8 a 10% dos casos familiais estão relacionados a mutações nos genes BRCA1 e BRCA2 e esta frequência nos casos esporádicos é ainda menor (3- 10%). Assim, os fatores relacionados à desregulação oncogênica em mulheres jovens com ou sem antecedentes familiares que apresentam testes genéticos negativos para essas mutações não estão suficientemente esclarecidos. Tais evidências sugerem que o câncer em mulheres muito jovens apresenta características biológicas especificas. Considerando que a expressão gênica é regulada em múltiplos níveis, alguns estudos recentes tem referido a importância dos microRNAs neste processo tanto na degradação de RNAs mensageiros como na repressão da tradução. A análise da expressão dos microRNAs em câncer de mama tem revelado perfis característicos de determinados níveis de progressão da doença. No presente trabalho, nosso objetivo foi determinar o perfil de expressão de microRNAs em tumores de mama de mulheres jovens (idade igual ou abaixo de 35 anos) não-portadoras de mutações nos genes BRCA1/2. As pacientes foram subdivididas em 2 grupos [familial (n=8) e não-familial (n=20)] e, em seguida, os dados de expressão foram comparados aos obtidos em amostras normais de mamoplastia. A quantificação da expressão dos microRNAs foi realizada pela reação de RT-PCR em tempo real, utilizando o sistema TaqMan microRNA Assay. As análises estatísticas mostraram 246 miRNAs de expressão diferencial entre os 3 grupos (normal, familial e não-familial) sendo que, destes, encontramos 137 miRNAs diferencialmente expressos entre os grupos familial e normal; 44 miRNAs entre os grupos não-familial e normal e 4 miRNAs entre os grupos familial e não-familial. Nossos dados demonstram que os tumores do grupo de pacientes familiais quando comparados aos normais apresentam um perfil de miRNAs globalmente reduzido. O perfil de expressão de miRNAs no grupo de pacientes com câncer de mama esporádico é pouco distinto do grupo com história familial. Muitos dos miRNAs com expressão reduzida estavam envolvidos, de acordo com a literatura, numa sinalização comum relacionada a mecanismos de proliferação, apoptose e invasão celular. Os 4 miRNAs identificados como diferencialmente expressos entre os grupos familial e nãofamilial embora relacionados com outros tipos de cancer precisam de uma melhor caracterização em cancer de mama / A rise in the incidence of breast cancer among young adult women (with age 35) was observed in recent decades. Breast cancer incidence in young woman has been correlated to poor survival and aggressive features. Due to different type of breast cancers in young woman, 8- 10% with familial history appears with mutation in BRCA1/2 genes, and similar proportion occurs in cases without familial history (3- 10%). However, early onset breast cancer patients with or without familial history, non carriers of BRCA1/BRCA2 mutations is not well elucidated. The deregulation by microRNAs has recently emerged as a major determinant of tumorigenesis. Because miRNAs function by targeting functionally important protein-conding genes, it is of outstanding interest to identify miRNAs involved in the molecular mechanism underlying aggressiveness in tumors of young patients that might represent biomarkers and therapeutic targets. This evidence suggests that breast cancer in young woman has specific biological characteristics. Understanding the patterns of miRNAs expression that potentially alter the regulation of key breast cancer genes we could give a better treatment to these patients. Thirty-two patients were selected: 8 with familial history of breast and ovarian suggestive of hereditary condition according to NCCN criteria and 20 without familial history. Patients of both groups were of non carriers of BRCA1/BRCA2 mutations. The determination of microRNA expression network between those 2 groups was performed by TaqMan microRNA Assay (Applied Biosystems) using as control 3 normal mamoplastia samples from wealthy young women. Data were normalized using endogenous miRNA presented in each array. Statistical comparisons were done using ANOVA and Student T test with adjusted FDR (5%). It was found that 246 miRNAs were differently expressed between the 3 groups. From the comparison of familial group against normal group it was found 137 miRNAs differently expressed. In the comparison between non familial and normal group it was found 44 miRNAs differently expressed. Finally the comparison between familial group and non familial group it was found 4 miRNAs differently expressed. Among these miRNAs are some which was well characterized in breast cancer with down-regulation such as: miR-125b, miR-126 and miR-100. Our findings suggested that tumors from familial or sporadic cases presented discrete differences of microRNA expression patterns. A global downregulation of 137 miRNAs in tumors from familial group of patients when compared to normal group was observed. Based on the literature, most of these miRNAs are related to mechanisms of proliferation, cells migration and apoptosis. To our knowledge, this is the first evidence of miRNA expression in tumors of early onset Breast Cancer patients, non carries BRCA1/2 mutation, providing insights that may lead to the detection of new conducts of treatment

The parathyroid hormone related protein (PTHrP) and Indian Hedgehog (IHH) are two secreted molecules, acting as paracrine factors during embryonic development and post-natal growth of endochondral bones. PTHrP and IHH are essential factors for the regulation of chondrocyte proliferation and differentiation. However, it has previously been shown that PTHrP and IHH are also expressed in the chick and mouse embryos intramembranous bones, which do not form through a cartilage intermediate and in which chondrocytes are absent. Similarly, the zebrafish orthologs, pthrp1 and ihha, are also expressed during the regeneration of the intramembranous bones of the fin rays of the zebrafish caudal fin. This surprising observation led us to further analyze the expression and function of pthrp1 and ihha in the regenerating fin rays. Gene expression analysis using in situ hybridization shows that pthrp1 is expressed in a stripe of cells located within the domain of expression of ihha in the newly differentiating osteoblasts in the regenerating fin rays. Also, pthrp1 expression is observed at the level of the joints between the bone segments forming the rays and co-localizes with the expression domain of evx1, a transcription factor that has been implicated in the formation of joints in the caudal fin. Furthermore, RT-PCR analyses show that pthrp2 and the pthrp receptors mRNA (pth1r, pth2r and pth3r) are also present in the fin regenerate. Finally, functional analysis shows that the knockdown of pthrp1 or ihha expression by electroporation of morpholinos induces a delay of the regenerative outgrowth of the fin. These results suggest that pthrp1 and ihha may be involved in the regulation of proliferation and differentiation of chondrocyte-like osteoblasts in the fin rays, playing a role similar to that described in the mammalian growth plate of endochondral bones. In addition, pthrp1 is possibly an important factor involved in the formation and maintenance of joints of the dermal bones of the fin rays.

[EN] Tomato spotted wilt virus is one of the most widespread and economically important viruses worldwide. It infects a large number of plant species, being the tomato and pepper the most affected. TSWV is transmitted from one plant to another by various species of thrips in a circulative and propagative manner, being Flankliniella occidentalis the main vector. The best strategy for disease control in tomato and pepper has been breeding resistant cultivars, but only tomato with the gene Sw-5 and pepper with gene Tsw have been effective against a wide spectrum of TSWV isolates, but resistance-breaking isolates often arise. To obtain a more effective and durable control is necessary: A) the genetic and biological characterization of conventional and resistance-breaking isolates of TSWV; B) the understanding the evolutionary and epidemiological factors involved in the emergence and dispersion of resistance-breaking isolates; C) the development of tools for viral detection and quantification; and D) evaluation of new sources of resistance (total or partial, estimated as the difficulty to viral infection or/and accumulation) or tolerance (total or partial, estimated as the difficulty to symptoms development and damage without affecting viral infection). In this work, TSWV isolates from Spanish tomato and pepper crops have been biologically and molecularly characterized. The complete genome of three TSWV isolates with different biotypes were sequenced: N (unable to infect Sw-5 resistant tomato and Tsw resistant pepper), T (tomato Sw-5 resistance-breaking), and P (pepper Tsw resistance-breaking). No correlation between genetic variation and the ability of overcoming resistance was found. These nucleotide sequences and others retrieved from the GenBank database were used to develop RT-qPCR, with high sensitivity and dynamic range. Primers and one TaqMan MGB were designed from conserved sequence stretches with the aim of detecting and quantifying any TSWV isolate. It was not possible to develop a molecular method to differentiate between conventional and resistance-breaking isolates since there is no correlation between genotype and biotype. Instead, two TaqMan MGB probes were designed to quantify the two main genotypes (according to the M segment) in mixed infections. RT-qPCR was used to evaluate TSWV accumulation in non-resistant tomato, non-resistant pepper and Datura stramonium (an important reservoir), as well as in the main vector F. occidentalis, which was considered to evaluate transmission efficiency. The results showed that resistance breakdown was not associated to a fitness cost (tradeoff) in the infectivity of susceptible hosts or transmissibility by thrips and that the resistance-breaking isolates have the same potential for dispersion in field as the conventional isolates. Finally, the information obtained from the previous studies and the RT-qPCR technique were used to evaluate the resistance and tolerance to TSWV of a new accession of Capsicum baccatum. In addition to considering the genetic and biological variation of TSWV, a new approach was used based on analysis of longitudinal data (those measured in the same individuals over time). The resistance level was estimated with two variables: A) absolute fitness, calculated from the variation in time of the viral titer, quantified by RT-qPCR; and B) infectition survival, the median time in which the virus was detected in a plant by ELISA. The tolerance level was estimated as symptom survival, the median time in which a plant developed severe symptoms. This analysis showed that this new accession is partially resistant and totally tolerant to TSWV conventional and resistance-breaking isolates and therefore is a good candidate for a pepper breeding program / [ES] Tomato spotted wilt virus (TSWV) es uno de los virus más extendidos y de mayor importancia económica del mundo. Infecta a un gran número de especies vegetales, siendo los cultivos de tomate y pimiento los más afectados. Este virus se transmite de una planta a otra por trips en una manera propagativa y circulativa, siendo Flankliniella occidentalis el más eficaz. El cultivo de variedades resistentes de tomate y pimiento permitió el control de la enfermedad, ya que estos genes inducen una respuesta hipersensible impidiendo la infección sistémica del virus. Sin embargo, con frecuencia aparecen aislados del virus capaces de superar estas resistencias. Para obtener un control de la enfermedad más eficaz y duradero es necesario: A) la caracterización genética y biológica de los aislados del TSWV que superan y no superan las resistencias; B) el estudio de los factores evolutivos y epidemiológicos implicados en la aparición y establecimiento de los aislados que superan las resistencias; C) el desarrollo de herramientas que permitan la detección y cuantificación de estos aislados virales; y D) la evaluación de nuevas fuentes de resistencia o tolerancia. En este trabajo se han caracterizado biológicamente y molecularmente diferentes aislados del TSWV procedentes de cultivos de tomate y pimiento de España. Se determinó la secuencia nucleotídica del genoma completo de tres aislados españoles correspondientes a tres biotipos: N (incapaz de infectar variedades resistentes de tomate o pimiento); T (supera la resistencia Sw-5 de tomate); y P (supera la resistencia Tsw de pimiento). No se encontró correlación entre la variación genética y la capacidad de superar la resistencia. Estas secuencias nucleotídicas y otras obtenidas de la base de datos Genbank se utilizaron para desarrollar una técnica basada en la RT-PCR cuantitativa (RT-qPCR) que permite detectar el virus con un alto grado de sensibilidad y cuantificar en un amplio rango dinámico. Se diseñaron los iniciadores y una sonda TaqMan MGB a partir de segmentos de secuencia conservados para que fueran válidos para todos los aislados del TSWV. También se desarrollaron dos sondas Taqman que permitían cuantificar diferencialmente variantes genéticas del virus en infecciones mixtas. La RT-qPCR se utilizó para evaluar la acumulación de varios aislados del TSWV en tomate sin Sw-5, pimiento sin Tsw y Datura stramonium, así como, en su principal vector F. occidentalis que se usó para evaluar la eficiencia de la transmisión. Se observó que la superación de la resistencia no suponía un coste en la eficacia biológica (fitness) tanto en la multiplicación del virus en estas plantas como en la transmisión por trips. Por tanto, los aislados que superan las resistencias tienen la misma capacidad de dispersión en campo que los aislados convencionales. Por último, se utilizó la RT-qPCR y la información obtenida para evaluar la capacidad de resistencia y tolerancia al TSWV de una accesión de Capsicum baccatum. El nivel de resistencia se estimó a partir de dos variables: A) la eficacia absoluta, calculada a partir de la variación en el tiempo del título viral, cuantificado por RT-qPCR; y B) la infectividad, como la mediana del tiempo que tarda el virus en ser detectado en una planta por ELISA. El nivel de tolerancia se estimó como la mediana del tiempo que tarda una planta en mostrar síntomas graves. Esta nueva accesión es parcialmente resistente y totalmente tolerante tanto para aislados del TSWV convencionales como para aquellos capaces de infectar e inducir síntomas severos en variedades de pimiento con el gen Tsw. Por tanto, esta nueva variedad de C. baccatum es un buen candidato para usarlo en programas de mejora genética de pimiento. / [CAT] Tomato spotted wilt virus (TSWV) és un dels virus més estesos i de major importància econòmica del món. Infecta a un gran nombre d'espècies vegetals i els cultius de tomaca i pebrot són alguns dels més afectats. Aquest virus es transmet d'una planta a una altra, per trips de manera propagativa i circulativa, i Flankliniella occidentalis és el més eficaç. El cultiu de varietats resistents de tomaca i pebrot (en els quals s'han introduït per millora genètica els gens Sw-5 i Tsw, respectivament) va permetre el control de la malaltia, ja que aquests gens indueixen una resposta hipersensible i impedeixen la infecció sistèmica del virus (resistència total). No obstant açò, amb freqüència apareixen aïllats del virus que són capaços de superar aquestes resistències. Per a obtenir un control de la malaltia més eficaç i durador és necessari: A) la caracterització genètica i biològica dels aïllats del TSWV que superen i no superen les resistències; B) l'estudi dels factors evolutius i epidemiològics implicats en l'aparició i establiment dels aïllats que superen les resistències; C) el desenvolupament d'eines que permeten la detecció i quantificació d'aquests aïllats virals; i D) l'avaluació de noves fonts de resistència (total o parcial, que dificulten la infecció i multiplicació viral) o tolerància (total o parcial, que dificulten l'aparició de símptomes i danys encara que no tinguen un efecte en la infecció viral). En aquest treball s'han caracteritzat biològicament i molecularment diferents aïllats del TSWV procedents de cultius de tomaca i pebrot d'Espanya. Es va determinar la seqüència nucleotídica del genoma complet de tres aïllats espanyols corresponents a tres biotips: N (incapaç d'infectar varietats resistents de tomaca o pebrot); T (supera la resistència Sw-5 de tomaca); i P (supera la resistència Tsw de pebrot). Els resultats van mostrar que no hi havia una correlació entre la variació genètica i la capacitat de superar la resistència. Aquestes seqüències nucleotídiques i altres obtingudes de la base de dades Genbank es van utilitzar per a desenvolupar una tècnica basada en la RT-qPCR que permet detectar el virus amb un alt grau de sensibilitat i quantificar en un ampli rang dinàmic. Es van dissenyar els iniciadors i una sonda TaqMan MGB a partir de segments de seqüència conservats perquè foren vàlids per a tots els aïllats del TSWV. Tambe es van desenvolupar dos sondes Taqman que permetien quantificar diferencialment variants genètiques del virus en infeccions mixtes. La RT-qPCR es va utilitzar per a avaluar l'acumulació de diversos aïllats del TSWV en tomaca sense Sw-5, pebrot sense Tsw i Datura stramonium així com, en el seu principal vector F. occidentalis que es va usar per a avaluar l'eficiència de la transmissió. Es va observar que la superació de la resistència no suposava un cost en l'eficàcia biològica (fitness) tant en la multiplicació del virus en aquestes plantes com en la transmissió per trips. Per tant, els aïllats que superen les resistències tenen la mateixa capacitat de dispersió en camp que els aïllats convencionals. Finalment, es va utilitzar la RT-qPCR i la informació obtinguda per a avaluar la capacitat de resistència i tolerància d'una accessió de Capsicum baccatum al TSWV. El nivell de resistència es va estimar a partir de dues variables: A) l'eficàcia absoluta, calculada a partir de la variació en el temps del títol viral, quantificat per RT-qPCR; i B) la infectivitat, com la mitjana del temps que es tarda a detectar el virus en una planta per mitjèr d'ELISA. El nivell de tolerància es va estimar com la mitjana del temps que tarda una planta a mostrar símptomes greus. Aquesta nova accessió és parcialment resistent i totalment tolerant tant per a aïllats del TSWV convencionals com per a aquells capaços d'infectar i induir símptomes severs en varietats amb el gen Tsw. Per tant, aquesta nova varietat d / Debreczeni, DE. (2015). Caracterización de aislados del virus del bronceado del tomate (TSWV) que superan las resistencias de los genes Sw-5 en tomate y Tsw en pimiento. Identificación de una fuente de tolerancia en pimiento [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51460 / TESIS

Acute lower respiratory tract infection (ALRI) is a major cause of paediatric morbidity and mortality, annually accounting for approximately 2 million childhood deaths worldwide of which up to 90% resides in the developing world. In 12-39% of ALRI cases no aetiological agent is identified, despite comprehensive investigations, thus suggesting that additional unknown agents may be involved. Since 2001 a number of new viruses have been identified that may account for some of these cases including human metapneumovirus (hMPV), human bocavirus (hBoV), and two new coronaviruses (hCoV) NL63 and HKU1. The contribution of the recently identified respiratory viruses to annual seasonal lower respiratory tract disease in Sub-Saharan Africa where human immunodeficiency virus infections may exacerbate respiratory infections is not fully understood. In addition, the role and disease association of many of these viruses as primary or co-infecting pathogens, as well as the underlying factors that may determine the pathogenesis of these viruses, is not yet well defined. Quantitative multiplex real-time RT-PCR assays were developed and validated for the detection of 13 well recognized and newly identified viral causes of ALRI, including respiratory syncytial virus (RSV), influenza viruses A and B, parainfluenza virus (PIV) types 1, 2, and 3, adenovirus, hMPV, hBoV and hCoV-NL63, -HKU1, -229E, and -OC43. The newly designed assays were subsequently used to facilitate the investigation of the contribution of respiratory viruses in patients requiring hospitalisation or attending outpatient visits in public sector hospitals serving the Pretoria area, South Africa. During 2006, the prevalence of the aforementioned respiratory viruses was determined by investigating the well recognized viruses previously diagnosed by routine immunofluorescence assays (IFA) in 737 respiratory specimens as well as viruses retrospectively detected by multiplex real-time RT-PCR in a sample group of 319 specimens. The epidemiology and disease association of these respiratory viruses in children who were predominantly less than 5 years of age with acute respiratory tract infections was investigated. Specimens were received from 2 public sector hospitals in Pretoria, South Africa. In addition, the disease association of each virus as a single or co-infection in human immunodeficiency virus (HIV) infected/exposed and HIV-uninfected children as well as the role of viral load was investigated. The multiplex assays could detect 2.5-25 recombinant plasmid DNA/RNA (in vitro transcribed) copies/μl, with a co-efficient of variation of less than 3.1%. Validation on 91 known positive respiratory specimens indicated similar specificity to IFA or single-round PCRs used in the initial identification of the viruses. Application of the multiplex assays to IFA negative specimens improved the detection of respiratory viruses by up to 44%. In children less than 5 years of age RSV was identified in 35.1%, followed by PIV 3 (8.3%); adenovirus (5.6%); influenza A (4.2%); hMPV (4.2%); hBoV (3.8%); hCoV-NL63 (1.6%); influenza B (1.0%); and PIV1, PIV2, hCoV-OC43, hCoV-229E, hCoV-HKU1 in less than 1% of cases. Co-infections were more common for the new viruses ranging from 58% of hMPV cases to 84% for hCoV-NL63 relative to 27% of RSV cases. Viruses were most frequently identified in children <1-year. RSV activity peaked in autumn and winter, PIV 3 in spring, while influenza A and B were mostly detected in winter. The observed seasonal distribution of hBoV and hMPV was less defined compared to traditional viruses, with both viruses showing variability over the two years. Comparable hospitalisation rates were observed for RSV, hMPV, PIV 3 and adenovirus, where approximately 60% of infected children were hospitalised. In addition, a high frequency of hospitalisations was observed in patients for both hMPV and hBoV in HIV-infected/exposed children. Co-infections occurred at higher frequencies with the new viruses, were more frequently associated with severe disease and were frequent in HIV-infected/exposed patients. Viral load was associated with severe RSV disease (p=0.014) however no significant association was observed for the new viruses as single infections. However, where hMPV occurred as a co-infection, higher viral loads of either hMPV or co-infecting agents occurred in severe cases. This association was also observed for hBoV. Most cases of hCoV-NL63 and hCoV-OC43 were co-infections in hospitalised patients. The newly developed multiplex assays demonstrates an improved sensitivity and scope of detecting respiratory viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the role of co-infections and viral load in respiratory virus pathogenesis. RSV remains the most significant viral cause of paediatric ALRI in South Africa. Viruses not currently included in routine diagnostic assays collectively contributed to 11% of ALRI cases in children <2-years in South African hospitals. / Dissertation (MSc)--University of Pretoria, 2010. / Medical Virology / unrestricted

MSc (Microbiology) / Department of Microbiology

Introducción: Las infecciones agudas del tracto respiratorio (IRA) son muy prevalentes, las IRAs bajas constituyen la cuarta causa de muerte a nivel mundial. Los agentes causales más comunes en niños son el Rinovirus (RV) y el Virus sincitial respiratorio (VSR). Existen tres especies de RV (A, B, C), estudios recientes han demostrado que la sintomatología y severidad de la enfermedad varía dependiendo de la especie de RV por la que hayan sido infectados los pacientes. Objetivo: Evaluar la prevalencia de Rinovirus en muestras de hisopado nasofaríngeo de niños con diagnóstico clínico de IRA en Lima, Perú durante el periodo 2009-2010. Materiales y métodos: Estudio retrospectivo de muestras de hisopado nasofaríngeo en niños, procesadas por la técnica RT-PCR para identificación de RV y sus especies. La población está compuesta por pacientes pediátricos en el Hospital Nacional Cayetano Heredia. Se analizó las variables por medio de la prueba de Chi cuadrado y Fischer. Resultados: RVA se detectó en 10.26%, RVB en 16.67%, RVC 73.9%. Grupo etario más prevalente fue de 0-5 meses. Signos y síntomas más comunes fueron tos, fiebre, rinorrea y dificultad respiratoria. Se encontró asociación entre sibilancias y RVA; tos, sibilancias e inyección conjuntival con RVC. Se halló pico de casos por RVC durante marzo, junio y noviembre. Conclusión: Se encontró alta prevalencia de infección por RVC en pacientes pediátricos, principalmente en pacientes de 0-5 meses. Distribución mensual muestra aumento de casos en marzo y junio. Se sugiere realizar vigilancia epidemiológica y estudios longitudinales para el estudio de este patógeno. / Introduction: Acute respiratory tract infections (ARTI) are a very prevalent group of diseases, lower ARTI represent the fourth cause of death worldwide. In children, the two most usual agents are Rhinovirus (RV) and Syncytial respiratory virus (SRV). RV is responsible for most is related with lower respiratory tract infections. Scientists have identified three RV species (A, B, C), recent studies have reported that symptomatology and severity vary within RV species. Objective: Asses the prevalence of Rhinovirus on nasopharyngeal swab samples of children with clinical diagnosis of ARTI in Lima, Peru during 2009-2010. Materials and method: Retrospective study about nasopharyngeal swab on children, which were processed through RT-PCR technique to identify RV and its species. The study population was pediatric patients, with clinical diagnosis of ARTI, at Hospital Nacional Cayetano Heredia. This investigation project will be revised by the ethics committee from Universidad Peruana de Ciencias Aplicadas. Results: RVA was detected in 10.26% of cases, RVB 16-67% and RVC in 73.9%. The most prevalent age group was the 0-5 months old. The most common signs and symptoms were cough, fever, rhinorrhea, and respiratory distress. The study found association between wheezing and RVA infection, cough, wheezing and conjunctival injection and RVC infection. There was a peak in RVC cases during the March, June, and November. Conclusion: We found a high prevalence for RVC infection, mainly in children between 0-5 months old. Monthly distribution showed an increase of RVC cases during March and June; epidemiological surveillance and longitudinal studies should be encouraged. / Tesis