Caracterização biológica e genética de isolados clínicos de dengue sorotipo 3

Orientadora : Claudia Nunes Duarte dos SantosTese (doutorado) - Universidade Federal do Paraná, Programa de Pós-Graduaçao em Biologia Celular e Molecular. Defesa: Curitiba, 30/07/2009Inclui bibliografia: f. 119-142A dengue é a principal arbovirose tropical transmitida ao homem por meio da picada das fêmeas de mosquitos hematófagos do gênero Aedes, doença de extrema importânciapara a saúde pública devido às epidemias observadas em áreas tropicais e subtropicais da Ásia, África, Austrália e Américas onde o vetor encontra-se amplamente disseminado. O vírus da dengue pertence à família Flaviviridae, gênero Flavivírus, possui quatro sorotipos antigenicamente distintos, designados de DENV1, DENV2, DENV3 e DENV4, subdivididos em genótipos. As manifestações da doença podem variar de inaparente ou doença febril suave até a doença hemorrágica severa e fatal. Nas últimas duas décadas, houve um aumento no número de infecções por dengue com manifestações clínicas atípicas, incluindo envolvimento do Sistema Nervoso Central (SNC). O objetivo deste trabalho é realizar a caracterização biológica e genética de isolados clínicos de DENV3 de pacientes exibindo diferentes formas clínicas, obtidos em contextos epidêmicos distintos das Regiões Norte (Acre e Rondônia) e Sudeste (RJ) do Brasil e do Paraguai. A caracterização biológica in vitro consistiu na infecção de diferentes substratos celulares (células de inseto e de mamíferos), a qual resultou na identificação de habilidades replicativas bastante divergentes entre os isolados clínicos. Visando identificar marcadores moleculares que pudessem ser associados a estes diferentes fenótipos, procedemos à caracterização genética, que compreendeu o sequenciamento genômico completo dos vírus. Os resultados obtidos permitiram a identificação de dois genótipos distintos (III e V) do DENV3 co-circulando no país, além de marcadores moleculares potencialmente envolvidos com diferenças fenotípicas observadas in vitro e in vivo entre os diferentes isolados clínicos. A falta de um modelo animal disponível que reproduza as formas clínicas observadas em humanos é um obstáculo para se estudar diferentes aspectos relacionados à patogenia da dengue. Com o objetivo de avaliar o comportamento dos isolados clínicos de DENV3 in vivo, utilizamos camundongos isogênicos como modelo. Parâmetros como replicação viral e produção de progênie viral foram avaliados, além de análises histopatológicas e imunohistoquímicas de tecidos dos animais inoculados. Os resultados mostraram que havia diferenças quanto à habilidade de replicação em diferentes áreas-alvo do SNC e consequentemente produção de doença, dependendo da cepa viral utilizada. Com o intuito de avaliarmos a resposta dos animais durante a infecção com os diferentes DENV3, foram realizadas análises por qPCR de alguns genes a partir de amostras de SNC. Genes relacionados às vias de sinalização por IFN, processamento e apresentação de antígenos, ativação do complemento e via tipo ubiquitinação presentaram-se modulados no SNC dos camundongos. Os resultados obtidos neste estudo vêm contribuir para o conhecimento sobre a patogênese dos vírus da dengue e, dessa forma, nortear o delineamento de possíveis estratégias antivirais.Dengue fever, the main arboviral infection transmitted to humans through the bite of hematophagous female mosquitoes from the genus Aedes, is one of the most important disease for public health due to observed explosive epidemics pattern in tropical and sub-tropical areas of the globe, where the mosquito vector is widely disseminated. The four antigenically distinct serotypes of dengue fever virus designated DENV1, DENV2, DENV3 and DENV4, belongs to the Flaviviridae family, genus Flavivirus. The clinical manifestations of dengue can range from unapparent or mild febrile disease, to the severe and fatal hemorrhagic fever. In the last two decades an increase in the number of atypical clinical manifestations related to dengue infection has been observed. The objective of this study was to characterize recent clinical DENV3 isolates obtained from patients exhibiting different clinical profiles and sampled in various epidemical contexts in North (Acre and Rondônia) and Southeast (Rio de Janeiro) regions from Brazil as well as in Assunção, Paraguay. The biological characterization consisted in the infection of different cellular substrates (from both insects and mammals origins), which resulted in the identification of a broadly diverging replicating capabilities among the different viral clinical isolates. Aiming to identify molecular markers which could be associated to these different phenotypes, we proceeded to the genetic characterization, which included the determination of the viral complete genomes. The obtained results enabled the identification of two distinct genotypes (III and V) of the serotype DENV3 co-circulating in Brazil, as well as molecular markers potentially involved in phenotypical differences observed in vitro and in vivo among the diverse clinical isolates. The lack of an available animal model capable of reproducing the clinical profile observed in humans is a hindrance in the research of varied aspects related to the pathogenesis of dengue fever. In order to evaluate the behavior of DENV3 clinical isolates in vivo, isogenic mice were utilized as models. Benchmarks such as viral replication and viral progeny production were evaluated, as were the histopathological and immunohistochemical analysis of Central Nervous System (CNS) tissues of the inoculated animals. The results demonstrated discrepancies in the replicating capability of the SNC in different target areas, and therefore in the production of the disease, depending on the virus type. With the purpose to assess the response of the animals during the infection with different strains of DENV3, qPCR analyses of selected genes were conducted. Genes related to the IFN signaling pathways, processing and presentation of antigens, complement activation and like-ubiquitin pathways were modulated in the SNC of the mice. These findings may contribute to the knowledge about dengue pathogenesis and eventually drive antiviral strategies

Red blood cells in cerebrospinal fluid as possible inhibitory factor for enterovirus RT-PCR

ABSTRACT The presence of hemoglobin in samples are considered an important inhibitory factor for polymerase chain reaction (PCR). The aim of this study was to examine the influence of red blood cells (RBC)s in cerebrospinal fluid (CSF) as an inhibitory factor to reverse transcription polymerase chain reaction (RT-PCR) for enteroviruses (EV). Forty-four CSF samples from patients showing characteristics of viral meningitis were assessed for EV by RT-PCR. Viral RNA extracted with guanidine isothyocianate buffer and virus detection was performed by in-house nested PCR. Positivity for EV RT-PCR was higher in CSF samples without RBCs than in samples with RBCs: 13(26%) and 36(9.2%), p = 0.001. In the group with positive EV RT-PCR, the mean + SD CSF RBC was 37 ± 183 cell/mm3; the group with negative results had 580 + 2,890 cell/mm3 (p = 0.007). The acceptable upper limit for CSF RBCs that could not influence RT-PCR was 108 cells/mm3. CSF samples with negative results for EV RT-PCR have more erythrocytes

Viral acute gastroenteritis: clinical and epidemiological features of co-infected patients

BACKGROUND: Acute gastroenteritis (AGE) is a common disorder that affects children worldwide. It is usually caused by viral agents, including rotavirus, enteric adenovirus, norovirus, and astrovirus groups. Currently, there are few reports about co-infection among these viruses, mainly in Brazil. METHODS: This is a retrospective study in which 84 rotavirus-positive samples from hospitalized patients at a teaching hospital in Southern Brazil, collected in the 2001-2010 period, were analyzed by polymerase chain reaction (PCR) and reverse transcription - polymerase chain reaction (RT-PCR), for the investigation of enteric adenovirus, astrovirus, and norovirus. RESULTS: In total, 12 of the 84 (14%) samples were positive to enteric adenovirus or norovirus. Clinical, laboratory, and demographic data showed statistically significant differences between mono and co-infected patients, including age and depletion rate. CONCLUSIONS: These findings highlight the need for implementation of other enteric virus detection assays in clinical diagnosis for a complete laboratory investigation of hospitalized pediatric patients with AGE, in order to understand the impact of these pathogens on disease severity, spread within hospital, and consequently, prevent the dissemination of nosocomial infections