Análise filoginética e genotipagem de amostras de vírus rábico através da técnica de seqüênciamento automatizado de produtos de RT-PCR

Dissertação (mestrado) - Universidade Federal de Santa Catarina. Programa de Pós-Graduação em Biotecnologia.O vírus rábico pertence à Ordem Mononegavirales e Família Rhabdoviridae, sendo constituído por um RNA de sentido negativo, não-segmentado e que codifica para cinco proteínas (N, NS, M, G e L). São conhecidos até o momento 7 genótipos virais, sendo o genótipo 1 formado pelo vírus rábico clássico, com distribuição mundial. A genotipagem de amostras do vírus é realizada mais comumente pela análise dos genes da nucleoproteína e glicoproteína, embora a região não-codificante G-L tenha sido sugerida para caracterização de amostras mais homogêneas. O presente trabalho descreve a genotipagem e a análise filogenética de amostras de vírus rábico isoladas em SC durante o ano de 2002, através do seqüenciamento automatizado de produtos de RT-PCR. A extração de RNA viral, transcrição para cDNA e amplificação via RT-PCR do gene da nucleoproteína e da região não-codificante G-L, são descritas em detalhes. Os resultados demonstraram que a porção 5´ do gene da nucleoproteína é altamente conservada entre as amostras estudadas e divergentes das amostras padrão de laboratório, sendo todas classificadas como genótipo 1. A comparação da seqüência nucleotídica destas amostras com outros isolados brasileiros disponíveis no GenBank, questiona a distribuição espécie-específica, sugerida por outros autores, levantando a possibilidade de que os agrupamentos por regiões geográficas possam ser relevantes. A análise da região não-codificante G-L revelou alto grau de variabilidade entre as amostras catarinenses, possibilitando a distribuição das mesmas em dois grupos. Nenhuma destas amostras apresentou o sinal clássico de parada de transcrição na extremidade 5´ do gene da glicoproteína, relatado no modelo de pseudogene viral. Além disso, três isolados também não apresentaram este sinal na extremidade 5´ da região não-codificante G-L, sugerindo uma possível transcrição bicistrônica entre o gene da glicoproteína e a polimerase viral. Outra possibilidade é que existam novos sinais de parada de transcrição (para o gene da glicoproteína e da região não-codificante G-L) e de início de transcrição (para o gene da polimerase) ainda não descritos. A relevância destes achados é discutida em detalhes

O uso de agentes inteligentes no apoio da interação do Bate-papo da CV-Muzar

Este artigo apresenta o Sistema Multiagente (SMA) proposto para monitorar a conversação no bate-papo da Comunidade Virtual do Muzar (CVMuzar). Para tanto, são analisados os marcadores conversacionais e sua aplicação em ambientes de conversação síncrona, com vistas a propor mecanismos de compensação para os déficits de comunicação identificados com objetivo de melhorar a interação. O SMA proposto conta com cinco agentes cooperativos que monitoram as conversas em tempo real e interferem na interface de conversação para aconselhar e, se necessário, advertir o participante

Macrophage Polarization in Leishmaniasis: Broadening Horizons

Leishmaniasis is a vector-borne neglected tropical disease that affects more than 700,000 people annually. Leishmania parasites cause the disease, and different species trigger a distinct immune response and clinical manifestations. Macrophages are the final host cells for the proliferation of Leishmania parasites, and these cells are the key to a controlled or exacerbated response that culminates in clinical manifestations. M1 and M2 are the two main macrophage phenotypes. M1 is a pro-inflammatory subtype with microbicidal properties, and M2, or alternatively activated, is an anti-inflammatory/regulatory subtype that is related to inflammation resolution and tissue repair. The present review elucidates the roles of M1 and M2 polarization in leishmaniasis and highlights the role of the salivary components of the vector and the action of the parasite in the macrophage plasticity

Genome-wide analyses reveal a highly conserved Dengue virus envelope peptide which is critical for virus viability and antigenic in humans.

Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans

Flavonoids as molecules with Anti-Zika virus activity

Zika virus (ZIKV) is an arthropod-born virus that is mainly transmitted to humans by mosquitoes of the genus Aedes spp. Since its first isolation in 1947, only a few human cases had been described until large outbreaks occurred on Yap Island (2007), French Polynesia (2013), and Brazil (2015). Most ZIKV-infected individuals are asymptomatic or present with a self-limiting disease and nonspecific symptoms such as fever, myalgia, and headache. However, in French Polynesia and Brazil, ZIKV outbreaks led to the diagnosis of congenital malformations and microcephaly in newborns and Guillain-Barré syndrome (GBS) in adults. These new clinical presentations raised concern from public health authorities and highlighted the need for anti-Zika treatments and vaccines to control the neurological damage caused by the virus. Despite many efforts in the search for an effective treatment, neither vaccines nor antiviral drugs have become available to control ZIKV infection and/or replication. Flavonoids, a class of natural compounds that are well-known for possessing several biological properties, have shown activity against different viruses. Additionally, the use of flavonoids in some countries as food supplements indicates that these molecules are nontoxic to humans. Thus, here, we summarize knowledge on the use of flavonoids as a source of anti-ZIKV molecules and discuss the gaps and challenges in this area before these compounds can be considered for further preclinical and clinical trials

Dengue virus pathogenesis in mouse central nervous system: studies on host response to dengue virus infection

Dengue virus (DENV) causes a self-limiting fever (DF) or severe hemorrhagic fever/shock-syndrome (DHF/DSS). Recently, clinical profile of DENV infection is changing, and neurological manifestations are becoming frequent. We previously demonstrated that mutations on E and NS3 proteins may account for DENV neurovirulence for mice. To validate the involvement of the observed mutations in the appearance of neurovirulent viral phenotypes, we constructed cDNA infectious clones encompassing the observed mutations. Aiming to determine host response to infection with neurovirulent and parental strains of DENV-1, we used a Mus musculus biochip (Virginia Commonwealth University) encompassing whole mouse genome. Newborn Swiss mice were infected intracerebraly with 8.000ffuC636 of FGA/89 (parental strain), FGA/NA a5c (neurovirulent strain) and mock. Animals infected with FGA/NA a5c show clinical signs of encephalitis around 9 days post-infection (dpi) and succumb to death (13.1 dpi ± 2.2), in contrast with animals infected with FGA/89 and mock. Kinetics of infection showed higher levels of viral RNA and progeny in FGA/NA a5c infected animals at 9 dpi. Microarray analyzes were carried out at 5, 6, 7 and 8 dpi in central nervous system (CNS) of mock, FGA/89 and FGA/NA a5c infected animals. Bioinformatic analysis revealed 149 genes up-regulated in CNS infection by DENV-1, at higher levels in animals infected with FGA/NA a5c. Analysis with the software Ingenuity Pathways Systems(IPA) showed that the main pathways modulated by DENV infection in the mouse CNS are IFN signaling, antigen presentation, complement cascades and protein ubiquitination. Additionally, 15 genes were selected by in silico analyses; of special interest are four genes encoding chemokines with chemoattractant activity, that are up-regulated following infection with the neurovirulent virus compared to parental virus. Nowadays we are performing the biological characterization of the selected genes and pathways

Relato e considerações sobre o desenvolvimento de uma ontologia para avaliação de sites da área de saúde

Este artigo descreve desenvolvimento de uma ontologia que visa avaliar a qualidade de sites/páginas com assuntos relacionados à área de saúde. O processo de desenvolvimento da ontologia foi realizado ao longo do segundo semestre de 2008 pelos alunos da disciplina CMP234 - Modelagem Conceitual e Ontologia do Programa de Pós-Graduação do Instituto de Informática da Universidade Federal do Rio Grande do Sul, ministrada pelo Professor Dr. José Palazzo Moreira de Oliveira. O desenvolvimento da ontologia foi feito com base na Metodologia 101 [1]. Assim, estrutura do documento observa, em grande parte, as etapas da Metodologia 101 e procura manter grande parte das informações e observações feitas ao longo do desenvolvimento visando o uso destas em futuras versões da ontologia. Para o desenvolvimento da ontologia foi utilizado o editor Protégé. As instâncias da ontologia foram criadas mediante extração dos dados dos sites/páginas, extração esta que foi feita manualmente pelos alunos da disciplina

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications