Laboratorio Of Pesquisas Em Virologia

São José do Rio Preto, Brazil

Laboratorio Of Pesquisas Em Virologia

São José do Rio Preto, Brazil
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Villabona-Arenas C.J.,University of Sao Paulo | Mondini A.,São Paulo State University | Bosch I.,Massachusetts Institute of Technology | Schimitt D.,Tufts University | And 2 more authors.
PLoS ONE | Year: 2013

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak. © 2013 Villabona-Arenas et al.


Morais A.T.S.,Laboratorio Of Pesquisas Em Virologia | Morais A.T.S.,São Paulo State University | Araujo G.C.,São Paulo State University | Vidotto A.,Laboratorio Of Pesquisas Em Virologia | And 4 more authors.
Protein and Peptide Letters | Year: 2014

The eukaryotic translation initiation factor 3, subunit L (eIF3L) is one of the subunits of the eIF3 complex, an accessory protein of the Polymerase I enzyme and may have an important role in the Flavivirus replication by interaction with a viral non-structural 5 protein. Considering the importance of eIF3L in a diversity of cellular functions, we have produced the recombinant full-length eIF3L protein in Escherichia coli and performed spectroscopic and in silico analyses to gain insights into its hydrodynamic behavior and structure. Dynamic light scattering showed that eIF3L behaves as monomer when it is not interacting with other molecular partners. Circular dichroism experiments showed a typical spectrum of α-helical protein for eIF3L, which is supported by sequence-based predictions of secondary structure and the 3D in silico model. The molecular docking with the K subunit of the eIF3 complex revealed a strong interaction. It was also predicted several potential interaction sites in eIF3L, indicating that the protein is likely capable of interacting with other molecules as experimentally shown in other functional studies. Moreover, bioinformatics analyses showed approximately 8 putative phosphorylation sites and one possible N-glycosylation site, suggesting its regulation by post-translational modifications. The production of the eIF3L protein in E. coli and structural information gained in this study can be instrumental for target-based drug design and inhibitors against Flavivirus replication and to shed light on the molecular mechanisms involved in the eukaryotic translation initiation. © 2014 Bentham Science Publishers.


Morais A.T.S.,Laboratorio Of Pesquisas Em Virologia | Morais A.T.S.,São Paulo State University | Terzian A.C.B.,Laboratorio Of Pesquisas Em Virologia | Duarte D.V.B.,Laboratorio Of Pesquisas Em Virologia | And 9 more authors.
Virology Journal | Year: 2013

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.


PubMed | Laboratorio Of Pesquisas Em Virologia
Type: Journal Article | Journal: BMC infectious diseases | Year: 2017

The 2009 revised World Health Organization (WHO) guidelines for dengue describe fever as the core symptom. Accordingly, the diagnosis of non-febrile patients is complicated. The aim of this study was to evaluate the importance of fever in patients with dengue according to the 2009 revised WHO classification.In this study, we assessed 30,670 dengue cases using enzyme-linked immunosorbent assay, detection of the non-structural protein 1, or polymerase chain reaction for diagnostic confirmation. Fishers exact test was used to evaluate associations between fever and related clinical manifestations. The Mann-Whitney U test was used to assess the association of dengue classification with fever and time to treatment. The effects of fever and time to treatment on the risk of progression were analyzed using an ordinal logistic regression to stereotype the model.Disease classification was found to associate significantly with both fever and time to treatment (both P<0.001). Non-febrile patients were nearly four-fold more likely to exhibit dengue without warning signs than severe dengue (odds ratio [OR]=3.74; 95% confidence interval [CI]: 3.20-4.36). Patients who received treatment within 7days were twice as likely to have dengue without warning signs as opposed to severe dengue when compared to those who waited >7days (OR=2.23; 95% CI: 1.78-2.80). However, this difference was negligible in the multivariate analysis (OR=1.02; 95% CI: 0.98-1.07).Fever is a risk factor for disease progression in patients with dengue. However, non-febrile patients should not be neglected because this may delay treatment and could lead to more severe disease.


PubMed | Laboratorio Of Pesquisas Em Virologia
Type: Journal Article | Journal: BMC infectious diseases | Year: 2016

The aim of this study was to evaluate the utility of the tourniquet test (TT) for dengue diagnosing. To our knowledge, no previous study with such a large sample, of this duration, with as many laboratory methods referenced, or relating the results of the TT to the 2009 WHO classification of severity has been conducted thus far.In this study, we analyzed the records of 119,589 suspected dengue cases in a Brazilian city, with 30,670 confirmed cases. The Cohens Kappa test was applied to evaluate the degree of agreement between the tests, and the sensitivity and specificity was calculated for the TT.Twenty-eight thousand six hundred thirty-five TT were performed. No association between the outcome of the TT and greater severity of infection, according to the 2009 guideline, was observed (P=0.28); furthermore, relevant agreement with the final diagnosis (=0.01; 95% CI=0.00 to 0.02) or individually with the IgM enzyme-linked immunoassay was not observed (=0.05; 95% CI=0.04 to 0.06), and was even lower with PCR (=0.27; 95% CI=0.06 to 0.49). Most importance of the TT was shown in relation to specificity (88.9%; 95% CI=0.88 to 0.89) and negative predictive value (70.3%; CI 95%=0.70 to 0.71).TT was more effective in detecting cases that were truly negative than positive. These results suggest that the TT should not be used as diagnosis of dengue.


Schmidt D.J.,University of Massachusetts Medical School | Schmidt D.J.,Tuft University Veternary School | Pickett B.E.,University of Alabama at Birmingham | Pickett B.E.,University of Texas Southwestern Medical Center | And 22 more authors.
Infection, Genetics and Evolution | Year: 2011

Dengue virus currently causes 50-100. million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level. © 2011.


Ferraz F.O.,Federal University of Minas Gerais | Bomfim M.R.Q.,University do Ceuma | Totola A.H.,Federal University of Säo João del Rei | Avila T.V.,Federal University of Minas Gerais | And 8 more authors.
Journal of Clinical Virology | Year: 2013

Background: Dengue is a widely spread arboviral disease in tropical and subtropical regions of the world. Dengue fever presents clinical characteristics similar to other febrile illness. Thus laboratory diagnosis is important for adequate management of the disease. Objectives: The present study was designed to evaluate the diagnostic performance of real-time PCR and serological methods for dengue in a real epidemic context. Study design: Clinical data and blood samples were collected from consecutive patients with suspected dengue who attended a primary health care unit in Belo Horizonte, Brazil. Serologic methods and real-time PCR were performed in serum samples to confirm dengue diagnosis. Results: Among the 181 consecutive patients enrolled in this study with suspected dengue, 146 were considered positive by serological criteria (positive NS1 ELISA and/or anti-dengue IgM ELISA) and 138 were positive by real-time PCR. Clinical criteria were not sufficient for distinguishing between dengue and non-dengue febrile illness. The PCR reaction was pre-optimized using samples from patients with known viral infection. It had similar sensitivity compared to NS1 ELISA (88% and 89%, respectively). We also evaluated three commercial lateral flow immunochromatographic tests for NS1 detection (BIOEASY, BIORAD and PANBIO). All three tests showed high sensitivity (94%, 91% and 81%, respectively) for dengue diagnosis. Conclusion: According to our results it can be suggested that lateral flow tests for NS1 detection are the most feasible methods for early diagnosis of dengue. © 2013 Elsevier B.V.


PubMed | Laboratorio Of Pesquisas Em Virologia, Federal University of Juiz de fora and Federal University of Alfenas
Type: Journal Article | Journal: Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] | Year: 2016

Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1-4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population.


Nogueira M.L.,Laboratorio Of Pesquisas Em Virologia | Nogueira M.C.L.,Laboratorio Of Pesquisas Em Virologia | Pacca C.,Laboratorio Of Pesquisas Em Virologia
Central Nervous System Agents in Medicinal Chemistry | Year: 2011

Encephalitis refers to an acute, usually diffuse, inflammatory process affecting the brain. The clinical hallmark of acute encephalitis is the triad of fever, headache, and altered mental status. The most common and important cause of encephalitis is the infection by a virus although other organisms can cause the disease. This article is a general overview of the most common viral encephalitides, divided into two families, Flavivirus and Alphavirus, and provides details about virus and RNA interference. More detailed descriptions of each viral family are provided bellow. © 2011 Bentham Science Publishers.

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