Juliano Bordignon

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) widespread 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.

Flow cytometry assay for intracellular rabies virus detection.

Following previous studies reporting microbiological diagnosis by flow cytometry, the possibility of using this method was examined to monitor infection of susceptible cell lines by a fixed rabies virus strain (Pasteur Virus strain-PV) or a wild rabies virus strain (WRS). Suspensions of BHK-21 and C6 cells were infected with viruses and a time course of virus infection was established. Sequentially, at several time points, infected and control uninfected cells were fixed, permeabilized, and stained with a rabies virus-specific antibody conjugate. This was achieved by resuspending cells in a solution containing p-formaldehyde in FACS lysis fluid, which allowed the detection of intracellular virus with flourescein-coupled antibodies by flow cytometry. A Becton Dickinson FACSCalibur flow cytometer was used to analyze the percentage of cells infected and the kinetics of the infection process was determined. As early as 12 h after inoculation with both rabies virus strains, significant levels (P<0.01) of infection (from 4.7 to 7.1%) were detected by flow cytometry. The maximum level of infection was obtained at 48 h in C6 cells (88%) with both viruses. The advantages of this method for examination of intracellular virus infection are discussed.

Phylogenetic characterization of hantaviruses from wild rodents and hantavirus pulmonary syndrome cases in the state of Paraná (southern Brazil).

Over 1,100 cases of hantavirus pulmonary syndrome (HPS) have occurred in Brazil since 1993, but little is known about Brazilian hantaviruses, and many of their rodent hosts remain unknown. The Araucaria hantavirus (ARAUV) was described recently from HPS patients from Paraná, in southern Brazil, but its host could not be identified. In this study, rodents were captured from regions with high HPS prevalence to address this issue. ARAUV RNA was detected in three distantly related rodent species: Oligoryzomys nigripes, Oxymycterus judex and Akodon montensis. Furthermore, a specimen of A. montensis was infected with a Jaborá-like virus, implying that A. montensis can be infected by at least two different hantaviruses. The presence of the same hantavirus strain in three different rodent species and the co-circulation of two different strains in the same rodent species highlight the potential for genomic reassortment, which could have an impact on hantavirus transmission dynamics in nature and on human epidemiology.

Production and characterization of monoclonal antibodies against the recombinant nucleoprotein of Araucaria hantavirus

Hantaviruses are rodent-borne RNA viruses that cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). From the first detection of infection in Brazil in 1993 until 2009, 1161 cases of HPS have been reported, with mortality rates of around approximately 40%. Currently, due to the absence of a vaccine or specific antiviral therapy, the only way to reduce mortality by hantavirus infection is a fast and precise diagnosis that allows for supportive clinical health care. To improve the detection of hantavirus infection, we developed monoclonal antibodies (Mabs) against the nucleoprotein (rNDelta85) of the Araucaria hantavirus strain (ARAUV). The specificity of generated Mabs for rNDelta85 was demonstrated by western blot, indirect immunofluorescence and immunohistochemistry. These are the first monoclonal antibodies to be produced and characterized against the South American hantavirus strain, and may be of special interest in the development of diagnostic assays and epidemiological studies.

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.

The citrus flavonone naringenin reduces lipopolysaccharide-induced inflammatory pain and leukocyte recruitment by inhibiting NF-κB activation

Lipopolysaccharide (LPS) is the major structural component of Gram-negative bacteria cell wall and a highly pro-inflammatory toxin. Naringenin is found in Citrus fruits and exhibits antioxidant and anti-inflammatory properties through inhibition of NF-κB activation but its effects in LPS-induced inflammatory pain and leukocyte recruitment were not investigated yet. We investigated the effects of naringenin in mechanical hyperalgesia, thermal hyperalgesia and leukocyte recruitment induced by intraplantar injection of LPS in mice. We found that naringenin reduced hyperalgesia to mechanical and thermal stimuli, myeloperoxidase (MPO, a neutrophil and macrophage marker) and N-acetyl-β-D-glucosaminidase (NAG, a macrophage marker) activities, oxidative stress and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production in the paw skin. In the peritoneal cavity, naringenin reduced neutrophil and mononuclear cell recruitment, and abrogated MPO and NAG activity, cytokine and superoxide anion production, and lipid peroxidation. In vitro, pre-treatment with naringenin inhibited superoxide anion and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production by LPS-stimulated RAW 264.7 macrophages. Finally, we demonstrated that naringenin inhibited NF-κB activation in vitro and in vivo. Therefore, naringenin is a promising compound to treat LPS-induced inflammatory pain and leukocyte recruitment.

Dengue Virus Type 3 Isolated from a Fatal Case with Visceral Complications Induces Enhanced Proinflammatory Responses and Apoptosis of Human Dendritic Cells

ABSTRACT A recent (2007 to 2009) dengue outbreak caused by dengue virus (DENV) in Paraguay presented unusual severe clinical outcomes associated with 50% mortality rates. Although it has been reported that inflammatory responses influence the severity of dengue virus infection (T. Pang, M. J. Cardosa, and M. G. Guzman, Immunol. Cell Biol. 85:43–45, 2007), there remains a paucity of information on virus-innate immunity interactions influencing clinical outcome. Using human dendritic cells from a major innate immune cell population as an in vitro model, we have investigated signature cytokine responses as well as infectivity-replicative profiles of DENV clinical isolates from either a nonfatal case of classical dengue fever (strain DENV3/290; isolated in Brazil in 2002) or a fatal case of dengue fever with visceral complications isolated in Paraguay in 2007 (strain DENV3/5532). Strain DENV3/5532 was found to display significantly higher replicative ability than DENV3/290 in monocyte-derived dendritic cells (mdDCs). In addition, compared to DENV3/290 results, mdDCs exposed to DENV3/5532 showed increased production of proinflammatory cytokines associated with higher rates of programmed cell death, as shown by annexin V staining. The observed phenotype was due to viral replication, and tumor necrosis factor alpha (TNF-α) appears to exert a protective effect on virus-induced mdDC apoptosis. These results suggest that the DENV3/5532 strain isolated from the fatal case replicates within human dendritic cells, modulating cell survival and synthesis of inflammatory mediators.

Evidence of circulation of Laguna Negra-like hantavirus in the Central West of Brazil: Case report

BACKGROUND Hantavirus pulmonary syndrome has been reported with increasing frequency in some Brazilian regions, but information about viral genetic identification is still limited. Recently, the state of Mato Grosso, in the Legal Amazon of Brazil, experienced a growing number of hantavirus pulmonary syndrome (HPS) cases but the genetic characterization of the causative hantavirus is still missing. OBJECTIVES Our goal was to identify the hantavirus strain involved in a fatal HPS case in the Central region of Brazil. STUDY DESIGN Nested RT-PCR was conducted on blood clot samples from an HPS patient from Mato Grosso. PCR-positive samples were sequenced, and the resulting sequences were compared with reference samples. Viral antigens were detected by immunohistological analyses in lung and liver tissues. RESULTS Analyses of the viral RNA isolated from the patient identified a Laguna Negra (LN)-like virus as the causative agent and histological analysis of lung sections were compatible with the genetic characterization. CONCLUSIONS This is the first report of circulation and human infection by a Laguna Negra-like hantavirus in Brazil.

Expression profile of interferon stimulated genes in central nervous system of mice infected with dengue virus Type-1

Dengue virus (DENV) infection can cause a self-limiting disease (dengue fever) or a more severe clinical presentation known as dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). Furthermore, data from recent dengue epidemics in Brazil indicate that the neurological manifestations are becoming more prevalent. However, the neuropathogenesis of dengue are not well understood. The balance between viral replication efficiency and innate immunity--in opposition during the early stages of infection--determines the clinical outcome of DENV infection. In this study, we investigated the effects of DENV infection on the transcription profile of the central nervous system (CNS) of mice. We observed in infected mice the up-regulation of 151 genes possibly involved in neuropathogenesis of dengue. Conversely, they may have a protective effect. Ingenuity Systems software analysis demonstrated, that the main pathways modulated by DENV infection in the mouse CNS are involved in interferon signaling and antigen presentation.

Calculating rabies virus neutralizing antibodies titres by flow cytometry

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton Dickinson FACSCalibur flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity.