Caracterização molecular de cepas do vírus respiratório sincicial isoladas de casos de infecção respiratória aguda na cidade de Belém, Pará, Brasil nos anos de 2000 a 2006

As doenças do trato respiratório são as principais queixas nos serviços de atendimento médico, sendo as infecções respiratórias agudas (IRA) as manifestações mais comuns, principalmente em crianças menores de cinco anos de idade. Em países em desenvolvimento as IRA constituem um sério problema de saúde pública. Em todo mundo estima-se que ocorram cerca de dois milhões de mortes devido as IRA a cada ano. Dentre os agentes causais das mesmas, destaca-se o Vírus Respiratório Sincicial (VRS), especialmente por causar doença grave em crianças menores de dois anos. Com o objetivo de gerar dados sobre a epidemiologia molecular deste vírus, foram analisadas amostras colhidas de pacientes com IRA no período de 2000 a 2006 na cidade de Belém, Pará. Foram utilizados testes de imunofluorescência indireta (IFI) para caracterização antigênica dos vírus isolados e RT-PCR para os genes codificadores das proteínas G e F, que foram em seguida parcialmente seqüenciados. Dentro do período estudado, 153 amostras positivas para VRS foram detectadas. A faixa etária de 0-4 anos foi a que concentrou maior número de casos (n=138; 90,19%). Em relação ao perfil sazonal, o pico de atividade do VRS ocorreu nos primeiros seis meses do ano, estando associado principalmente ao período de troca da estação chuvosa para um período de menor pluviosidade. Houve co-circulação dos subgrupos A e B nos anos de 2001 e 2003. Em 2000, 2005 e 2006 somente o subgrupo A circulou. Entretanto no ano de 2004 foi registrada a ocorrência somente do subgrupo B. Dentro do período estudado, genótipos distintos da proteína G do subgrupo A (GA2 e GA5) e do subgrupo B (SAB1 e SAB3) foram detectados, indicando o primeiro relato da circulação do genótipo SAB1 na América do Sul. Em 2004, um cluster diferenciado dos demais genótipos circulantes foi encontrado, sendo este denominado BRB1. A análise do gene codificador da proteína F permitiu a identificação de mutações na sequência nucleotídica resultando em trocas na cadeia aminoacídica da mesma. Este estudo representa o primeiro relato sobre dados da epidemiologia molecular do Vírus Respiratório Sincicial na região Norte do Brasil.

Influência do resveratrol sobre metabolismo energético, estresse oxidativo e expressão SIRT1 no miocárdio de ratos diabéticos

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

Cardiac Energy Metabolism and Oxidative Stress Biomarkers in Diabetic Rat Treated with Resveratrol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ineraction Model Between HRSV G-Protein and Flavonoids

Background: Acute respiratory infections (ARI) are the leading cause of infant mortality in the world, and human respiratory syncytial virus (HRSV) is one of the main agents of ARI. One of the key targets of the adaptive host immune response is the RSV G-protein, which is responsible for attachment to the host cell. There is evidence that compounds such as flavonoids can inhibit viral infection in vitro. With this in mind, the main purpose of this study was to determine, using computational tools, the potential sites for interactions between G-protein and flavonoids. Results: Our study allowed the recognition of an hRSV G-protein model, as well as a model of the interaction with flavonoids. These models were composed, mainly, of -helix and random coil proteins. The docking process showed that molecular interactions are likely to occur. The flavonoid kaempferol-3-O-α-L-arabinopyranosil-(2 → 1)-α-L-apiofuranoside-7-O-α-L-rhamnopyranoside was selected as a candidate inhibitor. The main forces of the interaction were hydrophobic, hydrogen and electrostatic. Conclusions: The model of G-protein is consistent with literature expectations, since it was mostly composed of random coils (highly glycosylated sites) and -helices (lipid regions), which are common in transmembrane proteins. The docking analysis showed that flavonoids interact with G-protein in an important ectodomain region, addressing experimental studies to these sites. The determination of the G-protein structure is of great importance to elucidate the mechanism of viral infectivity, and the results obtained in this study will allow us to propose mechanisms of cellular recognition and to coordinate further experimental studies in order to discover effective inhibitors of attachment proteins.

Estudo da Estrutura da Proteína SH do Vírus Sincicial Respiratório Humano: análise funcional da estrutura pentamérica por ferramentas de bioinformática

Pós-graduação em Biofísica Molecular - IBILCE

Role of Neutralizing Antibodies in Adults With Community-Acquired Pneumonia by Respiratory Syncytial Virus

Background. Respiratory syncytial virus (RSV) has been implicated in the etiology of adult community-acquired pneumonia (CAP). We investigated RSV infection in Chilean adults with CAP using direct viral detection, real-time reverse-transcription polymerase chain reaction (rtRT-PCR), and serology (microneutralization assay). Methods. RSV, other respiratory viruses, and bacteria were studied by conventional and molecular techniques in adults aged >= 18 years presenting with CAP to the healthcare facilities in Santiago, Chile from February 2005 through December 2007. Results. All 356 adults with CAP enrolled had an acute blood sample collected at enrollment, and 184 had a convalescent blood sample. RSV was detected in 48 cases (13.4%). Immunofluorescence assay and viral isolation each detected only 1 infection (0.2%), whereas rtRT-PCR was positive in 32 (8.9%) cases and serology was positive in 20 (10.8%) cases. CAP clinical characteristics were similar in RSV-infected and non-RSV-infected cases. RSV-specific geometric mean serum-neutralizing antibody titer (GMST) was significantly lower at admission in the 48 RSV-infected cases compared with 308 non-RSV-infected adults (GMST in log(2): RSV/A 8.1 vs 8.9, and RSV/B 9.3 vs 10.4; P < .02). Conclusions. RSV infection is frequent in Chilean adults with CAP. Microneutralization assay was as sensitive as rtRT-PCR in detecting RSV infection and is a good adjunct assay for diagnostic research. High RSV-specific serum-neutralizing antibody levels were associated with protection against common and severe infection. The development of a vaccine could prevent RSV-related CAP in adults.

Severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors

Abstract Background Lower respiratory tract infection (LRTI) is a major cause of pediatric morbidity and mortality, especially among non-affluent communities. In this study we determine the impact of respiratory viruses and how viral co-detections/infections can affect clinical LRTI severity in children in a hospital setting. Methods Patients younger than 3 years of age admitted to a tertiary hospital in Brazil during the months of high prevalence of respiratory viruses had samples collected from nasopharyngeal aspiration. These samples were tested for 13 different respiratory viruses through real-time PCR (rt-PCR). Patients were followed during hospitalization, and clinical data and population characteristics were collected during that period and at discharge to evaluate severity markers, especially length of hospital stay and oxygen use. Univariate regression analyses identified potential risk factors and multivariate logistic regressions were used to determine the impact of specific viral detections as well as viral co-detections in relation to clinical outcomes. Results We analyzed 260 episodes of LRTI with a viral detection rate of 85% (n = 222). Co-detection was observed in 65% of all virus-positive episodes. The most prevalent virus was Respiratory Syncytial Virus (RSV) (54%), followed by Human Metapneumovirus (hMPV) (32%) and Human Rhinovirus (HRV) (21%). In the multivariate models, infants with co-detection of HRV + RSV stayed 4.5 extra days (p = 0.004), when compared to infants without the co-detection. The same trends were observed for the outcome of days of supplemental oxygen use. Conclusions Although RSV remains as the main cause of LRTI in infants our study indicates an increase in the length of hospital stay and oxygen use in infants with HRV detected by RT-PCR compared to those without HRV. Moreover, one can speculate that when HRV is detected simultaneously with RSV there is an additive effect that may be reflected in more severe clinical outcome. Also, our study identified a significant number of children infected by recently identified viruses, such as hMPV and Human Bocavirus (HBov), and this is a novel finding for poor communities from developing countries.

Community-acquired pneumonia in Chile: the clinical relevance in the detection of viruses and atypical bacteria

Background Adult community-acquired pneumonia (CAP) is a relevant worldwide cause of morbidity and mortality, however the aetiology often remains uncertain and the therapy is empirical. We applied conventional and molecular diagnostics to identify viruses and atypical bacteria associated with CAP in Chile. Methods We used sputum and blood cultures, IgG/IgM serology and molecular diagnostic techniques (PCR, reverse transcriptase PCR) for detection of classical and atypical bacteria (Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumoniae) and respiratory viruses (adenovirus, respiratory syncytial virus (RSV), human metapneumovirus, influenza virus, parainfluenzavirus, rhinovirus, coronavirus) in adults >18 years old presenting with CAP in Santiago from February 2005 to September 2007. Severity was qualified at admission by Fine's pneumonia severity index. Results Overall detection in 356 enrolled adults were 92 (26%) cases of a single bacterial pathogen, 80 (22%) cases of a single viral pathogen, 60 (17%) cases with mixed bacterial and viral infection and 124 (35%) cases with no identified pathogen. Streptococcus pneumoniae and RSV were the most common bacterial and viral pathogens identified. Infectious agent detection by PCR provided greater sensitivity than conventional techniques. To our surprise, no relationship was observed between clinical severity and sole or coinfections. Conclusions The use of molecular diagnostics expanded the detection of viruses and atypical bacteria in adults with CAP, as unique or coinfections. Clinical severity and outcome were independent of the aetiological agents detected.

Die Rolle Prolin-reicher Sequenzmotive des Core-Proteins in der Morphogenese des Hepatitis-B Virus

Diese Arbeit hatte zum Ziel, den Ausschleusungsmechanismus des Hepatitis-B Virus zu beleuchten. Es ist bisher unbekannt, wie das virale Nukleokapsid umhüllt und das reife Virion aus der Leberzelle freigesetzt wird. Bei einigen RNA-Viren, beispielsweise HIV-1, Ebola oder RSV, vermitteln so genannte Late-Domänen im viralen Kapsid- oder Matrix-Protein die Knospung der Viren an intrazellulären Membranen oder der Plasmamembran. Da das HBV-Core-Protein ähnliche Sequenzen trägt, wurde in der vorliegenden Arbeit überprüft, welche Rolle diese im viralen Replikationszyklus spielen. Meine Ergebnisse zeigen, dass die beiden Prolin-reichen Sequenzen PPAY (129-132) und PPNAP (134-138), die retroviralen Late-Domänen ähneln, für die HBV-Morphogenese essentiell sind. Mutationen einzelner Aminosäuren innerhalb dieser Motive führen zu Phänotypen mit verändertem Kapsid-, Nukleokapsid- und Virus-Bildungs-Vermögen. Insbesondere sind die Aminosäure Tyrosin 132 des Motivs PPAY und die Prolinreste 134 und 135 des Motivs PPNAP erforderlich, da diese schon für die Bildung der Kapside unentbehrlich sind. Charakteristisch für beide Motive sind auch die hier gezeigten Interaktionen mit speziellen Wirtszellfaktoren, deren physiologische Funktion es ist, zelluläre Proteine in den endosomalen Sortierungsprozess einzuschleusen. Im Vordergrund stehen hier die E3 Ub-Ligase Nedd4, welche Proteine mit Ub konjugiert und diese so signifikant für die Einschleusung in das endosomale System markiert, und Tsg101, das als zentrale Komponente des ESCRT-I-Komplexes für die Erkennung von ubiquitinierten Proteinen zuständig ist und diese dadurch in die ESCRT-Kaskade des multivesikulären Endosoms einführt. Für die genannten Interaktionen ist das Motiv PPAY und hier wieder speziell das Tyrosin 132 des HBV-Core-Proteins für die Wechselwirkung mit Nedd4 notwendig. Hingegen vermittelt die L-Domänen-ähnliche Sequenz PPNAP die Assoziation von Core mit Tsg101, wobei die beiden Prolinreste 134 und 135 und auch das Asparagin 136 für die Interaktion essentiell sind. Sowohl Nedd4 als auch Tsg101 wirken im Zusammenhang mit Ubiquitin, weshalb eine Ubiquitinierung von Core, trotz bislang negativer Nachweise, wahrscheinlich ist. Zugunsten dieser Annahme spricht auch mein Nachweis, dass der Lysinrest an Position 96 des Core-Proteins, als potentieller Ub-Akzeptor, gerade in späten Schritten eine essentielle Rolle spielt. Weiterhin klärungsbedürftig ist auch die Frage, ob Core direkt mit Tsg101 und Nedd4 interagiert, oder ob andere Faktoren dazwischen geschaltet sind. Auch könnte mit Hilfe von siRNA-vermittelten Depletionsversuchen die physiologische Relevanz der Tsg101/Core-und Nedd4/Core-Interaktion weiterführend untersucht werden. Zudem zeigen meine Arbeiten, dass Core mit intrazellulären Membranen assoziiert, weshalb es interessant wäre, zu untersuchen, ob es sich hierbei um Membranen des endosomalen Systems handelt, an denen die finalen Schritte der Virus-Morphogenese stattfinden könnten.

Monocyte differentiation toward regulatory dendritic cells is not affected by respiratory syncytial virus-induced inflammatory mediators

Airway epithelial cells were shown to drive the differentiation of monocytes into dendritic cells (DCs) with a suppressive phenotype. In this study, we investigated the impact of virus-induced inflammatory mediator production on the development of DCs. Monocyte differentiation into functional DCs, as reflected by the expression of CD11c, CD123, BDCA-4, and DC-SIGN and the capacity to activate T cells, was similar for respiratory syncytial virus (RSV)-infected and mock-infected BEAS-2B and A549 cells. RSV-conditioned culture media resulted in a partially mature DC phenotype, but failed to up-regulate CD80, CD83, CD86, and CCR7, and failed to release proinflammatory mediators upon Toll-like receptor (TLR) triggering. Nevertheless, these DCs were able to maintain an antiviral response by the release of Type I IFN. Collectively, these data indicate that the airway epithelium maintains an important suppressive DC phenotype under the inflammatory conditions induced by infection with RSV.

Twelve years' detection of respiratory viruses by immunofluorescence in hospitalised children: impact of the introduction of a new respiratory picornavirus assay

Background Direct immunofluorescence assays (DFA) are a rapid and inexpensive method for the detection of respiratory viruses and may therefore be used for surveillance. Few epidemiological studies have been published based solely on DFA and none included respiratory picornaviruses and human metapneumovirus (hMPV). We wished to evaluate the use of DFA for epidemiological studies with a long-term observation of respiratory viruses that includes both respiratory picornaviruses and hMPV. Methods Since 1998 all children hospitalized with respiratory illness at the University Hospital Bern have been screened with DFA for common respiratory viruses including adenovirus, respiratory syncytial virus (RSV), influenza A and B, and parainfluenza virus 1-3. In 2006 assays for respiratory picornaviruses and hMPV were added. Here we describe the epidemiological pattern for these respiratory viruses detected by DFA in 10'629 nasopharyngeal aspirates collected from 8'285 patients during a 12-year period (1998-2010). Results Addition of assays for respiratory picornaviruses and hMPV raised the proportion of positive DFA results from 35% to 58% (p < 0.0001). Respiratory picornaviruses were the most common viruses detected among patients ≥1 year old. The seasonal patterns and age distribution for the studied viruses agreed well with those reported in the literature. In 2010, an hMPV epidemic of unexpected size was observed. Conclusions DFA is a valid, rapid, flexible and inexpensive method. The addition of assays for respiratory picornaviruses and hMPV broadens its range of viral detection. DFA is, even in the "PCR era", a particularly adapted method for the long term surveillance of respiratory viruses in a pediatric population.

Lack of an exaggerated inflammatory response on virus infection in cystic fibrosis

Respiratory virus infections play an important role in cystic fibrosis (CF) exacerbations, but underlying pathophysiological mechanisms are poorly understood. We aimed to assess whether an exaggerated inflammatory response of the airway epithelium on virus infection could explain the increased susceptibility of CF patients towards respiratory viruses. We used primary bronchial and nasal epithelial cells obtained from 24 healthy control subjects and 18 CF patients. IL-6, IL-8/CXCL8, IP-10/CXCL10, MCP-1/CCL2, RANTES/CCL5 and GRO-α/CXCL1 levels in supernatants and mRNA expression in cell lysates were measured before and after infection with rhinoviruses (RV-16 and RV-1B) and RSV. Cytotoxicity was assessed by lactate dehydrogenate assay and flow cytometry. All viruses induced strong cytokine release in both control and CF cells. The inflammatory response on virus infection was heterogeneous and depended on cell type and virus used, but was not increased in CF compared with control cells. On the contrary, there was a marked trend towards lower cytokine production associated with increased cell death in CF cells. An exaggerated inflammatory response to virus infection in bronchial epithelial cells does not explain the increased respiratory morbidity after virus infection in CF patients.

The prevalence of penicillin-non-susceptible Streptococcus pneumoniae among children aged < 5 years correlates with the biannual epidemic activity of respiratory syncytial virus

This study investigated whether the epidemiology of penicillin-non-susceptible pneumococci (PNSP) colonising small children correlated with the biannual epidemic activity of respiratory syncytial virus (RSV). Colonisation rates and the prevalence of PNSP among paediatric outpatients aged < 5 years was analysed between January 1998 and September 2003 using an established national surveillance network. Resistance trends were investigated using time-series analysis to assess the correlation with the biannual pattern of RSV infections and national sales of oral paediatric formulations of antibiotics and antibiotic prescriptions to children aged < 5 years for acute respiratory tract infections. PNSP rates exhibited a biannual cycle in phase with the biannual seasonal RSV epidemics (p < 0.05). Resistance rates were higher during the winter seasons of 1998-1999 (20.1%), 2000-2001 (16.0%) and 2002-2003 (19.1%), compared with the winter seasons of 1997-1998 (8.2%), 1999-2000 (11.6%) and 2001-2002 (9.5%). Antibiotic sales and prescriptions showed regular peaks during each winter, with no significant correlation with the biannual pattern of RSV activity and seasonal trends of PNSP. RSV is an important determinant of the spread of PNSP and must be considered in strategies aimed at antimicrobial resistance control.

Respiratory syncytial virus infection in 406 hospitalized premature infants: results from a prospective German multicentre database

Premature birth, chronic lung disease of prematurity (CLD), congenital heart disease and immunodeficiency predispose to a higher morbidity and mortality in respiratory syncytial virus (RSV) infection. This study describes the preterms hospitalised with RSV infection from the prospective German DSM RSV Paed database. The DMS RSV Paed database was designed for the prospective multicentre documentation and analysis of clinically relevant aspects of the management of inpatients with RSV infection. This study covers six consecutive RSV seasons (1999-2005); the surveillance took place in 14 paediatric hospitals in Germany. Of the 1,568 prospectively documented RSV infections, 26% (n=406) were observed in preterms [vs. 1,162 children born at term (74%)] and 3% (n=50) had CLD, of which 49 had received treatment in the last 6 months ('CLDplus'). A significantly higher proportion in the preterm group had congenital heart disease, nosocomial infection, and neuromuscular impairment. There were significantly more children older than 24 months in the preterm group. The attributable mortality was 0.2% (n=2) in children born at term vs. 1.2% (n=5) in the preterm group (p=0.015) [preterm plus CLD 8.0% (n=4 of 50); McIntosh grade 1, 8.6% (n=3 of 35) and McIntosh Grade 4, 15% (n=3 of 20)]. Eight patients were categorized as 'palivizumab failures'. In the multivariate analysis, premature birth, CLD(plus), and nosocomial infection were significantly and independently associated with the combined outcome 'complicated course of disease'. In conclusion, this is the first prospective multicentre study from Germany that confirms the increased risk for severe RSV disease in preterms, in particular in those with CLD treated in the last 6 months before the onset of the infection. From the perspective of our results, the statements of the German Society of Paediatric Infectious Diseases considering the use of passive immunisation (2003) seem reasonable.

Nosocomial infection: A risk factor for a complicated course in children with respiratory syncytial virus infection - Results from a prospective multicenter German surveillance study

BACKGROUND: Nosocomially acquired respiratory syncytial virus infections (RSV-NI) may cause serious problems in hospitalized paediatric patients. Hitherto, prospectively collected representative data on RSV-NI from multicenter studies in Germany are limited. METHODS: The DMS RSV Ped database was designed for the prospective multicenter documentation and analysis of clinically relevant aspects of the management of inpatients with RSV-infection. The study covered six consecutive seasons (1999-2005); the surveillance took place in 14 paediatric hospitals in Germany. RESULTS: Of the 1568 prospectively documented RSV-infections, 6% (n=90) were NI and 94% (n=1478) were community acquired (CA). A significantly higher proportion in the NI group displayed additional risk factors like prematurity, chronic lung disease, mechanical ventilation (med. history), congenital heart disease, and neuromuscular impairment. Of all NI, 55% occurred in preterms (30.6% of all RSV-infections in preterms with severe chronic lung disease of prematurity were NI). Illness severity as well as the total mortality, but not the attributable mortality was significantly higher in the NI group. In the multivariate analysis, NI was significantly associated with the combined outcome 'complicated course of disease'. CONCLUSION: This is the first prospective multicenter study from Germany, which confirms the increased risk of a severe clinical course in nosocomially acquired RSV-infection. Of great concern is the high rate of (preventable) NI in preterms, in particular in those with severe chronic lung disease or with mechanical ventilation due to other reasons.