Replicação de vírus Oropouche em macrófagosOropouche virus replication in macrophages

O vírus Oropouche (ORO) pertence à família Bunyaviridae, gênero Orthobunyavirus, sorogrupo Simbu, e é a segunda causa mais freqüente de arbovirose febril no Brasil, depois da dengue. Estima-se que mais de meio milhão de casos de febre do Oropouche tenham ocorrido no Brasil nos últimos 30 anos. Com o aquecimento global do planeta, desmatamentos e conseqüente redistribuição de insetos vetores e animais reservatórios, há risco de disseminação de vírus Oropouche para outras regiões do Brasil e da América do Sul. Para descrever a patogenia da infecção por Oropouche, o objetivo deste trabalho foi identifi car a ocorrência de replicação de vírus Oropouche em macrófagos de hamster in vitro. Neste experimento utilizou-se hamsters (Mesocricetus auratus) como fonte para obtenção de macrófagos peritoniais. Realizou-se estimulação/ativação (cavidade abdominal), sacrifício (48 horas), lavagem da cavidade abdominal e punção. Posteriormente, efetuou-se a cultura celular e infecção com vírus Oropouche da cepa de referência BeAn19991. Amostras foram coletadas em diversos tempos, semeadas em cultura de células Vero em diluições seriadas decimais. A leitura, pós-semeadura, foi verificada através de efeito citopático. Os resultados evidenciam que houve replicação do vírus Oropouche em macrófagos, concordando com outros estudos nos quais vários tipos de vírus são capazes de se replicar em macrófagos, inclusive da mesma família do Oropouche. Entretanto tais estudos foram apenas demonstrativos e não quantitativos. Apenas um estudo, também em in vitro, demonstrou a replicação do vírus Oropouche em células Hela, no qual foi obtido o título de 107,5 TCID50. O presente estudo obteve um título de 105,5 TCID50, demonstrando uma menor eficiência quando comparado ao estudo anterior.Abstract The Oropouche virus (ORO) belong to the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu, and is the second most frequent cause of arboviral febrile illness in Brazil, after dengue. Over 0.5 million cases of ORO fever happened in Brazil in the past 30 years. With the global warming, deforestation and redistribution of vectors and reservoir animals, have increased the risk of ORO virus dissemination to others areas of South America. For description ORO pathogenesis infection the purpose of this work was to identify in vitro the occurrence of Oropouche virus replication in macrophages. In this study to make used syrian hamsters (Mesocricetus auratus) as a source of peritoneal macrophages. Activation/stimulation was done (abdominal cavity), sacrifi ce (48 hours), peritoneal washing was performed and puncture. Cells recovered from the peritoneal lavage were layered and infected with ORO strain BeAn19991. Samples were collected at different times, sowed onto monolayer of Vero cells at serial decimal dilutions. Virus growth was detected by cytophatic effect. The results give evidence of ORO virus replication in macrophages, in accordance with others studies to which some types of virus are capable of reply in macrophages, also of the same family of ORO virus. However such studies had been only demonstrative and not quantitative. But a study, also in vitro, demonstrated ORO virus replication in HeLa cells in witch was obtained titers 107.5 TCID50. The present study obtained titers 105.5 TCID50 demonstrating a lesser effi ciency when compared with the previous study

Phylodynamics and Dispersal of HRSV Entails Its Permanence in the General Population in between Yearly Outbreaks in Children

Background: Human respiratory syncytial virus (HRSV) is one of the major etiologic agents of respiratory tract infections among children worldwide. Methodology/Principal Findings: Here through a comprehensive analysis of the two major HRSV groups A and B (n = 1983) which comprise of several genotypes, we present a complex pattern of population dynamics of HRSV over a time period of 50 years (1956-2006). Circulation pattern of HRSV revealed a series of expansions and fluctuations of co-circulating lineages with a predominance of HRSVA. Positively selected amino acid substitutions of the G glycoprotein occurred upon population growth of GB3 with a 60-nucleotide insertion (GB3 Insert), while other genotypes acquired substitutions upon both population growth and decrease, thus possibly reflecting a role for immune selected epitopes in linkage to the traced substitution sites that may have important relevance for vaccine design. Analysis evidenced the co-circulation and predominance of distinct HRSV genotypes in Brazil and suggested a year-round presence of the virus. In Brazil, GA2 and GA5 were the main culprits of HRSV outbreaks until recently, when the GB3 Insert became highly prevalent. Using Bayesian methods, we determined the dispersal patterns of genotypes through several inferred migratory routes. Conclusions/Significance: Genotypes spread across continents and between neighboring areas. Crucially, genotypes also remained at any given region for extended periods, independent of seasonal outbreaks possibly maintained by re-infecting the general population.FAPESPFAPESP [Nu 00/4205-6

Prospective surveillance study of acute respiratory infections, influenza-like illness and seasonal influenza vaccine in a cohort of juvenile idiopathic arthritis patients

Background\ud Acute respiratory infections (ARI) are frequent in children and complications can occur in patients with chronic diseases. We evaluated the frequency and impact of ARI and influenza-like illness (ILI) episodes on disease activity, and the immunogenicity and safety of influenza vaccine in a cohort of juvenile idiopathic arthritis (JIA) patients.\ud \ud Methods\ud Surveillance of respiratory viruses was conducted in JIA patients during ARI season (March to August) in two consecutive years: 2007 (61 patients) and 2008 (63 patients). Patients with ARI or ILI had respiratory samples collected for virus detection by real time PCR. In 2008, 44 patients were immunized with influenza vaccine. JIA activity index (ACRPed30) was assessed during both surveillance periods. Influenza hemagglutination inhibition antibody titers were measured before and 30-40 days after vaccination.\ud \ud Results\ud During the study period 105 ARI episodes were reported and 26.6% of them were ILI. Of 33 samples collected, 60% were positive for at least one virus. Influenza and rhinovirus were the most frequently detected, in 30% of the samples. Of the 50 JIA flares observed, 20% were temporally associated to ARI. Influenza seroprotection rates were higher than 70% (91-100%) for all strains, and seroconversion rates exceeded 40% (74-93%). In general, response to influenza vaccine was not influenced by therapy or disease activity, but patients using anti-TNF alpha drugs presented lower seroconversion to H1N1 strain. No significant differences were found in ACRPed30 after vaccination and no patient reported ILI for 6 months after vaccination.\ud \ud Conclusion\ud ARI episodes are relatively frequent in JIA patients and may have a role triggering JIA flares. Trivalent split influenza vaccine seems to be immunogenic and safe in JIA patients.This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ 308101/2003 to Dr. Ferriani), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES 56/2007-5 to Dr Carvalho) and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clinicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA 2534/2008 to Dr Carvalho)

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

Abstract\ud \ud \ud \ud Background\ud \ud 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.\ud \ud \ud \ud Methods\ud \ud 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.\ud \ud \ud \ud Results\ud \ud 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.\ud \ud \ud \ud Conclusions\ud \ud 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.This study was supported by Abbott Laboratórios do Brasil Ltda (academic grant), from an unrestricted investigator-generated proposal

Localização da replicação de rinovírus in vitro por hibridização in situ

BV UNIFESP: Teses e dissertaçõe

Epidemiology and population dynamics of HRSV in São Paulo.

<p>Bayesian skyline plots of HRSV genotypes prevalent in São Paulo (top) and seasonal distribution of HRSV cases in São Paulo during the 1995–2005 seasons are shown in the <i>x</i>-axis. GA1, GA3 GA7 and GB4 were excluded from the BSL analysis because of small sample size (n<10). The <i>y</i>-axis (on the left) represents a measure of relative genetic diversity presented as <i>Ne.g</i> reflecting the change in effective number of infections over time; where <i>g</i> is the average generation time. The <i>y</i>-axis (on the right) represents the number of samples in the study. (-) - Number of total samples collected during the period. (-) - Number of all HRSV positive cases identified with monoclonal antibodies and molecular characterization.</p

Global demographic history of HRSV genotypes.

<p>Bayesian skyline plots of complete HRSV sequences of HRSVA (n = 1203) and HRSVB (n = 780). The <i>y</i>-axis represents a measure of relative genetic diversity presented as <i>Ne.g</i> reflecting the change in effective population (a surrogate for number of infections) over time for the complete set of HRSV sequences for HRSVA (n = 1204) and HRSVB (n = 778). The dotted lines define the likelihood bounds corresponding to a 95% confidence interval (CI). (…) - Upper and lower limits for HRSVA; (—) - Upper and lower limits for HRSVB. The arrow represents a shift in dynamics between HRSVA and HRSVB.</p

Population dynamics and genetic diversity of HRSV.

<p>A) Bayesian skyline plots of HRSVA genotypes B) Bayesian skyline plots of HRSVB genotypes. Positively selected amino acid substitution sites are represented as previously described by Botosso et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041953#pone.0041953-Botosso1" target="_blank">[48]</a>. The <i>y</i>-axis represents a measure of relative genetic diversity presented as <i>Ne.g</i> reflecting the change in effective number of infections over time.</p