The His131Arg substitution in the FCGR2A gene (rs1801274) is not associated with the severity of influenza A(H1N1)pdm09 infection.

BACKGROUND: The virulence and pathogenicity of different influenza strains are responsible for a more or less severe disease. Recent studies have attempted to understand how host genetic factors may influence the clinical presentation of the disease. In the present study, the His131Arg (rs1801274) polymorphism was investigated in individuals from a Brazilian admixed population with a diagnosis of influenza A(H1N1)pdm09 infection. METHODS: In the present study, the influence of the His131Arg (rs1801274) polymorphism, a variant of the FCGR2A gene, was investigated in 436 patients with a diagnosis of influenza A(H1N1)pdm09, evaluated at health services in the northern and northeastern regions of Brazil between June 2009 and August 2010. Patients were divided into a group of non-hospitalized patients (n = 192) and a group of hospitalized patients (n = 244; 100 of them died). RESULTS: No significant difference in the allele or genotype frequencies of the rs1801274 polymorphism was observed between groups (p = 0.952 and p = 0.388). Multinomial logistic regression showed no effect of the rs1801274 polymorphism on severity or death of patients from the Brazilian admixed population (p = 0.368 and p = 0.469). CONCLUSIONS: The rs1801274 polymorphism is not associated with severe disease in patients infected with influenza A(H1N1)pdm09.

Dinâmica molecular dos vírus Influenza A (H1N1) pandêmico em cinco anos de circulação no Brasil / Molecular dynamic of influenza a (h1n1) pandemic viruses five years of circulation in Brazil

A primeira detecção do vírus Influenza A (H1N1)pdm09 no Brasil aconteceu em maio de2009, e foi seguida de uma extensa disseminação por toda a população brasileira, com grandeimpacto em morbidade e mortalidade. Para entender a dinâmica molecular do Influenza A(H1N1)pdm09 no país, a presente tese reuniu sete trabalhos que abordaram a análise filogenéticadeste agente viral durante e após o período pandêmico (2009 a 2014) e buscou indentificarpolimorfismos virais associados à virulência e à resistência ao antiviral Oseltamivir (OST). Para isso,as metodologias realizadas foram o sequenciamento dos genes de hemaglutinina (HA) eneuraminidase (NA) utilizando a metodologia de Sanger e a metodologia de pirosequenciamento paradetectar polimorfismos de base única (SNPs).Nossos resultados revelaram a circulação de nove grupos filogenéticos ao longo dos cincoanos do estudo, indicando uma substituição temporal dos grupos e ocasionalmente umaestratificação geográfica. No entanto, nenhum dos grupos filogenéticos identificados foramassociados com um pior prognóstico da infecção por influenza. Ao contrário do que foi observado emestudos anteriores, as mutações K-15E e Q310H no gene HA não se associaram ao aumento devirulência, mesmo na infecção de indivíduos imunocomprometidos. Por outro lado, polimorfismos noresíduo 222 da HA, que caracterizaram a presença de quasispecies virais, mostraram uma forteassociação com a gravidade da infecção, especialmente em gestantes. Nesta tese, tambémrealizamos a vigilância de marcadores de resistência no gene NA. Entre as amostras analisadasencontramos sete vírus com a mutação H275Y e dois com S247N, esses marcadores estãorelacionados com a diminuição de sensibilidade ao antiviral OST. Entre as amostras resistentes, agrande maioria foi detectada na região Sul do Brasil, em pacientes que não receberam OST. Istosugere uma possível transmissão sustentada do vírus resistentes no país...

The Influenza A (H1N1)pdm09 virus was first detected in May 2009 in Brazil and later resultedin an extensive spread throughout the Brazilian population with a severe impact on morbidity andmortality. To understand the molecular dynamic of (H1N1)pdm09 virus in Brazil this thesis groupedseven papers which approached the phylogenetic reconstruction of the virus during and after thepandemic period (2009 to 2014) and the genomic identification of viral polymorphisms associated withvirulence or antiviral resistance to Oseltamivir (OST). For this, we performed genome sequencing,focusing especially on the hemagglutinin (HA) and neuraminidase (NA) genes using conventionalSanger sequencing and PyroMark 96ID to detect single nucleotide polymorphisms (SNPs).Our results showed that in Brazil nine (H1N1)pdm09 phylogenetic groups circulated along thefive years of the study, indicating a temporal replacement of groups and ocasionally a geographicstratification. However, no phylogenetic group seemed to be associated with a worse clinical outcome.The increased virulence observed in previous studies with a 2009 group bearing the genetic markersK-15E and Q310H was not confirmed in our analyses, even evaluating an immunocompromisedpopulation. On the other hand, polymorphysms at position 222 of HA gene, which characterized thepresence of viral quasispecies, showed an association with increased virulence in brazilian samples,especially in pregnant women. In this study we also performed surveillance of resistance markers atthe NA gene. From the analysed samples we found seven viruses with H275Y and two with S247Nmutation, that diminish the sensibility to oseltamivir (OST). Among the resistant samples, the largemajority was detected in the Southern region of Brazil in patients that did not receive OST. Thissuggests a possible sustained transmission of resistant virus in the country...

On the estimation of the reproduction number based on misreported epidemic data.

Epidemic data often suffer from underreporting and delay in reporting. In this paper, we investigated the impact of delays and underreporting on estimates of reproduction number. We used a thinned version of the epidemic renewal equation to describe the epidemic process while accounting for the underlying reporting system. Assuming a constant reporting parameter, we used different delay patterns to represent the delay structure in our model. Instead of assuming a fixed delay distribution, we estimated the delay parameters while assuming a smooth function for the reproduction number over time. In order to estimate the parameters, we used a Bayesian semiparametric approach with penalized splines, allowing both flexibility and exact inference provided by MCMC. To show the performance of our method, we performed different simulation studies. We conducted sensitivity analyses to investigate the impact of misspecification of the delay pattern and the impact of assuming nonconstant reporting parameters on the estimates of the reproduction numbers. We showed that, whenever available, additional information about time-dependent underreporting can be taken into account. As an application of our method, we analyzed confirmed daily A(H1N1) v2009 cases made publicly available by the World Health Organization for Mexico and the USA.

Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero).

Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines. We generated a Vero cell line adapted to grow in suspension (sVero) in a serum-free medium and evaluated it for its potential as host cell for influenza vaccine production. Initially we studied the capacity of sVero cells to grow in the presence of incremental concentrations of trypsin. In comparison with adherent Vero cells (aVero), we found that sVero cells maintain their growth kinetics even with a three-fold increase in trypsin concentration. The influence of the conditions of infection on the yield of H1N1 produced in serum-free suspension cultures of sVero cells was investigated by a 2(2) full factorial experiment with center point. Each experiment tested the influence of the multiplicity of infection (m.o.i.) and trypsin concentration, on production yields at two levels, in four possible combinations of levels and conditions, plus a further combination in which each condition was set in the middle of its extreme levels. On the basis of software analysis, a combination of m.o.i. of 0.0066TCID(50%)/cell and trypsin concentration of 5µg/1.0×10(6) cells with a desirability of 0.737 was selected as the optimized condition for H1N1 production in sVero cells. Our results show the importance of proper selection of infection conditions for H1N1 production on sVero cells in serum-free medium.

Genetic and phylogenetic analyses of influenza A H1N1pdm virus in Buenos Aires, Argentina.

An influenza pandemic caused by swine-origin influenza virus A/H1N1 (H1N1pdm) spread worldwide in 2009, with 12,080 confirmed cases and 626 deaths occurring in Argentina. A total of 330 H1N1pdm viruses were detected from May to August 2009, and phylogenetic and genetic analyses of 21 complete genome sequences from both mild and fatal cases were achieved with reference to concatenated whole genomes. In addition, the analysis of another 16 hemagglutinin (HA), neuraminidase (NA), and matrix (M) gene sequences of Argentinean isolates was performed. The microevolution timeline was assessed and resistance monitoring of an NA fragment from 228 samples throughout the 2009 pandemic peak was performed by sequencing and pyrosequencing. We also assessed the viral growth kinetics for samples with replacements at the genomic level or special clinical features. In this study, we found by Bayesian inference that the Argentinean complete genome sequences clustered with globally distributed clade 7 sequences. The HA sequences were related to samples from the northern hemisphere autumn-winter from September to December 2009. The NA of Argentinean sequences belonged to the New York group. The N-4 fragment as well as the hierarchical clustering of samples showed that a consensus sequence prevailed in time but also that different variants, including five H275Y oseltamivir-resistant strains, arose from May to August 2009. Fatal and oseltamivir-resistant isolates had impaired growth and a small plaque phenotype compared to oseltamivir-sensitive and consensus strains. Although these strains might not be fit enough to spread in the entire population, molecular surveillance proved to be essential to monitor resistance and viral dynamics in our country.

Differential activation of host cell signalling pathways through infection with two variants of influenza A/Puerto Rico/8/34 (H1N1) in MDCK cells.

In cell culture-based influenza vaccine production, few efforts have been undertaken to characterise virus-host cell interactions in detail. Two influenza virus strains that grew to different virus titres, and differed in virus dynamics, apoptosis induction and proteome changes were observed. In order to elucidate biological mechanisms related to these differences, the induction of signalling cascades in adherent MDCK cells infected with two variants of influenza A/PuertoRico/8/34 (H1N1) was analysed. The pathways chosen for analysis are key components of the innate immune response and crucial for influenza A virus replication (NF-κB, IRF-3, PI3K-Akt, Jak-Stat, Raf/MEK/ERK, PKR/eIF2α). Interestingly, all investigated pathways were induced stronger by PR8-NIBSC than by PR8-RKI, the virus variant which results in higher virus titres. In particular, PR8-NIBSC infection lead to a higher induction of IFN-beta as well as IFN-stimulated gene expression, which was confirmed by Western blot as well as real-time PCR. Overall, results obtained clearly facilitate interpretation of observations regarding proteome changes and virus-induced apoptosis in cell culture-based vaccine manufacturing processes and support efforts towards design of improved host cell lines.

Influenza H1N1 2009: revisão da primeira pandemia do século XXI / H1N1 Influenza 2009: review of the first pandemic of the XXI century

Em 2009, ocorreu a primeira pandemia de influenza do século XXI e acarretou uma histeria generalizada, especialmente pela falta de informações concretas. Um sintoma comum e até menosprezado como a tosse tornou-se um problema de relacionamento humano. O presente trabalho faz uma revisão sob aspectos epidemiológicos, diagnósticos, terapêutico e de prevenção. O objetivo foi fornecer argumentos para que o clínico possa atuar precocemente frente à Gripe A H1N1 pandêmica

In 2009, there was the first influenza pandemic of the XXI century, which led to widespread hysteria, especially because of the lack of concrete information. A common, even belittled symptom such as cough became a problem in human relationships. This paper is a review on the epidemiology, diagnosis, therapy and prevention of H1N1 influenza. The aim was to provide arguments so that the clinician can act early against the Influenza H1N1 pandemic

Pandemia de Influenza A (N1H1): o que aprender com ela? / Pandemic Influenza A (N1H1): what to learn from it

Pandemias de gripe são eventos naturais que ocorrem periodicamente. O agente da pandemia atual, o vírus Influenza A (H1N1), foi identificado primeiramente no México em abril de 2009, disseminou-se rapidamente e tem causado óbitos principalmente entre adultos jovens. O objetivo deste manuscrito é apresentar os aspectos biológicos envolvidos na eclosão desta pandemia, bem como as estratégias de contenção populacional da pandemia de Influenza. Além das medidas populacionais, cuja eficácia tem sido descrita através de modelos teóricos, atualmente também dispomos de medicamentos com eficácia avaliada em alguns grupos de pacientes. Estes medicamentos reduzem moderadamente o tempo de duração e a gravidade dos sintomas, desde que iniciados precocemente. Esta pandemia, com um grande número de casos, mas causada por um vírus de baixa letalidade, poderia ser manejada preferentemente em Unidades de Atenção Primária à Saúde, que tratariam os casos leves e encaminhariam os graves aos hospitais. Contudo, o que ocorreu em inúmeras cidades foi a sobrecarga das emergências com situações de triagem, forçando os gestores a improvisar hospitais de campanha, containeres e tendas para abrigar o trabalho extra em serviços que já operavam no limite de estrutura física e de recursos humanos. A pandemia de Influenza expôs a fragilidade da nossa rede de atenção básica e a falta de leitos de UTI.

Influenza pandemics are natural events that occur periodically. The pandemic’s current agent, Influenza virus A (H1N1) was first identified in Mexico in April 2009, spread rapidly and has caused deaths mainly among young adults. The objective of this manuscript is to present the biological aspects involved in the outbreak of this pandemic, as well as population-control strategies for pandemic influenza. In addition to the population mitigation measures, whose efficacy has been described by theoretical models, today we also have drugs with efficacy valued in some patient groups. These drugs reduce moderately the duration and severity of symptoms, as long as they are started early. This pandemic, with a large number of cases, but caused by a virus of low lethality, could be managed preferably in Units of Primary Health Care, that would treat the wild cases and forward the severe ones to the hospitals. However, what occurred in numerous cities was the burden on emergency care with triage situations, forcing managers to improvise field hospitals, tents and containers to house the extra work in services that were already at the limit of physical infrastructure and human resources. Pandemic Influenza exposed the fragility of our network of primary care and lack of ICU beds.

A pandemia de Influenza no Rio Grande do Sul: estamos preparados para enfrentar uma segunda onda? / Pandemic Influenza in Rio Grande do Sul: we are prepared to face a second wave?

Devido à relevância do tópico na atualidade, dois artigos nesse número da Revista abordam a epidemia de influenza - “Bioética e Pandemia de Influenza” (3) e “Pandemia de Influenza A (H1N1): o que aprender com ela?” (4). Goldim, revisando aspectos bioéticos, afirma que em uma situação de pandemia, todos são co-responsáveis, não havendo possibilidade de se manter neutro. Dentro desse espírito, este editorial foi delegado a um cardiologista e a um geriatra que procuraram se “engajar no mesmo esforço solidário”, sem representar qualquer entidade, mas como cidadãos brasileiros

Anti-influenza virus activity of propolis in vitro and its efficacy against influenza infection in mice.

BACKGROUND: Propolis has been used worldwide as a dietary supplement to maintain and improve human health. We examined whether ethanol extracts of Brazilian propolis exhibit antiviral activity against influenza virus in vitro and in vivo. METHODS: Among 13 ethanol extracts screened in a plaque reduction assay, four showed anti-influenza virus activity. The anti-influenza efficacy of the four extracts was further examined in a murine influenza virus infection model. The mice were infected intranasally with influenza virus, and the four extracts were orally administered at 10 mg/kg three times daily for seven successive days after infection. RESULTS: In this infection model, only one extract, AF-08, was significantly effective at 10 mg/kg in reducing the body weight loss of infected mice. The doses of 2 and 10 mg/kg were also effective in prolonging the survival times of infected mice significantly, but 0.4 mg/kg was not. The anti-influenza efficacy of AF-08 at 10 mg/kg was confirmed in a dose-dependent manner in mice. AF-08 at 10 mg/kg significantly reduced virus yields in the bronchoalveolar lavage fluids of lungs in infected mice as compared with the control. The reduction of virus yields by AF-08 at 10 mg/kg significantly corresponded to those induced by oseltamivir at 1 mg/kg twice daily from day 1 to day 4 after infection. CONCLUSION: The Brazilian propolis AF-08 was indicated to possess anti-influenza virus activity and to ameliorate influenza symptoms in mice. AF-08 may be a possible candidate for an anti-influenza dietary supplement for humans.