Browsing by Issue Date, starting with "2017-09-10"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- Phenotypic and genotypic profile of susceptibility to neuraminidase inhibitors of influenza A(H3) circulating in Portugal during 2016/2017 seasonPublication . Guiomar, Raquel; Pechirra, Pedro; Costa, Inês; Conde, Patrícia; Cristóvão, Paula; Mendonça, Joana; Vieira, Luís; Borges, Vítor; Gomes, João PauloBackground: The influenza antiviral surveillance is one of the key areas for influenza control. The neuraminidase inhibitors (NAI) play an important role in flu treatment especially for high risk patients. This study aims to determine the NAI susceptibility profile of influenza A(H3) detected during 2016/2017 season in Portugal and to evaluate the emergence of new variants by deep sequencing analysis related to NAI resistance. Methods: Nasopharyngeal swabs were collected from patients with influenza like illness (ILI) selected in primary care settings in the scope of the National Influenza Surveillance Program during 2016/2017 season. For 134 A(H3) viruses, isolated in MDCK-Siat1 cells, the phenotypic antiviral drug susceptibility assay to NAI (Oseltamivir and zanamivir) was performed. Viral RNA was extracted directly from biological samples and after multiplex PCR amplification, the whole genome was sequenced for 84 influenza A(H3) viruses by deep sequencing on a MiSeq platform. The neuraminidase gene sequences were assembled using a in-house multi-software pipeline with a mean depth of coverage of 1144x. Multiple gene alignments and mutational analysis was performed on MEGA software 6.0. All neuraminidase sequences were submitted to Flusurver(http://flusurver.bii.a-star.edu.sg/) in order to detect any mutation associated with susceptibility to neuraminidase inhibitors. Results: All 134 A(H3) strains had IC50 compatible with susceptibility to both NAI. The IC50 mean values were 0,30 (IQR 0,22-0,36) for oseltamivir and 0,49 (IQR 0,40-0,59) for zanamivir. The IC50 were unchanged for viruses that belonged to both co-circulating clades 3C.2a (A/Hong Kong/4801/2014-like) and 3C.2a1 (A/Bolzano/7/2016-like) although amino acid replacements were observed in NA. By deep sequencing analysis no variations were identified in any of the known resistant markers for NAI. Intrahost single nucleotide variants (iSNV) (frequency above 20% and under 95%) were detected in 4/84 (5%) viruses in nucleotide positions not related with NAI susceptibility. A further synonymous iSNV was observed in 23/84 (27%),with a frequency range 97,8-100,0%, at position 666 (amino acid 222). Curiously, non-synonymous changes affecting this codon ( I222V/L substitution) have been associated with reduced susceptibility to oseltamivir and NA activity, although wild-type I222 was observed in 100% of viruses in this study. Majority of these viruses belonged to the 3C.2a1clade(A/Bolzano/7/2016-like). Conclusions: All of the tested A(H3)viruses were susceptible to oseltamivir and zanamivir. Isolates possessing iSNV`s expressed the wild-type aa residue and had IC50 compatible with NAI susceptible. NAI`s should be considered a good therapeutic options for influenza A(H3) infection, prioritized to high risk groups. Whole genome deep sequencing, directly from the biological samples, proved to be a good laboratory method to monitor the emergence of iSNV that could lead to a resistant genotype. This approach could be used in management of influenza severe infections under NAI treatment.
- Lower prevalence of protective antibodies for 2015/2016 influenza A(H1)pdm circulating strains comparing to seroprevalence for 2009 influenza A(H1)pdm virusPublication . Guiomar, Raquel; Conde, Patrícia; Cristóvão, Paula; Costa, Inês; Pechirra, Pedro; Portuguese Laboratory Network for the Diagnosis os Influenza InfectionBackground: Since 2009, influenza A(H1)pdm09 is circulating in the human population infecting in different ways specific age groups. The study aims to assess the seroprotection for A/California /07/2009 vaccine strain and evaluate the seroprotection for the circulating A(H1)pdm09 strains (clade 6B) in the Portuguese population. Seroepidemiological data can determine the vulnerable populations to disease and support intervention and action regarding vaccination programmes and other preventive measures, particularly in high-risk groups. Methods: To study influenza immunity a non-probabilistic sample was used. Samples were collected from people attending to hospital laboratories (n=13) for other reasons aside from influenza infection. We developed a cross-sectional study based on a convenience sample of 734 sera collected during July 2016, from all age groups (0–4; 5–14; 15–44; 45-64 and ≥65 years old), both genders, covering mainland and Atlantic islands. Sera were randomly selected. All samples were anonymized and recorded data: district residence/sample collection, gender and age. Antibody titers to A(H1)pdm09 virus strains [A/California /07/2009 and A/Lisboa/58/2015 (clade 6B)] were assessed by hemagglutination inhibition (HI) assay. HI titer>40 were considered protective. Seroprevalence estimates, overall and by age group, were calculated with 95% confidence intervals (95% CI). The HA1 subunit of the hemagglutinin gene from A(H1)pdm09 viruses used in HI were sequenced. Results: In July 2016, the prevalence of protective antibodies for influenza A/California/07/2009 was 38% (95% CI: 34–41) and for A/Lisboa /58/2015 was 23% (95% CI: 21–27). Highest seroprevalence was observed in 5-14 age group for both strains, 55% (95% CI: 47–63) for A/California/07/2009 and 42% (95% CI: 35–50) for A/Lisboa/58/2015. The lowest seroprevalences were detected in the 65+ age group for A/California/07/2009 (28%; 95% CI: 22–36) and in the 45-64 for the A/Lisboa /58/2015 (9%; 95% CI: 6-15). Was observed a reduced prevalence of protective antibodies for A/Lisboa /58/2015 for all age groups, although a higher decrease was seen in the adults 45-64 years old (24% drop in seroprevalence). The influenza A/Lisboa/58/2015 presented four amino acid substitutions in antigenic sites: S162N e K163Q (Sa), S185T (Sb) and S203T (Ca1), belonging to 6B.1clade. Conclusions: Although 38% of study population have demonstrated to have seroprotection for A(H1)pdm09 vaccine strain this could not represent seroprotection to the currently circulating A(H1)pdm09 strains. Individuals in the age group of 45-64 years old are more susceptible to infection by currently circulating influenza A(H1)pdm09 viruses. The presence of K163Q (Sa), S185T (Sb) substitutions are likely to be involved in antigenic drift, as previously described, allowing the virus to escape from immune response namely the one that could be induced by the vaccine. In future this event should be closely monitored, although WHO recommended a new A(H1)pdm09 strain to be included in the next season flu vaccine.
- Hazard assessment of benchmark metallic nanomaterials in alveolar epithelial cellsPublication . Saruga, Andreia; Louro, Henriqueta; Pinhão, Mariana; Silva, Maria JoãoThe fast development of nanotechnology has led to the manufacturing of a wide array of nanomaterials (NMs). Despite the number of studies addressing NMs toxicity, uncertainties about their safety remain, representing a challenge to regulatory authorities. This work intended to assess the toxicity of metallic NMs in alveolar epithelial cells and to relate the effects with their physicochemical properties. Benchmark NMs (JRC) - CeO2 (NM-212), TiO2 (NM-100) and BaSO4 (NM-220) - were dispersed and their properties in the culture medium were evaluated by DLS. A549 cells were exposed to each NM for cytotoxicity (MTT and plating efficiency assays) and genotoxicity (comet and cytokinesis-blocked micronucleus, CBMN, assays) assessment. A homogeneous dispersion that remained stable in culture medium was achieved for all NMs. The CeO2NM was the only one that decreased cells’ proliferative capacity after 8 days exposure. As to the genotoxicity, the TiO2NM significantly increased the level of DNA damage following 3h and 24h exposure, whereas the CeO2NM caused only a slight increase in DNA damage at 3h exposure. None of the NMs tested positive by the CBMN assay. BaSO4NM was neither cytotoxic nor genotoxic. In conclusion, this study contributed to the hazard assessment of different benchmark metallic NMs, disclosing diverse biological effects that will be interpreted considering the inherent physicochemical properties. The identification of key features and pathways that drive NMs’ toxicity is paramount to allow prediction of their adverse effects, avoiding the huge task of testing every new NM.
- A network integrative approach to unravel new links between NMD and mRNA processing pathwaysPublication . Nogueira, Gonçalo; Pinto, Francisco; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation termination codons (PTCs). This process has been associated with many genetic diseases and some forms of cancer caused by nonsense or frameshift mutations that introduce PTCs. Moreover, recent studies have shown that NMD is also involved in the regulation of a large number of transcripts, suggesting a major role in the control of gene expression. To further investigate the biological relevance of NMD and how this process can be modulated, we used a network analysis approach that integrates 1) protein-protein, 2) kinasetarget, 3) phosphatase-target, 4) miRNA-target, 5) transcription factors-target, 6) gene co-expression, 7) ubiquitination and 8) signaling interactions. The generated network was used to find novel NMD-associated proteins, prioritizing candidates with simultaneous interactions with different mRNA processing pathways (mRNA splicing, mRNA transport, mRNA translation and mRNA decay). Taking in account all information sources integrated in our network, we have created a scoring algorithm to identify new potentially important players in NMD, which can be essential to further understand the interplay between mRNA translation, PTC definition and NMD. Due to the diversity of regulatory links integrated in this workflow, we propose it can be applied to find molecular bridges between related biological processes and generate novel hypotheses about the molecular mechanisms co-regulating these phenomena.