Browsing by Author "Santos, L.A."
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- Different Mechanisms of Apoptosis by Influenza A and B VirusPublication . Santos, L.A.; Solá, S.; Correia, V.; Rodrigues, C.M.P.; Rebelo-de-Andrade, H.Background and Aims: The ability of Influenza A (infA) viruses to counteract and manipulate the host response to infection is well acknowledged. In addition, the activation of the PI3K/Akt survival pathway by the infA NS1 protein has been described as one of the strategies to delay the apoptotic response of the infected cell. As influenza B (infB) viruses differ genetically and phenotypically from infA viruses, namely at the NS protein level, we aimed to compare general apoptosis and survival pathways induced by each influenza type. Methods: MDCK-SIAT1 cells were infected with infA(H1N1)pdm09 virus A/Portugal/82/2009 (APT82) and infB virus B/Lisboa/08/2006 (BLx08). Activities of caspase-3 -7 -8 and -9 were measured at several time points post-infection (hpi). Total levels of Akt, pAkt, NF-κB, IκB, p53 and β-actin were also examined by Western blot. Results: Our results indicate that the apoptosis process induced by BLx08 was associated with activation of both intrinsic, caspase-9-dependent and extrinsic, caspase-8-dependent pathways as early as at 8hpi. In contrast, APT82-induced apoptosis only involved the activation of the intrinsic pathway, and occurred at 32hpi. Surprisingly, our data show that the activation of the survival pathway PI3K/Akt was significantly increased upon BLx08 infection when compared with APT82 infection. In fact, increased levels of pAkt were observed at the same time of caspase activation in the early phase of BLx08 infection. This, however, did not result in increased downstream NFκB activation, since its inhibitor IκB was also markedly upregulated. Increased p53 levels associated with APT82 infection may also explain the delayed apoptosis response in infA, assessed by caspase activity, as it may require transcriptional activation that it is deregulated and directed to viral replication. Conclusion: InfB and infA infection differs in time and levels of activation of apoptosis and survival signaling pathways. PI3K/Akt activation in infB is not sufficient to inhibit apoptosis. Further studies will clarify this difference and shed light into the use of cellular mechanisms as new ways to fight influenza.
- Evaluation and Characterization of Influenza Antiviral Drug Resistance in Portugal: Major Results and Achievements of a 5-Year StudyPublication . Correia, V.; Santos, L.A.; Giria, M.; Rebelo-de-Andrade, H.In 2007 started to be carried out for the first time in Portugal a study focused on influenza antiviral drug resistance. Three main objectives were established:(1)to determine the antiviral profile of influenza viruses to oseltamivir, zanamivir and amantadine;(2)to determine and monitor the baseline level of susceptibility along winter seasons and for each influenza sub(type);(3)to analyse and characterize the whole genome of viruses that showed phenotypic levels of inhibition to neuraminidase inhibitors(NAIs). NAIs profile was determined phenotypically, using a fluorescence MUNUNA assay, and genotypically by NA and HA sequencing. A total of 340 seasonal viruses(117 A(H3N2),93 A(H1N1),130 B) were tested for oseltamivir and of 297(112 A(H3N2),68 A(H1N1),117 B) for zanamivir. Additionally, 142 A(H1N1)pdm09 viruses were evaluated for both NAIs. Whole genome sequencing was performed in 27 of the A(H1N1)pdm09 viruses. Amantadine profile was determined through M2 pyrosequencing or conventional sequencing in a total of 205 seasonal A viruses(138 A(H3N2),84 A(H1N1)) and of 117 A(H1N1)pdm09 viruses. Main results are: -Resistance to oseltamivir in 27 A(H1N1) seasonal viruses(29%,N=93) from 2007/2008 and 2008/2009 and in one A(H1N1)pdm09 virus(0.7%,N=142) from 2010/2011. These viruses exhibited a highly reduced level of inhibition to oseltamivir by phenotypic analysis (170-650 IC50 fold-change) and NA H275Y mutation; -One suspected case of clinical resistance to oseltamivir with a mixed population of H275Y viruses(73,8%H275,26.2%Y275); -No resistance to zanamivir; -Dual reduced susceptibility to oseltamivir and zanamivir in one B virus(0,85%,N=117) and in two A(H1N1)pdm09 viruses(1,41%,N=142). These viruses exhibited a 2-4 IC50 fold-change level of inhibition to both NAIs. A mixed population of D197N viruses was found in the B virus(56%D197,44%N197) and the two A(H1N1)pdm09 viruses shared NA I223V and PB2 V480I mutations; -Resistance to amantadine in 49 A(H3N2) viruses(35,5%,N=138) from 2005/2006 to 2008/2009(46 S31N,3 S31N+V27A), and in all A(H1N1)pdm09 viruses(S31N). This 5-year study allowed to establish a technical platform for influenza antiviral drug resistance evaluation, to timely detect the emergence of resistant viruses, to acquire know-how on the natural variation of virus susceptibility, and to contribute for the management of cases suspected of clinical resistance. Additionally, it allowed the gathering of a large amount of data that will be used in more advanced studies, focused on evolutionary analysis and on detailed characterization of specific mutations.
- Selective pressure acting on influenza virus neuraminidase protein and relation with development of resistance to antiviral drugsPublication . Correia, V.; Santos, L.A.; Giria, M.; Rebelo-de-Andrade, H.Neuraminidase (NA) protein of influenza viruses has the particularity of being under antibody and antiviral drug selective pressure, as it is one of the main surface antigens and the target of neuraminidase inhibitors(NAIs). The aim of this study is to investigate the selective pressure(SP) acting on the NA of seasonal and pandemic influenza viruses. It comprises two objectives: (a)to evaluate the contribution of positive SP for the emergence of NAIs resistant viruses; and (b)to determine the impact of NAIs introduction into clinic and its wide use during pandemic on the SP acting on NA. For the 1st objective it will be analysed the SP acting on the sites associated with NAIs resistance or reduction in susceptibility. The 2nd objective implies a differential evolutionary pressure analysis according to time, with 3 sub-datasets of NA sequences being considered: (1)before worldwide introduction of NAIs into clinic(1999); (2)before wide use of oseltamivir during A(H1N1)2009 pandemic(2009); and (3)from 2009 to date. A large dataset of full-length NA coding sequences will be used for each (sub)type/variant, comprising sequences obtained at national level(since 2000/2001) and sequences available at GISAID and NCBI. A(H1N1)seasonal dataset was already created, including a total of 1523 sequences, from which 94 belong to 1st sub-dataset, 1094 to 2nd and 335 to 3rd. All SP analysis will be performed using the expertise acquired with this workshop. This study may contribute for understanding the role of antiviral drug selective pressure in NAIs resistance, patterns of emergency of resistant viruses and NA evolutio