Browsing by Author "Arraiolos, Ana"
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- Antigenic and genetic analysis of pandemic influenza A(H1N1) 2009 viruses from PortugalPublication . Pechirra, Pedro; Arraiolos, Ana; Conde, Patrícia; Gonçalves, Paulo; Cordeiro, Rita; Guiomar, RaquelBackround: The influenza AH1N1 2009 pandemic virus (AH1N1pdm) was first detected in Portugal in May 4th 2009. This virus had origin in a reassortment between a North American swine lineage (already a triple reassortant, circulating in pigs since the late 1990’s) and a Eurasian swine lineage. As the HA (North American swine lineage) continues to circulate in the human population, its antigenic sites will continue to be targeted by antibody-mediated selection pressure. Therefore it is important from a public health perspective, continue to characterise the HA and to monitoring the antigenic and genetic properties of the AH1N1pdm viruses in order to detect any changes and thus any necessity for selecting further vaccine candidates or changes in antiviral recommendations. In this study, is presented a genetic and antigenic characterisation of influenza AH1N1pdm viruses, isolated in Portugal over the 2009 influenza pandemic. Material and Methods: In Portugal, during the 2009 influenza pandemic, about 16500 clinical samples were tested by the National Influenza Reference Laboratory for the presence of influenza AH1N1pdm virus. From near 8000 AH1N1pdm-positive samples, 147 were isolated in MDCK-SIAT1 cell cultures and characterised antigenically by hemagglutination-inhibition assays (HI). Of these, 56 isolates were taken for sequence analysis of the HA1 gene segment. Results: Antigenically, the AH1N1pdm viruses are homogeneous, being similar to A/California/7/2009 and the later pandemic H1N1 viruses A/Bayern/69/2009 and A/Lviv/N6/2009. However, 12 of the isolated viruses show 4-fold or greater reductions in the HI titre against most of the HI sera panel. They react better with sera raised against the A/Bayern/69/2009 and A/Lviv/N6/2009. Three of these isolated viruses presented amino acid substitutions at hemagglutinin antigenic sites (G155E in epitope B; R205K in epitope D and E258D in epitope E of the HA1 subunit) and in one strain was observed the amino acid substitution V199I in the vicinity of epitope D. Changes in positions 153-157 of the HA have been highly associated with reduced HI titers with ferret antisera to the A/California/7/2009 vaccine virus. These changes usually emerge after virus propagation in cell cultures. Sequenced hemagglutinins of portuguese isolates show that these viruses, with two exceptions, belong to the clade 7, already described in the literature. As known, viruses from this clade have a S203T mutation. One of our strains, A/Lisboa/31/2009, belongs to clade 6, as presents the Q293H amino acid change. This viral strain was isolated from a patient that arrived from the USA (Boston, New York) in June 2009. Another viral strain, A/Lisboa/35/2009, belongs to an earlier clade (at least, previous to clade 4) because it don’t presents the S203T neither the Q293H in its hemagglutinin and it lacks also the V106I and N248D amino acid changes in its neuraminidase. Additionally, mutations P83S in HA present in all the portuguese isolated viruses and I321V in the majority of them have been observed in all the non-clade 1 isolates. Conclusions: The great majority of influenza AH1N1pdm viruses isolated in Portugal were similar to the vaccine strain A/California/7/2009. They were representative of the clade 7, except two strains with foreign travel history. Most of the observed amino acid changes in the HA were located at antigenic sites or in their vicinity.
- Characterization of influenza A/Fujian/411/2002(H3N2)-like viruses isolated in Portugal between 2003 and 2005Publication . Pechirra, Pedro; Gonçalves, Paulo; Arraiolos, Ana; Coelho, Anabela; Rebelo-de-Andrade, HelenaIn Portugal, influenza surveillance is achieved through the National Influenza Surveillance Programme (NISP), in close collaboration with other European and global surveillance networks. The NISP integrates epidemiological, clinical and virological data based on the information collected by a Network of Sentinel Medical Practitioners and by a network of Emergency Units of Hospitals and Health Care Centres. In this study, genetic and antigenic characterization of influenza A viruses of the A/Fujian/411/2002 lineage, isolated during the 2003/2004 and 2004/2005 influenza winter seasons, in the context of the NISP, are described. Antigenic analysis of A/ Fujian/411/2002-like viruses, first detected and isolated during the 2003/2004 winter season, revealed a close similarity with the reference strains A/Kumamoto/102/2002 and A/Wyoming/ 3/2003. Genetic analysis confirmed this similarity and revealed two different phylogenetic branches. The 2004/2005 influenza A(H3) isolates formed, both antigenic and genetically, a more homogeneous group and were closely related to A/Oslo/807/2004 and A/California/7/2004. During this season, the characterization of the influenza viral strains has shown continuous evolution to variants close related to A/Oslo/807/2004. The majority of amino acid substitutions detected in the haemagglutinin occurred at antigenic sites. This study reflects the contribution of individual countries for the surveillance and knowledge of the molecular epidemiology of the infection, essential for a concerted action towards the global monitoring of the disease. It also reflects the importance of constant monitoring of genetic and antigenic characteristics of circulating influenza strains, which will certainly be a major contribution to the formulation of influenza vaccines.
- Heterogeneous Selective Pressure Acting on Influenza B Victoria- and Yamagata-Like HemagglutininsPublication . Nunes, Baltazar; Pechirra, Pedro; Coelho, Anabela; Ribeiro, Carlos; Arraiolos, Ana; Rebelo-de-Andrade, HelenaAs a consequence of immune pressure, influenza virus hemagglutinin presents some of its amino acids under positive selection. Several authors have reported the existence of influenza A hemagglutinin codons under positive selective pressure (PSP). In this framework, the present work objectives were to demonstrate the presence of PSP and evaluate its effects on Victoria- and Yamagata-like influenza B viruses. Methodology adopted consisted in estimating the acceptance rate of nonsynonymous substitutions (ω = dN/dS) that describe the strength of selective pressure and identifying codons that may be positively selected, applying a set of continuous-time Markov chain codon-substitution models. Two groups of HA1 sequences (140 from Yamagata and 60 from Victoria lineage) were used. All the model maximum-likelihood estimates were obtained using codeml software application (PAML 3.15). The hypothesis of no existence of sites under PSP was rejected for both lineages (p<0.001), using likelihood ratio tests. These results demonstrate the presence of positive selection acting on hemagglutinin of both Yamagata- and Victoria-like influenza B viruses. Several different sites were identified to be under PSP on Yamagata and Victoria hemagglutinins. Sites found with a posterior probability >0.95 were codons 197 and 199 in both lineages, codon 75 in the Yamagata lineage, and codon 129 in the Victoria lineage. The detected amino acids are located at or near antigenic sites in influenza A virus H3 hemagglutinin.
- Immune response after vaccination or natural infection with pandemic influenza A (H1N1) in a Portuguese cohortPublication . Conde, Patrícia; Arraiolos, Ana; Pechirra, Pedro; Gonçalves, Paulo; Guiomar, Raquel
- Pandemic Influenza Virus Surveillance in Portugal: The Laboratory Network for the Diagnosis of Influenza A(H1N1)2009 InfectionPublication . Guiomar, Raquel; Pechirra, Pedro; Gonçalves, Paulo; Cordeiro, Rita; Conde, Patrícia; Arraiolos, Ana; Batista, Inês; Paixão, Eleonora; Nunes, Baltazar; Furtado (on behalf of the Laboratory Network for the Diagnosis of Influenza A(H1N1)2009 Infection), CristinaIn April 2009 a new influenza A(H1N1) virus of swine origin disseminated throughout the world, resulting in the first pandemic of the XXI century. To face the increasing number of diagnosis being requested, a National Laboratory Network for Influenza Surveillance of the new influenza A(H1N1)pandemic virus was activated in Portugal. This is a descriptive study of the Influenza-like Ilness (ILI) cases reported by this network. Association between the variables was evaluated by chi-squared test. Over 62089 ILI cases were notified, 25594 (41.2%) cases were laboratory confirmed A(H1N1)pdm virus, from week 17/2009 to week 15/2010. In the week 33 (summer) were detected 1039 (4.1%) positive cases for A(H1N1)pdm virus although the winter peak occurred in week 46 with 3131 (12.5%) A(H1N1)pdm positive cases. In the age group of 5-14 years old were detected the majority of positive cases 9983 / 15785 (63.0%) opposite in the elderly group (>65 years old) was detected the lower number of A(H1N1)pdm positive cases, 280/2361 (11.0%). The distribution by gender accounts 40.4% of female and 42.3% of male positive cases. The signs and/or symptoms present were analysed revealing that headache (49.7%), cough and myalgies (46.5%) and odinophagia (46.1%) were statistically associated with A(H1N1)pdm positive cases. The chronic pulmonary disease seemed to be more associated with laboratory confirmed A(H1N1)pdm cases. Ninety five strains were isolated and antigenically characterised, 45 were taken for genetic analysis (HA and NA gene). All the strains were antigenically and genetically like the pandemic vaccine strain. It was detected only one strain with the mutation H275Y in the neuraminidase, resistant to oseltamivir. This Laboratory network was an important tool to monitories and control the evolution of the pandemic.
- Seasonal and pandemic patterns of Influenza in PortugalPublication . Gonçalves, Paulo; Nunes, Baltazar; Paixão, Eleonora; Cordeiro, Rita; Pechirra, Pedro; Conde, Patrícia; Arraiolos, Ana; Furtado, Cristina; Guiomar, RaquelThe National Influenza Reference Laboratory has been collecting data on influenza activity in Portugal since 1957 through the National Influenza Surveillance Programme, including information on clinical and virological characteristics of the disease, allowing the estimation of weekly incidence rates for influenza-like illness (ILI). This information has not only been used by the National Health Authorities for the management of the disease, in its several aspects, but has also been contributing to the study of influenza by the World Health Organisation. Particularly during the past decade, the world had been preparing for a long awaited influenza pandemic, which characteristics could not been foreseen but was feared to have potentially devastating consequences. In April 2009 a new strain of influenza A(H1N1) virus of swine origin disseminated throughout the world, resulting in the first pandemic of the XXI century. To face the increasing number of diagnosis being requested, a Network of Associated Laboratories dedicated exclusively to the diagnosis of the new influenza A(H1N1) pandemic virus was activated in our country. The data on influenza collected over the past two influenza seasons, through the National Influenza Surveillance Programme and the Network of Associated Laboratories, is presented and compared. 2. Materials and methods Over 3000 ILI cases were notified to the National Influenza Reference Laboratory and to the Department of Epidemiology of the National Institute of Health, in the context of the National Influenza Surveillance Programme, from week 38/2008 through week 20/2010. The distribution by age group, gender and region, and the signs and/or symptoms present were analysed. Nasopharyngeal swabs were collected for virological characterisation of influenza viruses circulating during this period. The intensity and duration of the epidemic periods were described based on the weekly incidence rates for ILI and as a function of a defined baseline. From week 17/2009 through week 20/2010, over 53.800 ILI cases and respective biological specimens were also notified and analysed by the Network of Associated Laboratories. 3. Results Seasonal AH1, AH3 and B influenza viruses circulating in Portugal during the 2008/2009 season were replaced by the new influenza A(H1N1) pandemic virus since the beginning of the pandemic in the country on week 17/2009. When considering the data obtained though the National Influenza Surveillance Programme, lower ILI incidence rates, lower percentage of confirmed influenza cases (particularly in the population over 15 years) and fewer symptoms presented at the time of notification were observed during the 2009/2010 pandemic. The large volume of cases analysed through the Network of Associated Laboratories is currently under evaluation and will be presented at a later stage. 4. Conclusions Facing the circulation of a new virus and the threat that this could impose to the population and to the health care system, the total number of ILI cases reported and analysed in our country during the 2009/2010 winter boosted to numbers not seen in previous influenza seasons. It is a fact that the 2009/2010 pandemic has had a significant impact in Portugal in many areas, such as the adoption of health-care regulations, availability of health-care facilities, vaccination strategies, and public action/awareness. However, in terms of pattern of disease, the data collected through the National Influenza Surveillance Programme may reveal a different reality. The data analysed suggests that the 2009/2010 pandemic was milder than the previous influenza seasons. Preliminary analysis of the data collected through the Network of Associated Laboratories appears to corroborate these findings.
- Sequence analysis of resistance markers in pandemic influenza A (H1N1) 2009 viruses isolated in PortugalPublication . Pechirra, Pedro; Arraiolos, Ana; Conde, Patrícia; Gonçalves, Paulo; Cordeiro, Rita; Guiomar, RaquelBackround: In April 2009, a new influenza A (H1N1) virus (AH1N1pdm) was detected circulating in humans. Its rapid widespread transmission led to the declaration by the WHO of an influenza pandemic. This virus had origin in a reassortment between a North American swine lineage (already a triple reassortant, circulating in pigs since the late 1990’s) and a Eurasian swine lineage (which contributed to the AH1N1pdm with the neuraminidase (NA) and matrix (MP) gene segments). The NA inhibition assay is the base test to determine the susceptibility to NA inhibitors (NAI), however it requires virus isolation, and the consequent detection of a resistant viral strain requires confirmation by sequencing of the NA gene segment present in the original biological product. Material and Methods: In Portugal, during the 2009 influenza pandemic, about 16500 clinical samples were tested by the National Influenza Reference Laboratory for the presence of influenza AH1N1pdm virus. From near 8000 AH1N1pdm-positive samples, 153 were isolated in MDCK cells and of these 53 isolates were taken for sequence analysis of the NA and MP gene segments. The NA inhibition assays were performed by the WHO collaborating centre (London). Results: As all AH1N1pdm viruses, sequenced Portuguese isolates carried the S31N change in the M2 protein, which confers resistance to the adamantanes. The most common mutation in N1 neuraminidase associated with resistance to oseltamivir is the H275Y change. From our 53 sequenced isolates, only one carried this mutation. This was from an 8-years-old child with chronic diseases such as a DiGeorge syndrome diagnosed in 2004 and Evans Syndrome with auto-immune haemolytic anaemia diagnosed in 2006. Initially, this child was treated with an underdosing of oseltamivir, however, as the influenza-like syndrome persisted with a suspicion of viral pneumonia, led to the antiviral treatment dose review. The NA inhibition assay confirmed that this virus was resistant to oseltamivir. The NA inhibition assays performed in our viral isolates and accordingly to our sequence data, revealed that the majority of Portuguese AH1N1pdm viruses were sensitive to NAI. All neuraminidases presented the N248D substitution, except for A/Lisboa/35/2009, a viral strain from an early clade (at least, previous to clade 4) as it lacks also V106I amino acid change. Other amino acid substitutions, such as E119V, I223V, Q136K, known to reduce the susceptibility to NAI, were not found in our isolates. Conclusions: The use of conventional sequence analysis for monitoring the molecular markers of antiviral resistance in influenza A virus provides a valuable tool for an early detection of antiviral resistant strains.