Polymorphisms at Residue 222 of the Hemagglutinin of Pandemic Influenza A(H1N1)pdm09: Association of Quasi-Species to Morbidity and Mortality in Different Risk Categories

et al. (2014) Polymorphisms at Residue 222 of the Hemagglutinin of Pandemic Influenza A(H1N1)pdm09: Association of Quasi-Species to Morbidity and Mortality in Different Risk Categories. PLoS ONE 9(3): e92789. doi:10.1371/ journal.pone.0092789 Polymorphisms at Residue 222 of the Hemagglutinin of Pandemic Influenza A(H1N1)pdm09: Association of Quasi-Species to Morbidity and Mortality in Different Risk Categories Paola Cristina Resende 0 Fernando C. Motta 0 Maria de Lourdes A. Oliveira 0 Tatiana S. Gregianini 0 Sandra B. Fernandes 0 Ana Luisa F. Cury 0 Maria do Carmo D. Rosa 0 Thiago Moreno L. Souza 0 Marilda M. Siqueira 0 Toru Takimoto, University of Rochester Medical Center, United States of America 0 1 Laborato rio de V rus Respirato rios e do Sarampo, Instituto Oswaldo Cruz/ FIOCRUZ , Rio de Janeiro, Rio de Janeiro , Brazil , 2 Laborato rio Central de Sau de Pu blica do Estado do Rio de Grande do Sul -Fundac a o estadual de produc a o e pesquisa em sau de sec a o de virologia , Porto Alegre, Rio Grande do Sul, Brazil, 3 Laborato rio Central de Sau de Pu blica do Estado de Santa Catarina, Floriano polis, Santa Catarina , Brazil , 4 Laborato rio Central de Sau de Pu blica do Estado de Minas Gerais/Instituto Octa vio Magalha es e Fundac a o Ezequiel Dias , Belo Horizonte, Minas Gerais, Brazil, 5 Laborato rio Central de Sau de Pu blica do Estado do Parana , Curitiba, Parana , Brazil The D222G substitution in the hemagglutinin (HA) gene of the pandemic influenza A(H1N1)pdm09 virus has been identified as a potential virulence marker, because this change allows for virus invasion deeper into the respiratory tract. In this study, we analyzed D, G and N polymorphisms at residue 222 by pyrosequencing (PSQ). We initially analyzed 401 samples from Brazilian patients. These were categorized with respect to clinical conditions due to influenza infection (mild, serious or fatal) and sub-stratified by risky factors. The frequency of mixed population of virus, with more than one polymorphism at residue 222, was significantly higher in serious (10.6%) and fatal (46.7%) influenza cases, whereas those who showed mild influenza infections were all infected by D222 wild-type. Mixtures of quasi-species showed a significant association of mortality, especially for those with risk factors, in special pregnant women. These results not only reinforce the association between D222G substitution and influenza A(H1N1)pdm09-associated morbidity and mortality, but also add the perspective that a worse clinical prognosis is most likely correlated with mixtures of quasi-species at this HA residue. Therefore, quasi-species may have a critical and underestimated role in influenza-related clinical outcomes. - Funding: Brazilian Ministry of Health and Ministry of Technology and Science, CNPq and FAPERJ provided funding to conduct this research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The pandemic influenza A(H1N1)pdm09 virus was responsible for over 18,449 deaths worldwide from 2009 to 2010 [1]. Although increased mortality is generally associated with novel influenza viruses due to the lack of a preexisting immunity in the general population, several groups of patients at higher risk for complications due to the influenza infection were reinforced or identified in 2009, such as pregnant women, obese individuals, immunocompromised patients and, surprisingly, otherwise healthy young adults, among others [2]. In addition to hosts predisposed clinical conditions, some specific polymorphisms in the viral genome may enhance its virulence [3]. Circulating influenza A(H1N1)pdm09 viruses from 2009 to the present are virtually similar, and no major antigenic variation has been found in viral hemagglutinin (HA), which is the viral surface glycoprotein responsible for attachment and a major target of the hosts immune response [2]. However, mutations, such as D222G/E/N (H1 numbering without signal peptide), which are located within the HA receptor-binding domain (RBD), have been positively associated with worse clinical outcomes as early as during the 2009 pandemics; being therefore, a potential virulence marker [2,3]. The D222G mutation increases viral invasion to deeper areas of the respiratory tract because it amplifies viral tropism by allowing viral binding to sialic acid residues with the a2,6- (a2,6) and a2,3-linkages (a2,3) found in the upper and lower respiratory tract, respectively [4,5,6]. For RNA viruses, viral pathogenesis and evolution are two related processes because high mutation rates lead to the generation of quasi-species [7]. Although many of these novel variants may not be viable, others may evade hosts immune responses and/or be endowed with enhanced virulence [7]. Thus, by studying the heterogeneity of quasi-species at residue 222 of A(H1N1)pdm09 HA and its association with a worse clinical outcome, insights may be made as to what extent the active process of virus replication evolution influences A(H1N1)pdm09related morbidity and mortality. Therefore, the heterogeneity at residue 222 was analyzed and found to be associated with poor clinical outcomes of the Brazilian population. In our study, we found that A(H1N1)pdm09 mutants at any distribution at residue 222G/D/N are significantly associated with worse clinical conditions due to influenza and to risk factors than the D222G mutation alone. Moreover, we also found the heterogeneity of amino acids at residue 222 in a representative proportion of A(H1N1)pdm09-infected individuals in Brazil. Materials and Methods Data collection and ethical statement Our laboratory is the National Reference Laboratory for influenza and Respiratory Diseases for the Brazilian Ministry of Health (MoH) and we also integrate the World Health Organization (WHO) network for Influenza surveillance, as a National Influenza Center (NIC). As part of these surveillance systems, we continuously receive samples of suspected cases for virological surveillance, which includes different laboratory testing, such as subtyping, antigenic and genetic analyses. Samples are collected from individuals with respiratory influenza-like illness or severe acute respiratory infection (SARI), according to WHO and MoH case definitions [8]. Sociodemographic, clinical and epidemiological data are also collected by the epidemiological surveillance teams in the different Brazilian states, which includes basic information, such as gender, age, city/state of case notification and residence and dates of symptoms onset and sample collection. Personal information such as name and address are confidential and only assessed by the head of Laboratory. At the entrance, samples and forms are coded, in such a way that all analyses remain anonymous for all staff (maintaining confidentiality, in accordance to our e (...truncated)