Estudo molecular dos genes da hemaglutinina e neuraminidase de cepas de vírus influenza A circulantes no período de 2001 a 2013
Arquivos
Data
2015-07-28
Autores
Perosa, Ana Helena 
Orientadores
Bellei, Nancy Cristina Junqueira
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: O vírus influenza A é responsável por epidemias anuais e pandemias, associadas a níveis variados de morbidade e mortalidade. As glicoproteínas de superfície, hemaglutinina (HA) e neuraminidase (NA), desempenham papel importante na infecção e disseminação da doença e apresentam altas taxas de mutações, por serem o principal alvo da resposta imune humoral e o sítio de atuação das drogas mais utilizadas para tratamento de influenza. Objetivos: Caracterizar por métodos moleculares a HA e NA das cepas de influenza A que circularam durante a pandemia e nos períodos pré e pós-pandêmico e avaliar a presença de mutações em diferentes grupos de pacientes atendidos em um hospital terciário de São Paulo. Material e Métodos: Realizamos um estudo transversal com 2992 amostras respiratórias coletadas entre Junho de 2001 e Outubro de 2013 de adultos e crianças com diferentes co-morbidades, atendidos em diversas unidades de assistência à saúde do Complexo Hospital São Paulo/UNIFESP. Foi realizado RT-PCR em tempo real para detecção de influenza A, utilizando o protocolo do CDC para H1N1pdm09. As amostras positivas foram submetidas a um RT-PCR em tempo real com primers específicos para a subtipagem da HA, e posteriormente a RT-PCRs convencionais para amplificação e sequenciamento dos genes completos da HA e NA pelo método de Sanger. Foram construídas as árvores filogenéticas para cada gene de cada subtipo. Resultados: A positividade geral para influenza A foi 15,4% (462/2992), sendo 15,8% (176/1111) no período pré-pandêmico, 20,9% (184/879) durante a pandemia e 10,2% (102/1002) no período pós-pandêmico; a frequencia anual de influenza variou de 4 a 34% e foi mais alta em crianças (17,5% vs 14,2% - p<0,05). Antes da pandemia de 2009, o H1N1 sazonal e o H3N2 co-circularam, variando sua prevalência em cada temporada. O H1N1 sazonal foi o subtipo mais frequente em 2001 e 2008; a partir de 2008, apresentaram as mutações permissivas e em 2009, 100% das amostras possuíam a mutação H275Y. O H3N2 apresentou variações genéticas nas 12 temporadas analisadas, muitas vezes precedendo a cepa vacinal em 1-2 anos. As substituições D222G/N na HA do H1N1pdm09 foi identificada em apenas 2 pacientes. A mutação H275Y na NA, associada à resistência ao oseltamivir, foi identificada em uma amostra de H1N1pdm09, coletada em 2012 de um transplantado renal que veio a vii óbito mesmo após tratamento de 21 dias com oseltamivir e foi considerado o provável caso índice de um surto de H1N1pdm09 na unidade de transplante renal. Os outros pacientes deste surto não apresentaram a mutação de resistência, mas todos tinham as mutações permissivas V241I e N369K, presentes em todas as cepas de H1N1pdm09 a partir de 2012. Conclusões: Variações nos sítios antigênicos da HA são frequentes nos três subtipos de influenza A. O H1N1pdm09 com mutações permissivas na NA predominam desde 2012, o que poderia contribuir para a emergência de cepas resistentes com alto potencial de transmissibilidade nos humanos.
Influenza A virus is responsible for annual epidemics and occasional pandemics in humans with different morbidity and mortality rates. The surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), play important role in viral attachment and spread. These proteins are the major targets for immune response and drug activity, presenting high mutation rates. We described the genetic characteristics of HA and NA genes of influenza A viruses collected from different patient groups attended at a tertiary hospital from Sao Paulo, during the pandemic, pre and post-pandemic periods. A total of 2,992 respiratory samples were collected between June 2001 and October 2013, from adults and children with different comorbidities, attended at different health-care units of Sao Paulo Hospital. Samples were tested by CDC real-time RT-PCR for the presence of influenza A. Positive samples were analysed by real-time PCR to subtyped HA gene. The whole HA and NA gene were analyzed by Sanger sequencing and phylogenetic analyses of each gene and subtype were performed. The overall influenza A infection rate was 15.4% (462/2992); 15.8% (176/1111) in pre-pandemic period, 20.9% (184/879) during pandemic and 10.2% (102/1002) in post-pandemic period. The annual positivity rate ranged from 4 to 34% and children were more affected than adults (17.5% vs 14.2%, p<0.05). Seasonal H1N1 and H3N2 co-circulated before 2009 pandemic; seasonal H1N1 was prevalent in 2001 and 2008 and permissive mutations were observed since 2008. The most frequently change associated to oseltamivir resistance, H275Y, was observed in 100% of samples collected in 2009. Among H1N1pdm09 viruses sequenced, the D222G/N substitutions in HA, which had previously been associated with severe cases, was detected in only 2 patients. H275Y mutation in NA gene was identified in one sample collected from a kidney transplanted patient undergoing oseltamivir treatment. This patient was considered the likely index patient of an outbreak of influenza A(H1N1)pdm09 occurred in renal transplant ward during July 2012. All H1N1pdm09 samples from 2012 and 2013 had the double permissive mutations V241I / N369K. Changes in the HA antigenic sites were frequent in all subtypes of influenza A. The H1N1pdm09 with permissive mutations in NA predominate since 2012, which could contribute to the emergence of resistant strains with high potential for transmissibility in humans.
Influenza A virus is responsible for annual epidemics and occasional pandemics in humans with different morbidity and mortality rates. The surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), play important role in viral attachment and spread. These proteins are the major targets for immune response and drug activity, presenting high mutation rates. We described the genetic characteristics of HA and NA genes of influenza A viruses collected from different patient groups attended at a tertiary hospital from Sao Paulo, during the pandemic, pre and post-pandemic periods. A total of 2,992 respiratory samples were collected between June 2001 and October 2013, from adults and children with different comorbidities, attended at different health-care units of Sao Paulo Hospital. Samples were tested by CDC real-time RT-PCR for the presence of influenza A. Positive samples were analysed by real-time PCR to subtyped HA gene. The whole HA and NA gene were analyzed by Sanger sequencing and phylogenetic analyses of each gene and subtype were performed. The overall influenza A infection rate was 15.4% (462/2992); 15.8% (176/1111) in pre-pandemic period, 20.9% (184/879) during pandemic and 10.2% (102/1002) in post-pandemic period. The annual positivity rate ranged from 4 to 34% and children were more affected than adults (17.5% vs 14.2%, p<0.05). Seasonal H1N1 and H3N2 co-circulated before 2009 pandemic; seasonal H1N1 was prevalent in 2001 and 2008 and permissive mutations were observed since 2008. The most frequently change associated to oseltamivir resistance, H275Y, was observed in 100% of samples collected in 2009. Among H1N1pdm09 viruses sequenced, the D222G/N substitutions in HA, which had previously been associated with severe cases, was detected in only 2 patients. H275Y mutation in NA gene was identified in one sample collected from a kidney transplanted patient undergoing oseltamivir treatment. This patient was considered the likely index patient of an outbreak of influenza A(H1N1)pdm09 occurred in renal transplant ward during July 2012. All H1N1pdm09 samples from 2012 and 2013 had the double permissive mutations V241I / N369K. Changes in the HA antigenic sites were frequent in all subtypes of influenza A. The H1N1pdm09 with permissive mutations in NA predominate since 2012, which could contribute to the emergence of resistant strains with high potential for transmissibility in humans.
Descrição
Citação
PEROSA, Ana Helena Sitta. Estudo molecular dos genes da hemaglutinina e neuraminidase de cepas de vírus influenza a circulantes no período de 2001 a 2013. 2015. 163 f. Tese (Doutorado em Infectologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.