RESUMO
To investigate the genetic characteristics of human influenza viruses circulating in Chungbuk province, we tested 510 clinical samples of nasopharyngeal suction from pediatric patients diagnosed with respiratory illness between June 2007 and June 2008. Genetic characterization of the HA genes of H3N2 isolates indicated the relative higher similarity to A/Virginia/04/07 (99.6%) rather than that of A/Wisconsin/67/2005 (98.4%), a Northern Hemisphere 2007-2008 vaccine strain, based on amino acid sequences. We found several altered amino acids at the H3 HA1 antigenic sites compared with the vaccine strain; K140I at site A, K158R at site B, and K173N (H471) or K173Q, and S262N at site E, but there was no antigenic shift among the H3N2 viruses. Interestingly, A/Cheongju/H383/08 and A/Cheongju/H407/08 isolates had single amino acid substitution at D151G on the catalytic site of the N2 NA while A/Cheongju/H412/08 and A/Cheongju/ H398/07 isolates had one amino acid deletion at residue 146. Furthermore, we found that 25% (3 out of 12 isolates) of the H3N2 subtype viruses had the amino acid substitution at position 31 on the M2 protein (Aspartic acid to Asparagine) and confirmed their drug-resistance by biological assays. Taken together, the results of this study demonstrated continuous evolutions of human H3N2 viruses by antigenic drift and also highlighted the need to closely monitor antigenic drug resistance in influenza A viruses to aid in the early detection of potentially pandemic strains, as well as underscore the need for new therapeutics.
Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H3N2/genética , Adolescente , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Variação Antigênica , Linhagem Celular , Criança , Pré-Escolar , DNA Viral/análise , Cães , Inibidores Enzimáticos/farmacologia , Humanos , Lactente , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/metabolismo , Influenza Humana/genética , Influenza Humana/virologia , Coreia (Geográfico) , Lectinas/genética , Dados de Sequência Molecular , Nasofaringe/virologia , Neuraminidase/deficiência , Neuraminidase/genética , Oseltamivir/farmacologia , FilogeniaRESUMO
We found a relatively high frequency of unique amantadine-resistant H3N2 and H9N2 avian influenza viruses (Val27Ile on M2 protein) isolated from live poultry markets in South Korea and confirmed that a Val27Ile single substitution in the M2 protein is enough to acquire the amantadine resistance phenotype by using reverse-genetically created human-avian reassortant viruses.
Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Substituição de Aminoácidos/genética , Animais , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Coreia (Geográfico) , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Aves Domésticas , RNA Viral/genética , Vírus Reordenados/efeitos dos fármacos , Vírus Reordenados/genética , Análise de Sequência de DNA , Proteínas da Matriz Viral/genéticaRESUMO
We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.
