RESUMO
In this study, a PCR-DGGE protocol was standardized in order to distinguish Victoria and Yamagata influenza B lineages directly from clinical samples. After routine multiplex PCR characterization, amplicons of the haemagglutinin gene bearing a 40bp-length GC clamp were generated by nested-PCR and analyzed by electrophoresis in 6% polyacrylamide gel with a 25-45% urea-formamide gradient. The results showed a perfect correlation between DGGE and phylogenetic analyses for all compared samples, besides some distinct profiles in Victoria and Yamagata groups that could be used to infer variability inside these groups. In summary, this DGGE protocol for the haemagglutinin gene is rapid, useful and efficient, being an alternative for discrimination between the influenza B lineages.
Assuntos
Eletroforese em Gel de Gradiente Desnaturante/normas , Vírus da Influenza B/classificação , Vírus da Influenza B/genética , Influenza Humana/virologia , Reação em Cadeia da Polimerase/normas , Virologia/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza B/isolamento & purificaçãoRESUMO
BACKGROUND: In contrast to influenza A, minor influenza B viruses can co-circulate with the dominant strain during an epidemic allowing the re-emergence of old strains and reassortment between those different strains. The 2001-2002 influenza season in the northern hemisphere was distinguished by the re-emergence of the Victoria-lineage viruses, which replaced the Yamagata-lineage, after being restricted to East Asia throughout the 1990s. OBJECTIVES: To describe the antigenic and genetic characteristics of influenza B viruses detected in South and South East Brazil and determine their lineages. STUDY DESIGN: Influenza samples collected during epidemics between 1999 and 2002 were analyzed by indirect immunofluorescence assay (IFA). Positive results were confirmed through multiplex PCR and isolation in cell culture. Isolated viruses were antigenically characterized by hemagglutination inhibition. Fourteen hemagglutinin (HA) gene sequences obtained in this work were used for phylogenetic analysis. RESULTS: Brazilian isolates from 2002 were associated with the Victoria-lineage, diverging from the vaccine used throughout that influenza season in Brazil. CONCLUSIONS: These results indicate the reappearance of Sichuan/7/97-like samples in South and South East Brazilian Regions simultaneously. They indicate the need for neuraminidase gene evaluation and demonstrate the importance of influenza laboratory surveillance to establish which strains should be included in the influenza vaccine.
Assuntos
Surtos de Doenças , Vírus da Influenza B , Influenza Humana/epidemiologia , Brasil/epidemiologia , Técnica Indireta de Fluorescência para Anticorpo , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza B/classificação , Vírus da Influenza B/genética , Vírus da Influenza B/imunologia , Vírus da Influenza B/isolamento & purificação , Influenza Humana/virologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Because of the extensive genetic variability of the influenza viruses, new virus mutants arise worldwide. In the human population, some strains may become potentially epidemic after evading the immune response of the host. At present, molecular methods have made it possible to identify these variants. However, if a large number of samples need to be analyzed the identification of randomly mutated nucleotides cannot be achieved by sequencing analysis or restriction fragment length polymorphism (RFLP). In order to improve this process, a denaturing gradient gel electrophoresis (DGGE) protocol capable of discriminating between reference strains representative of different influenza seasons, some mutant strains, and five clinical isolates was standardized Ribonudeic acid (RNA) was isolated and submitted to a one-step RT-PCR that amplified the region codifying for the globular domain of the Haemagglutinin (HA) molecule. The amplicons were analyzed by electrophoresis in 6% polyacrylamide gel at 60 degreeC/150 V for 8 h, using a 31--41% urea--formamide gradient. This method was able to distinguish between closely related nucleotide sequences, confirming its suitability as screening methodology for the analysis of influenza virus epidemiology, by allowing a faster and more extensive evaluation of a large number of the variant strains detected in a specific region of the world.
Assuntos
Eletroforese em Gel de Poliacrilamida/normas , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/isolamento & purificação , Influenza Humana/diagnóstico , RNA Viral/análise , Sequência de Aminoácidos , Sequência de Bases , Variação Genética , Hemaglutininas/química , Humanos , Vírus da Influenza A/genética , Influenza Humana/epidemiologia , Dados de Sequência Molecular , Mutação , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The circulation of influenza C viruses in Rio de Janeiro, Brazil, was studied when significant levels of antibodies were detected (56. 7%) with hemagglutination inhibition test, used as a standard methodology for influenza virus studies.
