RESUMO
The circulation pattern and genetic evolution of respiratory syncytial virus (RSV) in Japan were examined based on 109 RSV field strains isolated over 20 seasons (1980-2002) in two cities, Sapporo and Tokyo. The second hypervariable region of the large glycoprotein (G) gene was amplified by RT-PCR and the products sequenced directly. The nucleotide sequences were compared to those representatives of RSV genotypes identified previously. Japanese group A and B isolates clustered into five and four genotypes defined previously, respectively. Another one group A and one group B genotypes, which could not be assigned to previous genotypes, were also identified. Although different genotypes usually co-circulated in each season, the isolates in proximate seasons from two communities were usually located in the same branches. Moreover, the strains with genotypes defined previously were usually isolated at the same time as each reference strain of Western countries. Several mutant group B strains with 1-20 longer amino acid G proteins were newly identified in Sapporo. These findings suggest that Japanese RSV strains underwent geographical and also temporal clustering while participating in RSV genetic evolution in a global setting. In addition, Japanese strains, especially group B, might have evolved individually in each community, sometimes changing the length of the G protein.
Assuntos
Filogenia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Sequência de Aminoácidos , Pré-Escolar , DNA Viral/química , Evolução Molecular , Genótipo , Humanos , Lactente , Recém-Nascido , Japão , Epidemiologia Molecular , Dados de Sequência Molecular , Vírus Sincicial Respiratório Humano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas do Envelope Viral/genéticaRESUMO
We used heteroduplex mobility assay (HMA) to determine the genetic variability of 118 respiratory syncytial virus (RSV) field isolates from 19 epidemics occurring in a Japanese urban area between 1980 and 2000. Nucleotides 1 to 584 of the attachment G glycoprotein gene were amplified by reverse transcription-PCR, and the PCR amplicons were analyzed by HMA by using the earliest isolate from 1980 as the reference throughout. We also performed PCR-restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis on the same nucleotide sequence. PCR-RFLP revealed 9 patterns, whereas HMA produced 31 distinct patterns. The RFLP patterns were divided into two to seven distinct HMA genotypes. Field strains with similar degrees of G gene nucleotide differences from the reference strain often showed distinct HMA types. The RSV genetic heterogeneity detected by direct sequencing of the PCR amplicon was usually identical to HMA analysis. Analysis of the molecular epidemiology of RSV subgroup A isolates obtained by HMA showed that new RSV variants emerged with each epidemic and that previously dominant variants seldom recurred in subsequent epidemics. HMA is useful in detecting genetic variants of RSV subgroup A and has some advantages over other conventional methods.
Assuntos
Análise Heteroduplex/métodos , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Virologia/métodos , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , Variação Genética , Genótipo , Humanos , Japão/epidemiologia , Epidemiologia Molecular , Filogenia , Polimorfismo de Fragmento de Restrição , Vírus Sincicial Respiratório Humano/classificação , Análise de Sequência de DNARESUMO
A new commercial rapid 10-min one-step immunochromatography (IC) test, SAS RSV test, was compared to another IC test, Directigen EZ RSV, employing RT-PCR as the "gold standard" for detecting respiratory syncytial virus. Of 102 clinical samples, 79 were positive by RT-PCR, 66 (82.5%) were positive with the SAS RSV test, and 55 (69.6%) were positive with Directigen EZ RSV. The specificity of the new test was 91.3% (21 of 23), similar to that of Directigen EZ RSV (100% [23 of 23]). This test performs well enough to be used for patient care.
Assuntos
Antígenos Virais/análise , Kit de Reagentes para Diagnóstico , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/isolamento & purificação , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de TempoRESUMO
In January 2001, 20 children among 40 residents under 2 years old at a nursery home in Sapporo, Japan had respiratory symptoms and were confirmed as having respiratory syncytial virus (RSV) infection by a conventional diagnostic kit. Nasopharyngeal aspirates were collected from four RSV-positive patients and total RNA was extracted directly from the specimens for the analysis of RSV grouping and genotyping. All four RSV strains had the same G protein gene sequence of subgroup B and were assigned to identical strains. Interestingly, the G protein gene had a duplication of 60 nucleotides at the C-terminal third of the G protein gene in which three nucleotides differed each other. The predicted polypeptide is lengthened by 20 amino acids. The clinical picture of these cases was not different from those of patients with other RSV strains. These novel mutations were thought to be introduced in vivo.