Genetic Variability and Molecular Epidemiology of Respiratory Syncytial Virus Subgroup A Strains in Japan Determined by Heteroduplex Mobility Assay

Abstract

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.Human respiratory syncytial virus (RSV) is the most important viral pathogen causing lower respiratory tract infections in infants, immunocompromised hosts, and the elderly (15,16,27). Epidemics of RSV occur every winter in temperate climates, during rainy seasons, or year round in tropical regions (28). RSV can infect the same individual repeatedly and infants under 6 months of age who still possess maternal antibodies against the virus (21). These findings prompted one hypothesis that RSV infection may not produce a sufficient immune response against different strains of the virus because of the extensive genetic variability of the strains circulating in a community and worldwide.RSV was initially found to have two distinct antigenic groups, designated A and B, by their different reactivity with monoclonal antibodies against the viral antigens (1, 20). Epidemiology studies of RSV demonstrated that both antigenic groups circulate concurrently or alternately in a community during epidemics (18,32). Among viral surface antigens, the attachment G glycoprotein (G protein) has the greatest antigenic diversity between these two groups and among strains within each group. The G-protein variability is concentrated in its ectodomain, which contains two hypervariable regions separated by a conserved 11-amino-acid motif (4,7,19,29). Recent molecular epidemiological studies suggest that many distinct RSV genotypes circulate worldwide and that similar genetic variants are clustered by time rather than by geographic location (6,8,17,26).Genetic variability of RSV has most often been studied by using restriction fragment length polymorphism (RFLP) analysis and DNA sequencing (4, 30). RFLP analysis is relatively easy to perform, although genotypes obtained by ...

RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy

“…RSV can be classified into two subgroups, serotypes A and B, and the sequences of all 11 genes identified differ between the two serotypes. Genotypes can vary from year to year, with certain serotypes dominating in some years, but the cocirculation of multiple RSV strains is commonly observed (28,41). Although RSV serotype A may produce greater viral loads in infants (29,46,49,74) and RSV serotype A may produce disease slightly more severe than that produced by RSV serotype B, both RSV serotype A and RSV serotype B produce a similar and clinically indistinguishable disease spectrum.…”

Estudio molecular y evolutivo del gen de la glucoproteína G del virus sincicial respiratorio y su relación con la circulación local y global en un período de seis años

“…En contraste, la proteína G ha sido exhaustivamente estudiada, principalmente porque esta proteína es el producto del gen con mayor variabilidad genética y antigénica entre aislamientos. Se han realizado numerosos estudios sobre la misma: ensayos de reactividad de AcMos; análisis de mutantes de escape frente a AcMos; ensayos de digestión con RNasa A de heterohíbridos ARN:ARN (Cristina et al, 1990); análisis de polimorfismos de longitud de fragmentos de restricción (RFLP) (Coggins et al, 1998;Choi and Lee, 2000); análisis de movilidad de heterodúplex (Kuroiwa et al, 2004) y determinación de secuencia nucleotídica para las elaboración de árboles filogenéticos (Cane, 2001). Todos han demostrado que la proteína G posee un ectodominio inusual debido a que un alto porcentaje de diferencias (51-58%) en nt en su región codificante dentro o entre los dos subtipos resulta en cambios aminoacídicos (sustituciones no sinónimas).…”

Caracterização molecular das cepas do Vírus Sincicial Respiratório identificadas nos anos de 2001 e 2002 em unidade de transplante de células tronco hematopoéticas

“…A variabilidade de isolados dentro dos grupos A e B do RSV foram inicialmente demonstradas com diferença nas reações com anticorpos monoclonais e depois usando métodos de clivagem com enzimas de restrição, ensaio com RNAse, ensaio de mobilidade heteroduplex e análise de seqüenciamento de nucleotídeos (Sullender et al, 1993;Cane, 2001;Kuroiwa et al, 2004). O seqüenciamento de nucleotídeos do gene G mostra que a variabilidade de aminoácidos é acima de 20% dentro do grupo A e 9% dentro do grupo B (Cane, 2001).…”

“…Kuroiwa e col. observaram na Coréia que um genótipo epidêmico em 1980 e 1981 ressurgiu 20 anos depois (Kuroiwa et al, 2004). (Cane et al, 1991;Choi & Lee, 2000).…”