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.

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.

Human respiratory syncytial virus (RSV) is the most important viral pathogen causing lower respiratory tract infections in infants, immunocompromised hosts, and the elderly (1, 2, 10). Epidemics of RSV infection occur every winter in temperate climates, during rainy seasons, or year-round in tropical regions (11). RSV can infect the same individual repeatedly as well as infants under 6 months of age who still possess maternal antibodies against the virus (7). Many of these infections are difficult to distinguish clinically from other respiratory viral infections and some bacterial infections. Laboratory diagnosis by cell culture and viral serology is usually necessary to identify the etiologic agent, although the final results of RSV isolation by tissue culture usually require several days. A more rapid diagnosis can be made by direct detection methods, such as enzyme immunoassay and immunochromatography (IC) testing with nasopharyngeal swabs or aspirates (4,6,9). Recently these test have been widely used; however, they are only moderately sensitive in detecting RSV in respiratory tract specimens. On the other hand, immunofluorescent-antibody tests are highly sensitive for detecting RSV; however, they need special equipment and take at least 1 h to complete (4).We evaluated a new IC test, the SAS RSV test (SA Scientific, San Antonio, Tex.), for the first time to assess its clinical usefulness in detecting RSV antigens in nasopharyngeal swabs from subjects with RSV respiratory tract infections. This test takes 10 min to perform and relies on two specific monoclonal antibodies against RSV antigen. The sensitivity, specificity, and convenience of the test were assessed and compared to those of an existing IC test, Directigen EZ RSV (BD Biosciences, San Jose, Calif.). As a "gold standard " for detecting RSV in clinical samples, we used a multiplex reverse transcription-PCR (multiplex RT-PCR) method that was developed by Stockton et al. (12) for detecting and subtyping RSV (groups A and B). Recent studies suggest that RT-PCR is more sensitive than viral culture for detecting respiratory viruses in clinical specimens (13).One hundred two patients from 8 days to 9 years old (median ϭ 11 months) were included; 9, 1, 52, 23, and 17 subjects were diagnosed as having upper respiratory tract infection, laryngitis, tracheobronchitis, bronchiolitis, and pneumonia, respectively. The patients were seen during 2003 to 2004 at three institutions. Nasopharyngeal swabs obtained from each subject were suspended in 2 ml of 2% fetal calf serum-minimal essential medium and were used for two IC tests. The other was stored at Ϫ20°C until further analysis.For RNA extraction, we used 280-l samples according to the spin protocol of the QIAamp viral RNA minikit (Qiagen, Valencia, Calif.). For cDNA synthesis, 22.2 l of RNA solution was added to a reaction mixture (17.8 l) containing random hexamer (Takara, Otsu, Japan) and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, Calif.). The reaction mixture was incubated at...

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 ...