The availability of tools for the discrimination of antigenic and genetic variability among RSV isolates provided an opportunity to extend the understanding of the epidemiology of these viruses. In the following sections, insights gained from antigenic and molecular studies of viral epidemiology are reviewed.
Early studies of antigenic variability performed by using cross-neutralization assays demonstrated variability among isolates. It was suggested that strains that vary antigenically circulate simultaneously in the world (32
). The circulation of distinct viruses during a single epidemic period was also described (22
The recognition of the two major antigenic groups of RSV and the variability that occurs within these groups provided an opportunity to better define the epidemiology of these viruses. The work of Anderson et al. (7
) showed that the two groups have existed for over 20 years and have a worldwide distribution and that both groups may occur during a single season. Group A viruses were identified more often than the group B viruses. Mufson et al. (85
) suggested that the two groups had evolved separately for a considerable period.
Hendry et al. (59
) analyzed isolates collected in Boston in 1981 to 1982 and in 1983 to 1984. The two antigenic groups were found to circulate concurrently. Temporal and geographic clustering of the groups was observed. The two groups were isolated with similar frequencies in one period, and the group A viruses predominated in the other period. In a later study, the prevalence of the two antigenic groups in six RSV outbreaks in Boston from 1981 to 1987 was assessed (58
). Of 981 viruses, 60% were group A and 39% were group B. In two of the six periods the groups were found in equal proportions, in three periods the group A viruses were dominant, and in one period the group B viruses were dominant. Heterogeneity among the group A viruses was noted.
Akerlind and Norrby (1
) found that both antigenic groups also circulate in Sweden. Group B viruses isolated in West Virginia were classified as B1 or B2 based on differences in G-protein MAb reactivity. The B2 viruses predominated in a single epidemic year and were not detected subsequently (2
). Similarly, a variant group A RSV with altered reactivity to F MAbs was identified in only one epidemic period (86
). The distributions of group A and B viruses during five epidemic periods from 1981 to 1986 in West Virginia were analyzed (83
). Of 211 viruses tested, 76% were group A and 24% were group B. Both groups were isolated each year, but the group A viruses were found more often in most epidemic periods. The studies in West Virginia were later extended to include the epidemic periods from 1978 to 1988. Of 405 viruses tested, 79% were group A and 21% were group B. The group B viruses were dominant in one epidemic (82
Storch and Park (109
) analyzed 114 viruses isolated from 1981 to 1986 in St. Louis, Mo., and found that the group A viruses were predominant (80% of the isolates), although both groups were found. Analysis of RSV isolates collected in Newcastle upon Tyne, United Kingdom, between 1965 and 1986 showed that the two antigenic groups cocirculated; group B viruses were less frequently isolated (80
). Viruses isolated during five epidemic periods in Sapporo, Japan, were analyzed (125
). The first and last periods showed a group A virus predominance, but during the three intervening periods the group B viruses were isolated more frequently. RSV isolates collected during three epidemic periods in Uruguay from 1987 to 1987 were analyzed; group A isolates were dominant in two epidemic periods, and group B isolates were dominant in one (100
RSV isolates collected in Tecumseh, Mich., between 1965 and 1971 and between 1976 and 1981 were classified into groups (79
). Group A and B viruses were present as remotely as 1965; overall, the group A viruses predominated. In Hawaii, RSV was isolated each month of the year, although the peak incidence was in the winter wet season (98
). Group A and B viruses cocirculated except during the months of lowest incidence.
The prevalence of the group A and B viruses in Caen, France, during eight epidemic periods from 1982 to 1990 was determined. Both subgroups occurred each year, and there was a gradual change in the predominant group over a period of about 5 years (45
). In Finland, a regular alteration of group prevalence was observed, suggesting that antigenic variation of RSV influenced the epidemiology of the virus (133
RSV isolates from Rochester, N.Y., spanning a 15-year collection period were classified into groups, with variants within the group being designated subgroups. Three patterns of group distributions were seen, group A predominance (9 years), equal proportions of group A and B (4 years), and group B predominance (2 years). Overall, the pattern was that 1 or 2 years of group A predominance was followed by a year in which group B viruses would make up at least 40% of the isolates. Within each of the groups, subgroups were identified. Among the group A viruses, subgroups A1 and A2 predominated; among the group B viruses, three subgroups were found in similar numbers (54
A total of 483 RSV isolates collected in 14 laboratories in the United States and Canada during two epidemic periods were analyzed. Group A viruses accounted for 63% of isolates, group B accounted for 24%, and 14% could not be grouped. Within the group A and B viruses, there were six and three subclassifications, respectively. Isolates varied among laboratories during the same year. The authors concluded that RSV outbreaks may be community or regional phenomena but were not national ones (6
). Antigenic and genomic diversity among group A viruses was assessed for 47 isolates collected during four RSV seasons in St. Louis. Based on reactivity with MAbs to the G protein, four subgroups within the group A viruses were identified. RNase protection patterns were similar with two of the antigenic subgroups and more diverse with one of the subgroups. Thus, this study also demonstrated that there is viral heterogeneity within a single epidemic period (107
The group classification was determined for 87 viruses isolated in Rio de Janeiro, Brazil, from 1982 to 1988. Both group A and B viruses were identified in each epidemic period, although their relative predominance varied. Four antigenic variants among the group A viruses and three variants among the group B viruses were recognized (105
). Among viruses isolated in Cleveland, Ohio, over three epidemic periods between 1985 and 1988, 176 were group A and 21 were group B (106
). A total of 53 isolates of group B RSV obtained in Sapporo, Japan, from 1980 to 1989 were tested for intragroup antigenic variability. Three different patterns of antigenic reactivity were reported (88
). Analysis of 613 viruses isolated in Vancouver, Canada, revealed that both group A and B viruses were circulating from 1987 to 1992. The group B viruses predominated during only one season (121
). RSV isolates from a single epidemic period in Texas were found to be almost equally distributed between A and B group viruses, and intragroup variability was observed (78
RSV strains from Australia and Papua New Guinea were characterized. In Australia the RSV season lasts from April through September (61
). In Papua New Guinea the RSV season is year-round. Both group A and B RSV isolates were identified. Group A viruses were found to replicate better than the group B viruses in cell culture. In Mexico City, RSV of both antigenic groups was identified, with a preponderance of group A viruses (122
Cane and Pringle analyzed viral isolates from a single epidemic period in Birmingham, United Kingdom (19
). PCR products were used for the partial sequencing of the SH-protein gene and restriction fragment analysis of a region of the N-protein gene. From this outbreak, four group A and two group B lineages were identified. Genetic variability among group A viruses isolated worldwide was also assessed. The viruses had been isolated in the United Kingdom, Germany, Malaysia, Finland, and Uruguay. This study showed that multiple RSV lineages can cocirculate and that viruses isolated on separate continents can be very similar (13
Five successive epidemics in Birmingham, United Kingdom, were assessed for variability among 187 isolates. PCR products from parts of the N-, SH-, and G-protein genes were analyzed by restriction digestion or nucleotide sequencing. Six group A and two group B lineages were identified; different lineages predominated in each epidemic. Interestingly, the isolation of some lineages increased for a few years and then declined. This result suggested the possibility that immunity interferes with the spread of particular viruses in the community (14
A total of 13 clinical isolates of group A RSV obtained at different times and places in the United States were analyzed by sequencing a variable region of the glycoprotein G gene. These results were compared to the sequences from three reference strains of RSV, and extensive genetic diversity was noted among the isolates. The genetic relationships had little correlation with the date or place of isolation or with the antigenic subgroupings based on MAb reactivity. These results showed that multiple RSV genotypes might circulate in individual communities. In addition, very similar genotypes may be found in geographically distant sites. The observation that half the nucleotide changes encoded amino acid changes suggested that the G protein may be subject to immune selection (101
The genetic and antigenic variability of the G protein among 76 group A viruses collected in Montevideo, Uruguay, and Madrid, Spain, during six epidemic periods was assessed (46
). RNase A mismatch cleavage, nucleotide sequencing, and MAb reactivity were used. A phylogenetic tree that included the sequences determined in this investigation and the sequences of isolates from Birmingham, United Kingdom, was constructed. Two main branches and several subbranches were identified. During a single epidemic, viruses from different branches were found. Very similar viruses could also be found in geographically distant locations and during different epidemic periods, indicating the ability of these viruses to spread globally. When G-protein MAbs with strain-specific patterns of reactivity were used, a close relationship was observed between genetic and antigenic relatedness. As was seen in earlier studies, changes accumulated in the two variable regions of the G protein, and the pattern of the changes suggested that there may be a selective pressure for change. The pattern of evolution for RSV was suggested to be similar to that for the influenza B viruses. However, the degree of divergence for the G protein was found to be the highest reported for any RNA virus gene product (46
Nucleotide sequence analysis and MAb reactivity for 48 group A viruses isolated during a 38-year period measured the variability of the attachment protein G. Nucleotide sequences were determined for the two variable regions of the G-protein gene. Phylogenetic analysis revealed that close clustering tended to be related to the date of isolation. However, in some years, two or more clusters might be distant from one another. Recent viral isolates were found to lie at the ends of the branches of the phylogenetic tree. There appeared to be an accumulation of amino acid changes with time; for the variable regions examined in this study, the rate of change was 0.5 to 1% per year. The reactivity of the viruses with G-protein MAbs in general mirrored the results of the genetic groupings. The pattern of evolution for RSV was said to be similar to that for influenza B virus, with cocirculation of multiple lineages and evidence for sequential evolution. The authors concluded that the current genotypes of RSV are widespread and that the genotypes are changing with time. Thus, vaccines modeled on historical prototype viruses may become antigenically less and less similar to currently circulating viruses (16
RSV isolates collected during three epidemics in Denmark were analyzed as to group (63
). The group B viruses were dominant during the first and last years, and the group A viruses were dominant during the intervening year. Restriction pattern analysis revealed diversity within the groups. Comparison of these results with previous work suggested that viruses similar in restriction patterns to the viruses found in Denmark had been found much earlier in Australia and the United States (117
). The authors commented that this apparent identity among isolates collected more than 20 years earlier in other parts of the world suggested that the globally circulating virus types might be relatively stable. They speculated that temporal fluctuations in the dominant viruses might be due to immune status favoring certain viruses from the circulating pool rather than the process of antigenic drift (63
Circulating RSV isolates collected in Liverpool, United Kingdom, during two epidemic periods were analyzed (43
). The viruses were categorized by restriction fragment analysis of N and G gene PCR products. Most (91%) of the isolates were members of group A. At least 10 different genotypes were found to cocirculate.
The analysis of a collection of RSV isolates from Cuba yielded fascinating results (126
). The G-protein genes of 23 viruses isolated in 1994 and 1995 had only five nucleotide differences from that of the Long strain virus isolated in 1956 in the United States. In addition, comparisons to viruses from later epidemic periods revealed that viruses with G proteins identical to those from 1994 to 1995 were present. No identical viruses had been reported from different epidemics previously. Thus, the Cuban isolates had unique features. The authors noted that previous studies had suggested that viruses from different lineages cocirculate and that over time there is an accumulation of changes among viruses in the same lineage. These studies were performed predominantly on viruses isolated in developed countries in temperate climates. The authors speculated that differences in the seasonality of RSV infections or restrictions on travel might have favored viral stability. The Cuban viruses demonstrate the uncertainties surrounding RSV epidemiology and provide an example of a lack of accumulation of change over time.
Most of the studies described above which assessed intragroup genetic variability to the level of nucleotide sequence determination analyzed only group A RSV isolates. Thus, until recently there was less information about the extent of variation among the group B viruses. Peret et al. (94
) characterized the molecular epidemiology of both antigenic groups of RSV in Rochester, N.Y., during five epidemic periods. The group A or B viruses predominated for 2 years each and were found in similar proportions in 1 year. The group B viruses were less variable than the group A viruses. Based on phylogenetic analyses of the nucleotide sequences of the gene encoding a region of the G protein, gene clades or genotypes were identified and were further classified into subtypes within the genotypes. Among the group A viruses, five genotypes and 22 subtypes and among the group B viruses four genotypes and 6 subtypes were described. Each year, one or two genotypes or subtypes accounted for at least half of the isolates. The predominant genotype or subtype was different each year. The authors hypothesized that strain differences affect protective immunity such that a novel virus may be transmitted more efficiently or may be more pathogenic. Thus, globally circulating strains could be introduced into a community and factors such as the levels of strain specific immunity would determine which strains might become epidemic strains (94
RSV strains isolated during three epidemic periods in Alabama were characterized by Coggins et al. (25
) as to group and intragroup differences by restriction pattern analysis and nucleotide sequencing of a variable region of the G-protein gene. Restriction fragment analysis revealed that the dominant pattern was different each year. Thus, even though the group A viruses were dominant for 2 years, the prevalent circulating virus was different each year. The deduced amino acid sequences for the variable region were compared, and it was found that among the group A viruses differences as great as 38% were found over the three epidemic periods. However, the viruses in one of the group A restriction patterns were noted to have no nucleotide differences observed over the three epidemic periods. The group B viruses displayed less variability than the group A viruses over this same period, with amino acid differences for the variable region being as great as 14%. The authors speculated that the limited variability among the group B viruses might contribute to a more restricted spread of these viruses, leading to the predominance of group A over group B viruses in many studies of RSV epidemiology. The group B viruses have the potential for greater variation than was seen among the Alabama isolates, as indicated by differences of up to 27% from a prototype group B virus. The percent nucleotide changes resulting in amino acid changes was calculated for the variable region analyzed in the study, and it was found that over 60% of the nucleotide changes resulted in amino acid changes among both the group A and B viruses. Thus, for both viral groups it is possible that there is a selective advantage, such as avoidance of the host immune response, associated with the G-protein changes.
Phylogenetic comparisons were performed for the Alabama isolates and compared to RSV G-protein gene sequences available through GenBank. As described for the studies discussed above, multiple lineages were evident among both group A and B viruses (25
). The dendrograms from this investigation were modified to show the corresponding genotype classifications described by Peret et al. (94
) (Fig. ). Not all of the isolates were classified, because they, or similar viruses, were not included in the earlier classification scheme. Such designations may be expected to undergo modification as more data become available and as formal phylogenetic comparisons are made among the growing number of available RSV gene sequences. The dendrograms show that viruses isolated on different continents several years apart may be phylogenetically very similar. Viruses from a single location and epidemic period may be placed on distant phylogenetic branches or may be phylogenetically very similar. These dendrograms may be compared to earlier work by other investigators (16
). The general groupings of the isolates are very similar among these different publications, suggesting that the broad outlines of these trees are likely to be maintained even as the specifics are refined. Among the group A viruses, the genotype designations of Peret et al. (GA1, GA2, etc.) correspond to the classifications of Cane et al. as follows, with branch designations for the G gene-based groupings and SHL for the classifications from the SH gene data: GA1 corresponds to branch 4 and SHL5; GA3 corresponds to branch 1 and SHL1, SHL3, and SHL4; and GA5 corresponds to branch 5 and SHL2 (16
FIG. 2 Group A (A) and B (B) RSV G-protein phylogenetic relationships. Partial G-protein sequences from RSV strains isolated in the Children's Hospital of Alabama were compared to published G-protein sequences available through GenBank (25). Viruses are identified (more ...)