JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 557-559
Vol. 29, No. 3
0095-1137/91/03557-03$02.00/0 Copyright X 1991, American Society for Microbiology
Antigenic Characterization of Respiratory Syncytial Virus Group A and B Isolates in Rio de Janeiro, Brazil MARILDA M. SIQUEIRA,l* JUSSARA P. NASCIMENTO,l AND LARRY J. ANDERSON2 Departmento de Virologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil,' and Respiratory and Enterovirus Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 303332 Received 21 June 1990/Accepted 12 December 1990
The antigenic characteristics of 87 strains of respiratory syncytial virus isolated in Rio de Janeiro, Brazil, from 1982 to 1988 were determined with a panel of monoclonal antibodies (MAbs) in an enzyme immunoassay. Four of these MAbs immunoprecipitated the fusion protein, and five immunoprecipitated the large glycoprotein. On the basis of the patterns of reaction of these MAbs to respiratory syncytial virus isolates in an enzyme immunoassay, we were able to separate isolates into the two major groups, A and B, and also to identify four variants within group A and three within group B. Strains from groups A and B were isolated each year, and the prevalence of the two groups varied over the seven study years.
Respiratory syncytial virus (RSV) is a major cause of lower-respiratory-tract illness in infants and young children worldwide (21), and reinfections are common (10, 15). Early studies with polyclonal animal sera in cross neutralization assays indicated antigenic heterogeneity among isolates of RSV (6). Recent studies of RSV isolates with a panel of monoclonal antibodies (MAbs) distinguished two major antigenic variants of RSV, originally designated groups 1 and 2 or subtypes A and B (5, 19). Investigators from different countries have reported that the two groups of RSV can cocirculate within the same community during the same RSV season and that their patterns of isolation can vary from year to year (1, 9, 11, 12, 16-18, 22, 25, 26). The most prominent antigenic differences between the two groups are found on the G protein, and the most prominent genetic differences are found on the G protein gene (13, 14, 22). Antigenic differences on the G protein have also been found between isolates within the two major groups, and these differences have made it possible to separate isolates within groups A and B into distinct subgroups (2, 5, 7, 11, 12, 22). In this study, we used MAbs to characterize both the group and the subgroup characteristics of RSV strains isolated in Rio de Janeiro, Brazil, from 1982 to 1988.
roughs Wellcome Co., Research Triangle Park, N.C.). When space was available, specimens were stored in liquid nitrogen; these specimens were the ones tested in this study. The remaining specimens, found positive for RSV by isolation or immunofluorescence, had been stored on dry ice or at -70°C and could not be recovered. MAbs. A panel of MAbs was used in an enzyme immunoassay (EIA) to categorize RSV isolates (Table 1). The antigenic sites and epitopes corresponding to the MAbs were determined by blocking antibody assays or isolate reaction studies (3-5). In brief, MAb 92-llc is specific for group A and MAb 102-lOb is specific for group B. MAbs 131-2a, 131-2g, and 133-lh react against all isolates. MAbs 143-5a, 130-Sf, 130-6d, and 130-9g react with different epitopes on the RSV G protein and differentiate isolates within the same group (Table 2) (24). Representative isolates from each of the subgroups have given consistent patterns of reaction against the MAbs after serial passages in tissue cultures including three or more limiting-dilution clonings. EIA. Polystyrene flat-bottomed microtiter plates (Immulon-2; Dynatech Laboratories, Inc., Alexandria, Va.) were used as the solid phase. The diluent used for the capture antibody was 0.25 M carbonate-bicarbonate buffer (pH 9.6). The buffer used throughout for component dilutions was 0.05 M phosphate-buffered saline (pH 7.2) containing 0.15% Tween 20 and 2% bovine serum albumin (PBST/BSA). The buffer used for washing steps was 0.01 M phosphate-buffered
MATERIALS AND METHODS Virus isolates. Nasopharyngeal secretions were collected from children under 5 years of age and suffering an acute respiratory illness from February to July of 1982 to 1988, when RSV activity is usually high in Rio de Janeiro (23). Samples were obtained in the first 7 days of illness by suction through a nasal catheter as described by Gardner and McQuillin (8). The nasopharyngeal secretions were immediately placed on wet ice and kept at 4°C for transport to the laboratory. Each specimen was split, and half was inoculated onto fresh monolayers of HEp-2 cells. The other half was centrifuged, and the cell pellet was tested for RSV antigen by immunofluorescence. The cell monolayers were observed for cytopathic effects for 21 days. The presence of RSV was confirmed by immunofluorescent staining of cell culture material with bovine anti-RSV hyperimmune serum and fluorescein-conjugated rabbit anti-bovine serum (Bur-
TABLE 1. Characteristics of MAbs against RSV" MAb
130-6d 143-Sa 130-Sf 130-9g 131-2g 133-lh 131-2a 102-lOb
92-llc *
Immunizing RSV strain
A2 82-776 A2 A2 A2 A2 A2 18537 Long
a Data are from Anderson et al. (3).
Corresponding author. 557
Epitope
EIA reaction of group:
A
B
G12 GSa G4 G3b G13 Fla F2 Flc
+ + + +
+ + +
+ + +
+ + + +
Flb
+
-
558
J. CLIN. MICROBIOL.
SIQUEIRA ET AL.
TABLE 3. RSV group A and B isolates, Rio de Janeiro, 1982 to 1988
TABLE 2. Categorization of RSV groups and subgroups by their reactivities with MAbs
No. of isolates
Reactivitya with MAb:
Yr
Subgroup
A/1
130-6d
143-5a
130-Sf
130-9g
92-llc
+ + +
+
-
-
A/2 A/3 A/4
-
+
-
-
-
-
+l
+
-
A/5
+
-
+ +
+
A/6
-
-
-
-
+ + + + + +
B/1
-
+
+
-
-
-
-
+
-
-
-
-
+
-
-
B/2 B/3
102-lOb
131-2a
-
+ + + + + + + + +
-
-
-
+ + +
a The EIA was done with a MAb as the capture, with virus, and with a biotinylated second MAb. +, Positive reaction in the EIA. A reaction was considered positive when PIN was >2.0 and when P - N was >0.150. t, Weakly positive reaction in most but not all tests. Subgroup B/1 isolates consistently gave weakly positive reactions against MAb 143-5a; this reaction pattern is distinct from the negative one for subgroups B/2 and B/3. Group B/3 isolates gave weakly positive or negative reactions against MAb 130-5f; this reaction pattern is distinct from the positive one for subgroups B/1 and B/2. -, Negative reaction in the EIA.
saline (pH 7.2) containing 0.05% Tween 20. Incubation steps performed in a humidified chamber to prevent drying in the peripheral wells. The substrate was 0.1 mg of 3,3', 5,5'-tetramethylbenzidine (Sigma Chemical Co., St. Louis, Mo.) per ml-1.3 RI of 3% H202 in 0.1 M citrate-phosphate buffer (pH 5.5) per ml. The plates were divided in rows. To the first five rows, 75 [L of MAb 131-2g at a 1:3,000 dilution was added, and to the remaining three rows, 75 ,ul of MAb 133-lh at a 1:1,000 dilution was added. These two MAbs were adsorbed to the solid phase overnight at 4°C. Next, the plates were washed, and 50 ,ul of virus diluted 1:5 in PBST/BSA was added and incubated overnight at room temperature. The plates were washed, and the biotinylated detecting MAb diluted in PBST/BSA was added and incubated for 1 h at 37°C. The plates were washed, and 75 ,ul of peroxidase-conjugated streptavidin (Amersham International, Amersham, United Kingdom) diluted 1:3,000 in PBST/BSA was added and incubated for 15 min at room temperature. The plates were washed, and the substrate was added and incubated for 15 min at room temperature. The reaction was stopped by the addition of 25 ,ul of 2 M H2SO4, and the A450 was read. A reaction was considered positive when P N was >0.150 and when PIN was >2.0, where P equals the absorbance of the strain against the respective MAb and N equals the absorbance of the respective MAb against uninfected HEp-2 cells. To assign a virus to a subgroup, we also required that it have a high titer of RSV antigens, that is, a P - N of >1.0 against MAb 92-llc, 102-lOb, or 131-2a. were
-
Totala
Group A
Group B
1982 1983 1984
56 73 11
1985 1986 1987 1988
18 5 18 47
1 6 3 7 3 4 39
4 1 2 4 1 9
2
1
a Only isolates stored in liquid N2 were teted in this study. The number isolated was the number stored in liquid N2 plus the number stored under dry ice or at -70°C; these latter isolates could not be reisolated and did not have sufficient antigen to group.
common (39 of 42) (P < 0.001, chi-square test). In 1988, one isolate was classified as intermediate (positive with MAbs 92-llc and 102-lOb); this isolate could not be recovered to determine whether it contained two isolates, one group A and one group B, or was truly an intermediate isolate. When we categorized isolates into subgroups, a great deal of diversity was seen even in the same year. For example, in 1983, 1985, 1987, and 1988, at least four distinct isolates circulated during the respective RSV outbreaks (Table 4). Over the seven study years, three subgroups were particularly common, A/i, A/4, and B/2; other subgroups were present, but less commonly. Two of the isolates were from the same patient. The first isolate from this patient was isolated in 1987 and was a group B strain; the second isolate was isolated in 1988 and was a group A strain.
DISCUSSION
This study is consistent with several other studies that showed the simultaneous circulation of the two major groups of RSV strains, A and B, and revealed antigenic variants within the two major groups (1, 7, 9, 11, 12, 16-18, 22, 25, 26). In this study, four antigenic variants within group A and three antigenic variants within group B were identified. Interestingly, strains from four subgroups, A/l, A/4, B/1, and B/2, were isolated over a period of at least 6 years, suggesting that these subgroups are stable strains and are not rare antigenic variants or variants that evolved sequentially over time; there appear to be several lineages of RSV strains evolving independently and not just the two representing groups A and B. A clearer understanding of the evolution of RSV strains, however, will require studies, such as sequence TABLE 4. Subgroups of RSV group A and B isolates, Rio de Janeiro, 1982 to 1988
RESULTS A total of 87 RSV isolates were available for the EIA strain characterization studies (Table 3), 32 from 1982 to 1986 and 55 during 1987 and 1988. During the first 3 years of the study, most, over 50%, of the isolates did not grow or had too little antigen to test. During the last 4 years, over 60% of the isolates were successfully tested. Both group A and group B isolates were identified in all seven study years, but their relative importance appeared to vary from year to year. For example, in 1987, group B isolates were most common (9 of 13), while in 1988, group A isolates were most
Intermediate
No. of isolates of the following subgroup:
Yr
1982 1983 1984 1985 1986 1987 1988a
A/1
A12
A/4
1 2 3 5
1
3
15
2 3 2 2
2 21
A/6
B/1
B/2
B/3
3
1 1 2 3
1
1 2 1
a One intermediate isolate was not included.
4 2
2
B/?
1
VOL. 29, 1991
ANTIGENIC CHARACTERIZATION OF RSV ISOLATES IN BRAZIL
studies, that provide a more comprehensive measure of the similarities and differences between RSV subgroups. When we compared our results with those of other published studies, we found that in 1987 some regions (such as Rio de Janeiro) had predominantly group B isolates, while other regions had predominantly group A isolates (11, 18, 22, 25, 26). This observation, as previously noted (11), suggests that RSV outbreaks are not caused by national or international outbreak strains, such as those which occur with influenza virus (20). The role that antigenic differences in strains play in the clinical and epidemiological characteristics of RSV is yet to be determined. Studies of strain differences, however, are providing important information about the epidemiology of RSV, including relationships between community and regional outbreaks as well as the nosocomial transmission of RSV (7). ACKNOWLEDGMENT This work was supported in part by the Brazilian National Research Council. REFERENCES 1. Akerlind, B., and E. Norrby. 1986. Occurrence of respiratory syncytial virus subtype A and B strains in Sweden. J. Med. Virol. 19:241-247. 2. Akerlind, B., E. Norrby, C. Orvell, and M. A. Mufson. 1988. Respiratory syncytial virus: heterogeneity of subgroup B strains. J. Gen. Virol. 69:2145-2154. 3. Anderson, L. J., P. Bingham, and J. C. Hierholzer. 1988. Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. J. Virol. 62:4232-4238. 4. Anderson, L. J., J. C. Hierholzer, Y. 0. Stone, C. Tsou, and B. F. Fernie. 1986. Identification of epitopes on respiratory syncytial virus proteins by competitive binding immunoassay. J. Clin. Microbiol. 23:475-480. 5. Anderson, L. J., J. C. Hierholzer, C. Tsou, R. M. Hendry, B. F. Fernie, Y. Stone, and K. McIntosh. 1985. Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. J. Infect. Dis. 151:626-633. 6. Coates, H. V., L. Kendrick, and R. M. Chanock. 1963. Antigenic differences between two strains of respiratory syncytial virus. Proc. Soc. Exp. Biol. Med. 112:958-964. 7. Finger, F., L. J. Anderson, R. C. Dicker, B. Harrison, R. Doan, A. Downing, and L. Corey. 1987. Epidemic infections caused by respiratory syncytial virus in institutionalized young adults. J. Infect. Dis. 155:1335-1339. 8. Gardner, P. S., and J. McQuillin. 1980. Rapid virus diagnosis: application of immunofluorescence, p. 55-124. Butterworth (Publishers) Inc., Boston. 9. Gimenez, H. B., N. Hardman, H. M. Keir, and P. Cash. 1986. Antigenic variation between human respiratory syncytial virus isolates. J. Gen. Virol. 67:863-870. 10. Henderson, F. W., A. M. Collier, W. A. Clyde, and F. W. Denny. 1979. Respiratory-syncytial-virus infections, reinfections and immunity: a prospective, longitudinal study in young children. N. Engl. J. Med. 300:530-534.
559
11. Hendry, R. M., L. T. Pierik, and K. McIntosh. 1989. Prevalence of respiratory syncytial virus subgroups over six consecutive outbreaks: 1981-1987. J. Infect. Dis. 160:185-190. 12. Hendry, R. M., A. L. Talis, E. Godfrey, L. J. Anderson, B. F. Fernie, and K. McIntosh. 1986. Concurrent circulation of antigenically distinct strains of respiratory syncytial virus during community outbreaks. J. Infect. Dis. 153:291-297. 13. Johnson, P. R., and P. L. Collins. 1989. The IB (NS2), 1C (NS1) and N proteins of human respiratory syncytial virus (RSV) of antigenic subgroups A and B: sequence conservation and divergence within RSV genomic RNA. J. Gen. Virol. 70:1539-1547. 14. Johnson, P. R., Jr., R. A. Olmsted, G. A. Prince, B. R. Murphy, D. W. Alling, E. E. Walsh, and P. L. Collins. 1987. Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity. J. Virol. 61:3163-3166. 15. Kim, H. W., J. 0. Arrobio, C. D. Brandt, B. C. Jeffries, G. Pyles, J. L. Reid, R. M. Chanock, and R. H. Parrott. 1973. Epidemiology of respiratory syncytial virus infection in Washington, D.C. I. Importance of the virus in different respiratory tract disease syndromes and temporal distribution of infection. Am. J. Epidemiol. 98:216-225. 16. Monto, A. S., and S. Ohmit. 1990. Respiratory syncytial virus in a community population: circulation of subgroups A and B since 1965. J. Infect. Dis. 161:781-783. 17. Morgan, L. A., E. G. Routledge, M. M. Willcocks, A. C. R. Samson, R. Scott, and G. L. Toms. 1987. Strain variation of respiratory syncytial virus. J. Gen. Virol. 68:2781-2788. 18. Mufson, M. A., R. B. Beishe, C. Orvell, and E. Norrby. 1988. Respiratory syncytial virus epidemics: variable dominance of subgroups A and B strains among children, 1981-1986. J. Infect. Dis. 157:143-148. 19. Mufson, M. A., C. Orvell, B. Rafnar, and E. Norrby. 1985. Two distinct subtypes of human respiratory syncytial virus. J. Gen. Virol. 66:2111-2124. 20. Murphy, B. R., and R. G. Webster. 1990. Orthomyxoviruses, p. 1091-1154. In B. M. Fields, D. M. Knipe, R. M. Chanock, M. S. Hirsch, J. L. Melnick, T. P. Monath, and B. Roizman (ed.), Fields virology, vol. I. Raven Press, Ltd., New York. 21. Pan American Health Organization. 1982. Acute respiratory infections in children. Pan American Health Organization, Washington, D.C. 22. Storch, G. A., and C. S. Park. 1987. Monoclonal antibodies demonstrate heterogeneity in the G glycoprotein of prototype strains and clinical isolates of respiratory syncytial virus. J. Med. Virol. 22:345-356. 23. Stutmoller, F., and J. P. Nascimento. 1983. Respiratory infections in Brazil: a status report. Pediatr. Res. 7(Suppl.):10381040. 24. Sullender, W. M., L. J. Anderson, K. Anderson, and G. W. Wertz. 1990. Differentiation of respiratory syncytial virus subgroups with cDNA probes in a nucleic acid hybridization assay. J. Clin. Microbiol. 28:1683-1687. 25. Taylor, C. E., S. Morrow, M. Scott, B. Young, and G. L. Toms. 1989. Comparative virulence of respiratory syncytial virus subgroups A and B. Lancet i:777-778. 26. Tsutsumi, H., M. Onuma, K. Suga, T. Honjo, Y. Chiba, S. Chiba, and P. L. Ogra. 1988. Occurrence of respiratory syncytial virus subgroup A and B strains in Japan, 1980 to 1987. J. Clin. Microbiol. 26:1171-1174.
Vol. 29, No. 3
0095-1137/91/03557-03$02.00/0 Copyright X 1991, American Society for Microbiology
Antigenic Characterization of Respiratory Syncytial Virus Group A and B Isolates in Rio de Janeiro, Brazil MARILDA M. SIQUEIRA,l* JUSSARA P. NASCIMENTO,l AND LARRY J. ANDERSON2 Departmento de Virologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil,' and Respiratory and Enterovirus Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 303332 Received 21 June 1990/Accepted 12 December 1990
The antigenic characteristics of 87 strains of respiratory syncytial virus isolated in Rio de Janeiro, Brazil, from 1982 to 1988 were determined with a panel of monoclonal antibodies (MAbs) in an enzyme immunoassay. Four of these MAbs immunoprecipitated the fusion protein, and five immunoprecipitated the large glycoprotein. On the basis of the patterns of reaction of these MAbs to respiratory syncytial virus isolates in an enzyme immunoassay, we were able to separate isolates into the two major groups, A and B, and also to identify four variants within group A and three within group B. Strains from groups A and B were isolated each year, and the prevalence of the two groups varied over the seven study years.
Respiratory syncytial virus (RSV) is a major cause of lower-respiratory-tract illness in infants and young children worldwide (21), and reinfections are common (10, 15). Early studies with polyclonal animal sera in cross neutralization assays indicated antigenic heterogeneity among isolates of RSV (6). Recent studies of RSV isolates with a panel of monoclonal antibodies (MAbs) distinguished two major antigenic variants of RSV, originally designated groups 1 and 2 or subtypes A and B (5, 19). Investigators from different countries have reported that the two groups of RSV can cocirculate within the same community during the same RSV season and that their patterns of isolation can vary from year to year (1, 9, 11, 12, 16-18, 22, 25, 26). The most prominent antigenic differences between the two groups are found on the G protein, and the most prominent genetic differences are found on the G protein gene (13, 14, 22). Antigenic differences on the G protein have also been found between isolates within the two major groups, and these differences have made it possible to separate isolates within groups A and B into distinct subgroups (2, 5, 7, 11, 12, 22). In this study, we used MAbs to characterize both the group and the subgroup characteristics of RSV strains isolated in Rio de Janeiro, Brazil, from 1982 to 1988.
roughs Wellcome Co., Research Triangle Park, N.C.). When space was available, specimens were stored in liquid nitrogen; these specimens were the ones tested in this study. The remaining specimens, found positive for RSV by isolation or immunofluorescence, had been stored on dry ice or at -70°C and could not be recovered. MAbs. A panel of MAbs was used in an enzyme immunoassay (EIA) to categorize RSV isolates (Table 1). The antigenic sites and epitopes corresponding to the MAbs were determined by blocking antibody assays or isolate reaction studies (3-5). In brief, MAb 92-llc is specific for group A and MAb 102-lOb is specific for group B. MAbs 131-2a, 131-2g, and 133-lh react against all isolates. MAbs 143-5a, 130-Sf, 130-6d, and 130-9g react with different epitopes on the RSV G protein and differentiate isolates within the same group (Table 2) (24). Representative isolates from each of the subgroups have given consistent patterns of reaction against the MAbs after serial passages in tissue cultures including three or more limiting-dilution clonings. EIA. Polystyrene flat-bottomed microtiter plates (Immulon-2; Dynatech Laboratories, Inc., Alexandria, Va.) were used as the solid phase. The diluent used for the capture antibody was 0.25 M carbonate-bicarbonate buffer (pH 9.6). The buffer used throughout for component dilutions was 0.05 M phosphate-buffered saline (pH 7.2) containing 0.15% Tween 20 and 2% bovine serum albumin (PBST/BSA). The buffer used for washing steps was 0.01 M phosphate-buffered
MATERIALS AND METHODS Virus isolates. Nasopharyngeal secretions were collected from children under 5 years of age and suffering an acute respiratory illness from February to July of 1982 to 1988, when RSV activity is usually high in Rio de Janeiro (23). Samples were obtained in the first 7 days of illness by suction through a nasal catheter as described by Gardner and McQuillin (8). The nasopharyngeal secretions were immediately placed on wet ice and kept at 4°C for transport to the laboratory. Each specimen was split, and half was inoculated onto fresh monolayers of HEp-2 cells. The other half was centrifuged, and the cell pellet was tested for RSV antigen by immunofluorescence. The cell monolayers were observed for cytopathic effects for 21 days. The presence of RSV was confirmed by immunofluorescent staining of cell culture material with bovine anti-RSV hyperimmune serum and fluorescein-conjugated rabbit anti-bovine serum (Bur-
TABLE 1. Characteristics of MAbs against RSV" MAb
130-6d 143-Sa 130-Sf 130-9g 131-2g 133-lh 131-2a 102-lOb
92-llc *
Immunizing RSV strain
A2 82-776 A2 A2 A2 A2 A2 18537 Long
a Data are from Anderson et al. (3).
Corresponding author. 557
Epitope
EIA reaction of group:
A
B
G12 GSa G4 G3b G13 Fla F2 Flc
+ + + +
+ + +
+ + +
+ + + +
Flb
+
-
558
J. CLIN. MICROBIOL.
SIQUEIRA ET AL.
TABLE 3. RSV group A and B isolates, Rio de Janeiro, 1982 to 1988
TABLE 2. Categorization of RSV groups and subgroups by their reactivities with MAbs
No. of isolates
Reactivitya with MAb:
Yr
Subgroup
A/1
130-6d
143-5a
130-Sf
130-9g
92-llc
+ + +
+
-
-
A/2 A/3 A/4
-
+
-
-
-
-
+l
+
-
A/5
+
-
+ +
+
A/6
-
-
-
-
+ + + + + +
B/1
-
+
+
-
-
-
-
+
-
-
-
-
+
-
-
B/2 B/3
102-lOb
131-2a
-
+ + + + + + + + +
-
-
-
+ + +
a The EIA was done with a MAb as the capture, with virus, and with a biotinylated second MAb. +, Positive reaction in the EIA. A reaction was considered positive when PIN was >2.0 and when P - N was >0.150. t, Weakly positive reaction in most but not all tests. Subgroup B/1 isolates consistently gave weakly positive reactions against MAb 143-5a; this reaction pattern is distinct from the negative one for subgroups B/2 and B/3. Group B/3 isolates gave weakly positive or negative reactions against MAb 130-5f; this reaction pattern is distinct from the positive one for subgroups B/1 and B/2. -, Negative reaction in the EIA.
saline (pH 7.2) containing 0.05% Tween 20. Incubation steps performed in a humidified chamber to prevent drying in the peripheral wells. The substrate was 0.1 mg of 3,3', 5,5'-tetramethylbenzidine (Sigma Chemical Co., St. Louis, Mo.) per ml-1.3 RI of 3% H202 in 0.1 M citrate-phosphate buffer (pH 5.5) per ml. The plates were divided in rows. To the first five rows, 75 [L of MAb 131-2g at a 1:3,000 dilution was added, and to the remaining three rows, 75 ,ul of MAb 133-lh at a 1:1,000 dilution was added. These two MAbs were adsorbed to the solid phase overnight at 4°C. Next, the plates were washed, and 50 ,ul of virus diluted 1:5 in PBST/BSA was added and incubated overnight at room temperature. The plates were washed, and the biotinylated detecting MAb diluted in PBST/BSA was added and incubated for 1 h at 37°C. The plates were washed, and 75 ,ul of peroxidase-conjugated streptavidin (Amersham International, Amersham, United Kingdom) diluted 1:3,000 in PBST/BSA was added and incubated for 15 min at room temperature. The plates were washed, and the substrate was added and incubated for 15 min at room temperature. The reaction was stopped by the addition of 25 ,ul of 2 M H2SO4, and the A450 was read. A reaction was considered positive when P N was >0.150 and when PIN was >2.0, where P equals the absorbance of the strain against the respective MAb and N equals the absorbance of the respective MAb against uninfected HEp-2 cells. To assign a virus to a subgroup, we also required that it have a high titer of RSV antigens, that is, a P - N of >1.0 against MAb 92-llc, 102-lOb, or 131-2a. were
-
Totala
Group A
Group B
1982 1983 1984
56 73 11
1985 1986 1987 1988
18 5 18 47
1 6 3 7 3 4 39
4 1 2 4 1 9
2
1
a Only isolates stored in liquid N2 were teted in this study. The number isolated was the number stored in liquid N2 plus the number stored under dry ice or at -70°C; these latter isolates could not be reisolated and did not have sufficient antigen to group.
common (39 of 42) (P < 0.001, chi-square test). In 1988, one isolate was classified as intermediate (positive with MAbs 92-llc and 102-lOb); this isolate could not be recovered to determine whether it contained two isolates, one group A and one group B, or was truly an intermediate isolate. When we categorized isolates into subgroups, a great deal of diversity was seen even in the same year. For example, in 1983, 1985, 1987, and 1988, at least four distinct isolates circulated during the respective RSV outbreaks (Table 4). Over the seven study years, three subgroups were particularly common, A/i, A/4, and B/2; other subgroups were present, but less commonly. Two of the isolates were from the same patient. The first isolate from this patient was isolated in 1987 and was a group B strain; the second isolate was isolated in 1988 and was a group A strain.
DISCUSSION
This study is consistent with several other studies that showed the simultaneous circulation of the two major groups of RSV strains, A and B, and revealed antigenic variants within the two major groups (1, 7, 9, 11, 12, 16-18, 22, 25, 26). In this study, four antigenic variants within group A and three antigenic variants within group B were identified. Interestingly, strains from four subgroups, A/l, A/4, B/1, and B/2, were isolated over a period of at least 6 years, suggesting that these subgroups are stable strains and are not rare antigenic variants or variants that evolved sequentially over time; there appear to be several lineages of RSV strains evolving independently and not just the two representing groups A and B. A clearer understanding of the evolution of RSV strains, however, will require studies, such as sequence TABLE 4. Subgroups of RSV group A and B isolates, Rio de Janeiro, 1982 to 1988
RESULTS A total of 87 RSV isolates were available for the EIA strain characterization studies (Table 3), 32 from 1982 to 1986 and 55 during 1987 and 1988. During the first 3 years of the study, most, over 50%, of the isolates did not grow or had too little antigen to test. During the last 4 years, over 60% of the isolates were successfully tested. Both group A and group B isolates were identified in all seven study years, but their relative importance appeared to vary from year to year. For example, in 1987, group B isolates were most common (9 of 13), while in 1988, group A isolates were most
Intermediate
No. of isolates of the following subgroup:
Yr
1982 1983 1984 1985 1986 1987 1988a
A/1
A12
A/4
1 2 3 5
1
3
15
2 3 2 2
2 21
A/6
B/1
B/2
B/3
3
1 1 2 3
1
1 2 1
a One intermediate isolate was not included.
4 2
2
B/?
1
VOL. 29, 1991
ANTIGENIC CHARACTERIZATION OF RSV ISOLATES IN BRAZIL
studies, that provide a more comprehensive measure of the similarities and differences between RSV subgroups. When we compared our results with those of other published studies, we found that in 1987 some regions (such as Rio de Janeiro) had predominantly group B isolates, while other regions had predominantly group A isolates (11, 18, 22, 25, 26). This observation, as previously noted (11), suggests that RSV outbreaks are not caused by national or international outbreak strains, such as those which occur with influenza virus (20). The role that antigenic differences in strains play in the clinical and epidemiological characteristics of RSV is yet to be determined. Studies of strain differences, however, are providing important information about the epidemiology of RSV, including relationships between community and regional outbreaks as well as the nosocomial transmission of RSV (7). ACKNOWLEDGMENT This work was supported in part by the Brazilian National Research Council. REFERENCES 1. Akerlind, B., and E. Norrby. 1986. Occurrence of respiratory syncytial virus subtype A and B strains in Sweden. J. Med. Virol. 19:241-247. 2. Akerlind, B., E. Norrby, C. Orvell, and M. A. Mufson. 1988. Respiratory syncytial virus: heterogeneity of subgroup B strains. J. Gen. Virol. 69:2145-2154. 3. Anderson, L. J., P. Bingham, and J. C. Hierholzer. 1988. Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. J. Virol. 62:4232-4238. 4. Anderson, L. J., J. C. Hierholzer, Y. 0. Stone, C. Tsou, and B. F. Fernie. 1986. Identification of epitopes on respiratory syncytial virus proteins by competitive binding immunoassay. J. Clin. Microbiol. 23:475-480. 5. Anderson, L. J., J. C. Hierholzer, C. Tsou, R. M. Hendry, B. F. Fernie, Y. Stone, and K. McIntosh. 1985. Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. J. Infect. Dis. 151:626-633. 6. Coates, H. V., L. Kendrick, and R. M. Chanock. 1963. Antigenic differences between two strains of respiratory syncytial virus. Proc. Soc. Exp. Biol. Med. 112:958-964. 7. Finger, F., L. J. Anderson, R. C. Dicker, B. Harrison, R. Doan, A. Downing, and L. Corey. 1987. Epidemic infections caused by respiratory syncytial virus in institutionalized young adults. J. Infect. Dis. 155:1335-1339. 8. Gardner, P. S., and J. McQuillin. 1980. Rapid virus diagnosis: application of immunofluorescence, p. 55-124. Butterworth (Publishers) Inc., Boston. 9. Gimenez, H. B., N. Hardman, H. M. Keir, and P. Cash. 1986. Antigenic variation between human respiratory syncytial virus isolates. J. Gen. Virol. 67:863-870. 10. Henderson, F. W., A. M. Collier, W. A. Clyde, and F. W. Denny. 1979. Respiratory-syncytial-virus infections, reinfections and immunity: a prospective, longitudinal study in young children. N. Engl. J. Med. 300:530-534.
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