Subgroup Characteristics of Respiratory Syncytial Virus Strains

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1987, p /87/ $02.00/0 Copyright C 1987, American Society for Microbiology Vol. 25, No. 8 Subgroup Characteristics of Respiratory Syncytial Virus Strains Recovered from Children with Two Consecutive Infections MAURICE A. MUFSON,l* ROBERT B. BELSHE,' CLAES ORVELL,223 AND ERLING NORRBY3 Department of Medicine, Marshall University School of Medicine, and Veterans Administration Medical Center, Huntington, West Virginia 25701,' and Department of Virology, National Bacteriological Laboratory3 and Department of Virology, Karolinska Institute, Stockholm, Sweden Received 24 February 1987/Accepted 28 April 1987 Respiratory syncytial virus strains from 13 children who had repeat infections at least 1 year apart were identified as either subgroup A or subgroup B according to reaction patterns with several monoclonal antibodies directed against the large surface glycoprotein (G), fusion protein (F), nucleoprotein (NP), and matrix protein (M). The virus strains were characterized by enzyme immunoassay, polyacrylamide gel electrophoresis, and immunofluorescence procedures. During the first infection, 10 children had subgroup A strains and 3 had subgroup B strains. Of the 10 children with subgroup A strains during their first infection, 6 had subgroup B and 4 had subgroup A strains during the second infection. Of the three children with subgroup B strains during their first infection, one had subgroup A and two had subgroup B strains during their second infection. No child experienced unusually severe respiratory tract illnesses during second infections with respiratory syncytial virus. Fourfold or greater rises in serum antibody as determined by enzyme immunoassay were as common after the first infection as after the second infection among the seven children tested. Thus, second infections with strains of either subgroup of respiratory syncytial virus did not potentiate respiratory illness, and infection with subgroup A strains of respiratory syncytial virus provided some protection from a second infection with the homologous, but not the heterologous, subgroup of the virus. Recently, two subgroups (designated subgroup A and subgroup B) of respiratory syncytial virus (RSV) have been identified by their different patterns of reactivity with monoclonal antibodies generated against the major structural components of the virus (2, 13). The two subgroups differ in at least four structural proteins, namely, the large glycoprotein (G), the fusion protein (F), the nucleoprotein (NP), and the phosphoprotein (P) (13). Molecular weight analyses and footprinting with proteases of RSV protein-antibody complexes bound to Sepharose A showed major differences of F and P proteins between subgroups A and B (7, 15). Both the Fl cleavage product of F protein and the P protein were slightly larger in subgroup A strains. Studies of the epidemiology of subgroup A and B strains in two communities showed that they circulate together in annual epidemics (1, 9). Subgroup A strains may occur more commonly than subgroup B strains, but individual variation is seen in different seasons (9). In previous studies, maternally derived circulating neutralizing antibody was shown to lessen the severity of primary RSV illness but not prevent the occurrence of infection and lower respiratory tract illness (LRI) (12). Since nearly all older children and adults possess circulating antibody to RSV, the isolation of RSV during acute respiratory tract illness from individuals in these age groups represents reinfection (3). In a study of day school children conducted during three epidemic seasons of RSV, reinfection occurred in three-fourths of the children during the second epidemic, and third infections occurred in two-thirds of the children during the third epidemic (8). The recognition of two subgroups of RSV raises questions about the relative role of each subgroup in consecutive infections of children * Corresponding author. and adults and further questions on the extent of crossimmunity between viruses representing the two subgroups. The key questions to be answered are (i) how often second infections represent a new infection with a virus of the alternate subgroup or reinfection with the same subgroup, (ii) whether the illnesses associated with second infections are severer because of possible immunopathological responses due to the existence of prior antigenic sensitization with the same or other subgroups, and (iii) how important it is to include both subgroups in any RSV vaccine or diagnostic reagent. In this report, we describe the occurrence of RSV strains of different subgroup characteristics in two infections at least 1 year apart in 13 children. MATERIALS AND METHODS Population. This study included only children with acute upper respiratory tract illness (URI) or LRI for whom RSV was isolated from pharyngeal swab specimens on two occasions at least 9 months apart, i.e., in two epidemiologic years. These children had been tested while participating in our ongoing studies of the etiology and epidemiology of acute respiratory tract diseases in Huntington, W.Va., among infants and children (5). Surveillance of RSV infections was begun in 1978, when we conducted a trial of a live RSV vaccine in children 1 to 4 years of age (4). The vaccine trial lasted 2 years through three epidemic seasons of RSV infections. During the study, all vaccinated children were tested for RSV infection each time they developed any symptoms or signs of respiratory tract illness which prompted their parents to bring them to a physician or the hospital or which required our staff nurses to visit them in their homes, at which time they obtained specimens for virus isolation. Between 1981 and 1986, surveillance of RSV 1535

2 1536 MUFSON ET AL. J. CLIN. MICROBIOL. infections focused mainly on infants and children who required hospitalization for acute respiratory tract illness. Virus isolation. Virus isolation was performed on pharyngeal swab specimens taken in veal infusion broth as previously described (4, 5). These specimens were inoculated into roller tube cultures of HEp-2, monkey kidney, and fetal diploid fibroblast cells and handled as described elsewhere (4, 5). RSV isolates were recognized by their characteristic cytopathic effect in HEp-2 cells, the specificity of 18 of the 26 strains was confirmed by immunofluorescence procedures, and all 26 strains were confirmed by enzyme-linked immunosorbent assay (ELISA). All RSV isolates were stored at -70 C until they were characterized for subgroup properties by ELISA, immunofluorescence assay, and radioimmune precipitation assay (RIPA). Each test included a reference subgroup A strain (the Long strain) and a subgroup B strain (the CH18537 strain, kindly provided by Robert M. Chanock, National Institute of Allergy and Infectious Diseases). Monoclonal antibodies. Eighteen monoclonal antibodies specifying 12 epitopes of RSV were selected from a collection of 31 monoclonal antibodies and used in the ELISA procedures. These antibodies included five anti-g antibodies specific for three epitopes (Gi, antibody C793; G2, antibodies B14, B18, and B23; G4, antibody B17), four anti-f antibodies specific for one epitope (Fl, antibodies C787, B46, B47, and B81), five anti-np antibodies specific for four epitopes (NP1, antibody B90; NP4, antibody B130; NP5, antibody B131; NP6, antibodies B60 and B62), three anti-m antibodies specific for three epitopes (Ml, antibody C781; M3, antibody B39; M4, antibody B50), and one anti-p antibody (C771) (1, 13). Eight monoclonal antibodies were used in the immunofluorescence assay and in RIPA (Gi, antibody C793; G2, antibody B18 in RIPA and antibody B23 in immunofluorescence assay, G3, antibody B158; Fl, antibody C787; F2, antibody B151; NP1, antibody B90; NP2, antibody B27; NP4, antibody B130 [1, 13]). ELISA procedures. For subgroup identification of RSV isolates by ELISA, virus isolates were passaged in HEp-2 cells grown in plastic flasks (Nunc) and harvested when approximately 50% of the cell sheet showed cytopathic effect. The cells were removed from the surface of the flask with a mixture of trypsin-edta, washed twice in phosphate-buffered saline, and dispensed into microtiter plates (50,000 cells per well). The plates were allowed to dry at room temperature, and they were stored at room temperature until used. The ELISA procedure has been described elsewhere (14). Briefly, dilutions of monoclonal antibodies prepared in mouse ascites fluid were added to wells and allowed to react overnight at 4 C. Four 10-fold dilutions starting at 1/1,000 were used. The plates were washed thoroughly, a 1/1,000 dilution of rabbit anti-mouse immunoglobulin G conjugated with peroxidase (DAKO, Santa Barbara, Calif.) was added, and the plates were incubated at room temperature for 30 min. After a further thorough washing, 5-aminosalicylate substrate was added, the plates were incubated at 37 C for 30 min, and the color which formed was quantitated in a microtiter spectrophotometer 30 min later. Usually an absorbance value of 0.2 or higher was considered positive. Values less than 0.2, which were considered significant, were at least three times the background value (control wells of infected cells were treated with all reagents except dilutions of monoclonal antibodies). Immunofluorescence. Identification of RSV subgroup by immunofluorescence was performed as previously described (13, 17). Briefly, HEp-2 cells were grown on glass microscopic slides, and when confluent, the monolayer was infected with RSV. When approximately 50% of the cell monolayers showed cytopathic effect, the slides were fixed in cold acetone at -20 C for 10 min and stored at -70 C until they were used. Monoclonal antibodies at a dilution of 1/20 were dispensed dropwise onto small circumscribed areas of the acetone-fixed monolayer and reacted at room temperature for 30 min. After the monolayer was washed, goat anti-mouse immunoglobulin G F(ab)2 fragment conjugated to fluorescein (Cooper Biomedical, Inc., West Chester, Pa.) was added to the same areas for 30 min at room temperature. Finally, the slides were washed, and a cover slip was mounted with glycerol. The monolayers were viewed with a UV microscope and an excitation filter, and the intensity of fluorescence was graded from + (minimum) to (maximum). Fluorescence of + or greater was considered a positive result. Controls consisted of uninfected HEp-2 cells treated in the usual manner and infected cells treated with all reagents except monoclonal antibodies. RIPA. The procedure for RIPA has been described previously (13, 17). Briefly, the RSV strains were radiolabeled with [35S]methionine (for precipitation of the F, NP, M, and P proteins) or [3H]glucosamine (for precipitation of the G and F proteins) and reacted with the appropriate monoclonal antibodies directed against the RSV structural proteins. Five monoclonal antibodies were used with [35S]methioninelabeled virus, and five monoclonal antibodies were used with [3H]glucosamine-labeled virus; two of these antibodies were used with both the [35S]methionine- and [3H]glucosaminelabeled viruses. The RSV-monoclonal antibody mixtures were adsorbed to Sepharose A beads, washed thoroughly, and dried overnight at 37 C. The beads were treated with sample buffer and fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis under reducing conditions. Clinical data. Details of the illnesses were abstracted from clinic charts or hospital records, as appropriate. Parents of children in the vaccine study were interviewed by the nurses who regularly conducted surveillance of acute respiratory disease among the study children. URI was defined as symptoms and signs localized to the upper airway passages, including rhinorrhea, cough, otitis media, and pharyngeal inflammation, but no fever. When fever higher than 100 F (37.8 C) was present, the illness was defined as a febrile URI. LRI was characterized by rales or wheezing, radiographic evidence of pneumonia or hyperinflation, or both clinical signs and radiographic abnormalities. RESULTS Between 1979 and 1985, 13 children were identified who had two RSV infections confirmed by isolation of the virus from pharyngeal swab specimens (Table 1). Of the 26 RSV isolates, 15 belonged to subgroup A and 11 belonged to subgroup B. The assignment of isolates to either subgroup A or B was based on their characteristic patterns of reaction with monoclonal antibodies directed against the F, G, NP, and M proteins of the virus (Table 2). For each of the 26 RSV strains, the results of subgroup characterization by ELISA, RIPA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunofluorescence tests were in complete agreement. Subgroup A strains reacted with all monoclonal antibodies, but subgroup B strains failed to react with five (B14, B18, B23, B158, and B17), one (C787), and one (B90) antibody directed against the G, F, and NP proteins, respectively.

3 VOL. 25, 1987 TABLE 1. Virus subgroup in and clinical data on children twice infected with RSV RSV subgroup Age (mo) Type of illness no. Sex Infec- Infec- Infec- Infec- Infec- Infection 1 tion 2 tion 1 tion 2 tion 1 tion 2 1 Female B A URI URI 2 Male A A URI URI 3 Male A A F-URIa LRI 4 Male A B F-URI URI 5 Female A A URI URI 6 Male A B URI LRI 7 Male B B LRI URI 8 Male A B 6 18 LRI LRI 9 Male A A 7 32 LRI LRI 10 Female B B LRI LRI il Female A B URI URI 12 Female A B URI URI 13 Male A B URI URI a F-URI, Febrile URI. Overall, second infections with the homologous subgroup were detected as often as second infections with the alternate subgroup. Of the 13 children, 6 had two infections of the same subgroup and 7 had two infections with different subgroups (Table 3). During the first infection, 10 of the 13 children had subgroup A strains, which represented a 3:1 ratio of A/B strains. This ratio of A/B strains in our community at large has been observed in each epidemic year since 1981, except from 1984 to 1985, when B strains occurred somewhat more often than A strains (M. A. Mufson, data to be published). By contrast, subgroup B strains occurred more often during second infections than A strains did (only one subgroup B strain of second infections occurred during the 1984-to-1985 epidemic year). Of the 10 children who had subgroup A strains during their first infection, 6 had B strains and 4 had A strains during the second infection. This finding TABLE 2. Characteristics of subgroup A and B strains of RSV isolated from children with two infections Reactions of RSV strains with Monoclonal monoclonal antibodies" Epitope antibody Subgroup A Subgroup B ELISA RIPA IF ELISA RIPA IF C793 Gi B14 G2 + ND ND - ND ND B18 G2 + + ND - - ND B23 G2 + ND + - ND - B158 G3 ND + + ND - - B17 G4 + ND ND - ND ND C787 Fl B46 Fl + ND ND + ND ND B47 Fl + ND ND + ND ND B81 Fl + ND ND + ND ND B151 F2 ND B90 NP B27 NP2 ND + + ND + + B130 NP B131 NP5 + ND ND + ND ND B60 NP6 + ND ND + ND ND B62 NP6 + ND ND + ND ND C781 Ml + ND ND + ND ND B39 M3 + ND ND + ND ND B50 M4 + ND ND + ND ND C771 P + ND ND + ND ND a IF, Immunofluorescence; +. positive; -, negative; ND, not determined. SUBGROUPS OF RSV IN CHILDREN 1537 TABLE 3. Pattern of occurrence of RSV subgroups in children with two infections No. of children with the RSV subgroup following interval between No. of children infections (yr): Infection 1 Infection A A A B B A i 1 B B 2 1 i represents significantly more subgroup B strains than expected by chance (X2 = 6.53, df = 1, P < 0.02). None of these 10 children had received the live RSV vaccine (only 1 of the 13 children studied was a vaccine recipient, and this child had two subgroup B infections). The data are consistent with an interpretation that infection with subgroup A strains conferred immunity to second infections with strains of the same subgroup, at least as reflected in the occurrence of second infections in the 2 years after the first infection. The ages of children at their first RSV infection were 6 to 36 months (mean, 24 months), and at their second infection, they were approximately 1 or 2 years older. No second infections were identified within the interval of a single annual epidemic. The illnesses associated with these RSV infections were characterized as febrile URI, URI, or LRI, which included pneumonia, bronchiolitis, or croup (Table 1). No difference was observed in the intensity of the primary infection with virus of either subgroup. The respiratory tract illnesses associated with second infections, whether subgroup A or B, were not severer than the illnesses observed during first infections. LRI occurred as often with first infections as they did with second infections. Serum sample pairs from both infections for 7 of the 13 children were tested for antibody by ELISA with the subgroup A Long strain as the antigen (Table 4). Four children had fourfold or greater rises during only one infection, and three had rises in antibody during both infections. Three of the four children who exhibited only one rise in antibody did so during their second infection. Rises in antibody were detected in five of eight instances of infection with virus of subgroup A and five of six instances with virus of subgroup B. The age of the child at the time of infection was unrelated to the development of a rise in antibody level. TABLE 4. Antibody responses of children twice infected with RSV RSV subgroup Titer of antibody to RSV measured by ELISA' Patient no. Infection 1 Infection 2 Infection 1 Infection 2 Acute Conva- Acute onvaphase lescent.ace descent phase pnasehphase 1 B A <2,500 <2,500 <2,500 10,000 3 A A 2,500 2,500 2,500 20,000 4 A B <2,500 5,000 5,000 40,000 5 A A <2,500 5,000 5,000 5,900 6 A B 2,500 5,000 <2,500 10,000 7 B B <2,500 20,000 <2,500 5,000 il A B 2,500 40,000 2,500 10,000 a Values in boldface represent fourfold or greater rises in antibody.

4 1538 MUFSON ET AL. DISCUSSION J. CLIN. MICROBIOL. The data from this study show that infection with subgroup A strains of RSV confers some measure of homologous immunity. Since the annual epidemics of RSV consist of three times as many A strains as B strains (Mufson, to be published), first infections among the 13 children in this study showed the same ratio and were predominantly infections with subgroup A strains. A similar ratio of A to B strains might have been expected during second infections on the basis of a random allocation of strains. However, more subgroup B strains developed during second infections of RSV. Of the 10 children who had subgroup A strains during their first infections, 6 had subgroup B strains in second infections. This ratio of B/A strains during second infections had a very low probability of occurring by chance. Too few children had first infections with subgroup B strains to allow for a similar analysis of these data. Further studies on the durability of homologous immunity seem warranted. Reinfection with RSV occurs commonly (3, 8). As virtually all older children and adults possess neutralizing antibody to RSV, infections in these groups reflect reinfection. Whereas severe respiratory tract disease associated with RSV infection occurs almost exclusively in very young children, adults usually experience only mild URI (10). Studies with adults would be important to document the influence of homologous or heterologous immunity to the two subgroups of RSV. From our limited data, there is no indication that the pathologic potentials of subgroup A and B strains of RSV differ. Second infections were not severer as a group than first infections with either subgroup of RSV, and LRI occurred as often during second infections as during first infections. Reinfection with RSV has not been associated with severer illness in children (8). Thus, there was no evidence of immunopathologic amplification of illness in cases of two consecutive infections with strains of different RSV subgroups. Our observations of consecutive infections with RSV represented different circumstances than the potentiation of disease observed after the administration of killed RSV vaccine (6, 11). The F glycoproteins of RSV play a major role in immune protection against infection (18, 19). The most striking differences in reaction patterns of subgroups A and B with a panel of nine monoclonal antibodies were observed with the G protein (13). However, these antibodies reacted with epitopes representing a single antigenic site. Monoclonal antibodies against the F protein also showed some differences between virus strains representing different subgroups. Furthermore, the F protein exhibited distinct molecular weight differences between viruses representing subgroups A and B (15). The occurrence of both subgroupunique and subgroup-shared structures in the G and F proteins may have consequences for the degree of cross protection between infection with viruses of different subgroup characteristics. In cotton rats, it was shown that replication of RSV in the respiratory tract stimulated the production of anti-f antibodies and provided protection against infection after an intranasal challenge with virus of the same subgroup (16). Recombinant vaccinia virus containing the mrna coding sequence of the F protein of RSV inoculated intradermally into cotton rats induced high levels of circulating anti-f neutralizing antibodies (16). When these rats were challenged by intranasal instillation of a high dose of infectious RSV, replication of the virus in the respiratory tract, especially the lungs, was significantly reduced. The effect was similar to intranasal immunization of rats with infectious RSV. A recombinant vaccinia virus possessing the mrna coding sequence of the G protein was much less effective in preventing replication of RSV after challenge (16). Our findings have implications for the composition of RSV vaccine since we have shown homologous immunity in subgroup A strains. Cross immunity between the two subgroups of RSV was not observed, we can speculate that both subgroups should be incorporated into any RSV vaccine to achieve the broadest possible representation of immunogens. Additional observations on subgroup-specific immunity are needed to assess the extent of cross immunity, if any, between the two subgroups and the importance of subgroup-specific immunity in modulating infection. ACKNOWLEDGMENTS Excellent technical assistance was provided by Dallas Hughey and Mariethe Ehnelund. This work was supported in part by a grant from the Swedish Medical Research Council (project B86-16X C) and the World Health Organization Programme for Vaccine Development. LITERATURE CITED 1. Âkerlind, B., and E. Norrby Occurrence of respiratory syncytial virus subtypes A and B strains in Sweden. J. Med. Virol. 19: Anderson, L. J., J. C. Hierholzer, C. Tsou, R. M. Hendry, B. F. Fernie, Y. Stone, and K. Mclntosh Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. J. Infect. Dis. 151: Belshe, R. B., J. M. Bernstein, and K. N. Dansby Respiratory syncytial virus, p In R. B. Belshe (ed.), Textbook of virology, 1st ed. PSG Publishing Co., Littleton, Mass. 4. Belshe, R. B., L. P. Van Voris, and M. A. Mufson Parenteral administration of live respiratory syncytial virus vaccine: results of a field trial. J. Infect. Dis. 145: Belshe, R. B., L. P. Van Voris, and M. A. Mufson Impact of viral respiratory diseases on infants and young children in a rural and urban area of southern West Virginia. Am. J. Epidemiol. 117: Chin, J., R. L. Magoffin, L. A. Shearer, J. H. Schieble, and E. H. Lennette Field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population. Am. J. Epidemiol. 89: Gimenez, H. B., N. Hardman, H. M. Keir, and P. Cash Antigenic variation between human respiratory syncytial virus isolates. J. Gen. Virol. 67: Henderson, F. W., A. M. Collier, W. A. Clyde, Jr., and F. W. Denny Respiratory syncytial virus infections, reinfections and immunity. A prospective longitudinal study in young children. N. Engl. J. Med. 300: Hendry, R. M., A. L. Talis, E. Godfrey, L. J. Anderson, B. F. Fernie, and K. Mclntqsh Concurrent circulation of antigenically distinct strains of respiratory syncytial virus during community outbreaks. J. Infect. Dis. 153: Johnson, K. M., H. H. Bloom, M. A. Mufson, and R. M. Chanock Natural reinfection of adults by respiratory syncytial virus. Possible relation to mild upper respiratory disease. N. Engl. J. Med. 267: Kim, H. W., J. G. Conchola, C. D. Brandt, G. Pyles, R. M. Chanock, K. Jensen, and R. H. Parrott Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine or by infection. Am. J. Epidemiol. 89: Lamprecht, C. L., H. E. Krause, and M. A. Mufson Role of maternal antibody in pneumonia and bronchiolitis due to respiratory syncytial virus. J. Infect. Dis. 134:

5 VOL. 25, Mufson, M. A., C. Orvell, B. Rafnar, and E. Norrby Two distinct subtypes of human respiratory syncytial virus. J. Gen. Virol. 66: Norrby, E., S. N. Chen, T. Togashi, H. Sheshberadaran, and K. P. Johnson Five measles virus antigens demonstrated by use of mouse hybridoma antibodies in productively infected tissue culture cells. Arch. Virol. 71: Norrby, E., M. A. Mufson, and H. Sheshberadaran Structural differences between subtype A and B strains of respiratory syncytial virus. J. Gen. Virol. 67: Olmsted, R. A., N. Elango, G. A. Prince, B. R. Murphy, P. R. Johnson, B. Moss, R. M. Chanock, and P. L. Collins Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus: comparison of the individual SUBGROUPS OF RSV IN CHILDREN 1539 contributions of the F and G glycoproteins to host immunity. Proc. Natl. Acad. Sci. USA 83: Sheshberadaran, H., S. N. Chen, and E. Norrby Monoclonal antibodies against five structural components of measles virus. I. Characterization of antigenic determinants on nine strains of measles virus. Virology 128: Taylor, G., E. J. Stott, M. Bew, B. F. Fernie, P. J. Cote, A. P. Collins, M. Hughes, and J. Jebbett Monoclonal antibodies protect against respiratory syncytial virus infection in mice. Immunology 52: Walsh, E. E., J. J. Schlesinger, and M. W. Brandriss Protection from respiratory syncytial virus infection in cotton rats by passive transfer of monoclonal antibodies. Infect. Immun. 43: ,