Isolation of Rhinovirus Intertypes Related to Either Rhinoviruses 12 and 78 or 36 and 58

INFECTION AND IMMUNITY, Apr. 1983, p. 213-218 0019-9567/83/040213-06$02.00/0 Copyright 1983, American Society for Microbiology Isolation of Rhinovirus Intertypes Related to Either Rhinoviruses 12 and 78 or 36 and 58 Vol. 40, No. 1 LAUREL M. HALFPAP AND MARION K. COONEY* Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98195 Received 14 July 1982/Accepted 3 January 1983 Many antigenic relationships have been demonstrated among the 90 rhinovirus serotypes. Among these are reciprocal cross-reactions between serotypes 12 and 78 and between serotypes 36 and 58. Neutralizing-antibody titers to homologous virus of the related pairs are generally 16- to 64-fold higher than to the heterologous member, and neutralization by heterologous anti in the pools is not seen with prototype viruses. However, a number of isolates were encountered which gave anomolous results when tested with the anti pools in fetal tonsil cells. When these strains were tested in fetal tonsil cells against the monospecific antisera composing the pools, it was shown that several isolates were apparently intertypes, neutralized equally by antisera to related types 12 and 78 or 36 and 58. Isolate 1104, an apparent intertype between serotypes 36 and 58, and isolate 9433, intermediate between serotypes 12 and 78, were selected to use as immunogens in rabbits. When tested in HeLa cells, anti prepared against isolate 1104 neutralized isolates 1104, 58, and 36 at titers of 1280, 640, and 40, respectively. The k values against isolates 1104, 58, and 36 were 356, 145, and 4, respectively, indicating a much closer relationship of isolate 1104 to type 58 than to type 36. Similar results were obtained with isolate 9433. The neutralizingantibody titer of anti-9433 was 160 against both 9433 and type 78 and was 20 against type 12. The k values of anti-9433 against 9433, 78, and 12 were 161, 111, and 2, respectively, indicating that 9433 and 78 were nearly identical. However, the respective neutralizing-antibody titers of anti-78 to type 78 and isolate 9433 were 640 and 80, and the respective k values were 172 and 85, demonstrating some antigenic differences. The discovery of intertypes confirms the antigenic variation among rhinoviruses, and the intertypes may represent links in the evolution of types. These observations also demonstrate that isolates in first or second passage in diploid cells may display an antigenic profile different from that seen in HeLa cells at high HeLa cell passage level. Rhinoviruses are the largest group of human viruses known at the present time; in this group, 89 serotypes and 1 subtype have been numbered, and at least 20 additional serotypes are believed to exist (13, 16, 17). A number of antigenic relationships have been shown, usually involving a reciprocal cross relationship between two rhinovirus serotypes, and occasionally among three. In our laboratories, rhinovirus isolates from specimens are typed by neutralization by using pools of rabbit anti-rhinovirus sera which include all 90 type-specific antisera (18). The pools are designed so that each type-specific anti is in only one, or at most two, of the pools. Prototype strains of rhinoviruses known to be related to other serotypes are not neutralized by anti pools used for typing which contain the heterologous related virus. Thus, isolates are neutralized by one or two anti pools, but we have observed occasional isolates 213 that are neutralized by three or four pools. These field strains show sufficient variation from the prototype virus to cause a problem in typing. When the identification of these isolates was resolved with monospecific antisera, most were found to be related to rhinoviruses 36 and 58 or to rhinoviruses 78 and 12. Both pairs are reciprocally neutralized at low dilutions (3, 23). These observations suggested considerable variation among wild-type rhinovirus strains, to the extent that intertypes might occur between related pairs of rhinoviruses. Isolates intermediate between two pairs of related rhinoviruses, types 36 and 58 and types 12 and 78, were chosen for further study of antigenic relationships to each other and to prototype strains. The results presented here illustrate considerable variation among wild-type strains, with some strains apparently equally related to both members of a related pair. These findings have implications

214 HALFPAP AND COONEY for the future classification of the rhinovirus group as well as for an explanation of the very large number of serotypes in this group. MATERIALS AND METHODS Viruses. Rhinovirus isolates 9433 and 1104, related to types 12 and 78 and to types 36 and 58, respectively, were selected from specimens collected during the Seattle Virus Watch, 1965-69 (14), and from children (5) during 1974-75. A diploid line of human fetal tonsil cells was used for rhinovirus isolation and typing. To ensure a pure strain of virus, the isolates were triply plaque purified and passed in HeLa cells to attain a high concentration of virus for immunogen preparation. Prototype strains of rhinoviruses 12, 78, 36, and 58 were originally obtained from V. V. Hamparian (Ohio State University), and all viruses were stored at -700C. Cell culture. Fetal tonsil cells were used for the initial isolation and early passages of specimens. M- HeLa cell cultures, cultures of a rhinovirus-sensitive cell line originally obtained from the Merck Institute, West Point, Pa., were used for propagation and plaque assay of rhinoviruses. The growth medium was Eagle minimum essential medium with 10% fetal bovine (Flow Laboratories, Inc., Rockville, Md.) plus 100 U of penicillin and 100,ug of streptomycin per ml. For virus propagation, 1% fetal bovine was used in minimum essential medium with 30 mm Mg2" (11). Immunization of rabbits. The procedure used by Cooney and Kenny (4) was followed to produce a hightiter immunogen. Briefly, the procedure included infection of HeLa cells with a high multiplicity of rhinovirus, harvesting infected cells into a small volume of medium before virus release, and homogenization of cells to allow virus release. The homogenate was centrifuged to sediment cell debris. The supernatant fluid was treated with fluorocarbon and constituted the immunogen. Each of two rabbits was injected with a total rhinovirus dose of 3.3 x 107 PFU of isolate 9433, 3.0 x 108 PFU of isolate 1104, and 1.6 x 108 PFU of prototype 58 (for the second lot of anti-58 ). Plaque assay. HeLa cell monolayers plated the previous day (four plates per sampling time) were inoculated with 0.2 ml of appropriately diluted virus. After absorption at room temperature for 1 h, monolayers were overlaid with 5 ml of minimum essential medium with 1% fetal bovine containing 30 mm MgCl2 and 0.4% agarose (Matheson Scientific, Inc., Norwood, Ohio). The plates were incubated at 33 C in a 2.5% CO2 incubator until plaques developed (3 to 5 days). The monolayers were stained with crystal violet, and the plaques were counted. Neutralizing-antibody determination. The procedure for the neutralizing-antibody assay has been described elsewhere (6). Briefly, twofold serial dilutions of anti were prepared in flat-bottomed microtiter plates in 0.025-ml volumes. An equal volume of the appropriate virus suspension, diluted to contain 100 50% tissue culture infectious doses (TICD50s), was added to the dilutions. After 1 h at room temperature, 0.05 ml of a HeLa cell suspension (20,000 cells) was added to each well. A virus titration and cell controls were included in each test. Plates were covered with Lucite INFECT. IMMUN. TABLE 1. Antigenic variation in rhinovirus isolates related to cross-reacting rhinovirus prototypes 58 and 36 Neutralizing-antibody titera No. or type of with: virus isolate Anti-58 Anti-36 1 20 80 1lb 40 20 lb 20 20 1 80 20 Prototype 58 640 80 Prototype 36 40 1,280 a Expressed as the reciprocal of the dilution which completely neutralized 30 to 300 TCID50s of virus in fetal tonsil cells. b Rhinovirus isolate selected for plaque purification. lids and incubated in 2.5% CO2 at 340C until controls showed the presence of 300 TCID50s of virus (usually at 3 days). Formalinized crystal violet was added to each well to inactivate virus and to stain remaining cells. Neutralizing-antibody titers were expressed as the reciprocal of the highest dilution which completely neutralized the virus dose. Determination of k values. To determine neutralization rate constants (k values) (1, 19), equal volumes of diluted and virus (approximately 10' PFU/ml) were mixed and incubated along with a virus control in a water bath at 37 C. The -virus mixture and the virus control were sampled at different time intervals (0, 2, 5, 10, and 15 min or 0, 5, 10, 15, and 30 min for homologous or heterologous neutralization, respectively). The mixture was diluted 1:100 in cold (0 C) diluent to stop neutralization, and 0.2 ml was plated on HeLa monolayers (four plates per sample) to assay the unneutralized virus (4). Each k value reported is the average of duplicate experiments. The k values (reciprocal of the dilution times slope) were based on the following equation: k = 2.3 x (Dlt) x loglo (VJ/V,), where D is the reciprocal of the final dilution, t is the time in minutes, and V0 and V, are the numbers of PFU of virus at time zero and time t, respectively (1). A value for the slope of the regression line was determined. A CDC 6600 computer, using a REGRESSION subprogram of the Statistical Package for the Social Sciences (Vogelback Computing Center, Evanston, Ill.) in a basic application of the general linear model (2), was used to calculate the slope for each k value by linear regression analysis. Only the points on the linear portion of the neutralization curve were calculated by regression analysis. RESULTS Antigenic variation in rhinovirus isolates related to prototype rhinoviruses 36 and 58. The prototype rhinoviruses 36 and 58 show an antigenic relationship when neutralized with rabbit anti (3). The low-level reciprocal neutralization between types 36 and 58 (Tables 1 and 2) is not equivalent in both directions. Anti-36 showed a higher titer for type 58 than anti-58 showed for type 36.

VOL. 40, 1983 ANTIGENIC VARIATION IN RHINOVIRUS ISOLATES 215 TABLE 2. Neutralizing-antibody titers and k values for prototypes 36 and 58 and strain 1104 Anti-36 Anti-1104 Anti-58 Rhinovirus Titer kb Titer k Titer k 36 1,280 295.4 40 3.7 40 3.0 1104 80 19.4 1,280 356.2 320 131.7 58 80 22.6 640 145.1 640 574.8 a Neutralization titer expressed as the reciprocal of the dilution which neutralized 30 to 300 TCID50s of virus in HeLa cells. b Neutalization rate constant calculated as described in the legend to Fig. 1. 300-0 ANTI-RHNOsRJUS 58vsuI04,Dz2OOO A ANTI-RHINOVIRUS S8 vs 36, D 40 0 ANTI-RHINOVIRUS58 vs58,d 2OOO Antisera to rhinoviruses 36 and 58 were titrated against isolates which appeared to be partially neutralized by pools containing both anti-36 and anti-58 sera. A range of antigenic variation among isolates was revealed. Two isolates (Table 1) were nearly equally or equally neutralized by the two antisera; the remaining two were antigenically closer to either type 36 or type 58. The isolates equally neutralized by the prototype antisera appeared to be intertype strains. The two isolates were plaque purified and passaged two to three times in HeLa cells. Cloned viruses from each isolate were equally neutralized by antisera to rhinoviruses 36 and 58. The cloned isolate that grew to the highest titer, isolate 1104, was selected to produce immunogen. The homologous neutralizing-antibody titer of the anti-58 used in the typing pools and titrated against the isolates was 640, and this was thus suitable for typing isolates. However, the homologous k value was only 75, indicating a low affinity, and the therefore was of questionable value as a reagent for comparing type 58 with the possible intertypes. Therefore, a pair of rabbits was immunized with a new preparation of type 58. The resulting anti possessed a high affinity (homologous k value = 574) and a neutralization titer similar to that of type 58 (640 to 1,280). This lot of anti also had a fourfold higher neutralization titer to isolate 1104 (320) than did the anti-36 (80), indicating that isolate 1104 was type 58. The high-affinity anti-58 was used in all subsequent experiments. The neutralization rate constant (k value) and the microneutralization assay were used to determine the relationship between the variant 1104 and the prototypes 36 and 58 (Table 2). The results of both assays indicated that isolate 1104 was a variant of type 58 and was reciprocally related to type 36. Variant 1104 and prototype 58 were neutralized by anti-36 at a dilution of 1:80. However, anti to prototype 58 neutralized 1104 and 58 at extremely different rates and titers, as illustrated by a sample experiment (Fig. 1). These results indicate that isolates 58 and 1104 are variants of the same type. Antigenic variation in rhinovirus isolates related to prototypes 12 and 78. The prototypes 12 and 78 have previously been shown to be antigenically related (3, 13). The neutralizing-anti- 200-100 K=156.~50 K-645 K=3 I0 I 0 2 5 10 15 Time in minutes FIG. 1. Comparison of rate of neutralization of homologous rhinovirus 58 and heterologous rhinoviruses 36 and 1104 by rabbit anti-rhinovirus 58. The k values (reciprocal of the dilution times slope) were based on the following equation: k = 2.3 x (Dit) x loglo (VJV,), where D is the reciprocal of the final dilution, t is the time in minutes, and VO and V, are the numbers of PFU of virus at time zero and time t, respectively. The slope was calculated by linear regression analysis for each neutralization constant. Only points on the linear portion of the curve were used. The r values (fit of the line) are 0.98 (0), 0.96 (A), and 0.91 (0).

216 HALFPAP AND COONEY TABLE 3. Antigenic variation in rhinovirus isolates related to cross-reacting rhinovirus prototypes 12 and 78 Neutralizing-antibody titer' No. or type of virus isolates Anti-12 with: Anti-78 4 40-80 320-640 5 640 40-160 5 160-320 80-160 3b 160 160 Prototype 12 1,280 10 Prototype 78 40 640 a Expressed as the reciprocal of the dilution which completely neutralized 30 to 300 TCID50s of virus. b Rhinovirus isolates selected for plaque purification. body titers and k values shown in Tables 3 and 4 demonstrate that anti-12 neutralizes type 78 at a higher titer and a faster rate than anti-78 neutralizes type 12. This unequal reciprocal neutralization is parallel to the antigenic relationship between prototypes 36 and 58. Titrations of anti-12 and anti-78 sera with selected isolates as the antigen were performed in fetal tonsil cells and revealed a range of antigenic variation among isolates. The isolates listed in Table 3 all demonstrate the crossreaction between types 12 and 78. However, four patterns of neutralization were observed. Four isolates were antigenically closer to rhinovirus 12, five were closer to rhinovirus 78, and five were nearly equally related and three were equally related to types 12 and 78. The three isolates which appeared to be intertypes between types 12 and 78 were plaque purified in HeLa cells and successively passaged in HeLa cells to maximize the titer. All of these clones were more closely related to type 78 than to type 12. Two of the cloned isolates were neutralized at a 16-fold-higher titer by anti-78 than by anti-12. The third clone of isolate 9433 was neutralized by anti-78 at an eightfold-higher titer than by anti-12 (Table 4); therefore, this clone of isolate 9433 INFECT. IMMUN. was selected to produce immunogen. Although the original reaction indicates that it was an intertype, the neutralization titers and k values of anti-9433 against isolates 9433, 78, and 12 (Table 4) reconfirmed the close antigenic relationship between 9433 and 78. Anti-9433 had the highest k value to the homologous virus; however, the neutralizing-antibody titers to isolate 9433 and type 78 were equivalent. The relationship of 9433 to type 12 appears to be similar to that of the prototype strain. Comparison of antisera to strain 1104 and to prototype 58. The virus isolates listed in Table 1 and 3 and other isolates from the Seattle Virus Watch (14) were used to compare anti to isolate 1104 with that to type 58 and anti to isolate 9433 with that to type 78. Neutralizingantibody titers of anti-1104 were consistently high against strain 1104, prototype 58, and other isolates typed as 58, whereas anti-58 titers varied (Table 5). Anti-1104 and anti-58 sera equally neutralized prototype 36 (titer, 40) and six other isolates typed as 36 (titer, 10 to 20). Anti-9433 had equal or lower titers than anti-78 to the isolates listed in Table 3 and to other isolates typed as 78. DISCUSSION Within the rhinovirus group, over 40 serotypes can be linked directly by one- or two-way crosses or indirectly through two or more serotypes; e.g., rhinoviruses 67 and 28 are linked via types 11, 13, and 32 (anti-11 neutralizes type 28, anti-13 neutralizes type 11, and anti-32 neutralizes both types 13 and 67) (7, 10, 20). Schieble et al. (23) reported that anti to rhinovirus 58 neutralized type 36; this has been confirmed and extended to a reciprocal cross in our laboratories (3). A reciprocal cross between rhinoviruses 12 and 78 demonstrated in rabbit antisera was among those reported by Fox (13) to make a total of 12 groups of related viruses. Cross-reacting types, strain variants, and prime strains of existing prototyes have evolved (6, 21). Stott and Walker (26) observed antigenic differences in strains of rhinovirus 51 isolated in different years, and TABLE 4. Neutralizing-antibody titers and k values for prototypes 12 and 78 and strain 9433 Anti-12 Rhinovirus Anti-9433 Anti-78 Titer" kb Titer k Titer k 12 1,280 207.0 20 1.5 10 1.2 9433 20 11.9 160 161.1 80 85.8 78 40 4.2 160 111.1 640 172.3 a Neutralization titer expressed as the reciprocal of the dilution which neutralized 30 to 300 TCID50s of virus in HeLa cells. b Neutralization rate constant calculated as described in the legend to Fig. 1.

VOL. 40, 1983 TABLE 5. Neutralizing-antibody titers of prototype 58 anti and strain 1104 anti against isolates typed as rhinovirus 58 Neutralizing-antibody titera Rhinovirus with: isolate Anti-1104 Anti-58 Group Pb 4700 640 160 13702 1,280 160 30059 640 160 30593 1,280 320 31158 1,280 320 Group II1 1235 1,280 320 1342 1,280 640 1104 1,280 320 Prototype 58 640 640 a Expressed as the reciprocal of the dilution which completely neutralized 30 to 300 TCIDos of virus in HeLa cells. b Virus isolates typed as 58. Virus isolates typed as intermediate between 36 and 58. they suggested that such strain variation of type 51 demonstrated the evolution of rhinovirus serotypes; however, no direction of evolution was indicated. Schieble et al. (24) discovered a "prime" strain of rhinovirus 22, and anti to that strain neutralized prototype 22, but the prime strain was not neutralized by prototype 22 anti. A reciprocal cross-neutralization between two prototypes offers a possibility of intermediate strains. However, rhinoviruses 21 and 22 cross-reacted in only one direction, and rhinovirus 51 had no cross-reactions; therefore, the extent of variation could be tested in relation to only one prototype virus. Now we have observed yet another phenomenon, rhinovirus intertypes. An intertype virus has the unique advantage of being related to two viruses, so antigenic variation can be measured relative to both prototypes. In addition to the constant antigenic evolution of rhinoviruses in the field, the manipulation of strains in the laboratory can induce apparent antigenic variations. Different cell lines vary in their susceptibility to rhinovirus infection, and an increased Mg2+ or Ca2+ concentration enhances virus yield and plaque production in HeLa cells (11). The initial observation that isolates 1104 and 9433 were equally neutralized by both of their respective prototype antisera was based on titrations done on fetal tonsil cells used for virus isolation (Tables 1 and 3). The results in Tables 2 and 4 are from experiments using HeLa cells. The change in cell lines could partially explain why both appear antigenically ANTIGENIC VARIATION IN RHINOVIRUS ISOLATES 217 closer to one of the two prototypes. However, a more likely possibility is that during plaque purification the single virus selected was slightly different antigenically from the entire population of viruses in the original specimen. An interesting experiment for future study would be to select a large number of plaque-purified virus clones from one specimen and to document the frequency of different antigenic variants. Another possible reason why variants 1104 and 9433 were not equally related to their respective pairs of prototypes was the dominant neutralization of anti-12 and anti-36 sera. An apparent intertype equally neutralized by anti-36 and anti-58 sera, for example, could be antigenically closer to type 58 for anti-58 to neutralize the virus at the same dilution as anti-36. The same possibility could explain why isolate 9433 was shown to be a variant of type 78, with an antigenic relationship to type 12. Neutralization kinetics provide the sensitivity required for discerning antigenic variation between virus strains and are the basis for this study. However, it is important to correlate the neutralizing-antibody titer with the neutralization rate. Occasionally, the neutralization titer will not reflect the affinity of the immunoglobulin; e.g., two lots of anti-58 anti discussed in this paper had titers of 640, but the k values reflecting the affinity of the antibody were very different, i.e., 75 and 574, respectively. Nine of twelve rhinovirus 58 strains recovered from field specimens (14) were identified by using the low-affinity, but three strains were only identified as type 58 by the highaffinity anti-58 or by anti to isolate 1104. The meaningful interpretation of k values depends on a precise statistical analysis. Calculation of the linear regression line and slope of each experiment is a marked improvement over the less accurate method of sketching a line visually through data points on a graph. Another advantage of linear regression analysis is that the r value (correlation coefficient) can be calculated. If the line perfectly fits all of the points on the graph, then r = 1. The average r value in 36 experiments was 0.935, with a standard deviation of 0.066. The differences between the three k values listed under each anti in Tables 2 and 4 range from 31 to 99%, except for the values obtained with rhinovirus 36 anti against isolates 1104 and 58, which differ by 16.5%. Dulbecco and Ginsburg (9) stated that "differences of about 20% are usually significant" for animal viruses. Anti-1104 neutralized a wider range of viruses typed as 58 and neutralized viruses intermediate between types 36 and 58 at a higher dilution than did prototype 58 anti. The

218 HALFPAP AND COONEY INFECT. IMMUN. total amounts of virus immunogen injected to produce anti-1104 and anti-58 sera were essentially the same (3.0 x 108 PFU of prototype 58). Clearly, anti-1104 would be a superior representative of type 58 and would improve typing efficiency. Isolate 9433 did not produce a high-titer anti (homologous neutralization titer, 160) compared with prototype 78 (homologous titer, 640). Consequently, anti-9433 had equal or lower neutralization titers to viruses intermediate between types 12 and 78 and to isolates typed as 78. Other isolates (Table 3) have the potential to be more representative of type 78. The major reason for the prevailing pessimism concerning a rhinovirus vaccine is the multiplicity of rhinovirus serotypes. As Fox (13) has noted, some rhinovirus serotypes persist in a population and cause more infections than others; hence, they would be the viruses of choice for a vaccine. 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