Antigenic Characterization of Measles and SSPE Virus Haemagglutinin by Monoclonal Antibodies

J. gen. Virol. (1981), 57, 357-364. Printed in Great Britain 357 Key words: measles/morbillivirus/monoclonal antibody Antigenic Characterization of Measles and SSPE Virus Haemagglutinin by Monoclonal Antibodies By VOLKER TER MEULEN,* SIEGLINDE L()FFLER, MICHAEL J. CARTER AND JOHN R. STEPHENSONt Institute of Virology and Immunobiology, Versbacher Strasse 7, D-8 700 Wiirzburg, Federal Republic of Germany (Accepted 5 August 1981) SUMMARY Hybrid cells secreting monoclonal antibodies directed against the haemagglutinin (H) protein of measles virus (Edmonston) were produced by fusion of mouse myeloma cells with spleen cells derived from immunized mice. Measles antibodies secreted by these cells were tested for their ability to react with measles virus in immunoprecipitation experiments and assays of binding, neutralization, haemagglutination inhibition and haemolysin inhibition. On this basis 21 out of 75 hybridomas could be defined and divided into five functional groups with different properties. However, when tested against other measles virus strains, including those isolated from subacute sclerosing panencephalitis (SSPE) patients, normalized radioimmunoassay (RIA) binding titres showed that the extent to which a given antibody bound could vary greatly with the virus strain examined. Moreover, the biological actions within a group were found to be very heterogeneous, even when high antibody binding titres were observed. These results suggest that different measles virus strains, which are not distinguishable by polyvalent sera, do in fact possess antigenic differences. Furthermore, the functional significance of a given virus epitope may vary from strain to strain. Hybridoma antibodies were also used to demonstrate the occurrence of antigenic changes within the H polypeptide of SSPE virus during the course of a non-productive, persistent infection in vitro. INTRODUCTION The association of measles virus with central nervous system (CNS) diseases such as measles encephalitis and subacute sclerosing panencephalitis (SSPE) has stimulated interest in the biological and biochemical properties of this agent. In both disorders measles virus has occasionally been isolated and compared with virus derived from acute measles (ter Meulen et al., 1972a, b; Agnarsdottir, 1977; Fraser & Martin, 1978). Although biological and biochemical differences between certain strains have been reported, the results obtained are not consistent and do not permit the unequivocal differentiation of SSPE viruses in general from wild-type measles viruses. However, any observed virological differences might reflect mutational changes leading to antigenic alterations. Antigenic analysis of measles and SSPE viruses by monoclonal hybridoma antibodies might help to define these agents (Birrer et al., 1981; Trudgett et al, 1981). In the present study monoclonal hybridoma antibodies were obtained against measles virus haemagglutinin and used to characterize this structural protein from different measles virus t Present address: PHLS Centre for Applied Microbiology and Research, Vaccine Research & Production Laboratory, Porton Down, Salisbury, Wiltshire SP40JG, U.K. 0022 1317/81/0000-4811 $02.00 1981 SGM

358 V. TER MEULEN AND OTHERS strains. It could be shown that the various measles and SSPE virus strains tested displayed antigenic changes or at least differences in the topography of the antigenic sites. Moreover, antigenic changes of the haemagglutinin were detected by this method in an experimentally established persistent infection in tissue culture. It is conceivable that similar changes develop during measles virus persistency in CNS tissue. METHODS Tissue culture cells and viruses. A continuous cell line derived from human prostate, MA 160 (Microbiological Associates, Bethesda, Md., U.S.A.) was used for growth of virus stocks and virus preparations for immunization according to Baczko & Lazzarini (1979). Measles virus radiolabelled antigens were prepared in Vero cells. The P3X63Ag8 mouse myeloma cell line was a gift of H. Koprowski, Wistar Institute, Philadelphia, Pa., U.S.A. The following measles virus strains were used: the vaccine strain Edmonston (EDM), two isolates; Woodfolk (WDF) and EVA, from cases of acute measles, three isolates; LEC, Mantooth (MAN), Halle, from SSPE cases and one isolate, Braxator (BRX), from a case of acute measles encephalitis (ter Meulen et al., 1972 a). A persistent infection with SSPE virus LEC was initially established by intracerebral inoculation of hamsters with LEC virus (plaque-purified) grown in Vero cells. Brains from animals with a subacute encephalitis were trypsinized and co-cultivated with Vero cells. The surviving cells were cloned and a persistently infected cell line was obtained which has been carried in our laboratory over a period of several years. Cells from passages 240 to 250 were used during the experiments described here. In this cell line infectious virus could never be detected or reactivated. Approx. 80% of the cells exhibited virus antigen by immunofluorescence. Immunization of animals. Measles virus Edmonston, grown in MA160 cells and purified from the medium as described by Bellini et al. (1979) was used for immunization. 109 p.f.u, of infectious virus, emulsified in Freund's complete adjuvant were inoculated intraperitoneally into 8- to 10-week-old female Balb/c mice (Jackson Laboratories, Bar Harbor, Me., U.S.A.). Four weeks later the animals were boosted intravenously with l0 s p.f.u, of virus. Three days later the animals were sacrificed. Production of hybrid cells. Spleens of immunized Balb/c mice were removed, processed and fused according to a method proposed by Koprowski et al (1977). When hybrid cells were observed, growth media were tested for the presence of measles antibodies by radioimmunoassay (RIA). Positive cells were cloned as described by Coffino & Scharff (1971) and then inoculated into Balb/c mice primed with Pristane (Flamand et al., 1980). Ascites fluid was collected and used in serological and immunoprecipitation experiments. In vivo protein labelling and immunoprecipitation. Vero cells infected with different measles viruses were labelled with [35S]methionine between 20 and 24 h post-infection as described previously (Stephenson & ter Meulen, 1979). Uninfected cells were similarly treated. Radioimmunoassay. The RIA was carried out based on the method previously described by D6rries & ter Meulen (1980). 125I-labelled anti-mouse F(ab')2 antibodies were used to detect monoclonal antibodies reacting with measles virus antigen. For a comparative analysis of reactivity of monoclonal antibodies with the various measles virus strains used in this study, the method described by Gerhard (1976) for influenza viruses was adapted to the above RIA system. The different measles virus strains, grown in MA160 cells, were titrated and equilibrated against a hyperimmune serum of measles virus Edmonston produced in mice which does not distinguish between measles and SSPE viruses (Fraser & Martin, 1978). The extent of monoclonal antibody binding to measles and SSPE virus antigens was determined using the RIA. The amount of antibodies binding to the Edmonston virus was taken as the reference value (100%) and antibody reacting with the other measles virus antigens was

Antigenic character&ation of measles viruses 359 expressed as a percentage thereof. This assay was performed three times using, on each occasion, four determinations for each monoclonal antibody preparation. In these experiments the extent of antibody binding per well was found to be very reproducible, and replicate titrations did not differ by more than 15 %. Serological assays. The haemagglutination inhibition (HI), the haemolysin inhibition (HLI) and the neutralization (NT) assays for measles virus were carried out as previously described by Norrby & Gollmar (1975). The immunofluorescence test was performed as previously published (ter Meulen et ai., 1970). Reagents. The anti-mouse F(ab') 2 antibodies were kindly provided by Dr W. Gerhard, Wistar Institute. FITC-labelled anti-mouse immunoglobulin antisera were purchased from Melloy. ~2Slodine and L-[3SS]methionine were obtained from Amersham International. RESULTS Characterization of measles antibodies produced by hybridoma cell cultures After several cell fusion experiments 21 out of 75 hybridoma cell cultures secreting measles antibodies were obtained which could be characterized (Table I). All of these selected hybridomas reacted in the RIA with the virus used for immunization and immunoprecipitated the haemagglutinin (H) polypeptide of the Edmonston virus. None of the clones inhibited the HLI reaction, whereas the biological activities in the other two assays were quite variable. The different reactions in the HI and in the NT tests permitted grouping of the hybridomas into five groups. In group 1, neither of the clones blocked the haemagglutination reaction nor were capable of neutralizing infectious Edmonston virus. The clones of group 2 exhibited HI activity but failed to neutralize virus. Group 3 consists of clones which showed similar activities in both HI and NT tests. The hybridoma antibodies of group 4 revealed a significantly higher activity in the HI than in the NT assay whereas the NT activity of the clones of group 5 exceeded their HI activity. All hybridoma antibodies reacted with Vero cells infected with the Edmonston virus as detected by indirect immunofluorescence using a FITC-labelled anti-mouse IgG serum. With a labelled anti-mouse IgM serum this reaction could not be visualized. Comparative antigenic analyses of the H polypeptide from several measles virus isolates by monoclonal antibodies The reactivity of monoclonal antibodies with different measles viruses measured in a radioimmunoassay is summarized in Table 2. Provided any comparisons of hybridoma antibodies are made between the patterns of binding relative to Edmonston virus and not based on the absolute extent of binding, such comparisons are independent of any variations in the total immunoglobulin content of the ascites fluids. All virus strains regardless of whether they had been derived from acute measles or CNS infections, reacted with all hybridoma antibodies tested. A common reaction pattern amongst the viruses of each group with the different hybridoma groups was not observed. In general, considerable variation occurred in the reaction of a given hybridoma antibody with different measles viruses. For example, hybridoma 154 of group 5 reacted equally well with the H polypeptide of measles virus EVA, Mantooth and Halle but to a reduced extent with Woodfolk, LEC and Braxator. Similar results were observed with other hybridoma clones. However, the LEC and Braxator strains showed reduced reactivity with all hybridoma clones except for clone 772, and 321 and 322. These latter hybridomas possibly recognize an antigenic structure common to all the haemagglutinin molecules of the various virus strains used. In contrast, hybridoma 34 exhibited quite a different pattern with the measles virus isolates obtained from CNS infection as compared to those from acute measles. This hybridoma antibody reacted only to 18 to

360 Table V. TER MEULEN AND OTHERS 1. Reaction of hybridoma myeloma antibodies with measles virus Edmonston in different serological assays and in immunoprecipitation* Hybridoma Group Clone Immunoprecipitation no. no. RIA HI HLI NT H polypeptide 1 172 10 4,5 <8 <16 <8 + 173 10 s <8 <16 <8 + 2 296 10-5 32 <16 <8 + 298 10-5 128 <16 <8 + 3 34 10-5 64 <16 64 + 299 10-4'5 64 <16 64 + 584 10 4,5 64 <16 128 + 585 10-5 128 <16 128 + 771 10-4 16 <16 16 + 772 10-4 32 <16 16 + 26 10-7.~ 512 <16 512 + 151 I0 -s 8192 <16 4096 + 4 153 10-7,5 8192 <16 2048 + 321 10-7,5 2048 <16 512 + 322 10 -s 16384 <16 1024 + 323 10 7,5 8192 <16 2048 + 5 131 10-6 128 <16 1024 + 152 10-7.5 512 <16 4096 + 154 10-7,5 1024 <16 8192 + 155 10-6 512 <16 2048 + Hyp 10-4'~ 256 64 128 + * In the HI, HLI and NT tests titres of hybridoma antibodies are expressed as the reciprocal of the highest twofold dilution neutralizing the virus or blocking haemagglutination and haemolysin activity; Hyp = hyperimmune mouse serum against measles virus Edmonston. 26 % with the CNS isolates in comparison to the other measles viruses, suggesting that antigenic differences exist between the H polypeptides of these two groups of viruses. Similar differences were observed by analysing the effect of the monoclonal antibodies upon the biological activities of the measles viruses. In the HI and NT tests great variation was observed among the antibody activities towards the different measles viruses (Table 3). These reactions ranged from high to low or zero activity in both tests as exemplified for clone 153 of group 4 in the HI and clone 584 of group 3 in the NT assay. Four features of these results are interesting. Firstly, the hybridomas of group 1 did not react with any of the viruses tested in the HI and NT assay despite the fact that a considerable reaction was detected in the RIA. This observation indicates that the measles antibodies secreted from these two clones react with antigenic sites on the H polypeptide of each measles virus which are not associated with haemagglutination or infectivity. Secondly, hybridoma antibodies which inhibited haemagglutination but failed to neutralize measles virus Edmonston reacted differently with the other measles viruses as demonstrated by the hybridomas of group 2. These clones (296 and 298) were effective in the neutralization of all CNS isolates and measles virus Woodfolk but ineffective against Edmonston and EVA. Thirdly, those clones, which revealed a high reactivity in RIA (clones 321 to 323 of group 4), also yielded high NI and NT titres for all viruses. Fourthly, clone 34 of group 3, which showed a reduced RIA reactivity for CNS-derived measles viruses, gave similar results in the HI and NT tests. In addition, calculation of the HUNT ratio revealed extensive variation within each hybridoma clone. The majority of hybridoma antibodies neutralized the LEC and Braxator viruses more efficiently than they blocked the haemagglutination reaction. Moreover, clone 26

Antigenic characterization of measles viruses 361 Table 2. Immunoreactivity of hybridoma myeloma antibodies with different measles and SSPE virus isolates in RIA * Hybridoma Isolate from acute measles CNS isolate Group Clone no. no. EDM WDF EVA MAN Halle LEC BRX 1 172 100 102 128 136 126 56 78 173 100 89 102 139 102 52 92 2 296 100 82 36 99 44 53 46 298 100 102 84 ll0 66 66 44 3 34 100 90 81 22 26 25 18 299 100 113 78 72 82 82 52 584 I00 62 88 103 74 86 87 585 100 87 125 100 119 97 133 771 100 64 50 83 56 45 51 772 100 64 109 124 88 105 107 26 100 137 125 133 126 54 130 151 100 92 48 134 53 55 33 4 153 100 143 117 157 113 57 64 321 100 126 108 123 95 102 128 322 100 133 133 148 122 112 106 323 100 130 103 153 100 80 76 581 100 93 49 134 53 55 33 5 133 100 130 128 133 124 85 90 152 100 117 104 103 111 66 43 154 100 87 109 114 100 47 61 155 100 119 95 120 119 67 92 * Results are expressed as percentage of reactivity to the virus used (Edmonston) for inducing the various hybridomas. Table 3. Haemagglutination inhibiting (HI) and neutralizing (NT) activity of hybridoma antibodies against measles and SSPE viruses* Isolate from acute measles CNS isolate Hybridoma r ~ ~ c ^ c ~ EDM WDF EVA MAN Halle LEC BRX Group Clone r ~'--'v ~" v ~" ~ r ~ -~r- z. ~ c ~ c ~" no. no. HI NT HI NT HI NT HI NT HI NT HI NT HI NT 172 <8 <8 <8 <8 <8 <8 <8 <8 173 <8 <8 <8 <8 <8 <8 <8 <8 296 32 <8 16 256 8 <8 16 32 298 128 <8 32 128 16 <8 128 16 <8 <8 <8 <8 <8 <8 <8 <8 <8 <8 <8 <8 16 128 8 1024 32 512 64 256 16 512 128 512 34 64 64 128 128 64 64 8 <8 <8 <8 <8 <8 <8 16 299 64 64 64 128 16 32 64 128 32 256 8 1024 32 512 584 64 128 64 <8 64 <8 64 <8 32 <8 8 512 32 I024 585 128 128 256 128 128 128 16 512 32 64 32 2048 128 5t2 771 16 16 16 <8 16 <8 32 <8 16 64 8 256 32 16 772 32 16 16 <8 16 <8 32 <8 16 <8 <8 128 8 16 26 512 512 1024 256 512 512 64 128 64 256 32 2048 64 512 151 8192 4096 8192 512 2048 256 256 64 2048 1024 1024 8192 512 16384 4 153 8192 2048 8192 256 1024 128 1024 512 2048 2048 64 8192 1024 16384 321 2048 512 512 512 512 512 512 512 256 512 2048 4096 256 1024 322 16384 1024 8192 1024 8192 1024 8192 512 8192 1024 8192 4096 8192 4096 323 8192 2048 16384 2048 8192 2048 32768 512 8192 1024 8192 8192 8192 4096 581 128 32 64 256 16 128 128 <8 128 256 <8 2048 32 1024 5 131 128 1024 512 256 128 128 128 128 64 256 256 8192 64 8192 152 512 4096 2048 256 128 128 512 64 256 512 32 4096 1024 16384 154 1024 8192 4096 128 1024 512 1024 32 512 256 64 8192 1024 16384 155 512 2048 2048 256 256 256 1024 <8 256 256 256 8192 256 16384 * Titres of hybridoma antibodies were expressed as the reciprocal ofthe highest twofold dilution neutralizing 50%ofthe p.eu. or causing completeinhibition of 4 HA units ofeach virus.

362 v. TER MEULEN AND OTHERS Table 4. Antigenic variation in measles H polypeptide during a persistent infection* t" Group no, Hybridoma Clone no. 3 585 26 151 4 323 5 131 152 SSPE serum Immunofluorescence assay on infected TC cells.a ( EDM LEC LEC persistent + + + + + -- + + -- + + + + + -- + + -- + + + * The biological properties of these hybridoma myeloma antibodies are listed in Table 3. The characterization of any hybridoma antibody as positive or negative was based on immunofluorescent studies at a variety of hybridoma antibody dilutions (10-1 to 10 3). In this manner pro-zone phenomena were excluded. and 585 (group 3) reacted with all isolates from CNS tissue more effectively in the NT test than in the HI assay, whereas no pronounced differences were observed with the other three measles virus strains. This finding may indicate differences in the functional importance of the antigenic sites involved in the biological reactions. Changes in the antigenicity of the haemagglutinin protein arising during a persistent infection Studies on persistent infections with other negative-stranded RNA viruses have shown that mutations arise during virus persistence (Holland et al, 1979). As measles virus appears to establish a persistent infection in the brain of SSPE patients, a cell line persistently infected with SSPE virus LEC was examined to see if such mutations were detectable as antigenic changes in the virus proteins. This cell line was compared with cells lytically infected with the original SSPE virus LEC by immunofluorescence, using haemagglutinin-specific monoclonal antibodies raised against measles virus. As shown in Table 4, all six hybridoma antibodies reacted with cells lytically infected with either Edmonston or SSPE virus LEC. However, the persistently infected cells gave a positive immunofluorescence with only two of the clones used. Therefore, it would appear that antigenic changes have arisen in the haemagglutinin of this virus during the course of a persistent infection. DISCUSSION The use of hybridoma monoclonal antibodies has provided a new tool for the characterization and antigenic mapping of virus proteins. Using this technique, important information has been obtained concerning the structure of the virus polypeptides, as well as the antigenic relatedness of particular strains within the influenza and rabies virus groups (Koprowski et al., 1977; Gerhard et al., 1980; Wiktor & Koprowski, 1978; Flamand et al., 1980). In the present study the structural polypeptides of measles and SSPE viruses, responsible for the biological activity of haemagglutination have been characterized by monoclonal antibodies in order to define the antigenic relationship between these virus strains. It is known that isolates from SSPE patients vary in their host range, growth rate, plaque size, type of haemagglutination, fluorescent antibody staining as well as neuropathogenicity but all of these changes have also been occasionally observed between 'regular' measles viruses (Agnarsdottir, 1977; Fraser & Martin, 1978). So far, no single stable property has been found which is solely a characteristic of measles virus isolates from CNS tissue, with the possible exception of reduced sensitivity to neutralization by immune sera (Payne & Baublis, 1973). Similar serological differences have been seen by a competition radioimmunoassay

Antigenic characterization of measles viruses 363 between measles and SSPE viruses which have been interpreted as the result of minor antigenic differences between these agents (Hall et al., 1978). Use of monoclonal antibodies has demonstrated a great variation in reactivity with the different measles and SSPE virus isolates. The data obtained in the binding assays indicate antigenic differences on the H polypeptide between these measles virus strains, whereas the results from the biological tests suggest differences in the topography of the various sites involved in the different biological activities of these agents. Such structural changes might explain the lack of correlation between the inhibiting effects of a given hybridoma antibody on the biological activities of the various measles viruses. For instance, clone 153 of group 4 exhibited a low haemagglutination inhibition activity for LEC virus and a high neutralizing titre for the same agent. However, with measles virus Woodfolk the opposite results were obtained. Furthermore, monoclonal antibodies were obtained which selectively inhibited haemagglutination by certain strains without an accompanying neutralization effect (group 2 hybridoma) or vice versa (clone 772 of group 3 for LEC virus). However, two clones might possibly react with antigenic sites common to, and functionally similar, in each virus. Clones 322 and 323 of group 4 inhibited haemagglutination and neutralized all viruses tested equally well. This is also reflected in the RIA results. A common monoclonal antibody reaction pattern, permitting differentiation between measles viruses isolated from acute measles or from CNS disease, was not observed. A possible exception was clone 34 of group 3. This monoclonal antibody reacted equally well in all assays with the measles virus strains Edmonston, Woodfolk and EVA but not at all, or to a significantly reduced extent with viruses of CNS origin. This suggests that the CNS isolates of measles virus may have lost antigenic sites on their H polypeptides possessed by the other measles viruses. Possibly, this is the result of persistent infection in CNS tissue. Such an interpretation is further supported by results obtained from the comparative analysis of tissue culture cells infected lytically and persistently with SSPE virus LEC. In the persistent infection, which had been derived from diseased hamster brain and kept in tissue culture over a period of several years, antigenic changes were detectable with the hybridoma antibodies employed (Table 4). Only two out of six monoclonal antibodies, which were capable of recognizing the H polypeptide by immunofluorescence in the lytic infection, stained this protein in persistent infection. The data presented here document that different strains of measles virus not only share common antigenic determinants but also reveal distinct antigenic or structural changes of the H polypeptide. Structural alterations need not affect the specificity of the individual monoclonal antibody binding site, but could lead to topographical changes in the distribution and location of these sites in relation to the biologically active regions of the molecule. Therefore, the biological effect of a monoclonal antibody is not only dependent on the affinity of the antibody but also on the importance of the specific antigenic site in the biological reaction. The monoclonal antibodies selected for this study did not show an anti-haemolysin effect in contrast to those used by Togashi et al. (1981). That group obtained monoclonal antibodies against measles virus which immunoprecipitated the H polypeptide but contained both HI and HLI activity. Hybrid clones with similar properties were also isolated in our laboratory but not included in this study, since it is likely that these are not truly monoclonal. The observation of antigenic changes in the measles virus H protein during persistency is of great interest in relation to SSPE. Holland and co-workers have shown, using vesicular stomatitis virus, that genome changes can accumulate during persistent infection. These were accompanied by virus protein changes as demonstrated by biochemical techniques (Holland et al., 1979; Rowlands et al., 1980). It is likely that such events change the antigenic structures of more than one structural protein. The availability of monoclonal antibodies

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