Monoclonal Immunoglobulin M Antibody to Japanese - NCBI

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May 6, 1983 - to a polypeptide of molecular weight 56,000 seen in nitrocellulose transfers of sodium dodecyl sulfate-polyacrylamide gels. Cross-reactivity with ...
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Vol. 41, No. 2

IMMUNITY, Aug. 1983, P. 774-779

0019-9567/83/080774-06$02.00/0 Copyright © 1983, American Society for Microbiology

Monoclonal Immunoglobulin M Antibody to Japanese Encephalitis Virus that Can React with a Nuclear Antigen in Mammalian Cells ERNEST A. GOULD,'* ALEXANDER C. CHANAS,' ALAN BUCKLEY,' AND CHRISTOPHER S. CLEGG2

Arbovirus Research Unit, Winches Farm Field Research Station, St. Albans,' and Special Pathogens Reference Laboratory, Porton Down, Salisbury, Wiltshire,2 United Kingdom

Received 28 December 1982/Accepted 6 May 1983

An immunoglobulin M (IgM) class monoclonal antibody raised against Japanese encephalitis virus reacted with an epitope on the nonstructural virus protein P74 (NV4 in the old nomenclature) of several flaviviruses and also with an antigen present in the nuclei of a variety of mammalian cell types. This antigen had a characteristic granular distribution by immunofluorescence and may correspond to a polypeptide of molecular weight 56,000 seen in nitrocellulose transfers of sodium dodecyl sulfate-polyacrylamide gels. Cross-reactivity with nuclear antigen was also occasionally observed in the IgM antibody fraction of mice early after infection with Japanese encephalitis virus and also in acute sera from some clinical cases of encephalitis containing IgM antibody to Japanese encephalitis virus. MATERIALS AND METHODS Cells and cell culture. The cells were grown in Leibovitz L15 medium containing 5% fetal calf serum, 10% tryptose phosphate broth, penicillin, and streptomycin. Vertebrate cells were incubated at 37°C, whereas invertebrate cells were incubated at 28°C. Vero, HEp-2, BHK-21, MDCK, CCL 9.1, and PS cells were obtained from the Microbiology Department of the London School of Hygiene and Tropical Medicine. MRC-5 cells were purchased from Flow Laboratories (Irvine, Scotland). Primary mouse embryo fibroblasts (strain TO or BALB/c) were prepared by trypsinization of embryos at or near full term. Primary chicken embryo fibroblasts were prepared by trypsinization of 10-day-old embryos. The skeletal cells were seeded at 106 cells per ml into sterile disposable tissue culture flasks. Mosquito (AP61) (24) and Xenopus laevis (XL2) (19) cell cultures were kindly supplied by C. J. Leake (Department of Entomology, London School of Hygiene and Tropical Medicine). Monoclonal antibody. A cloned hybridoma culture producing the monoclonal antibody designated 62.4a was isolated by standard fusion procedures (9) with the modifications previously described (2). Immune splenocytes were obtained from BALB/c mice given two intraperitoneal inoculations of live JE virus (Nakayama strain; 0.1 ml of 20% suckling mouse brain suspension) followed by one intravenous dose a week later. Fusion with the nonsecreting myeloma cell line P3-X63-Ag8-653 (8) was carried out 2 days later. The antibody was obtained as mouse ascitic fluid and had a titer of at least 1/10,000 by indirect immunofluorescence. Immunodiffusion. To identify the immunoglobulin class secreted by the cloned culture 62.4a, the supernatant culture medium was concentrated 10-fold with

Japanese encephalitis (JE) virus is a member of the mosquito-borne flaviviruses. It produces epidemics, often of a severe nature, in eastern Asia from Japan and the southeastern USSR to Indonesia and India (1). Its replication cycle in mammalian cells is thought to be confined to the cytoplasm, and nuclear fluorescence has never been reported (25). There is, however, evidence of a nucleus-associated process in its replicative cycle, since the yield of JE virus was severely depressed in enucleated chicken embryo cells (10). It was suggested that either loss of nuclear membranes during enucleation or loss of a hostmediated process involving modification of the viral genome could be responsible for the depressed yield of virus. On the basis of hemagglutination inhibition and neutralization tests, cross-reactivity has been detected between JE virus and several other flaviviruses (5, 17, 22); nevertheless, the significance of this antigenic cross-reactivity and its relevance to the pathogenesis of the flaviviruses are unknown. We are currently producing monoclonal antibodies against several of the flaviviruses. Here we describe the properties of one of the monoclonal antibodies prepared against JE virus. It produced bright nuclear immunofluorescence in both infected and noninfected vertebrate cell cultures as well as the characteristic cytoplasmic immunofluorescence seen with flaviviruses. The possible significance of virus-cell related antigens in the pathogenesis of flavivirus infections is discussed. 774

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Lyphogel (Gelman Sciences Ltd., United Kingdom). Double immunodiffusion tests were then performed against monospecific anti-mouse immunoglobulins. Immunofluorescence. Cells on glass cover slips were infected with JE virus at an estimated input multiplicity of 0.01 and incubated at 37°C for 48 h. They were washed in phosphate-buffered saline (PBS), fixed in chilled acetone, and stored dry at - 20°C. For immunofluorescence tests the appropriate dilution of antibody was incubated on the cells at 37°C for 40 min. They were then washed in PBS and treated with either fluorescein-conjugated anti-mouse immunoglobulin M [IgM(Fc)], immunoglobulin G (IgG; Nordic Immunological Reagents), or protein A (Pharmacia Fine Chemicals, Uppsala, Sweden). Western blot analysis. Confluent cultures of Vero cells were infected with JE virus at a multiplicity of 10 to 20. After 30 h of incubation, actinomycin D was added to the medium (20) at a final concentration of 1 ,ug/ml. At 48 h postinfection the cells were washed in warm methionine-free medium and incubated for 90 min in this medium supplemented with 2% dialyzed fetal calf serum and 10 j±Ci of [35S]methionine (1,100 Ci/mmol; Amersham International, Amersham, United Kingdom) per ml. The cells were then washed twice in cold PBS and lysed in 1% sodium dodecyl sulfate1% 2-mercaptoethanol-100 mM Tris-hydrochloride (pH 6.8)-15% glycerol-0.01% bromophenol blue (0.5 ml per 25-cm2 flask). The lysates were heated at 100°C for 2 min and pipetted rapidly to reduce the viscosity. Samples were analyzed on 8 to 15% polyacrylamide gradient gels (11), and the separated proteins were electrophoretically transferred to nitrocellulose sheet (23) at 200 to 300 mA for 12 to 16 h in 96 mM glycine12.5 mM Tris-20% isopropanol. The transferred protein was stained briefly in amido black (23) and destained in 10% acetic acid-10% isopropanol, and protein binding sites were blocked by incubation in PBS2.5% bovine serum albumin-0.05% NaN3 for 60 min at room temperature with constant agitation. The transfers were incubated with a 1/5,000 dilution of 62.4a ascitic fluid in PBS-2.5% bovine serum albumin-5% fetal calf serum-1% Triton X-100-0.1% sodium dodecyl sulfate for 2 h at room temperature with constant agitation. The protein was washed three times for 5 min each in PBS-0.05% Triton X-100 and then incubated with a 1/1,000 dilution of peroxidase-conjugated anti-mouse globulin (Miles Laboratories) for 1 h, washed again, and then incubated in 50 mM sodium acetate (pH 5.0) containing 200 ,ug of aminoethyocarbazole per ml and 0.015% H202 for 30 min in the dark. Viruses. The flaviviruses were obtained from either J. Porterfield (Sir William Dunn School of Pathology, University of Oxford) or from C. J. Leake. Each virus was prepared as a stock culture by intracerebral inoculation of newborn mice (virus diluted 1:100 in PBS). When 20 to 40% of the mice were moribund, a 20% suspension of the brains was prepared in PBS and stored at -70°C.

RESULTS Determination of the antibody subclass secreted by hybridoma 62.4a. Double-immunodiffusion tests were performed on concentrated hybridoma culture medium against monospecific mouse immunoglobulins. A single precipitation

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line was obtained with antibody 62.4a and antimouse IgM(Fc). No lines were obtained with either anti-IgGl, anti-IgG2a, anti-IgG2b, antiIgG3, or anti-IgA. The same result was obtained with hybridoma culture fluid obtained from cloned cultures that had been cloned again. Antigenic specificity of antibody 62.4a for both virus and cellular antigens. Indirect immunofluorescence experiments with 62.4a antibody on acetone-fixed Vero cells cultured on cover slips for 48 h after infection with JE virus (multiplicity of infection, 0.1) showed diffuse staining throughout the cytoplasm in approximately 10 to 15% of the cells. This corresponded to the known proportion of cells that contained JE virus-specific antigen. In addition, a granular or speckled type of fluorescent staining within the nuclei, but not the nucleoli, of approximately 95% of the cells was also observed (Fig. 1). These patterns of fluorescence were observed only when using anti-IgM(Fc) or whole antimouse globulin fluorescent conjugates, and these results were reproduced after recloning the hybridoma culture. Control, noninfected cells showed only the nuclear type of fluorescence. No cytoplasmic or nuclear fluorescence was observed when either anti-mouse IgG or fluorescein-conjugated protein A was used as the second-step reactant. When the 62.4a ascites fluid was titrated the cytoplasmic antigen was detectable at dilutions of 10-4, whereas the nuclear antigen usually had a titer of 1.0 to 2.0 log10 units less. Hybridoma culture fluid also produced both patterns of immunofluorescence and absorption of either 62.4a ascites fluid or hybridoma culture fluid with excessive quantities of acetone-fixed, noninfected Vero cells removed their ability to produce nuclear and cytoplasmic immunofluorescence. Indirect immunofluorescence tests were also carried out in Vero cells to determine the antigenic cross-reactivity of the monoclonal antibody with a selection of flaviviruses that are antigenically cross-reactive with JE virus. The 62.4a antibody was reactive (bright fluorescence in infected cells) with the following flaviviruses: JBE, KUN, USU, WN, ZIKA, TMU, DEN-1, DEN-2, and DEN-4. The 62.4a antibody was not reactive (no fluorescence) with LGT, LI, NEG, POW, CR, DB, MOD, MML, BSQ, KOK, SLE, WSL, YF, BAN, EH, UGS, or DEN-3. (The abbreviations used are as recommended in the Catalogue ofArthropod-Borne Viruses [1]). The characteristic diffuse virus-specific immunofluorescence was observed in the cytoplasm of 9 of 26 different flaviviruses tested. In addition, several different strains of JE and of yellow fever (YF) virus were tested, (data not shown); in each case the JE strains reacted with 62.4a, but there was no reaction with the YF virus strains.

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FIG. 1. Indirect immunofluorescence with JE virus-infected Vero cells and 62.4a monoclonal antibody. Noninfected cells show fluorescent dots only in the nuclei. Infected cells show both nuclear and cytoplasmic fluorescence.

To determine the specificity of the nuclear

staining, a broad spectrum of both vertebrate and invertebrate cell cultures was examined by indirect immunofluorescence with 62.4a monoclonal antibody and anti-mouse-IgM(Fc) conjugate. All cells of mammalian origin, which included human (MRC-5 and HEp-2) monkey (Vero), hamster (BHK-21), canine (MDCK), porcine (PS), and mouse (CCL 9.1, primary BALB/c, and primary TO) cultures were found to contain the same nuclear antigen (showed bright fluorescence in the nuclei of the cells). The percentage of positive cells varied from 25 to 95%, depending on the density of cells and their age since subculturing. Subconfluent cell monolayers generally showed a lower percentage of nuclear antigen-positive cells. An increase of positive nuclei was observed as cell density increased. Cultures of primary chicken embryo fibroblasts, mosquito (AP61), and X. laevis (XL-2) cells, on the other hand, did not produce nuclear fluorescence despite the fact that JE virus can replicate and produce cytoplasmic fluorescence in these cells. Determination of molecular specificity. The specificity of antibody 62.4a was determined by examining the ability of nitrocellulose transfers of electrophoretically separated proteins of cell lysates ("Western blots") to bind the antibody. Virus-specific proteins on the transfers were identified by comparing the proteins labeled in a 90-min pulse of [ 5S]methionine in infected and

uninfected cells. The larger virus-specific proteins were clearly visible against a background of continuing host protein synthesis (Fig. 2, tracks a and b). Antibody binding was tested under two sets of conditions: (i) with low levels of cell protein loaded on the gel (equivalent to about 2.5 x 104 cells), and high dilutions of monoclonal antibody (1/5,000 dilution of ascitic fluid), or (ii) with higher levels of cell protein (5 X 104 cells) and lower dilution of antibody (1/500). The only virus-specific protein capable of binding antibody under either set of conditions was the nonstructural protein P74 (previously known as NV4 [20]) (Fig. 2, tracks c and e). This protein is thus unequivocally identified as the virus-specific target for antibody 62.4a. The antibody was also capable of binding to two classes of cellular proteins which were found in infected and uninfected cells. Under conditions of high antigen and antibody concentration, several proteins appeared to bind antibody (Fig. 2, tracks c and d). However, with lower antigen and antibody concentrations, only a single cellular protein with an apparent molecular weight of 56,000 was able to bind observable quantities of antibody (Fig. 2, tracks e and f), indicating that this protein has a higher affinity for the antibody than the other cellular proteins. It was unusual in that it penetrated the nitrocellulose matrix, rather than binding to the surface as most proteins do, and did not bind monoclonal antibody 30.22, which is specific for Sindbis virus nucleo-

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ing. JE virus-specific IgG antibody producing only cytoplasmic fluorescence was detectable from day 4 postinfection (Fig. 3b). Finally, 18 of 26 human sera from cases of suspected JE encephalitis in Nepal produced IgM-specific nuclear fluorescence similar to that described above. This was shown not to be due to rheumatoid factor by using a Rheum-Wellcotest kit (Wellcome Laboratories, Beckenham, England).

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GP53(E) _'

a

b

c

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d

e

f

FIG. 2. Molecular specificity of antibody 62.4a determined by Western blot analysis. Infected (tracks a, c, and e) and uninfected (tracks b, d, and f) cells were labeled with [35S]methionine; the proteins were analyzed on an 8 to 15% polyacrylamide gradient gel and transferred to nitrocellulose. Radioactive proteins were revealed by autoradiography (tracks a and b). Antibody-binding proteins were located by incubation with a 1/500 dilution (tracks c and d) or a 1/5,000 dilution (tracks e and f) of ascitic fluid containing antibody 62.4a. Protein loaded on the gel corresponded to about 2.5 x 104 cells (tracks a, b, e, and f) or 5 x 104 cells (tracks c and d).

capsid protein (2) and also active on Western blots. These experiments suggest that the characteristic nuclear fluorescence seen with antibody 62.4a is probably associated with the 56,000-molecular-weight protein, but the possibility that the other cellular proteins with lower binding affinity may be involved cannot be ruled out.

Antibody production in mice infected with JE virus. Conventional JE virus hyperimmune antisera produce cytoplasmic fluorescence, but do not produce nonspecific nuclear staining of mammalian cells (data not shown). We have, however, qualitatively reexamined the chronological aspects of the antibody response in JE virus-infected mice. Adult BALB/c mice inoculated intraperitoneally with 103 PFU of JE virus were bled daily, and their sera were examined by immunofluorescence for IgM antibodies to JE virus. With JE virus-infected Vero cells, cytoplasmic fluorescence was first detected with anti-IgM antibody on the day 3 postinfection. The sera from 3 of 15 mice produced both nuclear and virus-specific cytoplasmic fluorescence 3 to 5 days postinfection (Fig. 3a). Sera from uninfected mice gave no fluorescent stain-

DISCUSSION The monoclonal antibody 62.4a described here gives a characteristic diffuse cytoplasmic fluorescence in cells infected with JE virus and with a number of other flaviviruses. In addition, the antibody gives rise to speckled nuclear fluorescence in uninfected or infected cells of mammalian origin. These two activities were not separable either by repeated cloning of the hybridoma line or by absorption experiments, suggesting that both reside on the same immunoglobulin, and thus that the virus-specified protein and the protein found in mammalian nuclei possess identical or closely related epitopes. The virus-specified protein carrying the epitope is definitively identified by Western blot analysis as P74, a protein found in abundance in JE virus-infected cells (3), but which does not form part of the extracellular virus structure. As far as we are aware this is the first monoclonal antibody directed at a nonstructural protein of a togavirus to be found. Identification of the host protein recognized by antibody 62.4a in nitrocellulose transfers of sodium dodecyl sulfate-polyacrylamide gels cannot be so positive, since the reaction with the candidate 56,000-molecularweight protein is much weaker, and other cellular proteins also bind the antibody, although with lower affinity. The relative weakness of the reaction with the 56,000-molecular-weight protein reflects the difference in apparent antibody titer for viral and nuclear antigen seen by immunofluorescence and may be due to relatively low levels of the host protein as compared with viral P74 in the cells or to a lower affinity of the host protein for the antibody due to differences in the epitope structure. The ability of the host protein to penetrate the nitrocellulose membrane also suggests that it does not bind efficiently and may be partially lost on transfer. The existence of monoclonal antibodies that react with more than one antigen is expected on theoretical grounds (4, 13), and host cell proteins sharing a common determinant with a viruscoded protein have indeed been discovered by the use of these reagents (4, 12). Whether or not proteins carrying such common determinants share some common function, as suggested by

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FIG. 3. Indirect immunofluorescence with JE virus-infected Vero cells and mouse serum (day 4 postinfection) treated either with fluorescein-conjugated anti-IgM antibody (a) or with anti-IgG antibody (b).

Lane and Hoeffler (12), or arise purely by chance remains to be determined, as does the significance of such antigenic similarities in the immunopathology of flavivirus infections. The cross-reactivity of antibody 62.4a with several other antigenically related flaviviruses that follow similar replicative pathways demonstrates that the antigen is not unique to JE virus. On the other hand, there is no correlation between the cross-reactivity with other flaviviruses and the serological subgrouping of them suggested by Porterfield (17). That the chicken, mosquito, and X. laevis cells did not contain detectable 62.4a-reactive nuclear antigen is noteworthy, since it is known that many flaviviruses readily establish noncyto-

pathic infections in these cell lines, particularly in mosquito cell cultures (15, 18). Finally, although 62.4a antibody was induced in response to a JE virus-specific antigen, its production also represents an autoantibody respone in BALB/c mice. IgM class autoantibodies have been detected after virus infection; these include rheumatoid factors as well as various antinuclear antibodies (6, 7, 14, 16, 21). Whether the appearance of antibody against nuclear antigens in experimentally infected mice or in humans infected with JE virus is significant cannot yet be determined; nevertheless it is hoped that the demonstration of virus-specific monoclonal antibodies which also react with cellular antigens could contribute toward under-

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standing how

some self-limiting autoantibody triggered and their possible role in viral pathogenesis.

responses are

ACKNOWLEDGMENTS Part of this work was supported by the Wellcome Trust. We are indebted to D. Newman for her excellent assistance and for typing the manuscript. LITERATURE CITED 1. Berge, T. 0. 1975. International catalogue of arboviruses, 2nd ed., U.S. Department of Health, Education and Welfare publication no. (CDC) 75-8301. Public Health Service, Washington, D.C. 2. Chanas, A. C., E. A. Gould, J. C. S. Clegg, and M. G. R. Varma. 1982. Monoclonal antibodies to Sindbis virus glycoprotein El can neutralize, enhance infectivity and independently inhibit haemagglutination or haemolysis. J. Gen. Virol. 58:37-46. 3. Clegg, J. C. S. 1982. Glycoprotein detection in nitrocellulose transfers of electrophoretically separated protein mixtures using concanavalin A and peroxidase: application to arenavirus and flavivirus proteins. Anal. Biochem. 127:389-394. 4. Crawford, L., K. Leppard, D. Lane, and E. J. Harlow. 1982. Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen. J. Virol. 42:612-620. 5. de Madrid, A. T., and J. S. Porterfield. 1974. The flaviviruses (group B arboviruses): a cross-neutralization study. J. Gen. Virol. 23:91-96. 6. Dresner, E., and P. Trombly. 1959. The latex-fixation reaction in nonrheumatic diseases. N. Engl. J. Med. 261:981-988. 7. Johnson, R. E., and A. P. Hall. 1958. Rubella arthritis: Report of cases studied by latex tests. N. Engl. J. Med. 258:743-745. 8. Kearney, J. F., A. Radbruch, B. Liesegand, and K. Rajewsky. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell-lines. J. Immunol. 123:1548-1550. 9. Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256:495-497. 10. Kos, K. A., B. A. Osborne, and R. A. Godsby. 1975. Inhibition of group B arbovirus antigen production and replication in cells enucleated with cytochalasin B. J. Virol. 15:913-917.

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11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 12. Lane, D., and W. K. Hoeffier. 1980. SV40 large T shares an antigenic determinant with a cellular protein of molecular weight 68,000. Nature (London) 288:167-170. 13. Lane, D., and H. Koprowski. 1982. Molecular recognition and the future of monoclonal antibodies. Nature (London) 296:200-202. 14. Langenhuysen, M. M. A. C. 1971. Antibodies against yglobulin after blood transfusion and cytomegalovirusinfection. Clin. Exp. Immunol. 9:393-398. 15. Leake, C. J., M. G. R. Varma, and M. Pudney. 1977. Cytopathic effect and plaque formation by arboviruses in a continuous cell line (XTC-2) from the toad Xenopus laevis. J. Gen. Virol. 35:335-339. 16. Markenson, J. A., C. A. Daniels, A. L. Notkins, J. H. Hoofnagle, J. Gerety, and L. F. Barker. 1975. The interaction of rheumatoid factor with hepatitis B surface antigenantibody complexes. Clin. Exp. Immunol. 19:209-217. 17. Porterfield, J. S. 1980. Antigenic characteristics and classification of togaviridae, p. 13-46. In R. W. Schlesinger (ed.), The togaviruses. Academic Press, Inc., New York. 18. Pudney, M., C. J. Leake, and S. M. Buckley. 1982. Replication of arboviruses in arthropod in vitro systems: an overview, p. 159-194. In K. Maramarosch and J. Mitsuhashi (ed.), Invertebrate cell culture applications. Academic Press, Inc., New York. 19. Pudney, M., M. G. R. Varma, and C. J. Leake. 1973. Establishment of a cell line from the South African clawed toad Xenopus laevis. Experientia 29:466-467. 20. Shapiro, D., K. Kos, and P. K. Russell. 1973. Protein synthesis in Japanese encephalitis virus-infected cells. Virology 56:95-109. 21. Svec, K. H., and J. H. Dingle. 1%5. The occurrence of rheumatoid factor in association with antibody response to influenza A2 (Asian) virus. Arthritis Rheum. 8:524-529. 22. Theiler, M., and W. G. Downs. 1973. The arthropod-borne viruses of vertebrates. Yale University Press, New Haven. 23. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76:4350-4354. 24. Varma, M. G. R., M. Pudney, C. J. Leake, and P. H. Peralta. 1976. Isolations in a mosquito (Aedes pseudoscutellaris) cell line (Mos 61) of yellow fever virus strains from original field material. Intervirology 6:50-56. 25. Westaway, E. G. 1980. Replication of flaviviruses, p. 531577. In R. W. Schlesinger (ed.), The togaviruses. Academic Press, Inc., New York.