Virion and Soluble Antigens of Japanese Encephalitis Virus

Transcription

1 INFECTION AND IMMUNrrY, May 1975, p Copyright 1975 American Society for Microbiology Vol. 1 1, No. 5 Printed in U.SA. Virion and Soluble Antigens of Japanese Encephalitis Virus KENNETH H. ECKELS,* FRANK M. HETRICK,' AND PHILIP K. RUSSELL Department of Hazardous Microorganisms, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington, D.C. 212 Received for publication 17 September 1974 Japanese encephalitis virions contain a 58 x 13-molecular-weight envelope glycoprotein antigen that can be solubilized with sodium lauryl sulfate and separated from other virion structural polypeptides and viral ribonucleic acid by gel filtration chromatography. The 58 x 13-molecular-weight envelope protein is the major antigen responsible for cross-reactivity of the virion in complement fixation tests with other closely related arboviruses. A naturally occurring soluble complement-fixing antigen is found in Japanese encephalitis mouse brain preparations after removal of particulate antigens. After partial purification by gel filtration and isoelectric focusing, the 53 x 13-molecular weight soluble complement-fixing antigen is more type specific than the Japanese encephalitis envelope antigen in complement fixation tests. Further, the Japanese encephalitis soluble complement-fixing antigen is stable to treatment with sodium lauryl sulfate and 2-mercaptoethanol, whereas virion complement-fixing antigens are unstable after this treatment. The classification of group B arboviruses is based on antigenic cross-reactivity measured by the hemagglutination inhibition test. Common, group antigens are also detected by complement fixation (CF), immunodiffusion, and neutralization tests as well (7, 8, 11, 12, 21, 29). Members of the Japanese encephalitis (JE) virus subgroup including Murray Valley encephalitis, West Nile encephalitis, St. Louis encephalitis, and Ilheus viruses exhibit extensive antigenic cross-reactivity by various serological tests. Each member could be considered a serotype with specific antigenic determinants that react only with homologous antibody. Clarke (6), using absorption techniques and the hemagglutination inhibition test, was able to show that each member of the subgroup possessed a specific antigen and one or more common group antigens. Crude mouse brain or cell culture suspensions of JE or dengue viruses contain two hemagglutinins and at least three CF antigen components (14, 22, 25, 26). The infectious virion and a noninfectious, subviral particle possess hemagglutinating and CF activity. A soluble CF (SCF) antigen found in dengue-infected mouse brain preparations is immunologically distinct from either hemagglutinin and appears to be a nonstructural antigen with a molecular weight of 39 x 13 (2, 3). The SCF antigen for each dengue serotype has antigenic type and group ' Present address: Department of Microbiology, University of Maryland, College Park, Md specificity determined by immunodiffusion analysis (2). Cells infected with JE and related viruses contain at least seven virus-specific polypeptides (22, 27, 28). Recently, Qureshi and Trent (17-19) extracted various soluble antigens from cells infected with JE subgroup and dengue viruses. An antigen with a molecular weight of 85 x 13 was found to be type specific when tested against other group B viruses and was immunologically and biophysically distinct from virion structural proteins. In addition to the nonstructural protein antigen, the major envelope virion polypeptide obtained from infected-cell extracts cross-reacted in serological tests and appeared to contain group-specific determinants. Preparations of highly characterized antigens, as those described for the JE and dengue subgroup viruses, are required to elucidate the complex antigenic relationships and ultimately the immunologic reactions resulting from infection with these viruses. This report describes the purification and antigenic characterization of the major envelope protein obtained from JE virions and a naturally occurring JE soluble antigen. MATERIALS AND METHODS Preparation of JE virion antigens. Radioactive JE virus was prepared in.5-gallon (about 1.9-liter) roller flasks containing approximately 2 x 18 BHK-21 cells. Growth medium consisted of Eagle 153

2 154 ECKELS, HETRICK, AND RUSSELL INFECT. IMMUN. basal medium containing 1% fetal bovine serum, 2 mm L-glutamine, 1 mm tris(hydroxymethyl)- aminomethane (Tris) (ph 9.), 1 U of penicillin per ml, and 1 yg of streptomycin per ml. A 28th suckling mouse brain passage seed of JE virus, strain Ml/311, was diluted in Earle balanced salt solution medium containing.25% human serum albumin, 2 mm L-glutamine, 8 mm Tris (ph 9.),.22% NaHCO,, normal strength Eagle basal medium vitamins,.5 strength Eagle basal medium amino acids, and antibiotics. Virus was inoculated in BHK-21 cell culture flasks to give a multiplicity of infection of 4 to 6. After adsorption of virus at 35 C for 1.5 h, the inoculum was removed and each flask was washed with 1 ml of Earle balanced salt solution. Six hours after the addition of 5 ml of Earle balanced salt solution medium, each flask received 5 uci of either a 3H-labeled amino acid mixture or [3H]glucosamine (New England Nuclear Corp.). Due to low yields of ribonucleic acid (RNA)-labeled JE virions from BHK-21 cells, primary chicken embryo cells were used for the preparation of doublelabeled JE virus. Cell monolayers were prepared by the method of Porterfield (16) in.5-gallon roller flasks and inoculated by using the same procedure as used for the preparation of JE virus in BHK-21 cells. Immediately after virus adsorption, 2 MCi of "Clabeled amino acids and 2 MCi of [3H]uridine were added to each cell culture flask. Labeled virus was harvested from BHK-21 cells at 3 h and from primary chicken embryo cells at 4 h postinoculation. Purification of JE virions followed the procedure used by Smith et al. (25) for dengue virus. Briefly, virus supernatant culture fluids were clarified by centrifugation at 1,5 x g for 2 min. The virus was pelleted from clarified culture fuids by centrifugation at 78, x g for 3 h followed by sedimentation in 5 to 25% sucrose gradients. Virioncontaining fractions were pooled after assaying 5 Ml of each fraction for radioactivity in 1 ml of scintillation fluid consisting of one part Triton X-1 (Packard) and two parts toluene containing Liquiflor scintillator (New England Nuclear Corp.). A Packard 2425 scintillation spectrometer was used for counting 3H- and "4C-labeled materials. The virion pool was diluted in an equal volume of.2 M Tris,.15 M NaCl,.1 M ethylenediaminetetraacetic acid (ph 8.2) buffer and centrifuged at 15, x g for 3 h. The pelleted virions were suspended in a suitable volume of buffer for chromatography or immunological tests. The preparation of mouse brain-derived JE virions also followed closely the procedure used for the isolation of dengue virus hemagglutinins (25). Yields of infectious virus from mouse brain were at least ten-fold higher than from cell culture, making it possible to characterize high-titer antigen preparations. Briefly, a 2% suspension of virus-infected brain homogenate in.2 M Tris buffer was clarified by centrifugation at 1,5 x g for 2 min. Host brain material was precipitated with 3 mg of protamine sulfate per ml by constant mixing at 4 C for 3 min. After centrifugation at 15, x g for 6 min, the supernatant fluid was removed and the virus was pelleted by centrifugation at 78, x g for 3 h. Hemagglutinins were separated by sedimentation through 5 to 25% sucrose gradients at 63, x g for 2.5 h and complete virions were located by the peak of hemagglutinin activity closest to the bottom of the centrifuge tube. If necessary, further concentration of virions was carried out by diluting the pooled gradient.' fractions in an equal volume of.2 M Tris buffer followed by centrifugation at 15, x g for 3 h. Gradient-purified JE virions, prepared from either mouse brain or cell culture, were solubilized with.5% sodium lauryl sulfate (SLS) in.1 M phosphate-buffered saline (PBS), ph 7.2. After heating at 35 C for 3 min, the sample was layered on a column (1 by 5 cm) filled with Sephadex G-2 and equilibrated with.1 M PBS (ph 7.2) buffer containing.5% SLS. The descending flow of eluant was adjusted by gravity feed to a rate of 5 ml per h and 1-ml fractions were collected at room temperature. Columns were calibrated with protein mixtures of bovine albumin, ovalbumin, chymotrypsinogen, and ribonuclease. Antigen-containing fractions were pooled and concentrated by freeze drying on a Virtis apparatus. Preparation of JE soluble antigen. After pelleting hemagglutinins at 78, x g for 3 h, SCF antigen for JE virus was prepared by methods previously described (3). Briefly, SCF antigen was precipitated from supernatant fluids by adding ammonium sulfate to a final concentration of 6% (wt/vol). After centrifugation at 15, x g for 6 min, the precipitate was suspended in 1/1 of the original volume using.1 M PBS (ph 7.2) and chromatographed on a Sephadex G-1 column (5 by 78 cm) equilibrated with.1 M PBS, ph 7.2. Fractions (15 ml) were collected and assayed for CF antigen activity. Fractions containing CF antigen were concentrated by pressure dialysis in an Amicon cell by using a PM-3 membrane. Further purification of the low-molecular-weight SCF antigen by isoelectric focusing (IEF) was carried out in ampholyte gradients ranging from ph 3 to 1 and stabilized in sucrose ( to 7%) with sulfuric acid as the cathode and ethanolamine as the anode. The sample was loaded in the 1 to 2% sucrose layer and focused with a current of 85 V. Focusing at a controlled temperature of 2 C was continued for approximately 48 h until the amperage dropped to 3 ma. Fractions (2 ml) were collected and the ph and CF antigen activity were determined. Complement fixation and hemagglutination tests. CF tests followed the procedure of Kent and Fife (13), using a microtiter modification. Dilutions for block CF tests were made in test tubes before addition to microtiter plates. Assay of column fractions for CF antigen activity was done in the presence of an excess of homologous antibody. Immune ascitic fluid preparations were usually diluted 1:1 or 1:2 for use against dilutions of CF antigens. Hemagglutination content of virion preparations was measured by a microtiter modification of the standard technique of Clarke and Casals (9). Virus assay. Monolayers of tlc-mk2 cells in 25-cm2 plastic flasks were used for plaquing JE virols. After virus inoculation and adsorption for 1.5 h, flasks received 7 ml of medium 199 containing 1% fetal bovine serum, 36 mm NaHCOs,.2% diethylamino-

3 VOL. 11, 1975 ethyl-dextran,.5% of 1 times concentrated Eagle basal medium vitamins and amino acids, 1% Noble agar, and antibiotics. After incubation at 35 C for 5 days, flasks received 4 ml of a second overlay containing 1% Noble agar and 1:6, neutral red in Hanks balanced salt solution. Plaque flasks were incubated ovemight at room temperature before plaques were counted. PAGE. Analysis of JE virion polypeptides by polyacrylamide gel electrophoresis (PAGE) using 8% acrylamide gels followed procedures previously described (22). Gel staining in Coomassie brilliant blue dye followed the methods of Maizel (15). Preparation of hyperimmune mouse ascitic fluid. The procedure for hyperimmunization of mice and the preparation of ascitic fluid followed that of Brandt et al. (1) with a modification introduced by Chiewsilp and McCown (5). RESULTS Purification of JE virion antigens. Complete solubilization of the JE virion was necessary to separate and characterize the proteins of the virion. Recovery of immunogenic and antigenic reactivity was also required in any solubilization procedure so that individual proteins could be identified and their antigenic specificity determined. Mild treatment of the JE virion with.5% SLS resulted in little or no loss of CF antigen activity (Table 1). Hemagglutinating activity and infectivity associated with the virion were substantially reduced after this treatment. This solubilization procedure al-,lowed protein subunits to be separated by gel filtration on Sephadex G-2 columns with good CF antigen recovery (7 to 8%). Typical chromatograms of JE virions derived from BHK-21 cells or suckling mouse brain after gel filtration in SLS are shown in Fig. 1. The peak eluting in the void volume of the column with a molecular weight of >25 x 1' had some CF antigen activity associated with it and probably contained incompletely solubilized or aggregated virion material. This peak could not be analyzed by PAGE since radioactivity levels were always very low. The major CF antigen activity was in a peak which eluted from Sephadex gel TABLE 1. Antigen activity after treatment of JE virions with SLS CF, HA JE virions PFU/mla units/ units/ml" ml Control 6.5 x 19 5,12 163,84 After.5% SLS at 35C, 2.5 x 12 5,12 <1,28 3 min apfu, Plaque-forming units. b HA, Hemagglutination. ANTIGENS OF JE VIRUS CELL CLTURE FRATI FIG. 1. Gel filtration chromatography of SLSsolubilized JE virions derived from cell culture and mouse brain. columns in fractions that contained a protein with a molecular weight of 58 x 13. A minor and variable CF antigen peak was calculated to have a molecular weight of 24 x 13. The approximate molecular weights were obtained from a standard curve constructed with known proteins treated with SLS and chromatographed identically to viral preparations. By PAGE analysis, the 58 x 13-molecularweight peak from Sephadex columns contained the JE envelope protein V-3, whereas the 24,-molecular-weight peak contained a mixture of the minor polypeptides V-2, NV-2, and V-1 (Fig. 2). Shapiro et al. (22) have designated V-3 and V-1 as envelope polypeptides and V-2 as the core protein of JE virions grown in chicken embryo cells. The peak labeled NV-2 was so designated since it appeared to correspond to a virus-specified, nonvirion protein formerly found only inside JE-infected cells. However, NV-2 has recently been found associated with the other structural proteins of intracellular JE virions and virions purified from cell culture fluids containing Tris as a maintenance buffer during viral replication (23). When PAGE analysis was repeated with chromatographed virions derived from mouse brain, a similar polypeptide pattern was observed when the major and minor CF antigen peaks were tested (Fig. 2). Stained bands resulting from PAGE of these peaks corresponded in molecular weight to labeled V-3 protein and to a mixture of labeled V-2 and V-1 proteins, respectively. To further characterize the two CF antigen o 155

4 156 ECKELS, HETRICK, AND RUSSELL INFECT. IMMUN. FIG. 2. Polyacrylamide gel electrophoresis of JE virions and Sephadex-separated polypeptides. All samples were heated at 1 C for 1 min in 1% SLS and 2-ME before PAGE. peaks separated by SLS chromatography, a JE virion preparation containing [3H ]glucosamine as a marker for viral glycosylated protein was chromatographed (Fig. 3a). The glycoprotein peak with a molecular weight of 58 x 13 corresponds to the major envelope polypeptide, V-3, as previously shown (Fig. 2). Another glycosylated peak from SLS chromatography was found with a molecular weight of approximately 24 x 13 and probably contained NV-2 protein. Of the minor virion structural polypeptides, only one, NV-2, is glycosylated (22). Simons and Kiiiiriiiinen (24) have used SLSgel filtration chromatography to split Semliki Forest virus into its major components of RNA, protein, and lipid. Similar results were found for the separation of RNA and protein in a double-labeled JE virion preparation chromatographed after solubilization with SLS. Viral [3H ]RNA eluted in the void volume of the column (Fig. 3b). Viral "4C-labeled protein eluted as previously shown with peaks having molecular weights of 58 x 1o and 24 x 13. Mouse brain virion preparations were monitored by absorption of ultraviolet light at 26 and 28 nm. A 26/28 ratio of 1.95 was found for the void volume fractions, whereas a 26/28 ratio of 1. was found for those fractions corresponding to the major CF antigen peak. JE SCF antigen purification. The supernatant fluids remaining after pelleting JE virions contained SCF antigens. Further purification and characterization of these antigens by gel *. filtration chromatography on Sephadex G-1 columns revealed the existence of two separable CF antigens in JE virus supernatant fluids (Fig. 4). A major CF antigen peak eluted in the void volume of the column, whereas the second CF antigen eluted in the molecular weight range of 45 x 1o to 67 x 13, and was labeled the SCF antigen. The JE SCF antigen from Sephadex columns was purified further by IEF in a ph gradient of 3 to 1. A major CF antigen peak shown in Fig. 5 was found with an isoelectric point (pi) of approximately 5.3, whereas several minor CF antigen zones were found at higher ph values. Separation of a major (pi 5.3) antigen from contaminating protein and minor CF antigen activity was considered a final purification of the SCF antigen. To determine a more exact molecular weight for the JE SCF antigen, the pi 5.3 antigen from IEF was rechromatographed on a Sephadex G-1 column. The molecular weight was found to be 53 x 13. The molecular size similarity of this soluble antigen to the major envelope polypeptide of JE virions stimulated further interest in the antigenic relationship and biophysical similarities between these two antigens. Dengue SCF antigens could be distinguished from antigens of the virion and the slowly sedimenting hemagglutinin by treatment with SLS and 2-mercaptoethanol (2-ME) (2). The JE SCF antigen was also stable under these conditions. The JE SCF antigen titer was reduced only twofold after this treatment, whereas the JE virion and envelope

5 VOL. 11, 1975 ANTIGENS OF JE VIRUS 157 Ic x c a- o -.- C c._ 2 ) C.cp n ol a._ cj x In Fraction Number FIG. 3. Gel filtration chromatography of SLSsolubilized JE virions labeled with (a) [3Hlglucosamine and (b) a double label of "4C-labeled amino acids () and [3Hluridine (). A Wang computer program was used for calculation of efficiency and spillover in the double label experiment. 46- Void Albumin Oalbubin Ij 4 ml, r JL FRACTION FIG. 4. Gel filtration chromatography of JE SCF antigens on a Sephadex G-1 column. Antigens were prepared from infected-mouse brain homogenates. CF antigens were substantially degraded with SLS and 2-ME (Table 2). These results parallel those obtained with dengue SCF and virion antigens. Immunological characterization of JE virion and SCF antigens. Cross-reactivity in CF tests due to common group antigens has been well documented for group B arboviruses. A high degree of cross-reactivity was evident for both JE virions and JE envelope antigen used as antigens in block CF titrations against subgroup antibody (Fig. 6). For JE virion antigen, the homologous reciprocal antibody titer was 128, whereas Murray Valley encephalitis and West Nile encephalitis antibody titers were 32 and 64, respectively, against this antigen. Similarly, the JE envelope antigen titered 64 against homologous antibody and 16 and 64 against Murray Valley encephalitis and West Nile encephalitis antibody. Although minor structural protein antigens of the virion could not be prepared and their immunologic specificity studied, the major envelope antigen containing the glycoprotein V-3 appeared to be responsible for a high degree of cross-reactivity in JE virion preparations. Type-specific determinants are also found on antigens contained in crude JE virus preparations. A Sephadex-purified JE SCF antigen was compared to a crude mouse brain-derive anti C o- I FRACTION NUMBER FIG. 5. Isoelectric focusing of a JE SCF antigen partially purified by Sephadex G-1 chromatography. TABLE 2. Complement-fixing antigen activity of JE virion, envelope, and SCF antigens after treatment with SLS and 2-ME Antigen Control SLS/2-MEa JE virion 32b <8 JE envelope 64 8 JE SCF 1,28 64 aantigen preparations were adjusted to 9 gg of protein per ml with bovine albumin followed by treatment with.5% SLS and.5% 2-ME at 35 C for 3 min. After 18 h dialysis against 5 volumes of.1 M PBS, the antigens were titrated for CF activity. Controls received.1 M PBS in place of SLS and 2-ME ḃcf units per milliliter.

6 158 ECKELS, HETRICK, AND RUSSELL INFECT. IMMUN. gen preparation by CF block titration (Fig. 7). The crude preparation consisted of particulate and soluble antigens, whereas the JE SCF antigen was free of cross-reactive antigen associated with the JE virion or slowly sedimenting hemagglutinin. An increase in type specificity for the SCF antigen was evident in cross-reactions with subgroup antibody. Specificity is further increased for the SCF antigen after isoelectric focusing purification of the pi 5.3 antigen. It is evident from Fig. 7 that the final step of purification removed some cross-reactive antigen and allowed for a high degree of type specificity for the JE SCF antigen in CF tests. ANTIGEN DILUTION JE ENV JE VIRION a) N) 9(D O 1M _ 32 1 qi~~~~~~2r _1 ~1128 ẕ > -I UZT132m FIG. 6. Antigenic similarity of JE virions and JE envelope antigen in CF block titrations using JE subgroup immune ascitic fluids. Before testing, JE virions were solubilized with.1% SLS. TABLE 3. CD z DISCUSSION A procedure for the solubilization and purification of JE virion antigens has been described that uses a detergent treatment followed by gel filtration chromatography. With this procedure, a major structural polypeptide was effectively separated from virion RNA and minor structural proteins and could be used for antigenic characterization without further purification. After mild treatment with SLS, hemagglutinating activity was lost and the major CF antigen activity was associated with a polypeptide having a molecular weight of approximately 58 x 13 and was identified by PAGE as the major JE envelope protein. Table 3 lists some characteris- z Um 8 JE g 32. '- 128t_ Ji w Li -, 128 z ANTIBODY MVE DILUTION WNE FIG. 7. Cross-reactivity of JE crude, SCF, and SCF-IEF-purified antigens in CF block titrations against JE, Murray Valley encephalitis and West Nile encephalitis immune ascitic tluids. Properties of currently characterized group B arbovirus structural and nonstructural antigensa Virus Antigen Mol wt Antigenic specificity SLS/2-ME Reference x 1O, stability JE Major envelope glycoprotein 58 Cross-reactive in CF tests Labile from virion JE Soluble CF (nonstructural?) 53 Type specific in CF tests Stable JE, SLE, Major virion glycoprotein 53 Cross-reactive in CF and ID Labile WNE, from cell extracts tests DEN-2 JE, SLE, Major intracellular, nonstruc- 85 Type specific in CF and ID Stable WNE, tural protein tests DEN-2 DEN-1,-2, Soluble CF, nonstructural Type specific in CF tests; Stable 2, 3, 2, 25-3, type- and group-specific determinants on same molecule a Abbreviations: SLE, St. Louis encephalitis; WNE, West Nile encephalitis; ID, immunodiffusion; DEN, dengue.

7 VOL. 11, 1975 ANTIGENS OF JE VIRUS tics of the JE envelope antigen and compares it to other currently characterized group B arbovirus structural and nonstructural antigens. Qureshi and Trent (19) have isolated a similar antigen from group B arbovirus-infected cells as listed in Table 3. Their antigenic intracellular polypeptide cross-reacted in CF and immunodiffusion tests with closely related viruses. The JE virion-derived major envelope antigen purified by us was antigenically identical to the whole virion and also cross-reacted with related viruses in CF tests. Although we found that two other structural proteins could be detected in JE virions derived from mouse brain, these played little if any role in crossreactions of the virion. Qureshi and Trent (17) found a nucleocapsid protein of St. Louis encephalitis which had at least one antigenic determinant that it shared with the major envelope protein. In our hands, a CF antigen with a molecular weight of approximately 24 x 13 and associated with the minor JE structural proteins could not be recovered consistently and used for immunologic characterization. Naturally occurring JE SCF antigens were found in mouse brain homogenates after removal of hemagglutinins. "Naturally occurring" refers to those antigens found in JEinfected mouse brain suspensions without the introduction of any specific physical or chemical treatment of cell membranes or particulate antigens that may give rise to artificially produced, soluble antigens. The Sephadex-purified JE SCF preparations contained a major antigen species with a pi of 5.3. The IEF pl 5.3 antigen was considered free of minor CF antigen contaminants and had a molecular weight of 53 x 13 when rechromatographed on Sephadex G-1 columns.. Although this antigen was similar in molecular weight to the major envelope antigen, the JE SCF antigen could be distinguished by its stability upon treatment with SLS and 2-ME. The JE SCF antigen after final purification by IEF was stable to SLS and 2-ME treatment when compared to JE virion antigens treated identically. Dengue-2 SCF antigen with a molecular weight of approximately 39 x 13 is similar to the JE SCF antigen in that it is stable to SLS and 2-ME treatment whereas the dengue hemagglutinins are not (2). These antigens could also be distinguished from dengue virion structural polypeptides by their electrophoretic mobility in acrylamide gels. Antigenically, they did not share major determinants with either of the two hemagglutinins (3) and the dengue SCF molecule contained both group- and type-specific determinants 159 (2). The dengue SCF antigens exhibited increased type specificity compared to crude mouse brain antigens in CF tests. The JE SCF antigens also showed increased type specificity against closely related Murray Valley encephalitis and West Nile encephalitis antibody, where cross-reactions were reduced in CF tests with an SCF antigen purified by IEF. Qureshi and Trent (19) have purified and characterized a type-specific antigen from St. Louis encephalitis and several other group B arbovirus-infected cell extracts with a molecular weight of 85 x 13 (Table 3). The highmolecular-weight antigen reacts only with homologous antibody in CF and immunodiffusion tests, does not inhibit neutralization of St. Louis encephalitis virions, and corresponds to the largest nonstructural polypeptide produced upon infection with these viruses. The dengue and JE SCF antigens also demonstrate immunological type specificity but are of much lower molecular weight. Upon infection with group B arboviruses, at least five nonstructural polypeptides are formed (22, 27, 28). The 85 x 13-molecular-weight antigen from group B-infected cell cultures and the dengue SCF antigen from mouse brain both appear to be nonstructural antigens that stimulate the production of specific antibody in an infected host. Antibodies to the 85 x 13- molecular-weight JE antigen have been found in convalescent human sera by CF and radioimmunoassay tests. It remains to be shown whether the lower-molecular-weight JE SCF antigen described here also induces specific antibody in human infections. It is also not clear whether the JE subgroup SCF antigens are distinctly nonstructural antigens or if they are aberrant forms of the major envelope polypeptide. Qureshi and Trent (17) separated four forms of the major envelope polypeptide of St. Louis encephalitis virions. The polypeptides, antigenically identical, were thought to be developmental stages in the formation of envelope protein found in mature virions. ACKNOWLEDGMENTS We thank W. E. Brandt, D. Shapiro, and V. R. Harrison for their interest and suggestions. Isoelectric focusing was kindly performed by J. M. Dalrymple, and C. M. Hampton provided excellent technical assistance. LITERATURE CITED 1. Brandt, W. E., E. L. Buescher, and F. M. Hetrick Production and characterization of arbovirus antibody in mouse ascitic fluid. Am. J. Trop. Med. Hyg. 16: Brandt, W. E., R. D. Cardiff, and P. K. Russell. 197.

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