Mengo virus capsid protein VP 1, representing residues. 259 to 277, can induce ... organization of the major capsid proteins of Mengo virus possesses close ...
Journal of General Virology(1991), 72, 1087-1092. Printedin Great Britain
Neutralizing antibody to Mengo virus, induced by synthetic peptides Susie Muir,*t Jory P. Weintraub, James Hogle and James L. Bittle Department of Molecular Biology, 10666 North Torrey Pines Road, Research Institute Scripps Clinic, La Jolla, California 92037, U.S.A.
A peptide from the carboxyl-terminal region of the Mengo virus capsid protein VP 1, representing residues 259 to 277, can induce serum neutralizing (SN) antibodies in both the mouse and guinea-pig. This peptide, termed F164, also induces high levels of protective neutralizing antibodies in mice subsequent
to immunization; 87 to 100% of mice are refractory to the effects of an intraperitoneal challenge of 100 LDso of Mengo virus. The mouse model discussed herein will prove useful for studying the immune response to Mengo virus and evaluating the immunogenicity of individual viral components.
location. VP4 is an integral component of the capsid shell and is cleaved from its precursor during the final stages of virus formation (Boege et al., 1986). The pentameric clusters of the VP1 polypeptides form a large proportion of the Mengo virus surface. Residues of VP1 together with those of VP3 line the narrow surface depressions ('pits') of the capsid. These pits are thought to be primarily responsible for the attachment of the virus to host cell receptors (Boege & Scraba, 1989) and are not accessible to antibodies. Although antisera to isolated denatured proteins do not often react with intact virus due to conformational changes in surface features, specific antisera against each of the isolated structural Mengo virus capsid proteins indicates that the most external and antigenic capsid protein is VP1 (Lund et al., 1977). Anti-VP2 serum, although able to cause immunoprecipitation and to produce a positive complement fixation reaction with intact virions, does not neutralize viral infectivity (Lund et aL, 1977). Neutralizing antigenic sites are currently being identified for Mengo virus. Utilizing monoclonal antibodies (MAbs) to select escape mutants, the existence of a single composite surface neutralization site on Mengo virus spanning residues of the VP3 'knob' and VP2 residues near the 'pit' has been proposed (U. Boege, S. Onodera, D. Scraba, G. Parks & A. Palmenberg, unpublished results). Structural analysis suggests as possible neutralizing antigenic sites several viral surface protrusions at amino acid residues 58 to 68 (a loop connecting #-barrel strands B and C of VP1), 78 to 83 (loop I connecting ]3barrel strands C and D of VP1), 93 to 105 (loop II connecting ]3-barrel strands C and D of VP1) and 153 to 162 (a surface-oriented loop connecting strands E and F of the #-barrel in VP2) (Fig. 1). These sites are in regions
Mengo virus is a small (30 nm diameter) spherical ssRNA animal virus in the Picornaviridae family. The organization of the major capsid proteins of Mengo virus possesses close physical similarities to rhinoviruses, hepatitis A virus, foot-and-mouth disease virus (FMDV) and other picornaviruses, such as poliovirus (Luo et al., 1987). Mice inoculated with the virus develop disease in the central nervous system (CNS) that, in many respects, mimics human enterovirus disease involving the CNS (Stringfellow et al., 1974). Mengo virus serves as the model virus for meningoencephalitides caused by enteroviruses; lethal Mengo virus infection of mice has been used as an in vivo test model to elucidate the prophylactic efficacy of antiviral compounds (Veckenstedt & Horn, 1974). Although strains of the virus have been isolated from humans, rhesus and African green monkeys, and the mongoose and mosquitoes, mice remain the species most susceptible to Mengo virus infection (Dick, 1948). In mice the virus can cause an acute lethal infection affecting not only the CNS, but also the salivary glands, exorbital glands, thymus, pancreas and kidneys. Animals die of a severe, diffuse meningoencephalitis (Veckenstedt, 1974; Guthke et aL, 1987). Mengo virus contains 60 copies of four major structural polypeptides: VP1, VP2, VP3 and VP4. Three of these polypeptides, VP1, VP2 and VP3, are to some degree exposed on the surface of the virus whereas the fourth polypeptide, VP4, has a completely internal t Present address: Institut Pasteur, UPMTG/Biotechnologies,25 rue du Dr Roux, 75015 Paris, France. 0000-9834 © 1991 SGM
S. M u i r and others
Methods Virus. The M plaque type variant of Mengo virus (Ellem & Colter, 1961 ; Boege et al., 1986) was used throughout these studies. Procedures for the production of the virus in cultured L cells and its purification have been described previously (Scraba et al., 1967). Peptides. Peptides were synthesized using both the simultaneous multiple peptide synthesis method (Houghten, 1985) and symmetrical anhydride chemistry (Merrifield, 1963; Hagenmaier & Frank, 1972) on automated ABI 430A synthesizers. Tetanus toxoid was used as the carrier protein in all coupling reactions, and peptides were coupled by using glutaraldehyde (Baron & Baltimore, 1982).
::~OC/ s e e 11~
Fig. 1. Four possible neutralizing antigenic sites of Mengo virus as predicted by Luo et al. (1987) (reprinted with permission from Science 235, 182-191) include amino acid residues (1) 58 to 68, (2) 78 to 83 and (3) 93 to 105 in VP1 and (4) 153 to 162 in VP2.
that correspond to the greatest frequency of changes between Mengo virus, Theiler's virus (TMEV) and e n c e p h a l o m y o c a r d i t i s v i r u s ( L u o et al., 1987). We have examined the capsid polypeptides of the M e n g o v i r i o n by a n a l y s i n g t h e s e r u m n e u t r a l i z i n g a b i l i t y o f m u r i n e a n t i s e r a to 140 o v e r l a p p i n g 10- to 1 9 - m e r s y n t h e t i c p e p t i d e s c o v e r i n g 100~o (47 p e p t i d e s ) o f t h e V P I r e g i o n ( M u i r & Bittle, 1989), a n d 1 0 0 K o f t h e V P 2 , V P 3 a n d V P 4 r e g i o n s ( 4 1 , 4 0 a n d 12 p e p t i d e s , r e s p e c t i v e ly). T h e C - t e r m i n a l p e p t i d e o f V P 1 , w h i c h g e n e r a t e d t h e most highly titred neutralizing anti-Mengo virus serum, w a s u t i l i z e d as an i m m u n o g e n in a m u r i n e c h a l l e n g e study.
Peptide immunization and bleeding of animals. Relevant regulations regarding safety, ethics and use of animals have been complied with during the course of this research project. (i) Mice. Groups of five female BALB/cByJ mice, 6 weeks of age (Research Institute of Scripps Clinic breeding colony) were injected intraperitoneally (i.p.) with complete Freund's adjuvant (CFA) emulsions containing 100 ~tg of one particular coupled peptide. Four weeks following the initial injection, two booster injections of peptide in incomplete Freund's adjuvant (IFA) emulsions were given at 2 week intervals, followed by a final boost 90 days from the primary injection. The first booster injection contained 100 ~tg of peptide, the second contained 50 ~tg of peptide, and the last contained 25 ~tg of peptide. All emulsions were made by mixing equal volumes of the peptide conjugate and either CFA or IFA as required. Animals were bled at 30, 60, 90 and 120 days subsequent to the primary injection. (ii) Guinea-pigs. Outbred female Hartley guinea-pigs were injected subcutaneously with a "cocktail" of 100 ~tg of each of five to six coupled peptides on a schedule similar to the i.p. murine injections. Only VP1 and VP4 peptides were injected into guinea-pigs. Serum was prepared from whole blood by allowing it to clot for at least 1 h at room temperature and then standing overnight at 4 °C prior to centrifugation at 5000 r.p.m, for 10 min. The serum was stored at - 20 °C without the addition of sodium azide. Solid-phase ELISA for the detection of antipeptide and antiviral antibodies. Microtitre plates (Costar) were coated overnight with either a purified Mengo virus preparation or uncoupled peptide in carbonatebicarbonate buffer. Plates were blocked for I h at 37 °C with 5 ~ nonfat dry milk in phosphate-buffered saline (PBS) prior to the addition of test sera. Sera were serially diluted from l : 10 to 1:1280 in PBS and plates were incubated for I h at 37 °C and washed with PBS containing 0.05~ Tween 20. Affinity-purified rabbit anti-mouse, and where appropriate, affinity-purified goat anti-guinea pig horseradish peroxidase IgG conjugate (Kirkegaard & Perry Laboratories) diluted 1 : 500 in PBS was then added and the plates were incubated as before. After washing, the enzyme substrate ABTS [2,2'-azino-di-(3-ethyl-benzthiazolin sulphonate ) diammonium salt (Kirkegaard & Perry Laboratories)] was added. After 10 rain incubation the absorbance was read at 492 nm. Four times the average of the negative control was taken as the cut-off point for the titre of the test sera. Serum-neutralizing (SN) assay (i) Preparation of target cells. Murine L929 cells were continuously cultured in 150 cm 2 flasks (Costar) in Dulbecco's MEM (DMEM) with 4.5 g glucose/litre (M.A. Bioproducts) to which was added 10~ foetal bovine serum (Flow Laboratories), and 1% solutions of penicillin/ streptomycin (Pen/Strep; 10000 units penicillin, I0000 ~tg streptomycin), 200mM-L-glutamine, 100raM-sodium pyruvate, 100 x nonessential amino acids (Flow Laboratories) and 100 × vitamins (Flow Laboratories). This was termed complete medium. Prior to assay cells were washed with PBS, subjected to treatment with 1×trypsin/EDTA (Flow Laboratories) and resuspended in complete medium as described above. Cells were plated at 1.5 × 104 cells/well in 96-well tissue culture
Synthetic peptides of Mengo virus
plates (Costar) and allowed to attach for at least 1 h at 37 °C in a 5% humidified chamber before being utilized in the SN assay. (ii) Titration of antiviral antibodies. In a separate 96-well plate duplicate twofold dilutions of each test serum and negative and positive control sera (1:2 to 1:256) were made in DMEM to which only Pen/Strep had been added (incomplete media). One-hundred TCIDso of Mengo virus was added to all wells of the plate containing sera and plates were incubated for 1 h at 37 °C in a humidified chamber. All serum samples had been heat-inactivated for 30 min at 56 °C prior to use. A pool of non-immunized BALB/cByJ sera served as negative control sera; Mengo virus-hyperimmunized Hartley guinea-pigs provided a source of positive control sera, In SN assays the positive control serum titre was between 1:1024 and 1:2048 whereas the negative control sera possessed no viral neutralizing antibodies. (iii) Procedure. One-hundred microlitres of each serum-virus mixture was added to the appropriate well of the target cell plate. As constant internal controls stock Mengo virus, diluted in incomplete medium, was titrated on each plate; additionally, tissue culture control wells were present on each plate. Plates were placed in a humidified chamber at 37 °C, 5 %00CO2 and, although allowed to incubate for 5 days prior to SN activity being recorded, were checked daily to ascertain cell status and the protective capacity of the serum.
Challenge model. Titrations of Mengo virus in both BALB/cByJ and C57/Blk6 mice (Jackson Laboratories) indicated that the virus was more lethal for the younger individuals of the latter strain; hence C57/Blk6 mice were utilized in the challenge model. C57/Blk6 mice 7 day-old (group A), 14 day-old (group B) and 21 day-old (group C) received primary i.p. injections of 100 lag of F164 peptide in PBS (groups A and B) and CFA (group C). Seven days later an i.p. booster injection of 100 gg of peptide in adjuvant was administered. Group C received IFA as adjuvant whereas group B received CFA and group A received the peptide in PBS. A final immunization followed 7 days later with both groups B and C receiving IFA as adjuvant and group A receiving CFA. Reduced and delayed antibody responses can occur when short immunization intervals are used, because B cell memory appears to require several weeks for full development. In these experiments the intervals between successive immunizations were deliberately short and chosen to ensure that the animals mounted an antibody response before their susceptibility to challenge waned. Seventeen days following the last booster, all immunized animals received an i.p. challenge of 100 LDs0 of Mengo virus. This LDs0 had been determined by an in vivo titration of Mengo virus in 3 week old C57/Blk6 mice. Both SN and antipeptide titres were measured 14 and 21 days following the initial immunization. Mice were also bled on the day of viral challenge and SN and antipeptide titres determined. When challenged, SN titres averaged at least 1:4 for each challenge group. Mice were observed for 10 days following viral challenge. Age- and sexmatched controls for all groups were given sham inoculations of PBS.
F130 F136 F149 F164
SPLGRFAVKS YDQLRPQRLT SSLSEGRTPQ PTSGDKIDMTPRAGVLMLE
Fig. 2. Location of the three tentative and the major neutralizing synthetic peptides within the VP1 sequence. The locations of the neutralizing synthetic peptides are denoted by the double dashes along the linear VP1 amino acid sequence for Mengo virus, M plaque-type variant. The amino acid sequence was determined from the nucleotide sequence (Luo et al., 1987). The amino acid residues are numbered with the amino terminus of VP1 designated 1 and residue 277 located at the carboxy terminus of VP1.
Table t. Immune response to Mengo virus F164 pooled antipeptide sera Days*
Murine SN titre
Murine ELISA titre
30 60 90 120
1:8 1:64 1:128 1:256
1:80 1:640 1:1600 1:1600
Guinea-pig SN titre
36 42 74
1:16 1:32 1:256
* Days following initial immunization of mice or guinea-pigs.
T a b l e 2. Mengo virus challenge o f F 1 6 4 immunized mice Group Age* Immunized No. S/Tt Survivors (~) Non-immunized No. S/T Survivors (%)
* Age, in days, at first immunization. t Number of survivors among total number of mice in the group. As groups D and E differed only in one immunization the same non-immunized mice were utilized as controls for both groups.
Results To identify the Mengo virus neutralization epitopes, protection of murine L929 cells from viral c.p.e, in an in vitro SN assay by anti-Mengo virus peptide antisera was measured. Examination of the capsid polypeptides via overlapping synthetic peptides covering the entirety of the capsid indicated one major neutralizing site named F164 and three tentative sites named F130, F136 and F149. All were in the VP1 region (Fig. 2). Murine antipeptide sera to the random overlapping peptides of
VP2, VP3 and VP4 did not possess Mengo virusneutralizing activity nor did guinea-pig antipeptide sera to peptides of the VP4 region. Only VP1 and VP2 peptides were injected into guinea-pigs. Peptide F164, corresponding to the 19 terminal carboxyl amino acids 259 to 277, was very strongly immunogenic in both mice and guinea-pigs, inducing significant SN and ELISA titres to Mengo virus and homologous peptide, respectively (Table 1). In contrast, the low SN titres of 1:2 and 1:4 induced by the other
S. Muir and others
three peptides might be regarded as artefactual even though these low titres were consistently elicited. Groups of mice hyperimmunized with peptide F164 were shown to be protected from the effects of Mengo virus at a statistically significant level (Table 2) over nonimmunized age- and sex-matched controls following challenge with live virus. Viral effects were observed as unilateral or bilateral hindlimb paralysis, tremors, and deaths beginning 4 days following viral challenge of nonimmunized controls. Some of the affected mice exhibited uni- or bilateral ocular serous discharge 3 to 4 days following challenge as well. Such a discharge can sometimes be seen with acute Mengo virus infection. All mice were challenged with 100 LDs0 of Mengo virus. Group A mice were challenged at 6 weeks of age. Of 15 immunized individuals two died within a 10-day observation period (13~) whereas 10 of 13 non-immunized controls (76~) died. Group B mice were challenged at 7 weeks of age; one of 19 immunized mice (5-2~) died during the observation period whereas in the control group eight of 13 mice (61 ~) died. Group C mice were challenged at 8 weeks of age. None of the 12 immunized controls died after viral challenge. However, only four of 14 non-immunized controls died (28~). An in vivo titration of Mengo virus in groups of 30 C57/Blk6 mice 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 and 24 weeks of age indicated that as mice age they appear to become more resistant to the effects of an i.p. Mengo virus challenge (personal observation). There was virtually no difference in the LDsos of Mengo virus titrated in 3, 4, 5 or 6 week old mice. It was noted that older mice, >8 to 10 weeks of age, became more refractory to the lethal effects of identical numbers of Mengo virion particles which killed younger mice 3 to 6 weeks of age. Twenty to 30~o of the older mice survived an i.p. challenge of Mengo virus which when titrated in 3 to 4 week old mice was 100K fatal. Although this may help to account for the high survival rate in mice challenged at 8 weeks of age it may not explain the entire phenomenon of survival in this age group. Two other groups of 14 day old mice were injected with the C-terminal peptide emulsified in CFA. One group (group D) received one injection (100 gg) of the peptide whereas the other group (group E) received both a primary (100 gg) and a secondary (50 gg) injection of peptide in IFA 2 weeks following initial immunization. A group of sex- and age-matched controls were shaminjected. All mice were i.p. challenged with 100 LDs0 of Mengo virus at 5 weeks of age. Ten percent of mice immunized once with the peptide died (1/10 mice); none of the 10 mice receiving two injections died; 12 of 19 noninjected. All mice were i.p. challenged with 100 LD50 of tion period. The results of this challenge experiment are in concordance with those results of the earlier immunization/challenge experiments in groups A and B.
Immunization/challenge data show that mice inoculated as young as 7 days old can mount an effective immune response to Mengo virus following immunization with peptide F 164. For the challenge experiments anti-virus particle antibody and anti-homologous peptide antibody titres as measured by an indirect ELISA were generally in accord with SN results. That is, those mice which possessed high ELISA titres had high SN titres and those with low ELISA titres had correspondingly low SN titres. Antibody titres to viral particles were from two- to 16-fold lower than titres to the C-terminal peptide. Consideration must be given to the possibility that the conformational nature of an epitope may undergo alteration as a consequence of virus particle distortion when virus is bound to the solid phase in ELISA (Ouldridge et al., 1984). Sites not normally available for antibody binding may be exposed; likewise, sites which are usually available for antibody interaction may become hidden. The antigen specificity of each of the 140 anti-peptide antisera was assayed by ELISA. Anti-homologous peptide results showed that each tetanus toxoid-coupled peptide was antigenic as all peptides evoked an antibody response. The vast majority of titres ranged between 1:320 to 1:1280. Antisera were quite specific in recognizing cognate peptide. Each peptide successfully competed its own homologous antisera in ELISA assays. Pre-absorbed sera had titres between 1:320 and 1 : 1280; post-absorption titres were less than 1:20. Neither mouse nor guinea-pig anti-virion serum possessed high levels of antibodies to the C-terminal region of VP1 as absorption of this serum with the F164 peptide did not affect the SN titre significantly. Immunization or infection with whole virus may not adequately present the C terminus of VP1 to the immune system such that a strong neutralizing antibody response is triggered. Alternatively, other viral epitopes may be processed preferentially and dominate the immune response. In contrast, when presented as an isolated epitope in the form of the synthetic peptide F164, the Cterminal region of VP1 was an excellent immunogen. The immune response to this region of VP 1 was vigorous with sufficient quantities of virus-neutralizing antibody produced to protect an animal from challenge. Immunoblots of Mengo virus screened with F164 peptideabsorbed anti-virion sera demonstrated that the Cterminal peptide was able to reduce the staining intensity of the VP 1 band of the virus (data not shown). Therefore, antibodies produced to F164 may be significant in establishing the virus-binding titre.
Discussion The synthetic peptide antibodies generated in this study defined an antigenic region within Mengo virus capsid
Synthetic peptides o f M e n g o virus
protein VP1, the carboxyl terminus, that will induce serum neutralizing antibodies in two mammalian species suggesting that the production of SN antibody is an inherent property of the peptide and not a consequence of the immunological idiosyncrasies peculiar to the host species. As both mice and guinea-pigs are susceptible to the effects of Mengo virus one might expect similar immune responses to a particular set of determinants. The potential antigenic sites in Mengo virus as suggested by Luo et al. (1987) have been predicted based upon the three-dimensional structure of Mengo virus and upon sequence comparisons between Mengo virus and other picornaviruses, particularly human rhinovirus 14, which have had their three-dimensional structures solved and/or have been sequenced. The predicted sites include amino acid residues 58 to 68, 78 to 83 and 93 to 105 in VP1 and 153 to 162 in VP2. In the present study, by employing antipeptide antibodies we have not been able to confirm any of these sites as neutralizing ones. The peptide which induced the strongest SN antibody response to Mengo virus was composed of VP1 amino acid residues 259 to 277 (F164) which encompass the C terminus. This peptide includes residues which are exposed on the surface of the virus and would be expected to be accessible to antibodies in the native infectious virions. These residues are disordered in the electron density map and could form a mobile region near the rim of the putative receptor-binding site; at present it is not possible, due to the fluidity of the C terminus, to make such a determination. The C-terminal region of VP1 is flexible in the crystal structure of the virus generating a disordered extension of approximately 15 amino acid residues somewhere around the 60 symmetrically distributed narrow depressions ('pits') on the virus surface. The pits are thought to be candidates for cell-receptor binding sites. Boege & Scraba (1989) have shown that during the maturation of Mengo virus the C-terminal ends of VP 1 undergo a postassembly trimming of three amino acids. They conclude that this modification has a dramatic effect on the dissociability of the virus capsid and suggest that the Cterminal region may play a role in the interaction of the Mengo virion with host cell receptors. The present findings, that antibodies to a C-terminal peptide are both neutralizing and protective, lend support to this idea. Although the carboxyl terminus is not predicted to be a neutralizing site for Mengo virus, the analogous residues have been shown to contain or to potentiate the immunogenicity of a neutralizing epitope in two other viruses: FMDV (DiMarchi et al., 1986; Meloen & Barteling, 1986; Strohmaier et al., 1982; Thomas et al., 1988) and TMEV (Ohara et al., 1988). These viruses are both picornaviruses with TMEV being closely related to the cardioviruses, of which Mengo virus is a member. Ohara et al. (1988), utilizing neutralizing MAbs
localized neutralizing epitopes to a site near the carboxyl end of VP1. TMEV escape variants have altered pathogenicity in mice and sequencing of RNA from TMEV mutants proves that the only amino acid changes are near the carboxyl end of VP1 suggesting that the C terminus epitope is important in viral persistence and disease. DiMarchi et al. (1986) demonstrate that a C-terminal peptide, comprising residues 200 to 213 of VP1, when coupled to residues 141 to 158 of VP1 strongly potentiated the induction of neutralizing antibodies to FMDV. Bittle et al. (1982) showed that this latter peptide is the major site for the induction of neutralizing and protective antibodies to FMDV challenge in guinea-pigs. In FMDV the C terminus peptide significantly increases the potency of residues 141 to 158 and may constitute an immunodominant antigenic domain (Parry et al., 1989) and be of importance in enhancing a protective response (Doel et al., 1988; Francis et al., 1985). Meloen & Barteling (1986) located what they term a minor 'neutralizing epitope' on the C-terminal end of isolated VP1 embracing residues 200 to 210 which induces neutralizing antibodies that correlate poorly with protection. This epitope may be related to the major neutralizing epitope, residues 141 to 158, as part of a discontinuous epitope (Meloen et al., 1988). This inference is supported by the work of Thomas et al. (1988) and agrees with previous reports (Parry et al., 1985; Xie et al., 1987). Additional neutralizing sites may be present in Mengo virus that do not reside within VP 1. Cross-neutralization tests with four MAbs to Mengo virion pentamers have identified a single composite neutralizing site on the capsid (U. Boege, S. Onodera, D. Scraba, G. Parks & A. Palmenberg, unpublished results). This site involves VP2 and VP3 with the VP2 portion aligning with NImlI (poliovirus site 2) and the VP3 site corresponding to site 3 of poliovirus type 1. Using overlapping synthetic peptides, no significant neutralizing sites were found in VP2, VP3 or VP4. One must recognize the limits of utilizing only synthetic peptides in determining neutralizing immunogenic sites for a particular virus. The present approach precluded the identification of discontinuous or conformational epitopes, i.e. those epitopes which have contact residues distributed along the primary structure which are brought together spatially in the natural folding of the polypeptide. Utilizing random overlapping synthetic peptides to cover the entirety of the capsid polypeptides allowed only for the identification of continuous (or sequential) immunogenic sites. The mapping and identification of immunogenic epitopes of Mengo virus based upon generation of small overlapping peptides, may introduce bias in the localization of a functional immunogenic site found on intact virus presented to a competent immune system.
S. Muir and others
It is premature to suggest that the C-terminal region is a sole dominant immunogenic site in Mengo virus for three reasons: the absorption by the C-terminal peptide of very low levels of neutralizing activity from antivirion serum was not statistically significant; the addition of just one or two amino acids can substantially increase the ability of a peptide to elicit neutralizing antibody (Francis et al., 1987), and thirdly, immunization with the C-terminal peptide does not grant 100 ~ protection to all vaccinated mice in all age groups. It would be interesting to investigate further the neutralizing sites described here utilizing the Pepscan method employed by Meloen et al. (1988). We thank Drs Douglas Scraba and Ulrike Boege for providing purified M variant Mengo virus and protocols for virus isolation. The technical assistance of Ms Danica Lerner, Mr Farid Karimi and Ms Trudie Muir is gratefully acknowledged.
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(Received 26 July 1990; Accepted 25 January 1991)