key: cord-0959784-dv6oeldo authors: Niesters, Hubert G.M.; Bleumink-Pluym, Nancy M.C.; Osterhaus, Albert D.M.E.; Horzinek, Marian C.; Van Der Zeijst, Bernard A.M. title: Epitopes on the peplomer protein of infectious bronchitis virus strain M41 as defined by monoclonal antibodies date: 1987-12-31 journal: Virology DOI: 10.1016/0042-6822(87)90145-0 sha: b7df37353c137e7045aa8d1027d548b2b8cbcb1b doc_id: 959784 cord_uid: dv6oeldo Abstract Sixteen monoclonal antibodies (Mcabs) were prepared against infectious bronchitis virus strain M41, all of them reacting with the peplomer protein. One of them, Mcab 13, was able to neutralize the virus and to inhibit hemagglutination. Competition binding assays allowed the definition of five epitopes, designated as A, B, C, D, and E, of which epitopes A and B are overlapping. Furthermore, the binding of Mcab 13 (epitope E) could be enhanced by the addition of Mcabs from group B, C, and D. A dot immunoblot assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. Only the binding of Mcab 13 was affected, indicating that the epitope involved in neutralization and hemagglutination is conformation dependent. The epitopes A to D were highly conserved among IBV strains, while epitope E was specific for strains M41 and D3896. In this last strain, however, this epitope was not involved in neutralization. Avian infectious bronchitis virus (IBV) is the etiological agent of a highly contagious respiratory disease of young chickens. The virus also causes a marked and rapid decline of egg production and quality in laying hens (Darbyshire, 1981) . IBV belongs to the coronaviruses, a group of positive-stranded enveloped RNA viruses. The virion contains three major structural proteins: the peplomer or spike (S), nucleocapsid (N), and matrix (M) proteins (Cavanagh, 1981; Stern and Sefton, 1982) . The M protein is present as polypeptide p23 and the glycosylated forms gp28, gp31, and gp36. The N protein has an apparent mol wt of 51 kDa (Macnaughton and Madge, 1977) . The characteristic surface projections of the virus are made up by the peplomer protein; the peplomer is composed of two molecules of each of the glycopolypeptides Sl (gp90) and S2 (gp84). Both contain highmannose, N-linked oligosaccharides. The S2 protein is anchored in the membrane while Sl is attached to S2 via a loose noncovalent association (Cavanagh, 1983a, b) . ' The peplomer protein is believed to be responsible for the induction of neutralizing antibodies and a protective immune response (Mockett et a/., 1984; Cavanagh et al., 1986) . In this study we describe the production of monoclonal antibodies (Mcabs) directed against the peplomer protein of IBV strain M41. To map the epitopes we performed reciprocal competitive binding assays. The Mcabs were used in a dot immunoblotting assay to analyze the sensitivity to denaturation of the epitopes responsible for antibody attachment. Furthermore, the variability of these epitopes in different IBV strains was analyzed. All IBV strains were obtained ~ METHODS from the Poultry Health Institute, Doorn, The Netherlands. Purified stocks were prepared from cloned virus (Niesters et a/., 1986) . Immunization of mice Gradient-purified M41 virus was disrupted in 1% Triton X-100 and diluted to 400 pglml in TESV buffer (0.02 M Tris-HCI, pH 7.4, 1 m/W EDTA, 0.1 ILI NaCI). Balb/c mice (from CPB, TNO, Zeist, The Netherlands) were primed intramuscularly with 100 pg virus in 0.5 ml complete Freund's adjuvant and boosted twice at weekly intervals with the same amount of antigen, in incomplete Freund's adjuvant. At least 6 weeks after the initial immunization and 3 days before the mice were sacrificed for the preparation of hybridomas, they were boosted intravenously with 10 pg purified intact virus (0.1 ml in TESV). Production of mouse hybridoma cell lines Hybridoma cell lines were prepared and cloned as previously described (Osterhaus el al., 1981) . Immune ascitic fluid was made in Balb/c mice primed with pristane (2,6,10,14-tetramethylpentadecane; Janssen Chimica, Belgium; Goding, 1980) . Hybridoma culture media and mouse body fluids were screened for antibodies in an ELISA. Microtiter plates (Inotech) were coated (3 hr at 37" followed by overnight incubation at 4') with 0.5 pg virus in 100 ~1 0.15 NI NaCl per well. Plates were washed four times with washing fluid (0.15 M NaCI, 0.05Ob Tween 20 (v/v)). All subsequent steps were performed at 37". Fifty microliters of V-buffer (0.05 /1/1 Tris-HCI, pH 7.4, 0.15 M NaCI, 1 mll/l EDTA, 0.1 Oh BSA (w/v), 0.05% Tween 20 (v/v), 109/o newborn calf serum) and 50 ~1 hybridoma supernatant or dilutions of ascitic fluid in V buffer were added to each well and incubated for 2 hr. After washing four times with washing fluid, peroxidase-conjugated rabbit anti-mouse IgG (H + L) (Nordic Immunological Laboratories) was added and incubated for 1 hr, followed by four washes with washing fluid. Peroxidase binding was visualized by incubation with 300 ~1 per well of a freshly prepared solution containing 50 mn/l citric acid (pH 4.0) 2.3 mlLl H202, 40 mM 2-2'-azinobis-3-ethylbenzthiazoline sulfonic acid (ABTS, Sigma). Absorption was measured at 405 nm in a multichannel photometer (Flow Laboratories). The immunoglobulin class of the hybridoma antibodies present in the culture media of the clones was determined by ELISA with rabbit antisera to mouse IgM (Miles), IgGl, lgG2, and lgG3 (Nordic) and goat anti-rabbit IgG serum conjugated with horseradish peroxidase (Nordic). Independent radioimmunoprecipitation tests with isotype-specific second antibody were carried out. Antigen consisted of purified virus, isolated as described above, or allantoic fluid from uninfected eggs. "Native antigen"-was prepared by dilution in TESV buffer or after treatment at 37" for 20 min with a final solution of 10 mM Tris-HCI, pH 7.4, 0.2 M NaCI, 0.5 M KCI, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2% (v/v) NP-40. "Denaturated antigen" was boiled for 2 min in a solution containing 29/o SDS and 59/o 2-mercaptoethanol (Talbot et al., 1984) . Native and denaturated viral antigens or proteins from allantoic fluid (1 pg in 2.5 ~1) were spotted onto nitrocellulose strips (Schleicher & Schuell, 0.45 pm pore size). The strips were dried under a heat lamp, saturated in gelatin buffer (500 mM NaCI, 10 mn/l sodium phosphate, pH 7.3, 10 mll/l EDTA, 0.259/o gelatin, 0.5% Triton X-100) and probed for 2 hr with Mcab solution. After three washes with gelatin buffer, peroxidase-conjugated rabbit anti-mouse IgG (H + L) was added and incubated for another 2 hr. The strips were washed with phosphate-buffered saline (PBS, pH 7.4). Binding was visualized by incubation with a freshly prepared substrate solution containing PBS, 0.5 mg/ml 3-3'-diaminobenzidinetetrahydrochloride (DAB, Serva), and 0.3% H202. The reaction was stopped by washing the strips with PBS (Roos et al., 1982) . Gradient-purified virus was labeled using lodogen (Pierce) to a specific activity of 1.9 &iIpg (Markwell and Fox, 1978) . Approximately 100,000 cpm 1251-labeled M41 virion proteins in 25 ~1 were immunoprecipitated with 2 ~1 ascitic fluid (Van der Zeijst et al., 1983) . Immunoprecipitates were analyzed by electrophoresis in 15% SDS-polyacrylamide gels (Rottier et al., 1981) . Virus neutralization was carried out according to Kusters et al. (1987) . Hemagglutination inhibition tests were performed with 8 hemagglutinating units in microtiter plates as described by Alexander et al. (1976) . Coupling of horseradish peroxidase (HRPO) to monoclonal antibodies lmmunoglobulin fractions were isolated from ascites fluids by precipitation at 4" for 2 hr with an equal volume of saturated ammonium sulfate (pH 7.0). After overnight dialysis against 0.01 11/1 sodium carbonate buffer (pH 9.5) antibodies were coupled to horseradish peroxidase (HRPO; Boehringer-Mannheim) by the periodate method (Wilson and Nakane, 1978 ) and stored at -70". Before carrying out the actual competition binding assays, the amount of binding in the ELISA was deter-mined for all Mcabs (peroxidase conjugated and nonconjugated). A series of twofold dilutions was reacted with antigen-coated plates. The resulting dose-response curve yielded the level of maximum binding (a measure for the relative avidity) and the Mcab concentration at which 50% binding occurred. For the competition assays, virus-coated plates were incubated for 1 hr at 37" with 200 ~1 per well of PBS containing 0.29/o Tween 20 and 10% newborn calf serum to saturate all binding sites on the plastic. The solution was removed and the plates were washed four times with washing fluid. A mixture of the peroxidase-conjugated Mcab (at twice the concentration giving half maximal binding) and the unconjugated, competing antibody at various concentrations from the linear part of the dose-response curve, were added simultaneously. Dilutions were made in PBS containing 1% newborn calf serum. The mixture was incubated for 1 hr at 37". The plates were then washed four times with washing fluid and binding of the peroxidase-conjugated Mcab was visualized as described above. Nonspecific binding to plates without antigen was taken to represent background. Binding of each conjugate to viral antigen in the absence of competitor represented 100% binding (0% inhibition), while the binding of the conjugate in the presence of excess of the same unlabeled Mcab represented 0% binding (100% competition). To define epitope groups, competition was considered to be positive only if it occurred symmetrically, i.e., when similar results were obtained when the antibody was used as competitor and as peroxidase-conjugated antibody. The percentage of competition usually reached a plateau. Competition was rated as strong if it was more than SO%, and significant if it was more than 35%. Our purpose was to generate and subsequently analyze Mcabs directed against the surface glycoproteins of IBV strain M41. To obtain predominantly Mcabs to surface proteins, immunized mice were boosted with intact sucrose-gradient-purified virions by intravenous injection a few days before the fusion. Spleen cells from two hyperimmunized mice were fused in independent experiments. The hybridoma cell lines secreting antiviral Mcabs were screened with the aid of an ELISA. The amount of viral antigen which provided maximum antibody binding and minimum background with polyvalent rabbit anti-M41 sera had been determined previously (0.5 pg sucrose-purified intact virions per well). These conditions were used throughout. Sixteen Mcabs directed against the peplomer protein were selected for further analysis (Table 1) . Their polypeptide specificities had been characterized by immunoprecipitation of lz51-radiolabeled purified virions; typical results are shown in Fig. 1 . No cross-reactivity was detected with allantoic fluid from uninfected eggs, both by ELISA and immunoprecipitation of '251-radiolabeled allantoic fluid. Cellular proteins, such as actin, transferrin, and ovalbumin, sometimes coprecipitated with the viral polypeptides (results not shown). These proteins may associate nonspecifically with the virion (Cavanagh, 1981; Lomniczi and Morser, 198 1; Wadey and Westaway, 198 1 Table 2. A direct relationship exists between the avidity and the maximal amount of Mcab bound to a given amount of antigen (Frankel and Gerhard, 1979; Bruck et al., 1982; Van Drunen Littel-van den Hurk et al., 1985) . The Mcabs were titrated in an indirect ELISA (Fig. 2. ) 1 lgG3 2 lgG3 3 IgGl 4 IgGl 5 lgG3 6 lgG2 7 lgG3 8 lgG3 9 IgGl 10 lgG3 11 lgG3 12 lgG3 13 lgG2 630,000 2560 14 IgGl 15 IgGl 16 IgGl a Neutralization assays were performed with 100 EIDSo per egg and serial lo-fold dilutions of the ascitic fluid. Only significant neutralization (> 100) is indicated. ' Inhibition was considered to be positive if the titer was 160 or more. and avidities were estimated using the plateau level absorbance values; they varied between 0.45 and 2.0. Mcabs 14, 15, and 16 did not reach a plateau level within the range studied. Mcab 13 behaved in a remarkable way: at concentrations up to 0.5 pg protein per well, this Mcab was able to saturate those viral antigen coated, while at concentrations higher than 2 pg per well, more antibodies could be bound (Fig. 2B) . Experiments were made to determine whether the Mcabs can be divided into clusters recognizing the same immunogenic region of the peplomer protein. All preparations except Mcabs 14, 15, and 16 retained their binding capacity after coupling to horseradish peroxidase. The avidity of the latter Mcabs apparently is too low in the ELISA. The remaining 13 Mcabs were used in the antibody competition assay. Dose-response curves for the peroxidase-conjugated (not shown) and the nonconjugated antibodies (Fig. 2) were plotted to determine the proper concentrations for the blocking assay. The relative avidity as well as the kinetics of self-competition were determined. Each antibody was used both as a competitor and as a peroxidase-conjugated preparation. Mcabs were considered to recognize the same epitope if use of reciprocal antibody preparations prevented binding. The percentage of competition was normalized to 100% in the homologous reaction. Five epitopes were found, which were designated A, B, C, D, and E. Epitopes A and B overlap, while a one-way competition was observed between Mcabs from group C and those of group D and between Mcab 1 (group A) and those of groups B and C (Fig. 3, Table 3 ). Besides competition, enhancement was also observed. The binding of peroxidase-conjugated Mcab 13 could be enhanced by the binding of Mcabs 4, 8, 9, 10, and 12 (Table 3 , Fig. 4) . Furthermore, the binding of peroxidase-conjugated Mcab 5 could be enhanced by Mcabs 9 and 12; the binding of peroxidase-conjugated Mcab 6 was enhanced by Mcab 12 (Table 3) . Enhancement was not observed in the reciprocal assays. Pairs of Mcabs which induced more than 50% enhancement are designated by arrows in Table 3 . The Mcabs were tested for their cross-reactivity with other IBV strains in an indirect ELISA (Table 2 ). All Nofe. Twice the amount of monoclonal antibody giving maximal binding to IBVM41 was used. Binding to the heterologous virus is expressed as percentage of the A 405 obtained with IBV-M41. Empty squares: 50 to 100%; crossed squares: 25 to 50%; full squares less than 25%. Table 1. strains belong to Dutch neutralization serotypes. The origin and serological relationships of the strains has been described before (Davelaar et al., 1984; Kusters eta/., 1987) . The M42 (Beaudette) strain is a laboratory strain of the same serotype as M4 1. The Mcabs l-l 2 of the epitopes A to D recognized most strains. An exception is Mcab 10 (epitope D) which failed to react with the M42 strain as well with D207, D212, D274, and D1466, while Mcab 6 and Mcab 12 did not recognize strains M42 and D207, respectively. Epitope E was only present in strains M41 and D3896; however, Mcab 13 did not neutralize D3896 (results not shown). In order to study the need for a native structure of the peplomer protein for antibody attachment, we performed a dot immunoblot analysis on native and denaturated virus. All Mcabs recognized the viral antigen in its native structure, i.e., in buffer with or without 2% NP-40. After denaturation by boiling in SDS and 2mercaptoethanol, the Mcabs recognizing the epitopes A to D still bound to the protein. The reactivity of Mcab 13, however, was fully abolished by denaturation. Representative results for each epitope are shown in Fig. 5 . In this paper we describe properties of Mcabs directed against the peplomer protein of IBV strain M41. Probably due to the immunization protocol in which mice were boosted intravenously with intact purified virions a few days before the fusion, only anti-peplomer protein, Mcabs were obtained. Similar observations have been made with influenza virus (Gerhard et a/., 1980) vesicular stomatitis virus (Lefrancois and Lyles, 1982a, b) , measles virus (Giraudon and Wild, 1981) , and Newcastle disease virus (lorio and Bratt, 1983) . Although the Mcabs do precipitate the peplomer protein Sl, we cannot exclude that this is the result of its association with S2, although the latter protein could never be precipitated as a clear band (Fig. 1) . The Mcabs did not recognize (glyco)proteins of the allantoic fluid in the ELISA, the dot blot analysis (see Fig. 5 ), or after immunoprecipitation of radiolabeled allantoic fluid. This indicates that the Mcabs are not directed to oligosaccharide side chains of the peplomer protein (Jackson et al., 1981) . All 13 antibodies were used in a blocking assay to identify the number of epitopes on the peplomer protein. The definition of epitopes by this assay is based on the assumption that a Mcab binding to a specific site hinders the attachment of another antibody to the same or a proximal site (Stone and Nowinski, 1980) . However, two antibodies directed to the same site will compete in a manner related to their relative avidities and concentrations. In practice, a spectrum of relative interference efficiencies will be obtained (Stone and Nowinski, 1980; Lefrancois and Lyles, 1982a, b .) Two-way competition is significant for the identification of Mcabs recognizing the same epitope, even when the degree of competition is low. Five epitopes (A to E) could be delineated on the peplomer protein, epitopes A and B overlapped. Furthermore there was extensive one-way competition between epitopes C and D and to a lesser extent between epitopes A and C. This does not necessarily indicate overlap of the epitopes, but may also be the result of conformational changes in the peplomeric protein induced by a Mcab against one epitope, leading to a reduced binding of a Mcab to another epitope. Cooperative effects among antibodies directed against different epitopes were observed. Binding of Mcab 13 in the ELISA was enhanced by Mcabs 4, 8, 9, 10, and 12 . These Mcabs, however, did not enhance neutralization or hemagglutination inhibition (HI). But neutralization and HI assays are not accurate enough to detect a twofold increase in titer. The binding of Mcab 5 and 6 was enhanced by Mcabs 9 and 12 and by Mcab 12, respectively. Enhancement of binding has been reported for Mcabs against VSV (Lefrancois and Lyles, 1982a, b) , yellow fever virus (Schlesinger et al., 1984) Semliki Forest virus (Boere eta/., 1984) and recently with another coronavirus, TGEV (Delmas et al., 1986) . It might be related to a more advantageous conformation of the epitope involved, thereby increasing the binding of the antibody. Enhancement was always a one-way process. Only epitope E is involved in both neutralization and hemagglutination inhibition. The corresponding Mcab 13 did not neutralize other IBV strains, but in the ELISA it reacted with one other IBV strain, 03896 (see Table 2 ). Two neutralizing Mcabs directed to Sl of IBV-M41 from Mockett et a/. (1984) were also strain specific. Apparently, neutralization epitopes are not shared by IBV strains. In contrast, Jimenez eta/. (1986) found that the critical epitopes involved in neutralization of TGEV were highly conserved. The epitopes A to D were not involved in any known biological function. From Tables 2 and 3 we can deduce that the epitopes A-C are conserved. Only four Mcabs 6, 10, 12, and 13 differentiated between IBV strains. Conservation of epitopes is not unexpected in view of the conserved amino acid sequences of IBV strains (Niesters et al., 1986 ; J. G. Kusters et a/., manuscript in preparation). With the use of the dot immunoblotting assay, we were able to characterize the role of the conformation of the protein in antibody attachment. When the tertiary structure of the protein is disrupted by SDS and 2-mercaptoethanol, attachment of only Mcab 13 is abolished, indicating that Mcab 13 requires the native structure of the protein for recognition, whereas epitopes A to D are conformation independent (Fig. 5) . Epitope E was also sensitive to SDS or NP-40/2-mercaptoethanol alone (data not shown). Talbot et al. (1984) found that two out of three neutralization relevant epitopes in MHV-JHM were completely sensitive to SDS denaturation, and one was partially sensitive. In the case of TGEV all six critical determinants were Mcabs 13 (epitope E). The assay was carried out as a normal competition assay. Unlike in Fig. 3 , the data have not been normalized to 100% competition in the homologous system. Instead, the direct percentage of A,,, in the control assay has been plotted. Approximately 1 pg purified virions were applied as a dot on nitrocellulose strips and probed with an amount of monoclonal antibody giving twice maximal binding. Rabbit anti-M41 pre-and hyperimmune serum were used as negative and positive controls, respectively. The binding was visualized with a peroxidase-labeled second antiserum as described under Materials and Methods. As negative control 2 wg allantoic fluid of uninfected eggs was applied. The monoclonals and the competition groups they belong to are indicated. sensitive to denaturation by SDS and 2-mercaptoethanol (Jimenez et al., 1986 ). It is not yet possible to define the number of epitopes involved in neutralization of IBV. Both our Mcab 13 and the Mcabs described by Mockett et al. (1984) neutralize no other IBV strains than M41. In contrast, polyclonal chicken antiserum against M41 does neutralize several IBV strains, but this does not imply that more than one epitope on Sl is involved in neutralization, as suggested by Mockett et a/. (1984) . Polyclonal antisera to one epitope contain a number of antibody species which could show cross-neutralization not observed with just one Mcab species. Recently, Koch eta/. (1986) found that some Mcabs to S2 were able to neutralize IBV. However, this was only observed with high concentrations of antibody and might be a consequence of steric hindrance by the excess of antibody. Additional high-titer neutralizing antibodies aganist IBV strains are needed to answer this question. Wege et al. (1984) identified six antigenic determinants on the peplomeric protein of MHV-JHM. Two overlapping determinants were involved in neutralization. Antibodies to one of these were able to protect mice in vivo against infection. Talbot and Buchmeier (1985) identified five epitopes on MHV strain 4 of which three were involved in neutralization. One of these three determinants was conserved among the strains tested. The nonneutralizing antibodies recognized all strains except MHV strain S. This strain was recognized only by one set of nonneutralizing antibodies. Delmas er a/. (1986) showed that there are four major and three minor antigenic determinants on the TGE virus peplomeric protein, of which two are involved in neutralization. It will be of interest to map our Mcabs on the peplomerit protein. We have cloned and sequenced the protein (Niesters et a/., 1986) , so the epitopes can be mapped both by the expression of small fragments of recombinant DNA clones (Stanley and Luzio, 1984) and by the PEPSCAN method (Geysen et al., 1984) . Mapping of the epitopes recognized by Mcab 13 by these methods is probably difficult, since the results of the dot immunoblot assay indicate that several distinct parts of the protein form the neutralization epitope. In this respect mapping of the Mcabs which enhance Mcab 13 might give useful information. A direct approach to map the neutralization epitope recognized by Mcab 13 is to isolate mutants escaping neutralization. Preliminary evaluation of the haemagglutination tests for avian infectious bronchitis virus Identification of distinct antigenic determinants on Semliki Forest virus by using monoclonal antibodies with different antiviral activities Topographical analysis by monoclonal antibodies of BLV-gp51 epitopes involved in viral functions Structural polypeptides of coronavirus IBV Coronavirus IBV: Further evidence that the surface projections are associated with two glycopolypeptides Coronavirus IBV: Structural characterization of the spike protein Coronavirus IBV: Virus retaining spike glycopolypeptide S2 but not Sl is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection Immunity to avian infectious bronchitis virus. ln Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the Netherlands Antigenic structure of transmissible gastroenteritis virus. II. Domains in the peplomer glycoprotein The rapid detection of binding constants for antiviral antibodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza virus Monoclonal antibodies against influenza virus Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid Monoclonal antibodies against measles virus Antibody production by hybridomas. 1. Immunol Monoclonal antibodies to Newcastle disease virus: Delineation of four epitopes on the HN glycoprotein Antigenic determinants of influenza virus haemagglutinin. VI. Antigenic characterization of the oligosaccharide side chains from HA1 of influenza virus haemagglutinins Critical epitopes in transmissible gastroenteritis virus neutralization Antigenic differentiation of avian infeCtious bronchitis variant strains employing monoclonal antibodies Molecular epidemiology of infectious bronchitis virus in the Netherlands The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. 1. Analysis of neutralizing epitopes with monoclonal antibodies The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. 2. Monoclonal antibodies to nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes Polypeptides of infectious bronchitis virus. 1. Polypeptides of the virion The polypeptide composition of avian infectious bronchitis virus particles Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril Monoclonal antibodies to the Sl spike and membrane proteins of avian infectious bronchitis coronavirus strain Massachusetts M41 The peplomer protein sequence of the M41 strain of coronavirus IBV and its comparison with Beaudette strains Monoclonal antibodies to poliowruses. Production of specific antibodies to the Sabin vaccine strain A molecular basis for the two locus model of human complement component C4 Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevis oocytes Analysis of 17D yellow fever virus envelope protein epitopes using monoclonal antibodies Construction of a new family of high efficiency bacterial expression vectors: Identification of cDNA clones coding for human liver proteins Coronavirus proteins: Biogenesis of avlan infectious bronchitis virus virion proteins Topological mapping of murine leukemia virus proteins by competition binding assays with monoclonal antibodies Antigenic variation among murine coronaviruses: Evidence for polymorphism on the peplomer glycoprotein E2 Western and dot immunoblotting analysis of viral antigens and antibodies: Application to murine hepatitis virus Viral proteins and RNAs in BHK cells persistently infected by lymphocytic choromeningitis virus Topographical analysis of bovine herpesvirus type-l glycoproteins: Use of monoclonal antibodies to identify and characterize functional epitopes Structural proteins and glycoproteins of infectious bronchitis virus particles labelled during growth in chick embryo cells Hybridoma antibodies to the murine coronavirus JHM: Characterization of epitopes on the peplomer protein (E2) Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In "lmmunofluorescence and Related Staining Techniques This work was supported by a research grant from Duphar BV, Weesp, The Netherlands. We thank Bar-t van Geffen for setting up the dot blot assay and Dr. 1. A. Lenstra for stimulating discussions during the preparation of this manuscript.