Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus.

When influenza virus monoclonal antibody-resistant (mar) mutants are selected by incubation in vitro with excess antibody, 90 to 99% of the mutants are not detectable. This observation may be explained by encapsidation of mar mutant RNAs within phenotypically wild-type envelopes. This phenotypic hiding can be revealed by selection of mar mutants in vivo after virus uncoating. Using experimental procedures appropriate to detect all viable mar mutants in a virus population, we determined precisely the mutation rates to the mar genotype by the fluctuation test for two nonoverlapping monoclonal antibodies.     

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

Identification of mar mutations in herpes simplex virus type 1 glycoprotein B which alter antigenic structure and function in virus penetration.

Analysis of six monoclonal antibody-resistant (mar) mutants in herpes simplex virus type 1 glycoprotein B identified two type-common (II and III) and two type-specific (I and IV) antigenic sites on this molecule. To derive additional information on the location of these sites, mar mutations were mapped and nucleotide alterations were identified by DNA sequencing. Each mutant carried a single amino acid substitution resulting from a G-to-A base transition. Alterations affecting antibody neutralization were identified at residues 473, 594, 305, and 85 for mutants in sites I through IV, respectively. Two clonally distinct site II antibodies each selected mar mutants (Gly to Arg at residue 594) that exhibited a reduction in the rate of entry (roe) into host cells. A site II mar revertant that regained sensitivity to neutralization by site II antibodies also showed normal entry kinetics. DNA sequencing of this virus identified a single base reversion of the site II mar mutation, resulting in restoration of the wild-type sequence (Arg to Gly). This finding demonstrated that the mar and roe phenotypes were the result of a single mutation. To further define structures that contributed to antibody recognition, monoclonal antibodies specific for all four sites were tested for their ability to immune precipitate a panel of linker-insertion mutant glycoprotein B molecules. Individual polypeptides that contained single insertions of 2 to 28 amino acids throughout the external domain were not recognized or were recognized poorly by antibodies specific for sites II and III, whereas no insertion affected antibody recognition of sites I and IV. mar mutations affecting either site II or III were previously shown to cause temperature-sensitive defects in glycoprotein B glycosylation, and variants altered in both these sites were temperature sensitive for virus production. Taken together, the data indicate that antigenic sites II and III are composed of higher-order structures whose integrity is linked with the ability of glycoprotein B to function in virus infectivity.     

Isolation, characterization, and physical mapping of a pseudorabies virus mutant containing antigenically altered gp50.

A pseudorabies virus variant ( mar197 -1) containing a mutation in a viral glycoprotein with a molecular weight of 50,000 ( gp50 ) was isolated by selecting for resistance to a neurtralizing monoclonal antibody ( MCA50 -1) directed against gp50 . This mutant was completely resistant to neutralization with MCA50 -1 in the presence or absence of complement, and was therefore defined as a mar (monoclonal-antibody-resistant) mutant. The mutation did not affect neutralization with polyvalent immune serum. The mar197 -1 mutant synthesized and processed gp50 normally, but the mutation prevented the binding and immunoprecipitation of gp50 by MCA50 -1. Thus, the mutation was within the structural portion of the gp50 gene affecting the epitope of the monoclonal antibody. The mutation was mapped by marker rescue with cloned pseudorabies restriction enzyme fragments to the short region of the pseudorabies genome between 0.813 and 0.832 map units. This is equivalent to a 2.1-kilobase-pair region.     

Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies.

Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced by polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected-cell extracts. Seven hybridomas producing antibodies specific for gC, one for gB, and one for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, one specific for gB and one specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 X 10(-4) in nonmutagenized stocks to 1 X 10(-2) in mutagenized stocks. Four gC and four gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The two other gC antigenic variants and the four gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The two mar C mutants were tested for resistance to neutralization with a panel of seven gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least two antigenic sites on glycoprotein gC.     

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity.

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.     

Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1.

Epidemiologic and genetic evidence suggests that influenza A viruses evolve more rapidly than other viruses in humans. Although the high mutation rate of the virus is often cited as the cause of the extensive variation, direct measurement of this parameter has not been obtained in vivo. In this study, the rate of mutation in tissue culture for the nonstructural (NS) gene of influenza A virus and for the VP1 gene in poliovirus type 1 was assayed by direct sequence analysis. Each gene was repeatedly sequenced in over 100 viral clones which were descended from a single virion in one plaque generation. A total of 108 NS genes of influenza virus were sequenced, and in the 91,708 nucleotides analyzed, seven point changes were observed. A total of 105 VP1 genes of poliovirus were sequenced, and in the 95,688 nucleotides analyzed, no mutations were observed. We then calculated mutation rates of 1.5 X 10(-5) and less than 2.1 X 10(-6) mutations per nucleotide per infectious cycle for influenza virus and poliovirus, respectively. We suggest that the higher mutation rate of influenza A virus may promote the rapid evolution of this virus in nature.     

Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

Spontaneous Mutation Rate of Measles Virus: Direct Estimation Based on Mutations Conferring Monoclonal Antibody Resistance

High mutation rates typical of RNA viruses often generate a unique viral population structure consisting of a large number of genetic microvariants. In the case of viral pathogens, this can result in rapid evolution of antiviral resistance or vaccine-escape mutants. We determined a direct estimate of the mutation rate of measles virus, the next likely target for global elimination following poliovirus. In a laboratory tissue culture system, we used the fluctuation test method of estimating mutation rate, which involves screening a large number of independent populations initiated by a small number of viruses each for the presence or absence of a particular single point mutation. The mutation we focused on, which can be screened for phenotypically, confers resistance to a monoclonal antibody (MAb 80-III-B2). The entire H gene of a subset of mutants was sequenced to verify that the resistance phenotype was associated with single point mutations. The epitope conferring MAb resistance was further characterized by Western blot analysis. Based on this approach, measles virus was estimated to have a mutation rate of 9 × 10⁻⁵ per base per replication and a genomic mutation rate of 1.43 per replication. The mutation rates we estimated for measles virus are comparable to recent in vitro estimates for both poliovirus and vesicular stomatitis virus. In the field, however, measles virus shows marked genetic stability. We briefly discuss the evolutionary implications of these results.     

Characterization of subtype-specific and cross-reactive helper-T-cell clones recognizing influenza virus hemagglutinin

The specificity and function of two T-cell clones derived from A/Memphis/1/71 (H3) influenza virus (Mem 71)-immune BALB/c spleen cells have been compared. One clone, X-31 clone 1, was subtype specific, proliferating in response to influenza strains of the H3 subtype only. The other, Jap clone 3, cross-reacted in proliferation assays with heterologous subtypes of influenza A, but not type B. Both clones recognized the HA1 chain of the hemagglutinin (HA) molecule and their proliferation in response to detergent-disrupted virus could be specifically inhibited by monoclonal antibodies to the HA. The T-cell clones were of the L3T4+ phenotype. Both recognized antigen in association with I-Ed, as indicated by studies with H-2 recombinant strains of mice and by blocking with monoclonal anti-I-E antibody. In vivo, both clones elicited a delayed-type hypersensitivity (DTH) reaction when inoculated into mouse footpads together with virus, X-31 clone 1 again displaying subtype specificity and Jap clone 3 being cross-reactive. The clones were also able to provide factor-mediated help in vitro to virus-primed B cells in an anti-HA antibody response. The cross-reactive T-cell clone provided help not only for B cells primed with influenza A subtype H3 and responding to H3 virus in culture, but also for H2 virus-primed B cells making anti-H2 antibody.     

Fine-epitope mapping of an antibody that binds the ectodomain of influenza matrix protein 2

Our previous work found that the monoclonal antibody 8C6, which recognized the epitope EVETPIRN on influenza A virus M2 protein, conferred protection against influenza virus challenge. In this study, 8C6 was used to screen the 7-mer phage peptide library in order to identify the crucial amino acid residues on the protective epitope EVETPIRN. Nine positive phage clones were selected by a test of dose-dependent binding activity to 8C6 after three rounds of panning. The phage clones exhibited a consensus motif (TXXR), which was found on the epitope EVETPIRN. Site-directed mutation analysis indicated that Thr and Arg on the epitope EVETPIRN played a key role in the recognition by 8C6. Furthermore, sequence alignment and analysis revealed that Thr and Arg on the epitope were highly conserved. Our results could provide useful information for influenza vaccine design based on M2 mimotope.     

Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach.     

The neuraminidase of influenza virus

It is the enzyme neuraminidase, projecting from the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3-dimensional structure of the epitopes was obtained by X-ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.     

Antigenic variation (mar mutations) in herpes simplex virus glycoprotein B can induce temperature-dependent alterations in gB processing and virus production.

Monoclonal antibody-resistant (mar) mutants altered in the antigenic structure of glycoprotein B (gB) of herpes simplex virus type 1, strain KOS-321, were selected by neutralization with each of six independently derived gB-specific monoclonal antibodies. Analysis of the reactivity patterns of these mar mutants with a panel of 16 virus-neutralizing monoclonal antibodies identified at least five nonoverlapping epitopes on this antigen, designated groups I through V. Multiple mar mutations were also introduced into the gB structural gene by recombination and sequential antibody selection to produce a set of mar mutants with double, triple, and quadruple epitope alterations. Group II (B2) and group III (B4) antibodies were used to select the corresponding mutants, mar B2.1 and mar B4.1, which in addition to carrying the mar phenotype were temperature sensitive (ts) for processing of the major partially glycosylated precursor of gB, pgB (Mr = 107,000), to mature gB (Mr = 126,000) and showed reduced levels of gB on the cell surface at high temperature (39 degrees C). These mutants were not, however, ts for production of infectious progeny. A recombinant virus, mar B2/4.1, carrying both of these alterations was ts for virus production and failed to produce and transport any detectable mature gB to the cell surface at 39 degrees C. Rather, pgB accumulated in the infected cell. Revertants of the ts phenotype, isolated from virus plaques at 39 degrees C, regained the B2 but not the B4 epitope and were phenotypically indistinguishable from the mar B4.1 parent. Finally, it was shown that group II (B5) and group III (B4) antibodies failed to immunoprecipitate pgB (39 degrees C) produced by ts gB mutants of herpes simplex virus type 1 which were not selected with monoclonal antibodies. Taken together, our findings indicate that (i) mar mutations can alter antigenic as well as other functional domains of gB, namely, the domain(s) involved in processing and infectivity, and (ii) group II and group III epitopes lie within an essential functional domain of gB which is a target for ts gB mutations.     

Analysis of the binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase from tern and whale using the BIAcore biosensor: effect of immobilization level and flow rate on kinetic analysis.

The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 microl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (K(A) = 6.3 +/- 1.3 x 10(7) M(-1)) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (K(A) = 4.3 +/- 0.7 x 10(6) M(-1)). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn(200), which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.     

Epitopes of herpes simplex virus type 1 glycoprotein gC are clustered in two distinct antigenic sites.

Epitopes of herpes simplex virus type 1 (HSV-1) strain KOS glycoprotein gC were identified by using a panel of gC-specific, virus-neutralizing monoclonal antibodies and a series of antigenic variants selected for resistance to neutralization with individual members of the antibody panel. Variants that were resistant to neutralization and expressed an antigenically altered form of gC were designated monoclonal antibody-resistant (mar) mutants. mar mutants were isolated at frequencies of 10(-3) to 10(-5), depending on the antibody used for selection. The epitopes on gC were operationally grouped into antigenic sites by evaluating the patterns of neutralization observed when a panel of 22 antibodies was tested against 22 mar mutants. A minimum of nine epitopes was identified by this process. Three epitopes were assigned to one antigenic site (I), and six were clustered in a second complex site (II) composed of three distinct subsites, IIa, IIb, and IIc. The two antigenic sites were shown to reside in physically distinct domains of the glycoprotein, by radioimmunoprecipitation of truncated forms of gC. These polypeptides lacked portions of the carboxy terminus and ranged in size from approximately one-half that of the wild-type molecule to nearly full size. Antibodies recognizing epitopes in site II immunoprecipitated the entire series of truncated polypeptides and thereby demonstrated that site II resided in the N-terminal half of gC. Antibodies reactive with site I, however, did not immunoprecipitate fragments smaller than at least two-thirds the size of the wild-type polypeptide, suggesting that site I was located in the C-terminal portion. Sites I and II were also shown to be spatially separate on the gC polypeptide by competition enzyme-linked immunosorbent assay with monoclonal antibodies representative of different site I and site II epitopes.     

Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions.

The influenza A virus M2 protein is an integral membrane protein of 97 amino acids that is expressed at the surface of infected cells with an extracellular N-terminal domain of 18 to 23 amino acid residues, an internal hydrophobic domain of approximately 19 residues, and a C-terminal cytoplasmic domain of 54 residues. To gain an understanding of the M2 protein function in the influenza virus replicative pathway, we produced and characterized a monoclonal antibody to M2. The antibody-binding site was located to the extracellular N terminus of M2 as shown by the loss of recognition after proteolysis at the infected-cell surface, which removes 18 N-terminal residues, and by the finding that the antibody recognizes M2 in cell surface fluorescence. The epitope was further defined to involve residues 11 and 14 by comparing the predicted amino acid sequences of M2 from several avian and human strains and the ability of the M2 protein to be recognized by the antibody. The M2-specific monoclonal antibody was used in a sensitive immunoblot assay to show that M2 protein could be detected in virion preparations. Quantitation of the amount of M2 associated with virions by two unrelated methods indicated that in the virion preparations used there are 14 to 68 molecules of M2 per virion. The monoclonal antibody, when included in a plaque assay overlay, considerably showed the growth of some influenza virus strains. This plaque size reduction is a specific effect for the M2 antibody as determined by an analysis of recombinants with defined genome composition and by the observation that competition by an N-terminal peptide prevents the antibody restriction of virus growth.     

Crystal structure of unliganded influenza B virus hemagglutinin

Here we report the crystal structure of hemagglutinin (HA) from influenza B/Hong Kong/8/73 (B/HK) virus determined to 2.8 Å. At a sequence identity of ~25% to influenza A virus HAs, B/HK HA shares a similar overall structure and domain organization. More than two dozen amino acid substitutions on influenza B virus HAs have been identified to cause antigenicity alteration in site-specific mutants, monoclonal antibody escape mutants, or field isolates. Mapping these substitutions on the structure of B/HK HA reveals four major epitopes, the 120 loop, the 150 loop, the 160 loop, and the 190 helix, that are located close in space to form a large, continuous antigenic site. Moreover, a systematic comparison of known HA structures across the entire influenza virus family reveals evolutionarily conserved ionizable residues at all regions along the chain and subunit interfaces. These ionizable residues are likely the structural basis for the pH dependence and sensitivity to ionic strength of influenza HA and hemagglutinin-esterase fusion proteins.     

Map location of the gene for a 130,000-dalton glycoprotein of bovine herpesvirus 1.

A bovine herpesvirus 1 variant (mar6) containing a mutation in a viral glycoprotein with a molecular weight of 130,000 (g130) was isolated by selecting for resistance to a neutralizing monoclonal antibody (130-6) directed against g130. Mar6 was completely resistant to neutralization by monoclonal antibody 130-6 in the presence and absence of complement, but was neutralized by polyvalent immune sera. The mar6 mutant synthesized and processed g130, but produced plaques which failed to react with monoclonal antibody 130-6 in an in situ immunoassay (black plaque). However, monoclonal antibody 130-6 was capable of binding and immunoprecipitating g130 from infected-cell extracts produced by lysis of mar6-infected cells with nonionic detergents. The mutation in mar6 was mapped by marker rescue with cloned bovine herpesvirus 1 restriction enzyme fragments to a 3.8-kilobase fragment at approximate map units 0.405 to 0.432. In addition, it was found that a DNA probe containing the glycoprotein B gene of herpes simplex type 1 hybridized uniquely to the same 3.8-kilobase fragment which was shown by marker rescue to contain the mutation site in the gene for bovine herpesvirus 1 g130.