Limits in Reliability of Glycoprotein G-Based Type-Specific Serologic Assays for Herpes Simplex Virus Types 1 and 2

Type-specific serologic assays for herpes simplex virus (HSV) types 1 and 2 based on glycoprotein G-1 (gG-1) (HSV-1) and gG-2 (HSV-2) discriminate between antibodies against HSV-1 and HSV-2. We previously developed a Western blot assay using gG-1 and gG-2 expressed in baculovirus, performed extensive validation studies, and determined that it was both sensitive and specific for type-specific detection of HSV antibody. Here we report that, among a cohort of Thai military recruits, the serostatus of some individuals changed from positive to negative over time (6.6% among those ever positive for HSV-1, and 14.9% among those ever positive for HSV-2). We tested a subset of these specimens in three other gG-based assays: an enzyme-linked immunosorbent assay, an immunoblot strip assay, and a Western blot assay. Positive-to-negative shifts occurred in every assay; the frequency of the shifts ranged from 6.1% to 21.2% of the specimen sets tested. There was only limited agreement among the assays concerning which individuals lost reactivity. This inaccuracy, exhibited by all of the assay protocols, was not predicted by validation studies employing specimens from cross-sectional studies and was most pronounced in HSV-2 testing. This argues for the inclusion of serial blood specimens in serologic assay validation procedures.     

Comparison of four enzyme immunoassays with a western blot assay for the determination of type-specific antibodies to herpes simplex virus

Most current enzyme immunoassays (EIAs) differentiate inadequately between types 1 and 2 herpes simplex virus (HSV) antibodies since significant cross-reactivity exists. We compared 4 IgG type-specific EIAs using a Western blot assay for resolution of discrepant results. The Diamedix had sensitivities of 100% for types 1 and 2 but specificities of only 71% and 61%, respectively. The cross-reactivity rate was 82% in positive samples tested. For HSV types 1 and 2, the Zeus sensitivities were 92% and 98%, respectively; specificities were 72% and 79%, respectively; the cross-reactivity rate was 54%. For HSV types 1 and 2, the Wampole sensitivities were 98% and 95%, respectively; specificities were 68% and 85%, respectively; the cross-reactivity rate was 47%. For HSV types 1 and 2, the Meridian sensitivities were 98% and 90%, respectively; specificities were 96% and 100%, respectively; no cross-reactivity was found between positive samples tested. While the Diamedix, Zeus, and Wampole assays showed good sensitivity, they lacked type specificity. The Meridian EIA offers the highest specificity along with no observed cross-reactivity. This EIA may be an easier, reliable alternative to Western blot for the determination of HSV type-specific antibodies.     

Underdiagnosis of genital herpes by current clinical and viral-isolation procedures.

BACKGROUND. The current clinical strategy for diagnosing genital herpes simplex virus (HSV) infection in women relies on clinical findings plus the selective use of viral culture. The effectiveness of this approach for identifying women with genital herpes is unknown. METHODS. We performed physical examinations, colposcopy, Pap smears, viral cultures, and HSV type-specific serologic assays of 779 randomly selected women attending a sexually transmitted disease clinic. RESULTS. Evidence of HSV type 2 infection was detected in 363 women (47 percent), and 9 others (1 percent) had positive cultures indicative of urogenital or anal infection with HSV type 1. Of these 372 women, only 82 (22 percent) had symptoms. Fourteen women (4 percent) had viral shedding without symptoms, 60 (16 percent) had formerly had symptomatic episodes, and 216 (58 percent) had antibodies to HSV-2 with neither viral shedding nor a history of clinical episodes. Characteristic ulcerations of the external genitalia were present in only two thirds of the 66 women with positive HSV cultures; the others had atypical genital lesions or asymptomatic viral shedding. Isolation of HSV from a genitourinary tract specimen was the most sensitive (77 percent) test for confirming a first episode of infection. The detection of HSV-2-specific antibodies was the most sensitive (97 percent) way to confirm symptomatic reactivations of HSV-2 infection. HSV-2 serologic testing also identified the 290 women with asymptomatic HSV-2 infections (37 percent), including 14 (5 percent) who were shedding virus asymptomatically on the day of the examination. CONCLUSIONS. The current strategy for diagnosing genital HSV infection in women misses many cases. Newly developed type-specific serologic methods can identify women with recurrent genital HSV-2 infection, as well as those with unrecognized or subclinical infection.     

Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types 1 and 2 in human sera.

Sera from patients with culture-proven genital herpes infections were tested for herpes simplex virus type 1 (HSV-1)- and HSV-2-specific antibodies by both a Western blot (immunoblot) technique (WBA) and immunodot enzyme assays (IEAs) specific for HSV-1 or HSV-2 glycoprotein G (gG). Of 137 serum samples tested, none was mistyped by either WBA or IEA. Both tests were most sensitive with sera obtained at least 21 days after onset of primary HSV-2 infections or sera drawn during recurrent HSV-2 genital episodes: 75 of 76 (99%) such serum samples were positive for HSV-2 antibody by WBA and 73 of 76 (96%) were positive by IEA. Of sera drawn earlier than 21 days from onset of primary genital HSV-2, antibodies to HSV-2 were detected in 25% by WBA and 8% by IEA. In patients with culture-proven primary genital HSV-1 infection, WBA detected antibodies to HSV-1 proteins in 16 of 17 (94%) serum samples drawn at least 21 days after onset of primary genital HSV-1 infection, compared with 9 of 17 (53%) serum samples tested for gG-1 by IEA. Both WBA and IEA are accurate and sensitive tests for HSV-2 antibody in patients convalescing from a first episode or having symptomatic or asymptomatic recurrent genital herpes. WBA was more sensitive than IEA in detecting seroconversion following primary HSV-1 genital herpes, although both assays may miss persons undergoing early seroconversion to HSV-2.     

Performance and use of a ribonucleotide reductase herpes simplex virus type-specific serological assay

In response to the increasingly evident need for herpes simplex virus (HSV) serotype-specific serologic assays that rely on proteins other than glycoprotein-G (gG), we developed a rapid serologic assay that is based on type-specific epitopes within the large subunit of HSV ribonucleotide reductase (R1). The assay (Au-2 enzyme-linked immunosorbent assay [ELISA]) uses an HSV type 2 (HSV-2) R1 peptide antigen. It provides a reliable method for detecting serotype-specific antibody to a protein other than gG-2. The Au-2 ELISA has high sensitivity and specificity as determined by direct comparison to Western blotting, a widely accepted "gold standard," and to ELISA with an HSV-1 R1 peptide (Au-1). The use of the Au-2 ELISA in conjunction with the gG-2-based assays will improve the sensitivity and specificity of serologic diagnosis and patient management.     

Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons

BACKGROUND: Most persons who have serologic evidence of infection with herpes simplex virus (HSV) type 2 (HSV-2) are asymptomatic. Historically, it has been assumed that these persons have less frequent viral reactivation than those with symptomatic infection. METHODS: We conducted a prospective study to investigate genital shedding of HSV among 53 subjects who had antibodies to HSV-2 but who reported having no history of genital herpes, and we compared their patterns of viral shedding with those in a similar cohort of 90 subjects with symptomatic HSV-2 infection. Genital secretions of the subjects in both groups were sampled daily and cultured for HSV for a median of 94 days. RESULTS: HSV was isolated from the genital mucosa in 38 of the 53 HSV-2-seropositive subjects (72 percent) who reported no history of genital herpes, and HSV DNA was detected by the polymerase-chain-reaction assay in cultures prepared from genital mucosal swabs in 6 additional subjects. The rate of subclinical shedding of HSV in the subjects with no reported history of genital herpes was similar to that in the subjects with such a history (3.0 percent vs. 2.7 percent). Of the 53 subjects who had no reported history of genital herpes, 33 (62 percent) subsequently reported having typical herpetic lesions; the duration of their recurrences in these subjects was shorter (median, three days vs. five days; P<0.001) and the frequency lower (median, 3.0 per year vs. 8.2 per year; P<0.001) than in the 90 subjects with previously diagnosed symptomatic infection. Only 1 of these 53 subjects had no clinical or virologic evidence of HSV infection. CONCLUSIONS: Seropositivity for HSV-2 is associated with viral shedding in the genital tract, even in subjects with no reported history of genital herpes.     

Use of densitometric analysis for interpreting HSV serologies based on Western blot

Western blot assays have been described for detecting antibodies to herpes simplex virus (HSV). A predominance of antibody binding to either the HSV-1 or the HSV-2-containing blot has been reported to indicate infection with HSV-1 or HSV-2, respectively. By densitometry, differential binding of total antibody on HSV-1 versus HSV-2 strips can be expressed as a ratio. To determine the clinical correlation of these ratios, sera from 81 patients with culture-proven oral or genital herpes were tested by Western blot (WB) and densitometry. Binding ratios accurately identified patients with primary HSV-2 infections. However, ratios on sera with HSV-1 antibody or dual antibody status showed considerable overlap. Densitometry was shown to amplify and clarify the band corresponding to the HSV-2 specific glycoprotein gG-2 and was useful, in this respect, for detecting HSV-2 antibody in the presence of HSV-1 antibody. Sera from 52 patients with asymptomatic HSV-1, HSV-2 or dual infection were also tested by WB. Typing results were confirmed by cross-adsorption of sera ("adsorption blot assay"). Ratios of HSV-2 to HSV-1 binding were higher in asymptomatic versus symptomatic patients with dual antibody (P less than 0.01). Ratios for those with HSV-1 or HSV-2 antibody types were not affected by disease expression.     

A prospective study of new infections with herpes simplex virus type 1 and type 2. Chiron HSV Vaccine Study Group.

BACKGROUND AND METHODS: Herpes simplex virus (HSV) infections are endemic, but the clinical characteristics of newly acquired HSV type 1 (HSV-1) and HSV type 2 (HSV-2) infections in adults have not been rigorously defined. We monitored 2393 sexually active HSV-2-seronegative persons for clinical and serologic evidence of new HSV infection. Of the participants, 1508 were seropositive for HSV-1 and 885 were seronegative. Charts were reviewed in a blinded manner for classification of those with genitourinary or oropharyngeal symptoms. Charts were also reviewed for all 174 persons with HSV seroconversion. RESULTS: The rates of new HSV-1 and HSV-2 infections were 1.6 and 5.1 cases per 100 person-years, respectively. Of the 155 new HSV-2 infections, 57 (37 percent) were symptomatic, 47 of which (82 percent) were correctly diagnosed at presentation. Among the 74 patients given a clinical diagnosis of genital HSV-2 during the study, 60 were given a correct diagnosis and 14 were given an incorrect diagnosis of genital herpes, for a ratio of true positive results to false positive results of 4:1. Among the 98 persons with asymptomatic HSV-2 seroconversion, 15 percent had genital lesions at some time during follow-up. Women were more likely than men to acquire HSV-2 (P<0.01) and to have symptomatic infection. Previous HSV-1 infection did not reduce the rate of HSV-2 infection, but it did increase the likelihood of asymptomatic seroconversion, as compared with symptomatic seroconversion, by a factor of 2.6 (P<0.001). Of the 19 new HSV-1 infections, 12 were symptomatic. The rates of symptomatic genital HSV-1 infection and oropharyngeal HSV-1 infection were the same (0.5 case per 100 person-years). CONCLUSIONS: Nearly 40 percent of newly acquired HSV-2 infections and nearly two thirds of new HSV-1 infections are symptomatic. Among sexually active adults, new genital HSV-1 infections are as common as new oropharyngeal HSV-1 infections.     

Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.     

Performance and Use of a Ribonucleotide Reductase Herpes Simplex Virus Type-Specific Serological Assay

In response to the increasingly evident need for herpes simplex virus (HSV) serotype-specific serologic assays that rely on proteins other than glycoprotein-G (gG), we developed a rapid serologic assay that is based on type-specific epitopes within the large subunit of HSV ribonucleotide reductase (R1). The assay (Au-2 enzyme-linked immunosorbent assay [ELISA]) uses an HSV type 2 (HSV-2) R1 peptide antigen. It provides a reliable method for detecting serotype-specific antibody to a protein other than gG-2. The Au-2 ELISA has high sensitivity and specificity as determined by direct comparison to Western blotting, a widely accepted “gold standard,” and to ELISA with an HSV-1 R1 peptide (Au-1). The use of the Au-2 ELISA in conjunction with the gG-2-based assays will improve the sensitivity and specificity of serologic diagnosis and patient management.     

Detection of herpes simplex virus type 2-specific antibody with glycoprotein G.

A recently described herpes simplex virus (HSV) type 2 (HSV-2)-specific glycoprotein (gG-2) was purified on an immunoaffinity column prepared with monoclonal antibody. This purified antigen was used in an immunodot enzymatic assay on nitrocellulose paper for the detection of HSV-2 antibodies in human serum. The test was very sensitive in that HSV-2 antibodies were detected in the convalescent sera of 132 of 134 patients with recurrent genital infections in which HSV-2 had been isolated earlier. Antibodies to gG-2 were detected in 17% of sera obtained within 10 days after the onset of a primary HSV infection and in 95% of sera obtained more than 10 days after onset. The specificity of the immunodot assay was demonstrated by testing sera from 245 HSV-seronegative adults, 344 children, 29 nuns, and 13 patients with primary genital HSV-1 infections. None of these 631 sera was reactive with the gG-2 antigen. When compared with a microneutralization test, the immunodot assay was found to be more specific in detecting HSV-2 antibodies. Reproducibility of the gG-2 assay, obtained by retesting 391 sera, was 95%. Thus, this assay has the sensitivity, specificity, and reproducibility necessary for the measurement of HSV-2 antibodies in seroepidemiological studies.     

Performance and use of HSV type-specific serology test kits

Herpes simplex virus type-specific serology tests based on glycoprotein gG-1 and/or gG-2 are important diagnostic tools to establish aetiology of genital symptoms and identify patients with unrecognized genital herpes. Clinicians can now select from three very different Food and Drug Administration-licensed kits. Diagnology's POCkittrade mark HSV-2 is a point of care test for herpes simplex virus type 2 (HSV-2) antibodies. Clinicians can perform this test while the patient waits. Focus Technologies' HerpeSelect enzymelinked immunosorbent assays (ELISAs) for herpes simplex virus type 1 (HSV-1) and HSV-2 are more traditional tests that can be semiautomated for high throughput at low cost in laboratories. Focus Technologies' HerpeSelect Immunoblot is a novel strip immunoblot that can be used for low volume testing in laboratories or even by healthcare facilities that are accredited for moderately complex testing. This review summarizes the performance data of these tests and describes how to interpret their results. Finally, situations that warrant follow-up testing are described along with suggested strategies for such testing.     

A novel glycoprotein for detection of herpes simplex virus type 1-specific antibodies

A novel herpes simplex virus type 1 (HSV-1)-specific glycoprotein reactive with monoclonal antibody H1379 was purified by affinity chromatography. This glycoprotein, provisionally designated as gG-1, forms two sets of bands with molecular weights of 40-44,000 and 60-88,000. When used in an immunodot enzymatic assay, gG-1 reacted strongly with rabbit antisera to HSV-1, but not with sera hyperimmune to HSV-2. Specificity of the assay was further established by the lack of reactivity of convalescent sera collected from 20 patients with primary genital HSV-2 infections, and from 100 sero-negative individuals. In contrast, antibodies to gG-1 were detected in 9 of 10 patients with primary HSV-1 infection, and in 63/67 patients with culture-positive, recurrent oral or genital HSV-1 infection. Reproducibility of the gG-1 immunodot assay for HSV-1 antibody detection was 96%. Serological assay with purified gG-1, done in parallel with the assay using purified gG-2 described in an earlier report, provides simple and reliable methods to detect type-specific HSV-1 and HSV-2 antibodies for seroepidemiological studies.     

Cellular immune response in genital herpes simplex virus infection.

We studied the relations between the cellular immune response, pre-existing complement-fixing antibody and virus type with duration of virus excretion in genital herpes simplex virus (HSV) infection. Thirty-six patients (seven with HSV-1 and 29 with HSV-2) with genital herpes underwent serologic testing, sequential viral cultures and weekly determination of lymphocyte-transformation stimulation index with inactivated HSV antic n. The duration of virus excretion was shortest in those with pre-existing complement-fixing antibody, was unrelated to virus type, and was inversely correlated with the magnitude of the mean peak stimulation index (r = -0.69, P less than 0.001). Prolonged virus excretion occurred in patients with a delayed and diminished peak index. Recurrent episodes had a higher peak index (29.4 compared to 14.5) (P less than 0.02), an earlier development of the peak during recurrences (9.1 vs. 25.8 days) (P less than 0.01) and a briefer duration of viral shedding than initial episodes. Thus, the temporal course and magnitude of the stimulation index correlate with and may determine the duration of genital HSV infection.     

Performance of the focus and Kalon enzyme-linked immunosorbent assays for antibodies to herpes simplex virus type 2 glycoprotein G in culture-documented cases of genital herpes

Glycoprotein G-based herpes simplex virus type 2 (HSV-2) enzyme-linked immunosorbent assays from Focus and Kalon were performed with specimens from 118 patients with culture-documented genital herpes episodes, and their results were compared. Sensitivity was 52% by Kalon and 86% by Focus for first HSV-2 episodes and 100% (for each of the two tests) for recurrent HSV-2. Median times to seroconversion were 120 days by the Kalon assay, 21 days by the Focus assay, and 68 days by Western blotting assay. Values for specificity were 100% (Kalon) and 93% (Focus).     

A comparison of PCR with virus isolation and direct antigen detection for diagnosis and typing of genital herpes

Patients attending the genitourinary medicine clinic at Watford General Hospital, UK, were examined for clinical signs of genital herpes infection. Genital swabs were taken from 194 patients (126 female, 68 male) who presented with genital ulceration or symptoms which were suggestive of genital herpes infection. Swabs from these patients were tested by three methods: (i) Detection of herpes simplex virus (HSV) antigen by direct HSV enzyme immunoassay (EIA), (ii) HSV isolation in Vero cell culture and (iii) HSV polymerase chain reaction (PCR). HSV was detected in 76 patients (39%) by EIA, in 93 (48%) by isolation in cell culture, and in 115 (59%) by PCR. Isolation by cell culture has been considered as the “gold standard” for the detection of HSV in genital lesions, but in this study HSV PCR was significantly more sensitive. Comparison of the three methods was as follows: Cell culture vs. PCR: Sensitivity 93/115 (80.9%), Specificity 79/79 (100%). HSV EIA vs. PCR: Sensitivity 75/115 (65.2%), Specificity 78/79 (98.7%). HSV EIA vs. Cell culture: Sensitivity 75/93 (80.7%), Specificity 100/101 (99%). EIA was less effective in detecting HSV among recurrent than among first episode infections, in comparison to culture or HSV PCR. This is the first comparison of HSV PCR with two other routine diagnostic methods for confirming genital herpes infection in a symptomatic population. The infecting HSV type was identified by restriction digestion of 108 HSV amplicons: HSV-1: 37/108 (34%), HSV-2: 71/108 (66%). In this population HSV-1 causes a significant proportion of genital herpes cases, and HSV-1 genital infection was detected in significantly more first episode infections (40.3%) than among recurrent infections (22.2%). J. Med. Virol. 55:177–183, 1998. © 1998 Wiley-Liss, Inc.     

Case study: type-specific HSV serology and the correct diagnosis of first-episode genital herpes during pregnancy

It is now known that the physical presentation of genital herpes simplex (HSV) infection can be misleading in making the diagnosis of genital herpes. An incorrect diagnosis can be particularly damaging in pregnancy where it may result in extended exposure of the fetus to antiviral agents, an inappropriate route and timing of delivery and a significant increase in fetal exposure to HSV during labour and delivery. Case 1 describes a 32-year-old woman at 30 weeks in her first pregnancy who had the appearance and clinical course typically ascribed to primary genital HSV infection. In contrast, Case 2, a 24-year-old woman at 34 weeks' gestation, had the physical appearance of a recurrent episode. Type-specific serological testing revealed that what Case 1 was actually experiencing was the first symptomatic reactivation of genital herpes, whereas Case 2 had a true primary genital HSV-2 infection that was accompanied by minimal symptoms. Had serology testing not been available, Case 1 would probably have delivered unnecessarily by Caesarean section, and Case 2 would have been managed as a recurrent infection and allowed to deliver vaginally with potentially disastrous results. These cases illustrate the usefulness of a type-specific serology in diagnosing genital herpes in pregnant women.     

The use of peptides from glycoproteins G-2 and D-1 for detecting herpes simplex virus type 2 and type-common antibodies.

BACKGROUND: identification and discrimination of latent herpes simplex virus (HSV) infection relies on antibody identification. The inclusion of synthetic peptides with HSV glycoproteins provides means for stable and discriminatory assays for population studies. OBJECTIVE: to determine whether virus-specific synthetic peptides might identify HSV type 2 (HSV-2) antibodies in the presence of the cross-reactive and more common HSV type 1 (HSV-1) antibodies. STUDY DESIGN: the capacity of synthetic peptides as HSV antigens was analyzed in enzyme immunoassay (EIA) using well characterized human serum cohorts. The HSV peptide assays were evaluated in comparison with two commercial HSV-2 assays. RESULTS: a combination of two C-terminal HSV-1 glycoprotein D (gD-1) peptides detected type-common HSV immunoglobulin G (IgG) with high sensitivity (95%) and specificity (93%). Peptides derived from the C-terminus of HSV-2 glycoprotein G (gG-2) had a high HSV-2 type-specificity. Inclusion of both gD-1 and gG-2 peptides gave a sensitivity for human anti-HSV-2 IgG that was similar to that of assays including different amounts of native gG-2. With western blotting as a standard, the sensitivity of the peptide assay ranged between 86% for HSV-2 seropositive persons and 61% for HSV-2 seroconverters. Addition of a small amount of native gG-2 to the peptide assay tended to increase the specificity. CONCLUSION: HSV gG and gD peptides show promise as type-specific and type-common HSV antigens. These peptides are more stable and reproducibly prepared than native or recombinant glycoproteins and may be considered for inclusion in future HSV serodiagnostic assays.     

Use of routine viral cultures at delivery to identify neonates exposed to herpes simplex virus

We obtained specimens for viral culture from mothers, infants, or both at the time of 6904 deliveries, without regard to the mothers' history of genital herpes. Herpes simplex virus (HSV) was recovered in cultured specimens from 14 of the 6904 deliveries (0.20 percent); all 14 mothers were asymptomatic. All viral isolates were herpes simplex virus type 2 (HSV-2). Only 1 of the 14 women (7 percent) had a history of genital herpes, whereas 12 (86 percent) had serologic evidence of a previous infection with HSV-2. None of the infants born to these 12 women contracted neonatal herpes. However, one of the two infants born to women with serologic evidence of a primary HSV infection at the time of delivery contracted neonatal herpes. Our findings show that most infants at risk of exposure to HSV at delivery will not be identified if concern about asymptomatic shedding of virus is limited to women with a history of genital herpes infection. Most neonatal exposure to an asymptomatic maternal HSV infection at delivery is not predictable or preventable. Therefore, physicians caring for newborns need to consider neonatal herpes in the differential diagnosis when infants become ill during the first weeks of life, regardless of the presence or absence of identifiable risk factors for HSV infection.     

Discrimination of antibody to herpes B virus from antibody to herpes simplex virus types 1 and 2 in human and macaque sera

The antigenic cross-reactive characteristics of herpes B virus and herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are responsible for false-positive diagnoses by serological assays in humans and macaques. In the present study, we developed a fluorometric indirect enzyme-linked immunosorbent assay (ELISA) with recombinant herpes B virus glycoprotein D (gD) and HSV-1 and HSV-2 gG (gG-1 and gG-2, respectively) to discriminate between the three primate herpesvirus infections. The secreted form of gD, gDdTM, was used to detect antibody to herpes B virus gD. Sera positive for herpes B virus, HSV-1, and HSV-2 showed specific reactions to gD, gG-1, and gG-2, respectively. Sera collected from humans and rhesus macaques were investigated for the presence of antibodies to the recombinant proteins of the three herpesviruses. The results suggested that the approach is able to discriminate between herpes B virus and HSV infections. The ELISA was also found to be able to detect infections with multiple primate herpesviruses and may have the potential to identify a subsequent infection in individuals that have already been infected with another herpesvirus. In addition, we found evidence of a greater cross-reactivity of herpes B virus with HSV-1 than with HSV-2. It is suggested that the ELISA with the recombinant antigens is useful not only for the serodiagnosis of primate herpesvirus infections but also for elucidation of the seroprevalence of herpesviruses in humans and primates.