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1.  Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses 
Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycoprotein G-2 (gG-2) is blocked by HSV-2-specific antibody. The Chiron RIBA HSV-1 and -2 strip immunoassay (SIA) utilizes HSV-1- and HSV-2-specific or cross-reactive antigens immobilized on nitrocellulose strips (HSV gB-1 and HSV gG-1 peptide bands specific for HSV-1 antibody, HSV-2 gG-2 band specific for HSV-2 antibody, and HSV gD-2 band cross-reactive for HSV-1 and HSV-2 antibodies). A total of 1,612 sera were tested by MAb-blocking EIA for HSV-2 antibody and by SIA for HSV-1 and HSV-2 antibodies. By EIA, 541 (33.6%) sera were positive for HSV-2 antibody and 1,068 sera were negative for HSV-2 antibody; 3 sera gave equivocal results. HSV-2 antibody was detected in 555 (34.4%) sera by SIA; 144 (26%) of these sera possessed only HSV-2 antibody, and 411 (74%) sera contained both HSV-1 and HSV-2 antibodies. SIA detected HSV-1 antibody in 1,155 (71.6%) sera; 744 (64%) of these sera contained HSV-1 antibody alone. Sixteen sera contained antibody against HSV but could not be typed by SIA. A total of 512 sera were positive for HSV-2 antibody by both the EIA and SIA. We concluded that the blocking EIA and SIA showed a high level of agreement in detecting HSV-2 antibody in this population. In contrast to the SIA, the blocking EIA is a useful tool for large epidemiological studies, though the SIA proved to be slightly more sensitive once sera with discrepant results were further tested.
PMCID: PMC95927  PMID: 10882665
2.  First episodes of genital herpes in a Swedish STD population: a study of epidemiology and transmission by the use of herpes simplex virus (HSV) typing and specific serology 
Sexually Transmitted Infections  2000;76(3):179-182.
Objectives: To determine the proportion of herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2) in first episodes of genital herpes. To evaluate the use of HSV specific serology for classifying first episodes of genital herpes and for defining HSV serostatus in the patients' sexual partners.
Methods: 108 consecutive patients with first episodes of genital herpes seen at three STD clinics in Sweden from 1995 to 1999 were included in the study. HSV culture and typing were performed and serum was tested for antibodies against a type common HSV antigen and a type specific HSV-2 antigen, glycoprotein G2 (gG2). A structured interview including questions about sexual behaviour and sexual partners was taken. "Steady" partners were offered a blood test for HSV serology and counselling.
Results: Of 108 patients, 11 had a negative HSV culture. Of the 97 who were HSV culture positive, 44% (43/97) were typed as HSV-1 and 56% (54/97) as HSV-2. For 86 of these 97 patients, HSV serology from the initial visit was available. Of 52 primary infections, thus initially seronegative, 64% were HSV-1 infections and of 19 female primary infections 16 (84%) were HSV-1. In 17% the first episode of genital herpes corresponded to the first clinical recurrence of an infection acquired earlier in life. There was a significant correlation between having orogenital sex and being infected with HSV-1 and also a history of labial herpes in the partner. Only 20% of partners of patients with an HSV-2 infection had a history of genital herpes.
Conclusions: Almost half of first episodes of genital herpes are caused by HSV-1. In young women with a primary genital infection, HSV-1 is much more frequent than HSV-2. Besides HSV typing, we found specific HSV serology of value for classifying first episodes and for diagnosing a subclinical HSV-2 infection in partners. Anamnestic data supported the suggestion that the orogenital route of transmission was common in genital HSV-1 infections.
Key Words: genital herpes; herpes simplex virus
doi:10.1136/sti.76.3.179
PMCID: PMC1744160  PMID: 10961194
3.  Effects of Genital Ulcer Disease and Herpes Simplex Virus Type 2 on the Efficacy of Male Circumcision for HIV Prevention: Analyses from the Rakai Trials 
PLoS Medicine  2009;6(11):e1000187.
Ron Gray and colleagues analyze data from two circumcision trials in Uganda to assess how HSV-2 status and genital ulcer disease affect the procedure's ability to reduce HIV infection.
Background
Randomized trials show that male circumcision (MC) reduces the incidence of HIV and herpes simplex virus type 2 (HSV-2) infections, and symptomatic genital ulcer disease (GUD). We assessed the role of GUD and HSV-2 in the protection against HIV afforded by MC.
Methods and Findings
HIV-uninfected men were randomized to immediate (n = 2,756) or delayed MC (n = 2,775) in two randomized trials in Rakai, Uganda. GUD symptoms, HSV-2 status, and HIV acquisition were determined at enrollment and at 6, 12, and 24 mo of follow up. Ulcer etiology was assessed by PCR. We estimated the prevalence and prevalence risk ratios (PRRs) of GUD in circumcised versus uncircumcised men and assessed the effects of HSV-2 serostatus as a risk-modifying factor for GUD. We estimated the proportion of the effect of MC on HIV acquisition that was mediated by symptomatic GUD, and by HSV-2 infection. Circumcision significantly reduced symptomatic GUD in HSV-2-seronegative men (PRR = 0.51, 95% [confidence interval] CI 0.43–0.74), HSV-2-seropositive men (PRR = 0.66, 95% CI 0.51–0.69), and in HSV-2 seroconverters (PRR = 0.48, 95% CI 0.30–0.79). The proportion of acute ulcers due to HSV-2 detected by PCR was 48.0% in circumcised men and 39.3% in uncircumcised men (χ2 p = 0.62). Circumcision reduced the risk of HIV acquisition in HSV-2 seronegative men (incidence rate ratio [IRR] = 0.34, 95% CI 0.15–0.81), and potentially in HSV-2 seroconverters (IRR = 0.56, 95% CI 0.19–1.57; not significant), but not in men with prevalent HSV-2 at enrollment (IRR = 0.89, 95% CI 0.49–1.60). The proportion of reduced HIV acquisition in circumcised men mediated by reductions in symptomatic GUD was 11.2% (95% CI 5.0–38.0), and the proportion mediated by reduced HSV-2 incidence was 8.6% (95% CI −1.2 to 77.1).
Conclusions
Circumcision reduced GUD irrespective of HSV-2 status, but this reduction played only a modest role in the protective effect of circumcision on HIV acquisition.
NIH Trial registration
ClinicalTrials.gov NCT00425984
Gates Foundation Trial registration
ClinicalTrials.gov NCT00124878
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Acquired immunodeficiency syndrome (AIDS) has killed more than 25 million people since 1981, and more than 30 million people (22 million in sub-Saharan Africa alone) are now infected with the human immunodeficiency virus (HIV), which causes AIDS. There is no cure for HIV/AIDS. Consequently, prevention of HIV transmission is extremely important. Because HIV is most often spread through unprotected sex with an infected partner, individuals can reduce their risk of becoming infected with HIV by abstaining from sex, by having one or a few partners, and by always using a male or female condom. In addition, three large trials in sub-Saharan Africa (including one in Rakai, Uganda) recently reported that male circumcision—the removal of the foreskin, a loose fold of skin that covers the head of the penis—can halve HIV transmission rates in men. Thus, as part of its HIV prevention strategy, the World Health Organization now recommends that male circumcision programs be scaled up in countries where there is a generalized HIV epidemic and where few men are circumcised.
Why Was This Study Done?
It is still not clear why male circumcision reduces HIV acquisition in men. Certainly, the foreskin contains many cells that HIV can infect and the foreskin's delicate lining is thought to be particularly vulnerable to HIV infection partly because intercourse can cause small tears in it through which HIV can enter the body. But male circumcision also reduces genital ulcer disease—sores on the penis and other genital organs caused by infection with several sexually transmitted organisms including the herpes simplex virus type 2 (HSV-2). Genital ulcer disease, particularly when caused by HSV-2, is thought to increase a person's risk of acquiring HIV, so could male circumcision reduce HIV transmission rates because of its beneficial effects on genital ulcer disease rather than through its removal of foreskin tissue with its rich source of HIV target cells? In this study, the researchers investigate this question by re-analyzing data collected in two Ugandan trials of male circumcision for HIV prevention.
What Did the Researchers Do and Find?
In the Ugandan trials, the researchers randomly assigned about 5,500 HIV-uninfected men to immediate circumcision or to circumcision 24 months later. At enrollment, they asked the men whether they had any symptoms of genital ulcer disease (for example, a painful penile sore or genital itching), examined the men's genital areas, and took blood samples to test for HSV-2 infection. The researchers repeated these examinations and tests at 6 months, 12 months, and 24 months and tested the study participants for HIV infection. The researchers' statistical analysis of these data shows that circumcision approximately halved symptomatic genital ulcer disease in the study participants irrespective of their HSV-2 infection status. Circumcision reduced the risk of HIV acquisition in men without HSV-2 infection by two-thirds but did not affect HIV acquisition among men infected with HSV-2 at enrollment. Among the men who became infected with HSV-2 during the study, circumcision reduced the risk of HIV acquisition but this reduction in risk was not statistically significant. That is, it could have happened by chance. Finally, the researchers calculated that 11.2% of the observed reduction in HIV acquisition associated with circumcision was mediated by reductions in symptomatic genital ulcer disease and 8.6% was mediated by reductions in HSV-2 infections.
What Do These Findings Mean?
The findings of this study are limited by the small number of people in some of the subgroups analyzed and by genital ulcer disease being self-reported. Furthermore, the validity of some of the findings may be compromised because the analysis described here was not specified in the original trial protocol. Nevertheless, these findings suggest that the reduction of genital ulceration following circumcision plays only a minor part in the ability of male circumcision to reduce HIV acquisition in men. They also suggest that circumcision reduces genital ulcer disease primarily by reducing the rate of nonherpetic ulceration, including sores caused by mild trauma during intercourse. Thus, the researchers conclude, most of the reduction in HIV acquisition provided by male circumcision may be attributable to the removal of vulnerable foreskin tissue containing HIV target cells.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000187.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS
Information is available from Avert, an international AIDS charity on many aspects of HIV/AIDS, including information on HIV and AIDS in Uganda, and on circumcision and HIV (in English and Spanish)
More information about male circumcision is available from the Clearinghouse on Male Circumcision
Information on the Rakai HIV prevention trial is available
The MedlinePlus Encyclopedia has a page on male genital sores (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases provides information about genital herpes
The US Centers for Disease Control and Prevention also provides information on genital herpes (in English and Spanish)
doi:10.1371/journal.pmed.1000187
PMCID: PMC2771764  PMID: 19936044
4.  Mutations in the N Termini of Herpes Simplex Virus Type 1 and 2 gDs Alter Functional Interactions with the Entry/Fusion Receptors HVEM, Nectin-2, and 3-O-Sulfated Heparan Sulfate but Not with Nectin-1 
Journal of Virology  2003;77(17):9221-9231.
Multiple cell surface molecules (herpesvirus entry mediator [HVEM], nectin-1, nectin-2, and 3-O-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for nectin-1. Each of these three substitutions in HSV-1 gD enhanced fusion with cells expressing human nectin-2 (ordinarily low for wild-type HSV-1 gD), but the same substitutions in HSV-2 gD were without effect on the already high level of cell fusion observed with the wild-type protein. The Q27P or Q27R substitution in either HSV-1 and HSV-2 gD, but not the L25P substitution, significantly reduced cell fusion and binding activity for both human and mouse HVEM. Each of the three substitutions in HSV-1 gD, as well as the deletions mentioned above, reduced fusion with cells bearing 3-O-sulfated heparan sulfate. Thus, the N terminus of HSV-1 or HSV-2 gD is not necessary for functional interactions with nectin-1 but is necessary for all of the other receptors tested here. The sequence of the N terminus determines whether nectin-2 or 3-O-sulfated heparan sulfate, as well as HVEM, can serve as entry/fusion receptors.
doi:10.1128/JVI.77.17.9221-9231.2003
PMCID: PMC187404  PMID: 12915538
5.  Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples 
Journal of Clinical Microbiology  1999;37(6):1941-1947.
We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.
PMCID: PMC84990  PMID: 10325351
6.  Left out but not forgotten: Should closer attention be paid to coinfection with herpes simplex virus type 1 and HIV? 
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are among the most common coinfections seen in individuals infected with HIV-1. Most research on HSV-HIV coinfection has focused on HSV-2, and in particular, on its impact on HIV transmission. HSV-2 is associated with micro- and macroulcerations in genital mucosal surfaces, increased numbers of HIV target cells in genital mucosal tissue and increases in plasma HIV viral load of up to 0.5 log10 copies/mL, such that HSV-2 infection increases the risk of both HIV acquisition and transmission. Because plasma HIV RNA levels are a major determinant of rates of CD4 cell decline, HSV-2 coinfection may also adversely affect the progression of HIV disease. Anti-HSV medications have in fact been associated with reciprocal decreases in HIV viral load in short-term studies. These findings have led to the development of several clinical trials of HSV-2 suppression as strategies for preventing HIV transmission and slowing the rate of HIV disease progression. HSV-1 coinfection has largely been ignored from this growing body of research, yet there are several reasons that this coinfection remains an important issue for study. First, the seroprevalence of HSV-1 is consistently higher than that of HSV-2 among both HIV-infected and HIV-uninfected populations, underscoring the relevance of HSV-1 coinfection to the majority of HIV-infected persons. Second, pre-existing HSV-1 antibodies in individuals may modulate the course of subsequently acquired HSV-2 infection; the implications of such changes on HSV-HIV coinfection remain unexplored. Third, HSV-1 and HSV-2 are closely related viruses that share 83% genetic homology. Their virological and pathobiological similarities suggest that their implications on HIV pathogenesis may be similar as well. Finally, HSV-1 is becoming increasingly relevant because the incidence of genital HSV-1 has risen. Although genital herpes is traditionally associated with HSV-2, recent studies have shown that the majority of serologically confirmed primary genital herpes in some settings is attributable to HSV-1. Because the genital tract is an important site of biological interaction between HSV and HIV, this epidemiological change may be clinically important.
PMCID: PMC2690523  PMID: 20190881
Coinfection; Genital herpes; HIV; HSV; Orolabial herpes
7.  Performance of HerpeSelect and Kalon Assays in Detection of Antibodies to Herpes Simplex Virus Type 2▿  
Journal of Clinical Microbiology  2008;46(6):1914-1918.
The performances of commercial enzyme-linked immunosorbent assays (ELISAs) in detecting herpes simplex virus type 2 (HSV-2) antibodies have been inconsistent for African and human immunodeficiency virus (HIV)-positive populations. We compared the performances of the HerpeSelect and Kalon glycoprotein G2 ELISAs for patients with genital ulcer disease in Ghana and the Central African Republic. Sera from 434 women were tested with the HerpeSelect assay, and a subsample (n = 199) was tested by the Kalon assay. Ulcer swabs and cervicovaginal lavage samples were tested for HSV-2 DNA by PCR. HSV-2-seronegative women with detectable genital HSV-2 DNA were retested for HSV-2 antibodies 14 and 28 days later by the two ELISAs. A total of 346 (80%) women were positive by HerpeSelect at baseline, and 225 (54%) had detectable genital (lesional or cervicovaginal) HSV-2 DNA. Sixty-six (19%) HerpeSelect-positive samples had low-positive index values (1.1 to 3.5), and 58% of these samples had detectable genital HSV-2 DNA. Global agreement between the two serological assays was 86%. Concordance was high (99%) for sera that were negative by HerpeSelect or had high index values (>3.5). Defining infection detected by HSV-2 DNA PCR and/or Kalon assay as true infection, 71% of sera with low-positive index values were associated with true HSV-2 infection. Twenty-five women were identified as having nonprimary first-episode genital HSV-2 infection. Rates of HSV-2 seroconversion at day 14 were 77% (10/13 patients) by HerpeSelect assay and 23% (3/13 patients) by Kalon assay, with four additional seroconversions detected by Kalon assay at day 28. HIV serostatus did not influence assay performance. Low index values obtained with the HerpeSelect assay may correspond to true HSV-2 infection, in particular to nonprimary first episodes of genital HSV-2 infection, and need to be interpreted in the context of clinical history.
doi:10.1128/JCM.02332-07
PMCID: PMC2446869  PMID: 18385443
8.  Variability of the Glycoprotein G Gene in Clinical Isolates of Herpes Simplex Virus Type 1 
Glycoprotein G (gG) of herpes simplex virus type 1 (HSV-1) has been used as a prototype antigen for HSV-1 type-specific serodiagnosis, but data on the sequence variability of the gene coding for this protein in wild-type strains are lacking. In this study, direct DNA sequencing of the gG-1 genes from PCR products was performed with clinical HSV-1 isolates from 11 subjects as well as with strains Syn 17+, F, and KOS 321. The reference strains Syn 17+ and F showed a high degree of conservation, while KOS 321 carried 13 missense mutations and, in addition, 12 silent mutations. Three clinical isolates showed mutations leading to amino acid alterations: one had a mutation of K122 to N, which is a gG-1–to–gG-2 alteration; another contained all mutations which were observed in KOS 321 except two silent mutations; and the third isolate carried five missense mutations. Two clinical isolates as well as strain KOS 321 showed a mutation (F111→V) within the epitope of a gG-1-reactive monoclonal antibody (MAb). When all viruses were tested for reactivity with the anti-gG-1 MAb, the three strains with the F111→V mutation were found to be unreactive. Furthermore, gG-1 antibodies purified from sera from the two patients carrying strains mutated in this epitope were less reactive when they were tested by an HSV-1-infected-cell assay. Therefore, our finding that the sequence variability of the gG-1 gene also affects B-cell epitope regions of this protein in clinical isolates may have consequences for the use of this protein as a type-specific antigen for serodiagnosis.
PMCID: PMC95783  PMID: 10548571
9.  Evaluation of Three Multiplex Flow Immunoassays Compared to an Enzyme Immunoassay for the Detection and Differentiation of IgG Class Antibodies to Herpes Simplex Virus Types 1 and 2▿  
The diagnosis of herpes simplex virus (HSV) infections is routinely made based on clinical findings and supported by laboratory testing using PCR or viral culture. However, in instances of subclinical or unrecognized HSV infection, serologic testing for IgG class antibodies to type-specific HSV glycoprotein G (gG) may be useful. This study evaluated and compared the performances of three multiplex flow immunoassays (AtheNA Multi-Lyte [Zeus Scientific], BioPlex 2200 [Bio-Rad Laboratories], and Plexus HerpeSelect [Focus Diagnostics]) for the simultaneous detection of gG type-specific IgG antibodies to HSV types 1 and 2 (HSV-1 and HSV-2). Serum specimens (n = 505) submitted for routine gG type-specific HSV IgG testing by enzyme immunoassay (EIA) (HerpeSelect; Focus Diagnostics) were also tested by the three multiplex flow immunoassays. Specimens showing discordant results were tested by HSV type-specific Western blotting (WB). For HSV-1 IgG, the AtheNA, BioPlex, and Plexus assays demonstrated agreements of 94.9% (479/505 specimens), 97.8% (494/505 specimens), and 97.4% (492/505 specimens), respectively, with the results of EIA. For HSV-2 IgG, the AtheNA, BioPlex, and Plexus assays showed agreements of 87.9% (444/505 specimens), 97.2% (491/505 specimens), and 96.8% (489/505 specimens), respectively, with EIA results. Timing studies showed that the AtheNA, BioPlex, and Plexus assays could provide complete analysis of 90 serum specimens in 3.1, 1.5, and 2.9 h, respectively, versus 3.1 h by EIA. These findings suggest that the gG type-specific HSV IgG multiplex immunoassays may be beneficial to high-volume clinical laboratories experiencing significant increases in the number of specimens submitted for HSV serologic testing. The evaluated systems provide comparable results to those of EIA, while reducing hands-on time and eliminating the necessity to aliquot specimens prior to testing.
doi:10.1128/CVI.00325-09
PMCID: PMC2815519  PMID: 20007366
10.  Conservation of Type-Specific B-Cell Epitopes of Glycoprotein G in Clinical Herpes Simplex Virus Type 2 Isolates 
Journal of Clinical Microbiology  2000;38(12):4517-4522.
Glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) is cleaved to a secreted amino-terminal portion and to a cell-associated, heavily O-glycosylated carboxy-terminal portion that constitutes the mature gG-2 (mgG-2). The mgG-2 protein is commonly used as a type-specific antigen in the serodiagnosis of HSV-2 infection. As the amino acid sequence variability of mgG-2 in clinical isolates may affect the performance of such assays, the gG-2 gene was sequenced from 15 clinical HSV-2 isolates. Few mutations were identified, and these were mostly localized outside the epitope regions described earlier. Five isolates were identical to different laboratory strains, indicating that the gG-2 gene is highly conserved over time. In the search for HSV-2 isolates harboring mutations within the immunodominant region of mgG-2, a pool of 2,400 clinical HSV-2 isolates was tested for reactivity with two anti-mgG-2 monoclonal antibodies (MAbs). Ten MAb escape HSV-2 mutants, which all harbored structurally restricted single- or dual-point mutations within the respective epitopes explaining the loss of binding, were identified. Sera from corresponding patients were reactive to mgG-2, as well as to a peptide representing the immunodominant region, suggesting that the point mutations detected did not diminish seroreactivity to mgG-2. The conservation of the gG-2 gene reported here further supports the use of mgG-2 as a type-specific antigen in the diagnosis of HSV-2 infections.
PMCID: PMC87630  PMID: 11101589
11.  Efficacy Results of a Trial of a Herpes Simplex Vaccine 
Background
Two previous studies of a herpes simplex virus type 2 (HSV-2) subunit vaccine containing glycoprotein D in HSV-discordant couples revealed 73% and 74% efficacy against genital disease in women who were negative for both HSV type 1 (HSV-1) and HSV-2 antibodies. Efficacy was not observed in men or HSV-1 seropositive women.
Methods
We conducted a randomized, double-blind efficacy field trial involving 8323 women 18 to 30 years of age who were negative for antibodies to HSV-1 and HSV-2. At months 0, 1, and 6, some subjects received the investigational vaccine, consisting of 20 μg of glycoprotein D from HSV-2 with alum and 3-O-deacylated monophosphoryl lipid A as an adjuvant; control subjects received the hepatitis A vaccine, at a dose of 720 enzyme-linked immunosorbent assay (ELISA) units. The primary end point was occurrence of genital herpes disease due to either HSV-1 or HSV-2 from month 2 (1 month after dose 2) through month 20.
Results
The HSV vaccine was associated with an increased risk of local reactions as compared with the control vaccine, and it elicited ELISA and neutralizing antibodies to HSV-2. Overall, the vaccine was not efficacious; vaccine efficacy was 20% (95% confidence interval [CI], −29 to 50) against genital herpes disease. However, efficacy against HSV-1 genital disease was 58% (95% CI, 12 to 80). Vaccine efficacy against HSV-1 infection (with or without disease) was 35% (95% CI, 13 to 52), but efficacy against HSV-2 infection was not observed (−8%; 95% CI, −59 to 26).
Conclusions
In a study population that was representative of the general population of HSV-1– and HSV-2–seronegative women, the investigational vaccine was effective in preventing HSV-1 genital disease and infection but not in preventing HSV-2 disease or infection. (Funded by the National Institute of Allergy and Infectious Diseases and GlaxoSmithKline; ClinicalTrials.gov number, NCT00057330.)
doi:10.1056/NEJMoa1103151
PMCID: PMC3287348  PMID: 22216840
12.  Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blot (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections. 
Journal of Clinical Microbiology  1993;31(12):3157-3164.
Western blots (immunoblots) for the detection of immunoglobulin M (IgM) antibodies specific for herpes simplex virus type 1 (HSV-1) and HSV-2 in patients' sera were developed. The locations of the type-specific glycoprotein G (gpG-2) of HSV-2 (92- and 140-kDa forms) and glycoprotein C of HSV-1 (gpC-1), which carries mostly type-specific antigenic epitopes, were checked with specific monoclonal antibodies. Western blot assays for IgM antibody to gpC-1 or gpG-2 were performed after depletion of IgG by precipitation with anti-human IgG. In patients with primary HSV-2 genital infections, seroconversion of IgM and IgG antibodies to both the 92- and 140-kDa forms of gpG-2 was observed, although both antibodies appeared in convalescent-phase serum after the first week. IgM and IgG antibodies to low-molecular-size polypeptides (40 to 65 kDa) were the first antibodies observed in patients with primary infection, but these antibodies were cross-reactive with HSV-1 and HSV-2. However, in patients with recurrent HSV-2 infections, IgG antibodies to both forms of gpG-2 and the low-molecular-size polypeptides were found no matter how early after onset the patient was bled, and IgM to gpG-2 did not appear. In patients with nonprimary initial genital HSV-2 infections, IgG antibody to HSV-1 was demonstrated in the first serum specimen, and HSV-2-specific IgM was found in 39% of the serum specimens. Hence, the Western blot assay can be used to test for IgM antibody to gpG-2, allowing for the retrospective diagnosis of inital HSV-2 infections and its use as a supplementary test to the gpG-2 IgG enzyme-linked immunosorbent assays developed elsewhere. In contrast, IgM antibody to gpG-2 is not usually detected in patients with recurrent HSV-2 infections.
Images
PMCID: PMC266368  PMID: 7508453
13.  Difference in Incidence of Spontaneous Mutations between Herpes Simplex Virus Types 1 and 2 
Spontaneous mutations within the herpes simplex virus (HSV) genome are introduced by errors during DNA replication. Indicative of the inherent mutation rate of HSV DNA replication, heterogeneous HSV populations containing both acyclovir (ACV)-resistant and ACV-sensitive viruses occur naturally in both clinical isolates and laboratory stocks. Wild-type, laboratory-adapted HSV type 1 (HSV-1) strains KOS and Cl101 reportedly accumulate spontaneous ACV-resistant mutations at a frequency of approximately six to eight mutants per 104 plaque-forming viruses (U. B. Dasgupta and W. C. Summers, Proc. Natl. Acad. Sci. USA 75:2378–2381, 1978; J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. T. Gelep, and P. A. Schaffer, Virology 132:26–37, 1984). Typically, these resistance mutations map to the thymidine kinase (TK) gene and render the virus TK deficient. To examine this process more closely, a plating efficiency assay was used to determine whether the frequencies of naturally occurring mutations in populations of the laboratory strains HSV-1 SC16, HSV-2 SB5, and HSV-2 333 grown in MRC-5 cells were similar when scored for resistance to penciclovir (PCV) and ACV. Our results indicate that (i) HSV mutants resistant to PCV and those resistant to ACV accumulate at approximately equal frequencies during replication in cell culture, (ii) the spontaneous mutation frequency for the HSV-1 strain SC16 is similar to that previously reported for HSV-1 laboratory strains KOS and Cl101, and (iii) spontaneous mutations in the laboratory HSV-2 strains examined were 9- to 16-fold more frequent than those in the HSV-1 strain SC16. These observations were confirmed and extended for a group of eight clinical isolates in which the HSV-2 mutation frequency was approximately 30 times higher than that for HSV-1 isolates. In conclusion, our results indicate that the frequencies of naturally occurring, or spontaneous, HSV mutants resistant to PCV and those resistant to ACV are similar. However, HSV-2 strains may have a greater propensity to generate drug-resistant mutants than do HSV-1 strains.
PMCID: PMC89907  PMID: 10817703
14.  Cervicovaginal Neutralizing Antibodies to Herpes Simplex Virus (HSV) in Women Seropositive for HSV Types 1 and 2 
Antibodies to herpes simplex virus type 1 (HSV-1) and HSV-2 of the immunoglobulin G (IgG) and IgA isotypes were detected in the cervicovaginal secretions (CVS) of 77 HSV-1- and HSV-2-seropositive but clinically asymptomatic African women by type-specific enhanced chemiluminescence Western blotting (ECL-WB). Of the 77 subjects, 34 were HIV negative, shedding HSV-2 DNA in their genital secretions; 20 were HIV positive, shedding HSV-2 DNA; and 23 were HIV negative, not shedding HSV-2 DNA. HSV-specific IgG was detected in CVS of nearly 70% of the women studied. HSV-specific IgA was found in CVS of 50% of the women studied. The distribution of CVS HSV-specific antibodies to each HSV type was highly heterogeneous, with a slight predominance of detectable IgG to HSV-1 (59%) over IgG to HSV-2 (41%), whereas the frequency of detectable IgA to HSV-1 (39%) was similar to that of IgA to HSV-2 (36%). The presence of detectable HSV-specific antibodies was inversely associated with HSV-2 DNA genital asymptomatic shedding but was not affected by HIV seropositivity. In addition, 13 of 77 (17%) CVS samples showed neutralizing activity against HSV-2, as assessed by an HSV-2 in vitro infectivity reduction assay. Neutralizing activity in CVS was associated with the presence of IgG and/or IgA antibodies to HSV-1 and/or to HSV-2 by ECL-WB. Among women whose CVS showed HSV-2-neutralizing activity, the specific activity of HSV-specific neutralizing antibodies was substantially (fivefold) higher in HSV-2 DNA shedders than in nonshedders. In conclusion, HSV-specific antibodies are frequently detected in CVS of asymptomatic African women seropositive for HSV-1 and HSV-2. A subset of these women had functional neutralizing activity against HSV-2 in their CVS. The origin of these antibodies and their role in HSV-2 disease of the female genital tract remain to be determined.
doi:10.1128/CDLI.10.3.388-393.2003
PMCID: PMC154966  PMID: 12738636
15.  Rapid Polymerase Chain Reaction Assay to Detect Herpes Simplex Virus in the Genital Tract of Women in Labor 
Obstetrics and gynecology  2010;115(6):1209-1216.
Objective
To develop a rapid quantitative real-time polymerase chain reaction (PCR) to detect herpes simplex virus (HSV) in the genital secretions of women that may be used in labor.
Methods
Samples of genital secretions from women in labor, swabs of active genital lesions, and swabs of buffer solution were analyzed using a newly developed rapid HSV PCR assay to detect HSV glycoprotein B gene and quantitate virion copy number. A previously validated TaqMan PCR to detect HSV glycoprotein B gene was performed as the comparator gold standard. Positivity determination that optimized sensitivity and specificity was determined with receiver operating characteristic (ROC) curves.
Results
The median time to result for rapid HSV PCR was 2 hours (range 1.5–3.5 hours). A positivity determination rule that required both wells of the rapid test to detect 150 copies or greater of HSV per ml maximized specificity (96.7%) without appreciable loss of sensitivity (99.6%). Among positive samples, the correlation between the rapid test and TaqMan for the quantity of HSV isolated was excellent (R=0.96, p<.001). The rapid test had a positive predictive value of 96.7% and a negative predictive value of 99.6% in a population with HSV shedding prevalence of 10.8%, based on the prevalence of genital HSV previously found among HSV-2 seropositive women in labor.
Conclusion
Rapid HSV PCR provides results with excellent sensitivity and specificity within a timeframe that could inform clinical decision making for identifying infants at risk of neonatal HSV infection.
doi:10.1097/AOG.0b013e3181e01415
PMCID: PMC3034453  PMID: 20502292
16.  Detection, quantification and genotyping of Herpes Simplex Virus in cervicovaginal secretions by real-time PCR: a cross sectional survey 
Virology Journal  2005;2:61.
Background
Herpes Simplex Virus (HSV) Genital Ulcer Disease (GUD) is an important public health problem, whose interaction with HIV results in mutually enhancing epidemics. Conventional methods for detecting HSV tend to be slow and insensitive. We designed a rapid PCR-based assay to quantify and type HSV in cervicovaginal lavage (CVL) fluid of subjects attending a Genito-Urinary Medicine (GUM) clinic. Vaginal swabs, CVL fluid and venous blood were collected. Quantitative detection of HSV was conducted using real time PCR with HSV specific primers and SYBR Green I. Fluorogenic TaqMan Minor Groove Binder (MGB) probes designed around a single base mismatch in the HSV DNA polymerase I gene were used to type HSV in a separate reaction. The Kalon test was used to detect anti-HSV-2 IgG antibodies in serum. Testing for HIV, other Sexually Transmitted Infections (STI) and related infections was based on standard clinical and laboratory methods.
Results
Seventy consecutive GUM clinic attendees were studied. Twenty-seven subjects (39%) had detectable HSV DNA in CVL fluid; HSV-2 alone was detected in 19 (70%) subjects, HSV-1 alone was detected in 4 (15%) subjects and both HSV types were detected in 4 (15%) subjects. Eleven out of 27 subjects (41%) with anti-HSV-2 IgG had detectable HSV-2 DNA in CVL fluid. Seven subjects (10%) were HIV-positive. Three of seven (43%) HIV-infected subjects and two of five subjects with GUD (40%) were secreting HSV-2. None of the subjects in whom HSV-1 was detected had GUD.
Conclusion
Quantitative real-time PCR and Taqman MGB probes specific for HSV-1 or -2 were used to develop an assay for quantification and typing of HSV. The majority of subjects in which HSV was detected had low levels of CVL fluid HSV, with no detectable HSV-2 antibodies and were asymptomatic.
doi:10.1186/1743-422X-2-61
PMCID: PMC1236615  PMID: 16095535
17.  Detection of type-specific antibody to herpes simplex virus type 1 by radioimmunoassay with herpes simplex virus type 1 glycoprotein C purified with monoclonal antibody. 
Infection and Immunity  1983;40(1):184-189.
Herpes simplex virus type 1 (HSV-1) and HSV-2 specify at least four glycoproteins designated gA/gB, gC, gD, and gE. Previous studies have shown that gC produced by HSV-1 is antigenically distinct from the corresponding HSV-2 glycoprotein. With the exception of gC, the glycoproteins of both serotypes share antigenic sites. Standard serological assays fail to differentiate the antibody to the shared antigenic determinants from the type-specific antibody. In this paper, we describe a procedure for purifying gC from HSV-1-infected cell extracts with an immunoadsorbent prepared with an HCL monoclonal antibody. When used in a solid-phase radioimmunoassay, gC proved to be a type-specific antigen for quantitation of antibody to HSV-1. Among individuals who had no antibody to HSV at the onset of infection, all of those with primary HSV-1 infection developed antibody to gC. Subjects with primary HSV-2 infection failed to develop antibody reactive with gC of HSV-1 (P less than 0.01). Both immunoglobulin G and M antibodies against gC were detected in sera from subjects with either primary or recurrent HSV-1 infection. Higher antibody titers to gC were found in sera from individuals with recurrent infection than in sera from those with primary HSV-1 infection.
Images
PMCID: PMC264834  PMID: 6832831
18.  Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies. 
Journal of Virology  1983;45(2):672-682.
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.
Images
PMCID: PMC256462  PMID: 6187935
19.  Dichotomy of Glycoprotein G Gene in Herpes Simplex Virus Type 1 Isolates 
Journal of Clinical Microbiology  2002;40(9):3245-3251.
Herpes simplex virus type 1 (HSV-1) encodes 11 envelope glycoproteins, of which glycoprotein G-1 (gG-1) induces a type-specific antibody response. Variability of the gG-1 gene among wild-type strains may be a factor of importance for a reliable serodiagnosis and typing of HSV-1 isolates. Here, we used a gG-1 type-specific monoclonal antibody (MAb) to screen for mutations in the immunodominant region of this protein in 108 clinical HSV-1 isolates. Of these, 42 isolates showed no reactivity to the anti-gG-1 MAb. One hundred five strains were further examined by DNA sequencing of the middle part of the gG-1 gene, encompassing 106 amino acids including the immunodominant region and epitope of the anti-gG-1 MAb. By phylogenetic comparisons based on the sequence data, we observed two (main) genetic variants of the gG-1 gene among the clinical isolates corresponding to reactivity or nonreactivity to the anti-gG-1 MAb. Furthermore, four strains appeared to be recombinants of the two gG-1 variants. In addition, one strain displayed a gG-1-negative phenotype due to a frameshift mutation, in the form of insertion of a cytosine nucleotide. When immunoglobulin G reactivity to HSV-1 in sera from patients infected with either of the two variants was investigated, no significant differences were found between the two groups, either in a type-common enzyme-linked immunosorbent assay (ELISA) or in a type-specific gG-1 antigen-based ELISA. Despite the here-documented existence of two variants of the gG-1 gene affecting the immunodominant region of the protein, other circumstances, such as early phase of infection, might be sought for explaining the seronegativity to gG-1 commonly found in a proportion of the HSV-1-infected patients.
doi:10.1128/JCM.40.9.3245-3251.2002
PMCID: PMC130675  PMID: 12202560
20.  Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations 
Background
The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1.
Methods
A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases.
Results
Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture.
Conclusions
This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.
doi:10.1186/1471-2334-2-7
PMCID: PMC113270  PMID: 12019036
21.  Seroepidemiology of Herpes Simplex Virus Type 1 and 2 in Northern Iran 
Background:
Herpes simplex virus (HSV) type 1 and 2 are common infectious agents worldwide. Data on prevalence of HSV-1 and HSV-2 infection are limited in Asia, especially in Iran. The aim of this study was to determine the seroprevalence of HSV type 1 and 2 based on age, gender, marital status, education, living area, job, symptoms and history of disease variables.
Methods:
The study population included 800 randomly selected persons from laboratories in Gilan Province, Iran, from 2010 to 2011. Demographic data gathered by a well-designed questionnaire and for serological studies, blood samples were collected and centrifuged. ELISA HSV-1, 2 and HSV-2 specific ELISA kits were used to determine IgG type specific antibodies in sera samples. Person’s chi-square test was applied to compare HSV-1 and HSV-2 seropositivities.
Results:
HSV-1 and HSV-2 IgG antibodies were positive in 467 (58.4%) and 28 (3.5%) subjects, respectively. There was significant correlation between age, marital status, job, symptoms, history of disease and HSV seroprevalence (P<0.05).
Conclusion:
Our findings were in agreement with prior studies in which HSV-1 infections was more prevalent than HSV-2 and seropositivity increased with age.
PMCID: PMC3469027  PMID: 23113228
HSV-1; HSV-2; ELISA; Prevalence; Iran
22.  Novel Approach for Specific Detection of Herpes Simplex Virus Type 1 and 2 Antibodies and Immunoglobulin G and M Antibodies 
Recently a few new herpes simplex virus (HSV) type-specific serological diagnostic tests have been introduced to the commercial market, but these tests have some limitations. Moreover, it is not yet clear which commercial test can be regarded as a “gold standard” for the serodiagnosis of HSV infections. In order to improve the clinical diagnostic value of serological tests for the detection of HSV infections, we developed novel, competition-based enzyme-linked immunosorbent assays for the specific determination of HSV type 2 antibodies (SeroHSV2) and HSV type 1 antibodies (SeroHSV1) and two complementary tests for the detection of HSV immunoglobulin M (IgM) and IgG antibodies (SeroHSV IgM and SeroHSV IgG). These four new kits were evaluated in comparison with some commercial kits for the detection of HSV antibodies that are commonly used at present in Israeli clinical laboratories. The results indicate that SeroHSV2 is highly sensitive (>92%) and highly specific (>94%). SeroHSV2 does not cross-react with other alphaherpesvirus antibodies. SeroHSV1 is highly sensitive (>94%) and specific (>91%) compared to four commercial available kits. SeroHSV IgM is highly specific (>92%) in comparison with other commercial HSV IgM tests. The sensitivity of SeroHSV IgM ranges between 50 and 70% compared to these tests. Further investigation of the discrepant results obtained by using in-house competition tests indicated that SeroHSV IgM is more sensitive. SeroHSV IgG was also found to be highly sensitive (>94%) and highly specific (>92%) compared to the other commercial HSV IgG tests.
PMCID: PMC95983  PMID: 11063496
23.  Locations of herpes simplex virus type 2 glycoprotein B epitopes recognized by human serum immunoglobulin G antibodies. 
Journal of Virology  1996;70(5):2950-2956.
Herpes simplex virus type 2 (HSV-2) glycoprotein B (gB-2) gene segments were expressed as recombinant proteins in Escherichia coli. gB-2 recombinant proteins were reacted with human serum immunoglobulin G (IgG) antibodies in Western immunoblot assays. Initially, samples were tested for the presence of HSV-1-specific antibodies and HSV-2-specific antibodies by using HSV-infected cell lysates as antigen targets in Western blot assays. Serum samples that contained HSV-2-specific IgG (n = 58), HSV-1-specific IgG (n = 33), or no detectable HSV antibodies (n = 31) were tested for reactivities with the gB-2 recombinant proteins. In 58 of 58 samples that contained HSV-2-specific IgG, antibodies were present that reacted strongly with a gB-2 amino-proximal segment between amino acids (aa) 18 and 75. Three of 33 serum samples that contained HSV-1- and not HSV-2-specific IgG (as defined by the HSV lysate Western blot assay) reacted with this segment. Both HSV-2 antibodies and HSV-1 antibodies reacted strongly with a carboxy-terminal gB-2 segment between aa 819 and 904; a second minor cross-reactive region was mapped to a gB-2 segment between aa 564 and 626. The gB-2 segment from aa 18 to 75 may constitute a useful reagent for the virus type-specific serodiagnosis of HSV-2 infections. Further studies will be required to determine the relative sensitivities and specificities of the assay for gB-2 aa 18 to 75, HSV gG assays, and HSV lysate Western blot assays for detecting virus type-specific antibody responses in acute and chronic HSV-2 infections.
PMCID: PMC190153  PMID: 8627770
24.  Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates. 
Journal of Virology  1986;60(2):470-475.
The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo.
PMCID: PMC288914  PMID: 3021981
25.  Evaluation of Three Glycoprotein G2-Based Enzyme Immunoassays for Detection of Antibodies to Herpes Simplex Virus Type 2 in Human Sera 
Journal of Clinical Microbiology  1999;37(5):1242-1246.
Three new glycoprotein G-based enzyme immunoassays (ETI-HSVK-G 2, Sorin Diagnostics Biomedica [assay A]; HSV Type 2 Specific IgG ELISA, Gull Laboratories, Inc. [assay B]; Cobas Core HSV-2 IgG EIA, Roche [assay C]) for the detection of herpes simplex virus (HSV) type 2 (HSV-2)-specific antibodies were evaluated. By testing sera from 25 individuals with culture-proven HSV-2 infection, the assays showed a sensitivity of 96%. The specificities, evaluated with sera from 70 HSV antibody-negative children, 75 HSV antibody-positive children, and 69 HSV antibody-negative adults, were 100% for assay A, 96.2% for assay B, and 97.8% for assay C, respectively. Discrepant results by any of the three assays, i.e., reactivity of a specimen in only one or two assays, occurred with similar frequencies for HSV-seronegative individuals as well as HSV-seropositive children and adults. For sera with discrepant results, the positive reactivity was mostly low. Thus, for determination of the prevalence of HSV-2 antibodies, only concordantly positive results were considered. On the basis of the results obtained with sera from 41 adults with culture-proven HSV-1 infection and from 173 HSV-antibody-positive pregnant women, the HSV-2 seroprevalence was 9.8%. The results show that the new glycoprotein G2-based enzyme immunoassays are useful tools for the detection of type-specific HSV-2 antibodies. However, if only one assay is performed, careful interpretation of the results is indicated, especially if the exhibited reactivity is low, and for determination of the definitive HSV-2 serostatus, confirmatory assays may still be necessary.
PMCID: PMC84740  PMID: 10203464

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