Discovery of the youngest sex chromosomes reveals first case of convergent co-option of ancestral autosomes in turtles.

Most turtle species possess temperature-dependent sex determination (TSD), but genotypic sex determination (GSD) has evolved multiple times independently from the TSD ancestral condition. GSD in animals typically involves sex chromosomes, yet the sex chromosome system of only 9 out of 18 known GSD turtles has been characterized. Here, we combine comparative genome hybridization (CGH) and BAC clone fluorescent in situ hybridization (BAC FISH) to identify a macro-chromosome XX/XY system in the GSD wood turtle Glyptemys insculpta (GIN), the youngest known sex chromosomes in chelonians (8-20 My old). Comparative analyses show that GIN-X/Y is homologous to chromosome 4 of Chrysemys picta (CPI) painted turtles, chromosome 5 of Gallus gallus chicken, and thus to the X/Y sex chromosomes of Siebenrockiella crassicollis black marsh turtles. We tentatively assign the gene content of the mapped BACs from CPI chromosome 4 (CPI-4) to GIN-X/Y. Chromosomal rearrangements were detected in G. insculpta sex chromosome pair that co-localize with the male-specific region of GIN-Y and encompass a gene involved in sexual development (Wt1-a putative master gene in TSD turtles). Such inversions may have mediated the divergence of G. insculpta sex chromosome pair and facilitated GSD evolution in this turtle. Our results illuminate the structure, origin, and evolution of sex chromosomes in G. insculpta and reveal the first case of convergent co-option of an autosomal pair as sex chromosomes within chelonians.

A review of zoonotic disease surveillance supported by the Armed Forces Health Surveillance Center.

The Armed Forces Health Surveillance Center (AFHSC), Division of Global Emerging Infections Surveillance and Response System conducts disease surveillance through a global network of US Department of Defense research laboratories and partnerships with foreign ministries of agriculture, health and livestock development in over 90 countries worldwide. In 2010, AFHSC supported zoonosis survey efforts were organized into four main categories: (i) development of field assays for animal disease surveillance during deployments and in resource limited environments, (ii) determining zoonotic disease prevalence in high-contact species which may serve as important reservoirs of diseases and sources of transmission, (iii) surveillance in high-risk human populations which are more likely to become exposed and subsequently infected with zoonotic pathogens and (iv) surveillance at the human-animal interface examining zoonotic disease prevalence and transmission within and between human and animal populations. These efforts have aided in the detection, identification and quantification of the burden of zoonotic diseases such as anthrax, brucellosis, Crimean Congo haemorrhagic fever, dengue fever, Hantaan virus, influenza, Lassa fever, leptospirosis, melioidosis, Q fever, Rift Valley fever, sandfly fever Sicilian virus, sandfly fever Naples virus, tuberculosis and West Nile virus, which are of military and public health importance. Future zoonotic surveillance efforts will seek to develop local capacity for zoonotic surveillance focusing on high risk populations at the human-animal interface.

Examination of a miniaturized funnel trap for Aedes aegypti (Diptera: Culicidae) larval sampling.

Funnel traps are often used to sample for the presence of Aedes aegypti (L.) (Diptera: Culicidae) larvae in subterranean aquatic habitats. These traps are generally > or = 15 cm in diameter, making them impractical for use in subterranean sites that have narrow (10-cm) access ports, such as those in standard-sized septic tanks. Recent research indicates septic tanks may be important habitats for Ae. aegypti in Puerto Rico and the Caribbean. To sample mosquito larval populations in these sites, a miniaturized funnel trap was necessary. This project describes the use of a smaller funnel trap for sampling larval populations. The effects of larval instar (third and fourth) and population density on trap efficacy also are examined. The trap detected larval presence 83% of the time at a larval density of 0.011 larvae per cm(2) and 100% of the time at densities > or = 0.022 larvae per cm(2). There was a significant trend of increasing percentage of recaptured larvae with higher larval population densities. Although the miniaturized funnel trap is less sensitive at detecting larval presence in low population densities, it may be useful for sampling aquatic environments with restricted access or shallow water, particularly in domestic septic tanks.

Induction of herpes simplex virus gB-specific cytotoxic T lymphocytes in TAP1-deficient mice by genetic immunization but not HSV infection.

Loading of most endogenous peptides on major histocompatibility complex class I molecules is conditional on their transport into the endoplasmic reticulum (ER) by the peptide transporter TAP. We describe an HSV-2/1 cross-reactive cytotoxic T-cell (CTL) epitope that is processed in a TAP1-independent manner in vivo following immunization of TAP1-/- mice with naked DNA or a recombinant vaccinia virus. These data indicated that TAP1-independent processing of endogenous proteins is sufficient to prime CTLs in vivo. TAP1-independent processing of this epitope was not due to ER targeting by signal sequences and exogenous loading of MHC-I molecules and was not influenced by the amino acids flanking this epitope. In contrast, TAP1-/- mice infected with HSV-2 or HSV-2 mutants did not mount a CTL response against this epitope.

Antibodies to the major linear neutralizing domains of cytomegalovirus glycoprotein B among natural seropositives and CMV subunit vaccine recipients.

The gB protein (gpUL55) of human cytomegalovirus (CMV) contains C-terminal (AD-1) and N-terminal (AD-2) linear immunodominant neutralizing domains. To measure antibodies to these epitopes, a modified protein (delta-gB) lacking heavily glycosylated intervening regions, the transmembrane domain, and the cytoplasmic domain, was expressed in recombinant baculovirus-infected cells. Eighty-six percent of 600 naturally CMV-seropositive individuals and 93% of 121 gB vaccine recipients had antibodies to delta-gB as detected by enzyme-linked immunosorbent assay (ELISA). The antibody level in vaccinees (median optical density [OD] = 1.73) exceeded that in natural seropositives (median OD = 0.94; p < .0001). Eleven percent of 95 natural seropositives and 7% of 120 gB vaccinees lacked A-gB antibodies but had neutralizing activity. Among subjects with delta-gB antibody, there were weak correlations between antibody level and neutralizing titer. These data suggest that antibodies to linear neutralizing gB domains are highly prevalent in naturally-infected individuals and regularly develop in gB vaccinees. However, for some individuals, discontinuous and/or linear epitopes not represented on delta-gB may be more important in the generation of neutralizing responses.

Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Limited antibody-dependent cellular cytotoxicity antibody response induced by a herpes simplex virus type 2 subunit vaccine.

The humoral response to a herpes simplex virus (HSV) type 2 subunit vaccine containing recombinant glycoproteins B (gB2) and D (gD2) was tested in 3 groups of patients. These included HSV-seronegative, HSV-1-seropositive, and HSV-2-seropositive individuals. There were excellent antibody responses, as measured by gB2- and gD2-specific ELISAs and HSV-2 neutralization assays. However, in 2 HSV-2 antibody-dependent cellular cytotoxicity (ADCC) assays, there were relatively low antibody responses, especially among HSV-seronegative individuals. The low ADCC responses may be associated with the poor efficacy of this vaccine observed in clinical trials.

Prevalence and incidence of herpes simplex virus type 2 infection among male Zimbabwean factory workers.

Stored sera from a cohort of 2397 male factory workers in Harare, Zimbabwe, were screened for herpes simplex virus type 2 (HSV-2)-specific antibodies, to estimate the prevalence and incidence of genital herpes infection and to assess the relation between HSV-2 and human immunodeficiency virus (HIV) acquisition. The prevalence of HSV-2 at enrollment was 39.8%. Correlates of HSV-2 seropositivity were HIV seropositivity, marital status, history of sexually transmitted disease (STD), older age, and higher income. The incidence of HSV-2 seroconversion during follow-up was 6.2/100 person-years. Correlates of HSV-2 seroconversion were enrollment while HIV-positive or seroconversion during follow-up, reported genital ulcer, history of STD, and number of sex partners. No evidence was found that HSV-2 infection was more likely to precede HIV or vice versa. HSV-2 and HIV seropositivity are strong markers for high-risk sexual behavior. Improved interventions targeted to populations in which the incidence of either viral infection is high are needed.

Effects of antigen dose and immunization regimens on antibody responses to a cytomegalovirus glycoprotein B subunit vaccine.

The purpose of this phase I study was to evaluate the safety and immunogenicity of 2 doses of cytomegalovirus glycoprotein B (CMV gB)/MF59 vaccine at 3 different immunization schedules. Ninety-five volunteers were randomized to 6 groups. Antibodies to gB represent the majority of the CMV-specific neutralizing response. Three groups received 5 microgram of gB antigen combined with MF59 (a proprietary adjuvant) and 3 groups received a 30-microgram dose at 0, 1, and 2 months; 0, 1, and 4 months; or 0, 1, and 6 months. The vaccine was well tolerated, and there was no significant difference in antibody production between the 2 doses. The vaccine induced highest antibody titers when given at 0, 1, and 6 months. A low dose of CMV gB/MF59 may be the preferred dose for future studies.

A subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein B and a new adjuvant.

A phase I randomized, double-blind, placebo-controlled trial was done with a cytomegalovirus (CMV) vaccine based on the envelope glycoprotein, gB, combined with a novel adjuvant, MF59. Participants received CMV gB vaccine with MF59 or CMV gB with alum or placebo at 0, 1, and 6 months. A fourth vaccine was given at 12 months to a subgroup. Levels of neutralizing antibody and antibody to gB 2 weeks after the third dose of vaccine exceeded those in seropositive control subjects. the formulation with MF59 was more immunogenic than that with alum. The optimal dose of gB appeared to be between 5 and 30 microg. The fourth dose produced a prompt rise in antibody level. There were no serious adverse events associated with vaccine. Local and systemic reactions were generally mild and, except for pain at the injection site, occurred with similar frequency in recipients of placebo and CMV vaccine.

Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: two randomized controlled trials. Chiron HSV Vaccine Study Group.

CONTEXT: In the last 3 decades, herpes simplex virus type 2 (HSV-2) infection seroprevalence and neonatal herpes have increased substantially. An effective vaccine for the prevention of genital herpes could help control this epidemic. OBJECTIVE: To evaluate the efficacy of a vaccine for prevention of HSV-2 infection. DESIGN: Two randomized, double-blind, placebo-controlled multicenter trials of a recombinant subunit vaccine containing 30 microg each of 2 major HSV-2 surface glycoproteins (gB2 and gD2) against which neutralizing antibodies are directed, administered at months 0, 1, and 6. Control subjects were given a citrate buffer vehicle. Participants were followed up for 1 year after the third immunization. SETTING AND PARTICIPANTS: We enrolled 2393 persons from December 10, 1993, to April 4, 1995, who were HSV-2 and human immunodeficiency virus seronegative. One trial with 18 centers enrolled 531 HSV-2-seronegative partners of HSV-2-infected persons; the other, with 22 centers, enrolled 1862 persons attending sexually transmitted disease clinics. A total of 2268 (94.8%) met inclusion criteria and were included in the analysis with 1135 randomized to placebo and 2012 to vaccine. MAIN OUTCOME MEASURE: Time to acquisition of HSV-2 infection, defined by seroconversion or isolation of HSV-2 in culture during the study period by randomization group. RESULTS: Time-to-event curves indicated a 50% lower acquisition rate among vaccine vs placebo recipients during the initial 5 months of the trial; however, overall vaccine efficacy was 9% (95% confidence interval, -29% to 36%). Acquisition rates of HSV-2 were 4.6 and 4.2 per 100 patient-years in the placebo and vaccine recipients, respectively (P =.58). Follow-up of vaccine recipients acquiring HSV-2 infection showed vaccination had no significant influence on duration of clinical first genital HSV-2 episodes (vaccine, median of 7.1 days; placebo, 6.5 days; P>.10) or subsequent frequency of reactivation (median monthly recurrence rate with vaccine, 0.2; with placebo, 0.3; P>.10). The vaccine induced high levels of HSV-2-specific neutralizing antibodies in vaccinated persons who did and did not develop genital herpes. CONCLUSIONS: Efficient and sustained protection from sexual acquisition of HSV-2 infection will require more than high titers of specific neutralizing antibodies. Protection against sexually transmitted viruses involving exposure over a prolonged period will require a higher degree of vaccine efficacy than that achieved in this study.

Intranasal immunization with recombinant gD2 reduces disease severity and mortality following genital challenge with herpes simplex virus type 2 in guinea pigs.

The ability of a genetically detoxified mutant of heat labile enterotoxin (LTK63) to act as a mucosal adjuvant following intranasal immunization with recombinant gD2 has previously been reported in mice [Ugozzoli M, O'Hagan DT, Ott GS. Intranasal immunization of mice with herpes simplex virus type 2 recombinant gD2: the effect of adjuvants on mucosal and serum antibody responses. Immunol 1998;93:563-571.]. In the current studies, these observations were extended to the guinea pig model. Immunized guinea pigs were subsequently challenged intravaginally with HSV-2. Intranasal immunization with gD2 and LTK63 induced a significant reduction in disease severity and a reduction in mortality. However, only intramuscular immunization with a potent adjuvant (MF59) induced protection against the incidence of disease.

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.

Local periocular vaccination protects against eye disease more effectively than systemic vaccination following primary ocular herpes simplex virus infection in rabbits.

Vaccination of experimental animals can provide efficient protection against ocular herpes simplex virus type 1 (HSV-1) challenge. Although it is suspected that local immune responses are important in protection against ocular HSV-1 infection, no definitive studies have been done to determine if local ocular vaccination would produce more efficacious protection against HSV-1 ocular challenge than systemic vaccination. To address this question, we vaccinated groups of rabbits either systemically or periocularly with recombinant HSV-2 glycoproteins B (gB2) and D (gD2) in MF59 emulsion or with live KOS (a nonneurovirulent strain of HSV-1). Three weeks after the final vaccination, all eyes were challenged with McKrae (a virulent, eye disease-producing strain of HSV-1). Systemic vaccination with either HSV-1 KOS or gB2/gD2 in MF59 did not provide significant protection against any of the four eye disease parameters measured (conjunctivitis, iritis, epithelial keratitis, and corneal clouding). In contrast, periocular vaccination with gB2/gD2 in MF59 provided significant protection against conjunctivitis and iritis, while ocular vaccination with live HSV-1 KOS provided significant protection against all four parameters. Thus, local ocular vaccination provided better protection than systemic vaccination against eye disease following ocular HSV-1 infection. Since local vaccination should produce a stronger local immune response than systemic vaccination, these results suggest that the local ocular immune response is very important in protecting against eye disease due to primary HSV-1 infection. Thus, for clinical protection against primary HSV-1-induced corneal disease, a local ocular vaccine may prove more effective than systemic vaccination.

Cervical antibody responses to a herpes simplex virus type 2 glycoprotein subunit vaccine.

Effective vaccines against genital herpes simplex virus type 2 (HSV-2) may need to induce genital tract immune responses. To determine local antibody responses to HSV-2 glycoproteins gB2 and gD2 in an intramuscular subunit vaccine, cervical secretions from HSV-seronegative women and HSV-1-seropositive women were tested for IgG and IgA to gB2 and gD2 by enhanced chemiluminescence Western blot. Most (94%) of the seronegative subjects developed cervical IgG to gB2, IgG to gD2, and IgA to gB2; 72% developed IgA to gD2. All HSV-1-seropositive subjects had cervical IgG responses to vaccine gB2 and gD2, 85% had IgA responses to gB2, and 50% had IgA responses to gD2. Responses were more rapid and titers more consistently sustained in the HSV-1-seropositive women. Further, vaccination resulted in cervical IgG and IgA titers comparable to those to HSV-2 gB2 and gD2 in response to recurrent HSV-2 genital infection.

Limited variability of glycoprotein gene sequences and neutralizing targets in herpes simplex virus type 2 isolates and stability on passage in cell culture.

Nucleotide sequence analyses of polymerase chain reaction-amplified genes were performed to determine whether adaptation of herpes simplex virus type 2 to replication in cultured cells or in internal organs during neonatal disseminated disease results in selection of variants with altered forms of three glycoproteins (gB, gC, or gD) that influence virus entry into cells. No variations in sequence were noted as a consequence of in vitro passage or replication in different organs. Five viruses from different subjects differed with respect to gB, gC, and gD gene sequences, expressing four distinct forms of gB, three of gC, and two of gD. These differences did not confer resistance to neutralization by guinea pig or human antisera from subjects immunized with recombinant gB or gD vaccines and may not be consequential for vaccine development.

A therapeutic vaccine that reduces recurrent herpes simplex virus type 1 corneal disease.

PURPOSE: To investigate the therapeutic efficacy of periocular vaccination with herpes simplex virus (HSV) recombinant glycoprotein D from HSV-1 (gD1) or HSV-2 (gD2) in decreasing HSV-induced recurrent dendritic keratitis and HSV-induced recurrent ocular shedding in rabbits latently infected with HSV-1. METHODS: Rabbits latently infected with HSV-1 were vaccinated periocularly (by subconjunctival injection) with gD1 and adjuvant, gD2 and adjuvant, or adjuvant alone. Eyes were examined daily for 49 days for recurrent herpetic keratitis and for recurrent infectious HSV-1 shedding. RESULTS: In both vaccinated groups, a significantly decreased number of eyes exhibited recurrences of herpetic keratitis compared with recurrences in adjuvant-treated control eyes (gD1 group, 27/1372, [2%]; gD2 group, 24/1274, [2%]; and control, 54/1274 [4%]; P < 0.005). Eyes in the gD1-vaccinated group (44/1308 [3.4%]; P = 0.01), but not those in the gD2-vaccinated group (71/1274 [5.6%]; P = 0.93), had significantly decreased viral shedding (positive cultures compared with total cultures) compared with eyes in the adjuvant-treated control group (69 of 1275 [5.4%]). CONCLUSIONS: Recurrent HSV-1 corneal disease was significantly reduced by therapeutic local periocular vaccination. The vaccine may be more efficacious against HSV-1-induced recurrent corneal disease than against recurrent HSV-1 ocular shedding. Its efficacy against corneal disease appeared to be longer lasting than its efficacy against recurrent spontaneous shedding. The heterotypic gD2 vaccine was as efficacious as the homotypic gD1 vaccine against recurrent corneal disease, whereas the homotypic vaccine was much more efficacious than the heterotypic vaccine against recurrent HSV-1 shedding. This is the first report in any animal model of a successful therapeutic vaccine against recurrent HSV-1-induced corneal disease. These results support the concept that development of a therapeutic vaccine for ocular HSV-1 recurrence in humans may be possible.

Association between human immunodeficiency virus and herpes simplex virus type 2 seropositivity among male factory workers in Zimbabwe.

To determine the seroprevalence of herpes simplex virus type 2 (HSV-2), to identify correlates of infection, and to describe the correlation with human immunodeficiency virus (HIV) seropositivity, 224 HIV-negative and 191 HIV-positive male factory workers in Zimbabwe were screened for HSV-2-specific antibodies. HSV-2 seroprevalence was 35.7% among HIV-negative subjects and 82.7% among HIV-positive subjects. The weighted estimate of HSV-2 seroprevalence in this population is 44.6%. The correlation between HIV and HSV-2 remained significant after controlling for multiple sex partners, paying for sex, and history of sexually transmitted disease (adjusted odds ratio, 8.0; 95% confidence interval, 4.8-13.1). If the association between HSV-2 and HIV is causal, then the high seroprevalence of HIV and HSV-2 suggests that suppressive HSV-2 treatment should be considered as a strategy to reduce HIV transmission in this population. HSV-2 seroconversion may be a suitable surrogate end point to evaluate HIV prevention interventions.

Therapeutic periocular vaccination with a subunit vaccine induces higher levels of herpes simplex virus-specific tear secretory immunoglobulin A than systemic vaccination and provides protection against recurrent spontaneous ocular shedding of virus in latently infected rabbits.

Rabbits latently infected with herpes simplex virus type 1 (HSV-1) were vaccinated either periocularly or systemically with a subunit vaccine (gB2 + gD2) plus adjuvant or adjuvant alone. Tear films were collected daily to measure recurrent infectious HSV-1 shedding. After systemic vaccination, the latently infected rabbits were not protected against recurrent ocular viral shedding (HSV-1-positive tear film cultures/total cultures) compared with either the systemic or periocular adjuvant controls (systemic vaccination = 49 of 972, 5.0%; systemic control = 46 of 972, 4.7%; periocular control = 43 of 930, 4.6%; P > 0.8). In contrast, latently infected rabbits vaccinated periocularly with the same vaccine had significantly reduced recurrent shedding (20 of 1026, 2.0%) compared with controls (P < 0.001) or systemic vaccination (P = 0.0002). Thus, recurrent HSV-1 shedding was significantly reduced by therapeutic local periocular subunit vaccination but not by therapeutic systemic subunit vaccination. Neutralizing antibody titers in the serum of systemically and ocularly vaccinated rabbits was similar. In contrast, HSV-specific tear secretory immunoglobulin A was significantly higher in the ocularly vaccinated group (P < 0.01). These results strongly suggest that in the rabbit, and presumably in humans, the local ocular (mucosal) immune response is much more important than the systemic immune response for therapeutic protection against recurrent ocular HSV-1. Thus development of a therapeutic vaccine against recurrent ocular HSV-1 should be directed at enhancing the local ocular (mucosal) immune response.

Disulfide bonds of herpes simplex virus type 2 glycoprotein gB.

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity and cell penetration. Recombinant forms of gB being used for the development of subunit vaccines are able to induce virus-neutralizing antibodies and protective efficacy in animal models. To gain structural information about the protein, we have determined the location of the disulfide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by expression in Chinese hamster ovary cells. The purified protein, which contains virtually the entire extracellular domain of herpes simplex virus type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptides were characterized by using mass spectrometry and amino acid sequence analysis. The conditions of cleavage (4 M urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three different HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule were found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys-571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to be similar in the corresponding gBs from other herpesviruses because the 10 cysteines listed above are always conserved in the corresponding protein sequences.