Evolution of antibody titres against Epstein-Barr virus and human herpesvirus 6A/B and expression of multiple sclerosis-associated retrovirus in the serum of pregnant multiple sclerosis patients.

Epstein-Barr virus (EBV), human herpesvirus 6A/B (HHV-6A/B) and multiple sclerosis (MS)-associated retrovirus (MSRV) have been described as possible MS triggers. We analysed antibody titres against EBV and HHV-6, and MSRV envelope (env) mRNA expression, in the serum of pregnant multiple sclerosis patients (P-MS) to study their possible link to the clinical activity of MS during pregnancy and postpartum and their possible role as relapse predictors. For that purpose, serum samples were collected from 71 pregnant women (50 pregnant MS and 21 pregnant healthy controls-P-HC) during pregnancy and postpartum. Relating to antibody titres, IgM antibody titres against HHV-6A/B were significantly higher in P-MS than in P-HC both in each pregnancy trimester and in the postpartum period. Moreover, IgM antibody titres against HHV-6A/B were higher in P-MS who suffered a relapse during the postpartum. Regarding MSRV env mRNA expression, the prevalence in the first trimester of pregnancy was significantly higher in P-MS who suffered relapses during pregnancy. Summing it up, high IgM antibody titres against HHV-6A/B and MSRV env mRNA expression during the first trimester of pregnancy could act as relapse predictors for the gestation/postpartum periods.

Cytomegalovirus Genetic Diversity Following Primary Infection.

BACKGROUND: Infection with multiple cytomegalovirus (CMV) strains (mixed infection) was reported in a variety of hosts. As the virus genetic diversity in primary CMV infection and the changes over time remain incompletely defined, we examined CMV diversity and changes in diversity over time in healthy adolescent females who participated in a phase 2 CMV gB/MF59 vaccine trial. METHODS: CMV genetic diversity was determined by genotyping of 5 genes-gB (UL55), gH (UL75), gN (UL73), US28, and UL144-in urine, saliva, and plasma samples from 15 study subjects. RESULTS: At the time of primary infection, 5 of 12 (42%) urine samples had multiple virus strains, and 50% of vaccine recipients were infected with gB1 genotype (vaccine strain). Mixed infection was documented in all 15 subjects within 3 months after primary infection, and the majority had different CMV genotypes in different compartments. Changes in genotypes over time were observed in all subjects. CONCLUSIONS: Infection with multiple CMV genotypes was common during primary infection and further diversification occurred over time. Infection with gB1 genotype in vaccine recipients suggests a lack of strain-specific protection from the vaccine. As only 5 polymorphic genes were assessed, this study likely underestimated the true genetic diversity in primary CMV infection.

Cloning, expression & evaluation of potential immunogenic recombinant capsid premembrane protein of West Nile virus.

Background & objectives: West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV. Methods: The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection. Results: The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively. Interpretation & conclusions: The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks.

Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects.

Herpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passage in vitro All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.IMPORTANCE Despite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

An ultra-sensitive capacitive microwire sensor for pathogen-specific serum antibody responses.

Detection of viral infection is commonly performed using serological techniques like the enzyme-linked immunosorbent assay (ELISA) to detect antibody responses. Such assays may also be used to determine the infection phase based on isotype prevalence. However, ELISAs demonstrate limited sensitivity and are difficult to perform at the point of care. Here, we present a novel technique for label-free, rapid detection of ultra-low concentrations of virus specific antibodies. We have developed a simple, robust capacitive biosensor using microwires coated with Zika or Chikungunya virus envelope antigen. With little discernable nonspecific binding, the sensor can detect as few as 10 antibody molecules in a small volume (10 molecules/30 µL) within minutes. It can also be used to rapidly, specifically, and accurately determine the isotype of antigen-specific antibodies. Finally, we demonstrate that anti-Zika virus antibody can be sensitively and specifically detected in dilute mouse serum and can be isotyped using the sensor. Overall, our findings suggest that our microwire sensor platform has the potential to be used as a reliable, sensitive, and inexpensive diagnostic tool to detect immune responses at the point of care.

Genetic Analysis of Serum-Derived Defective Hepatitis C Virus Genomes Revealed Novel Viral cis Elements for Virus Replication and Assembly.

Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly.IMPORTANCE HCV DVGs have been identified in vivo and in vitro, but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We identified a highly conserved secondary structure (SL750) within the core-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed previously unexpected novel cis elements critical for HCV replication and morphogenesis.

Serologic Evidence of Fruit Bat Exposure to Filoviruses, Singapore, 2011-2016.

To determine whether fruit bats in Singapore have been exposed to filoviruses, we screened 409 serum samples from bats of 3 species by using a multiplex assay that detects antibodies against filoviruses. Positive samples reacted with glycoproteins from Bundibugyo, Ebola, and Sudan viruses, indicating filovirus circulation among bats in Southeast Asia.

IFN-α-mediated Base Excision Repair Pathway Correlates with Antiviral Response Against Hepatitis B Virus Infection.

Previous studies identified APOBEC deaminases as enzymes targeting hepatitis B virus (HBV) DNA in the nucleus thus affecting its persistence. Interferon (IFN)-α treated chimpanzees and hepatitis C patients showed elevated APOBEC expression. We thus hypothesized that the responses to IFN-α treatment of chronic hepatitis B (CHB) patients is influenced by IFN-induced base excision repair (BER). CHB-treatment naïve patients, patients treated with PEGylated IFN-α, and patients with sequential treatment of Entecavior and PEGylated IFN-α were recruited. Blood and liver biopsy samples were collected before treatment and at treatment endpoint. BER genes were assessed by quantitative RT-PCR. BER gene expression levels and IFN treatment responses were correlated in patient liver biopsies. APOBEC3A, -B, -C, -D/E, and-G mRNA levels were up-regulated in IFN-treated patients. APOBEC3A expression was significantly higher in IFN-responders than in non-responders. BER genes NEIL3 was down-regulated in IFN-treated patients. APOBEC3 and BER gene expression at treatment endpoints partially correlated with the corresponding absolute DNA level or degree of HBsAg and HBV DNA decline. Our study suggests that the expression of APOBEC3A positively correlates with IFN-treatment responses in CHB patients, while NEIL3 shows negative correlation. These genes may involve to IFN mediated viral suppression and serve as biomarkers for CHB disease management.

Emergence of West Nile virus in West Bengal, India: a new report.

Background: The ICMR virus unit in Kolkata functions as an Appex Referral Laboratory for the detection of dengue (DENV) and chikungunya (CHIKV) infections in the eastern part of India. In spite of efforts for confirmatory diagnosis, some samples remain undiagnosed every year. West Nile virus (WNV) infection may mimic either dengue (flavivirus) or chikungunya (alphavirus) like illness. WNV is endemic in the tropical region where its principal/potential vectors are Aedes and Culex. Methods: We explored the existence of WNV within undiagnosed samples to identify the emergence of a new public health problem. Results: Of 1278 sera samples, 574 were negative for DENV and CHIKV either by ELISA or by reverse transcriptase (RT)-PCR. Of these 574 negative samples, 83 (14.5%) and 141 (24.56%) were positive for WNV by ELISA and RT-PCR, respectively; no samples were positive for WNV by both methods. After assembling raw sequencing data, partial envelope genome sequence of West Bengal isolates, WNV was compared through BLAST with other WNV Indian strains and 98% homology detected. Phylogenetic analysis of one West Bengal isolates (Accession No. KY421790) and 28 Indian isolates available in GenBank, indicated close clustering. Conclusions: The serological and molecular approaches have clearly established the emergence of WNV in West Bengal. Hence, for proper case management, detection of WNV in common febrile illness is strongly recommended.

Genotypes of hepatitis B virus identified in patients tested prior to endoscopy from a Teaching Hospital in the Central Province of Sri Lanka.

The present study was carried out to identify the hepatitis B virus (HBV) genotypes in six patients attending the surgical clinic who were positive for hepatitis B surface antigen. DNA was extracted from the serum of patients and subjected to a modified nested PCR to detect a 585 bp region within the S gene of the HBV genome. Positive PCR products were purified and sequenced via a cycle sequencing method. The sequence data were analyzed with reference sequences in the HepSEQ database to identify the particular HBV genotype of the samples. Nested PCR for the S gene of the HBV genome was positive in 2 out of 6 samples. The genotyping and sequence analysis of the PCR products showed HBV genotype A with a homology of 98% to the reference sequences in the HepSeq database.

Envelope specific T cell responses & cytokine profiles in chikungunya patients hospitalized with different clinical presentations.

BACKGROUND & OBJECTIVES: Since the 2006 massive outbreaks, chikungunya (CHIK) is a major public health concern in India. The aim of this study was to assess envelope specific immune responses in patients with chikungunya infection. METHODS: This study included 46 hospitalized patients with chikungunya virus infection (encephalitis, n=22, other systemic involvement, OSI, n=12, classical, n=12) and six controls from Ahmedabad city, Gujarat, India. T cell responses and the levels of Th1, pro/ anti-inflammatory cytokines against the CHIK virus envelope antigens were assessed by lymphocyte proliferation assay and by cytometric bead array in flow cytometry, respectively. RESULTS: Lymphoproliferative response was uniform among the patients. Comparisons of cytokines revealed significantly higher levels of interleukin (IL)-4 and IL-5 in encephalitis, OSI and classical patients versus controls. The levels of tumour necrosis factor (TNF)-α were higher in classical patients categories compared to the controls. Interferon (IFN)-γ levels were lower in encephalitis patients versus control. INTERPRETATION & CONCLUSIONS: Our findings showed recognition of T cell epitopes on the envelope region of chikungunya virus by all patient categories. Lower level of IFN-γ may be associated with the severity of disease in these patients.

Perinatal transmission of hepatitis C antigens: envelope 1, envelope 2 and non-structural 4.

BACKGROUND: Perinatal exposure to hepatitis C virus (HCV) antigens during pregnancy may affect the developing immune system in the fetus. We aimed to study the perinatal transmission of HCV structural and non-structural antigens. METHODS: Sera from 402 pregnant mothers were tested for anti-HCV antibody and HCV RNA. HCV antigens were determined in sera from 101 HCV-infected mothers and their cord blood. RESULTS: In both serum and cord blood samples, HCV NS4 (non-structural 4) at 27 kDa, E1 (envelope 1) at 38 kDa and E2 (envelope 2) at 40 kDa were identified, purified and quantified using western blotting, electroelution and ELISA. Maternal sera and neonate cord blood samples had similar detection rates for NS4 (94.1%), E1 (90.1%) and E2 (90.1%). The mean maternal serum levels (optical density, OD) of HCV NS4 (0.87 ± 0.01), E1 (0.86 ± 0.01) and E2 (0.85 ± 0.01) did not differ significantly (p > 0.05) from those of neonatal cord blood (0.83 ± 0.01, 0.87 ± 0.01 and 0.85 ± 0.01, respectively). Also, strong correlations (p < 0.0001) were shown between sera and cord blood sample levels of HCV NS4, r = 0.77; E1, r = 0.76 and E2, r = 0.80. The vertical transmission of these antigens in vaginal delivery did not differ significantly (p > 0.05) from those in caesarean section. CONCLUSIONS: These findings indicate that vertical transmission of HCV NS4, E1 and E2 antigens was very high. Thus, exposure to these antigens may influence the developing immune responses to natural infection or future vaccination.

Detection of Hepatitis B Virus Large Surface Protein Using a Time-Resolved Immunofluorometric Assay.

BACKGROUND: To establish a novel method based on time-resolved immunofluorometric assay (TR-IFMA) with higher sensitivity and a broader detection range for detecting serum hepatitis B virus large surface protein (L protein). METHODS: The precision, sensitivity, specificity, coefficient of recovery, and stability of the assay were evaluated and comparison with the classical enzyme-linked immunosorbent assay (ELISA) was also executed. RESULTS: The precision, specificity, and sensitivity of the TR-IFMA were clearly better than ELISA. Particularly, the sensitivity was 0.1 ng/ml; moreover, the specificity was 100%, 96%, 92.5%, 96.9%, 97.8%, and 100% in the sera of healthy blood donors, systemic lupus erythematosus (SLE) patients, rheumatoid arthritis (RA) patients, hepatitis C virus (HCV) patients, cytomegalovirus (CMV) infection patients, and pregnant patients, respectively. Meanwhile, we observed that the established TR-IFMA kit has a wider acceptable linear range of 0.63-10,367 ng/ml rather than the regular commercial ELISA kit having range of only 10.12-1095.9 ng/ml. Subsequently, correlation coefficient between the TR-IFMA and ELISA was 0.8009. The intra- and interassay precision rates were less than 5% for three different concentrations. The average recovery rate for L protein was 101.17%. In sum, the established assay kit performed better in terms of stability than the commercial ELISA kit. CONCLUSION: The TR-IFMA that we developed for L protein presented a higher sensitivity and wider detecting range than regular commercial ELISA. Therefore, this TR-IFMA has promising value both in the screening of HBV and monitoring of antiviral therapy.

The current hepatitis C virus prevalence in China may have resulted mainly from an officially encouraged plasma campaign in the 1990s: a coalescence inference with genetic sequences.

In this study, we investigated hepatitis C virus (HCV) molecular epidemiology and evolutionary dynamics. Both E1 and NS5B sequences were characterized in 379 of 433 patients in southern China and classified into five major subtypes: 1b in 256 patients, 6a in 67 patients, 2a in 29 patients, 3a in 14 patients, and 3b in 13 patients. Using the E1 sequences obtained, along with those from other studies using samples from China, we inferred the HCV epidemic history by means of coalescence strategies. Five Bayesian skyline plots (BSPs) were estimated for the five subtypes. They concurrently highlighted the rapid growth in the HCV-infected population size from 1993 to 2000, followed by an abrupt slowing. Although flanked on both sides by variable population sizes, the plots showed distinct patterns of rapid HCV growth. Coincidently, 1993 to 2000 was a period when contaminated blood transfusions were common in China due to a procedural error in an officially encouraged plasma campaign. The abrupt slowing in 1998 to 2000 corresponded to the central government outlawing paid blood donations in 1998. Using a parametric model, the HCV population growth rates were estimated during 1993 to 2000. It was revealed that the 6a rate was the highest, followed by those of 1b, 2a, 3b, and 3a. Because these rates differed significantly (P < 1e-9) from each other, they may help explain why 6a is increasingly prevalent in southern China and 1b is predominant nationwide. These rates are approximately 10-fold higher than those reported elsewhere. These findings suggested that during the plasma campaign, certain barriers to efficient viral transmission were removed, allowing wide HCV dissemination.

Immunocapture couples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid detection of type 1 dengue virus.

A facile method for accurate detection of type 1 dengue virus (DV1) infection from complex biological mixtures, using type specific immunocapture coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. The biological mixtures were treated with magnetic particles coated with a monoclonal antibody directly against type 1 dengue virus. After immunocapture purification, the DV1 was eluted with 30% acetic acid, directly spotted with seed-layer method, and analyzed by MALDI-TOF MS for DV1 capsid protein. The detection limit of the assay was ∼10(5)pfu/mL by MALDI-TOF MS. The immunocapture could unambiguously differentiate the DV1 from other serotypes of the dengue viruses and Japanese encephalitis virus, and could be used as a specific probe to detect DV1 from complex biological mixtures.

Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein.

Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. The immune mechanisms, which control viral multiplication and dissemination, are not yet known. Here, we studied the antibody response against the CHIKV surface antigens in infected patients. With plasma samples obtained during the early convalescent phase, we showed that the naturally-acquired IgG response is dominated by IgG3 antibodies specific mostly for a single linear epitope 'E2EP3'. E2EP3 is located at the N-terminus of the E2 glycoprotein and prominently exposed on the viral envelope. E2EP3-specific antibodies are neutralizing and their removal from the plasma reduced the CHIKV-specific antibody titer by up to 80%. Screening of E2EP3 across different patient cohorts and in non-human primates demonstrated the value of this epitope as a good serology detection marker for CHIKV infection already at an early stage. Mice vaccinated by E2EP3 peptides were protected against CHIKV with reduced viremia and joint inflammation, providing a pre-clinical basis for the design of effective vaccine against arthralgia-inducing CHIKV and other alphaviruses.

Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization.

Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non-structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV-infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs. Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (P = 0.0511 and 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (P = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for one woman, while statistical methods were consistent with PBMC compartmentalization for six women. Evidence of compartmentalization within a non-structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence.

Seroprevalence of herpes simplex virus type 1 and type 2 in Thuringia, Germany, 1999 to 2006.

The prevalence of herpes simplex virus (HSV) type-specific IgG was determined in sera taken in 1999 to 2006 from 1,100 children aged 0­18 years, 800 blood donors and 200 pregnant women in Thuringia, Germany, using tests based on the HSV glycoproteins (g) gG. By the age of 10­12 years, HSV-1 IgG prevalence reached 57.3%, rising to 69.3% by the age of 16­18 years and to 78.0% by the age of 28­30 years. Between 2.7% and 4.7% of the children aged up to 15 years had HSV-2 antibodies, increasing to 7.3% at the age of 16­18 years and to 13.6% among adults. The prevalence of HSV-1 antibodies among girls was significantly lower than among boys and a significantly higher prevalence of HSV-2 IgG in women than in men was detected. The reduced incidence of HSV-1 infections during childhood, especially in girls, has to be followed up since a higher number of primary HSV-2 infections may result. Between 2.7% and 4.7% of all children tested seemed to acquire HSV-2 by intrauterine or neonatal infection. We also compared the use of gG-1 with gC-1: the agreement of 97.2% between the two ELISAs suggests that gG-1 and gC-1 can be considered equivalent antigenic targets.

Hepatitis B virus large surface protein in serum as a candidate biomarker for evaluating hepatitis B virus infection.

AIM: To develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of serum hepatitis B virus large surface protein (HBV-LP), and study the clinical value of HBV-LP. METHODS: Serum HBV-LP levels and a panel of other HBV markers were investigated in a large population of patients with chronic HBV. The clinical value of HBV-LP was evaluated by comparing the coincidence of detection of HBV markers and the change of serum HBV-LP level during antiviral therapy. RESULTS: The ELISA was found to be sensitive and specific for the detection of HBV-LP. Serum HBV-LP level was positively correlated with HBV DNA (r=0.743) in HBV patients. Among the five HBV markers tested, HBV-LP displayed the highest coincidence rate (94.7%) with HBV DNA. CONCLUSIONS: Serum HBV-LP was strongly correlated with HBV DNA. This ELISA therefore offers a promising approach for the diagnosis and treatment monitoring of HBV patients.

Prevalence and risk factors for herpes simplex infection among patients at high risk for HIV infection in Brazil.

BACKGROUND: Herpes simplex infection is responsible for substantial morbidity in patients with HIV infection. Data from less-developed countries analyzing risk factors within this population are largely unavailable. AIMS: Investigate the incidence and seroprevalence of HSV-1 and HSV-2 infection in populations at high and low risk for HIV infection. MATERIALS AND METHODS: A prospective cohort study was performed in a population at high risk for STDs composed of 170 HIV seronegative male homosexuals and bisexuals (group A). The population at low risk for STDs was composed of 155 volunteer male blood donors (group B). All blood samples were screened using a type specific ELISA to HSV-1 and HSV-2 glycoprotein G (gG). RESULTS: The prevalence of HSV-1 and HSV-2 infection among all the 325 patients was 83.5% and 63.4%, respectively. Annual incidence of HSV-1 and 2 among group A were 0.053% and 0.08%, respectively. Among group B, the incidence for HSV-1 was 0.04% and for HSV-2 was 0.02%. Educational parameters (P<0.001), irregular use of condoms (P<0.001), and percentage of previous receptive anal intercourse (P<0,012) were significantly associated with seropositivity to HSV-2. About 8.4% of the HSV-1 seronegative subjects presented recurrence episodes of herpes labialis as well as 7.6% of the HSV-2 seronegative patients had genital herpes in the past. DISCUSSION: The high seroprevalence detected suggests that routine screening for HSV should be performed in populations at high risk for STDs, especially in HIV-infected patients. CONCLUSION: Educational campaigns, with particular focus on the transmission of HSV, and the regular use of condoms are important measures to reduce the HSV dissemination among patients with less advanced educations and at high risk for STDs.