An evaluation of herpes simplex virus antigenic markers in the study of established and developing cervical neoplasia

Previous reports from our laboratory have shown that antiserum to "pure" AG-e, a type-common HSV antigen, specifically stains atypical cervical cells in indirect immunofluorescence. These observations have been confirmed and extended. Antisera were prepared against the two protein components of pure AG-e, designated ICP 12 (M. W. = 140,000) and ICP 14 (M. W. = 130,000), and were purified to radiochemical homogeneity by SDS-acrylamide gel electrophoresis. The antisera reacted as well as antiserum to pure AG-e in immunofluorescence with HSV-2-(G)-infected cells, and their reactivity was adsorbed with pelleted HSV-2 (G) virions. Unlike antiserum to pure AG-e, the antisera to ICP 12 and ICP 14 were nonreactive in immunodiffusion, and only antiserum to ICP 12 showed complement fixation with soluble viral antigenic mixtures. Antisera to pure AG-e, ICP 12 and ICP 14 specifically stained exfoliated cervical cells from patients with herpetic cervicitis and atypical cells from patients with atypia, carcinoma in situ (CIS) or invasive cancer. However, both the number of patients with a positive response and the number of staining atypical cells were greater with antiserum to pure AG-e than with antisera to ICP 12 or ICP 14, suggesting that AG-e is a superior marker. Cells staining with antiserum to pure AG-e, individually identified, were classified as atypia (mild to marked), CIS or cancer. The ability of the antiserum to pure AG-e to identify atypical cervical cells was compared to cytopathologic screening in a blind study of 26 patients. A good correlation (80% to 93.8%) was observed, indicating that pure AG-e is a sensitive and specific marker for the identification of atypical cells.     

Intact microtubules support adenovirus and herpes simplex virus infections

Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-end-directed motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole (NOC) to depolymerize microtubules and measured viral gene expression at different times and multiplicities of infections. Our results indicate that in NOC-treated cells, viral transgene expression was significantly reduced at up to 48 h postinfection (p.i.). A quantitative analysis of subcellular capsid localization indicated that NOC blocked the nuclear targeting of Ad2 and also HSV-1 by more than 90% at up to 7 h p.i. About 10% of the incoming Texas Red-coupled Ad2 (Ad2-TR) was enriched at the nucleus in microtubule-depleted cells at 5 h p.i. This result is consistent with earlier observations that Ad2-TR capsids move randomly in NOC-treated cells at less than 0.1 micro m/s and over distances of less than 5 micro m, characteristic of Brownian motion. We conclude that fluorophore-tagged Ad2 and HSV-1 particles are infectious and that microtubules play a prominent role in efficient nuclear targeting during entry and gene expression of species C Ads and HSV-1.     

Detection of asymptomatic herpes simplex virus infections after vaccination.

Twenty-two volunteers seronegative for antibodies to herpes simplex virus (HSV) were enrolled in a trial to determine tolerance and immunogenicity of an HSV-2 glycoprotein subunit vaccine. Vaccine was administered at days 0, 28, and 140, and sera were obtained on days 0, 7, 14, 21, 28, 35, 49, 56, 140, 147, and 365 for determination of HSV neutralizing antibody activity and antibody-dependent cell cytotoxicity (ADCC). Sera were also tested by immunoprecipitation of radiolabeled HSV-2-infected cell proteins and polyacrylamide gel electrophoresis to identify the viral proteins which elicited antibody responses in vaccine recipients. After vaccination two male volunteers presented with atypical first-episode genital herpes: patient 1 with a culture-negative genital lesion at day 53 and patient 3 with urethritis at day 68. Seroconversion to wild-type viral proteins not present in the vaccine was detectable by radioimmunoprecipitation-polyacrylamide gel electrophoresis within 10 days in both patients. Two additional volunteers, one a sex contact of patient 1, seroconverted asymptomatically to nonvaccine proteins during the trial. All four vaccine breakthrough patients were indistinguishable from the other volunteers in the time required to develop neutralizing and ADCC antibodies, in the titer of these antibodies, and the time to seroconversion to gB and gD vaccine proteins. However, only one of the four breakthrough patients had antibodies to g80 (a complex of gC-2 and gE) after vaccination as compared with 15 of the other 18 volunteers (P = 0.05). Neither neutralizing antibody nor ADCC titers consistently identified acquisition of wild-type viral infection; therefore, protein-specific serologies were required to detect wild-type antibodies in these four patients. These data underscore the importance of using serologic assays which will distinguish naturally acquired infection from the immune response to vaccination.     

Monoclonal antibodies in the diagnosis of infections caused by the herpes simplex virus

A series of hybridomas producing monoclonal antibodies (McAb), specifically interacting with Herpes simplex virus (HSV) proteins, types 1 and 2, has been obtained. McAb 7c4 and 4f6 have been shown to be highly active in the solid-phase enzyme immunoassay (EIA) and to produce no reaction with HSV antigen in the indirect immunofluorescent assay (IIFA). McAb 2b6, 3e5, 4A, 2C effectively detect McAb in IIFA, but not EIA, while McAb 3d 10 exhibit activity in both biochemical assays. Moreover, as established in this investigation, McAb 4A are active against the protein of HSV capsid, McAb 3d10 and 2b6 detect two individual epitopes on the molecule of ribonucleohydreductase, McAb 2C are specific with respect to surface glycoprotein gB, McAb 7c4 and 416 recognize one or two overlapping epitopes of protein gD. McAb 2C are capable of completely neutralizing the infectious activity of HSV in the in vitro cell system. As determined by IIFA, McAb 4A and 4e5 stain specific inclusions in the nucleus of HSV-infected cells, while McAb 2C stain HSV protein, localized in the cytoplasm. All above-mentioned McAb are active against two common antigenic determinants of HSV 1 and HSV 2. The data obtained in this investigation suggest that the series of McAb under study may serve as the basis for the development of diagnostic test systems for the detection of HSV, types 1 and 2, by EIA and IIFA techniques.     

Detection of the markers of herpes simplex virus and cytomegalovirus in newborns and infants

A total of 111 children suspected for herpesvirus infection were examined. In blood and urine samples the infectious activity of herpes simplex virus (HSV) and cytomegalovirus (CMV) was detected by the rapid culture method (RCM) and the presence of virus DNA--by the polymerase chain reaction (PCR). HSV and/or CMV were detected by two laboratory methods in 57 examined children (51%). Of these, in 18 children (16.2%) both HSV and CMV were detected. The coincidence of the results of the detection of HSV and CMV by these two methods was observed in 72.4% and 75.2% of cases respectively. The comparative analysis of the detection of anti-CMV IgG and IgM was made with the use of commercial test systems produced bythe following manufacturers: Vector-Best and Bioservice (Russia), HUMAN and Boehringer (Germany). The effective detection of both anti-CMV (IgG and IgM) was ensured by the test systems Boehringer. The test system Vector-Best for anti-CMV IgG proved to be not inferior as regards sensitivity and specificity. The German test systems demonstrated the highest specificity in the detection of low-avid antibodies. The data obtained in this study indicate that the detection rate of HSV and CMV markers in newborns and infants suspected for herpesvirus infection was, on the average, 20 - 40%. Reliable diagnostics in newborns and infants is possible only in the presence of the combination of at least 2 serological tests (the determination of antivirus IgM and IgG avidity) and 2 methods for the detection of direct herpesvirus markers (PCR and RCM).     

Isoprinosine in the treatment of herpes virus infections in children with leukaemia and malignant lymphoma

Isoprinosine was given to 15 children with lymphoblastic diseases and 3 adults during infections with herpes viruses - herpes simplex (HSV) and varicella-zoster viruses (VZV). The control group included 7 children who had previously suffered from infections with HSV and VZV without being treated with isoprinosine. The clinical observations showed a significant inhibitory effect of isoprinosine on viral infections, with the duration of the disease being shortened considerably. The authors think isoprinosine treatment to be justified in viral infections, especially in children with neoplastic diseases, because of the immunostimulating action of the drug, rapid clinical improvement and good tolerance of the drug.     

Prevalence and risk factors for herpes simplex virus type 2 infections in East Croatia

The aim of the study was to establish the seroprevalence of herpes simplex virus type 2 (HSV-2) among sexually active adults from East Croatia and to evaluate its correlates with demographic and behavioural risk factors. This cross-sectional study was conducted from the 1st June until the 30th September 2005 at the Department of Transfusion Medicine, Osijek University Hospital Center. The study included 423 blood donors composed of 366 (86.5%) men and 57 (13.5%) women. Demographic data and data on the risky sexual behaviour during the lifetime were collected with usage of an anonymous questionnaire consisting of 15 questions. Western blot testing revealed that 3.3% (14/423) subjects were positive for the presence of IgG antibodies to herpes simplex virus 2, i.e. 10 (2.7%) men and 4 (7.0%) women. A higher HSV-2 seroprevalence was associated with female sex, older age, marital status (with single subjects been under the greater risk), irregular condom use on sexual intercourses with new partners and with higher number (five or more) of sexual partners during lifetime. Among determined risk factors in the study population, only the connection between the HSV-2 positivity and the older age was statistically significant. The study has showed that the demographic rather than the behavioural risk factors are associated with higher prevalence of HSV-2 antibodies in the observed population.     

Tobacco mosaic virus and the study of early events in virus infections

In order to establish infections, viruses must be delivered to the cells of potential hosts and must then engage in activities that enable their genomes to be expressed and replicated. With most viruses, the events that precede the onset of production of progeny virus particles are referred to as the early events and, in the case of positive-strand RNA viruses, they include the initial interaction with and entry of host cells and the release (uncoating) of the genome from the virus particles. Though the early events remain one of the more poorly understood areas of plant virology, the virus with which most of the relevant research has been performed is tobacco mosaic virus (TMV). In spite of this effort, there remains much uncertainty about the form or constituent of the virus that actually enters the initially invaded cell in a plant and about the mechanism(s) that trigger the subsequent uncoating (virion disassembly) reactions. A variety of approaches have been used in attempts to determine the fate of TMV particles that are involved in the establishment of an infection and these are briefly described in this review. In some recent work, it has been proposed that the uncoating process involves the bidirectional release of coat protein subunits from the viral RNA and that these activities may be mediated by cotranslational and coreplicational disassembly mechanisms.