Herpes simplex virus in postradiation cervical smears. A morphologic and immunocytochemical study.

From January 1987 to August 1988, cytomorphologic criteria of both herpes simplex virus (HSV) and radiation effects were observed in Papanicolaou smears from 3 of 1,340 patients who had received radiotherapy for squamous cell carcinoma of the cervix. Avidin-biotin immunoperoxidase staining, using a rabbit IgG polyclonal HSV antibody, confirmed the presence of HSV antigen in those three postradiation smears. Both multinucleated molded cells and epithelial cells that lacked cytopathic effects were positive for HSV. Three other postradiation smears from these cases were similarly positive for HSV antigen; the one preradiation smear was negative. In situ hybridization and immunoperoxidase studies on sections from the preradiation biopsies were negative: severely altered neoplastic cells showed no reactivity. The absence of HSV markers in the preradiation specimens suggests that the HSV infections were secondary to the radiotherapy; further studies are needed to prove this association and to assess the possible mechanisms. These cases clearly indicate that the overlapping features of radiation and viral effects (such as multinucleation) may be present simultaneously.     

Diagnosis of herpes simplex virus in routine smears by an immunoperoxidase technique

Routine Papanicolaou-stained cervicovaginal smears from 59 patients were cytologically screened for herpetic infection. Forty-one of the smears were positive for herpes, 2 were suspicious and 16 were negative. All 59 slides were then destained and restained by a commercial immunoperoxidase kit for the detection of herpes simplex virus (HSV). The immunoperoxidase stain was positive in 23 of the 41 cytologically positive slides. One of the 2 cytologically suspicious slides was also immunoperoxidase positive, as was 1 of the 16 cytologically negative slides. This study indicates that immunoperoxidase staining is very specific but not quite as sensitive as routine Papanicolaou-stained smears in the detection of HSV. The immunoperoxidase method is thus recommended for the confirmation of HSV cases rather than for the routine diagnosis of HSV infection.     

Immunoperoxidase staining for the detection of herpes simplex virus antigen in cervicovaginal smears

Destained cervicovaginal smears from eight patients with herpes simplex virus (HSV) infections were stained by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate the presence of the HSV type 2 (HSV-2) antigen. Positive results were obtained in six of the eight cases, with intense staining for the HSV-2-specific antigen throughout the cytoplasm and nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Virus isolation was successfully performed for the HSV-2-positive case that also had a histologically confirmed squamous-cell carcinoma of the cervix. The combined use of cytology and the PAP staining technique was of great value in the demonstration of cervical HSV infections.     

Cytodiagnosis of herpes simplex keratitis by means of an immunoperoxidase technique. A case report

Typical herpes simplex keratitis that developed in a 5-year-old boy was initially diagnosed cytologically in Papanicolaou-stained samples. Subsequently, an immunoperoxidase staining technique was used to identify the specific type of herpes simplex virus (HSV) in the destained cellular samples. The positive staining helped to establish the diagnosis of a type 1 HSV infection, permitting early treatment with acyclovir and subsequent complete recovery from the ocular herpetic infection. Emphasis is placed on the value of the immunoperoxidase technique for the rapid and specific diagnosis of cases of suspected HSV infection.     

Prevalence of Herpes Simplex Virus Type 1- and 2- Specific Antibodies among the Acute,Recurrent, and Provoked Types of Female Genital Herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P< 0.005), and 55% for the recurrent type (P<0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

HSV neutralization by the microbicidal candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.     

Congenital herpes simplex virus infection diagnosed by cytology of aspirated tracheobronchial material

Aspirated tracheal secretions from a ten-day-old newborn having signs of sepsis showed small clusters of cells with cytopathic changes consistent with herpes simplex virus (HSV) infection. The presence of type 2 HSV was confirmed by an immunoperoxidase procedure on the aspirated bronchial mucus and at necropsy in most of the viscera. Since prompt antiviral chemotherapy may favorably affect the outcome of HSV infections, early cytologic studies of tracheobronchial secretions may prove useful for rapid diagnosis.     

Comparison of several ELISA tests for detecting the presence of IgG and IgM against herpes simplex viruses

Four enzyme-linked immunosorbent assays designated test 1 (ETI-HSVK-G 1/2); test 2 (ETI-HSVK-M 1/2); test 3 (ETI-HSVK-G 2), and test 4 (BioElisa HSV2 IgG) were studied to evaluate different stages of herpes simplex virus (HSV) infection. Samples (50 sera and 14 cerebrospinal fluid) were included in four groups. Group 1 consisted of samples from patients with primary HSV infections; group 2 comprised samples from patients with recurrent HSV infections; group 3 were samples nonreactive to HSV; and group 4 were samples from patients with infections by other herpes viruses (4a, chickenpox; 4b, herpes zoster; and 4c, infectious mononucleosis by Epstein-Barr virus). The percentages of agreement between tests 1 and 2 were 100 and 72.1%, respectively. The total diagnostic values of tests 1 and 2 were: 100 and 50% sensitivity, respectively; and 100 and 89% specificity, respectively. Few positive results for HSV-2 infection were found, and so, tests 3 and 4 were not evaluated. The results of tests 3 and 4 for a chickenpox patient, and a herpes zoster patient were not in agreement.     

T cell immunity to herpes simplex viruses in seronegative subjects: silent infection or acquired immunity?

During the course of investigating T cell responses to HSV among volunteers entering trials of investigational genital herpes vaccines, 6 of the 24 immunocompetent subjects with no prior history of oral/labial or genital herpes possessed HSV-specific T cell immunity but, by multiple determinants of even the most sensitive serological assays, remained seronegative to HSV-1 and -2. Of these six immune seronegative (IS; HSV-seronegative with HSV-specific T cell responses) subjects, two had transient HSV-specific T cell responses, while four had CD4(+) and CD8(+) T cell responses directed at HSV that persisted for up to 4 years. CD4(+) T cell clones were isolated that recognized and had high binding affinities to epitopes in HSV-2 tegument proteins. All six IS subjects had potential sexual exposure to an HSV-2-infected sexual partner. Oral and genital mucosal secretions were sampled and tested for the presence of infectious HSV and HSV DNA. No evidence of HSV was detected in >1500 samples obtained from these IS subjects. The identification of persistent T cell responses to HSV in seronegative subjects is a novel finding in the herpesvirus field and suggests either undetected infection or acquired immunity in the absence of infection. Understanding the basis of these acquired immune responses may be critical in developing effective vaccines for genital herpes.     

Virus Type-Specific Thymidine Kinase in Cells Biochemically Transformed by Herpes Simplex Virus Types 1 and 2

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.     

Infection and inhibition of human cytotoxic T lymphocytes by herpes simplex virus.

The effect of herpes simplex virus type 1 (HSV-1) infection on human cytotoxic T-lymphocyte (CTL) lytic function was assessed. All HSV-infected CTL populations tested were significantly inhibited in lysing target cells. The inhibition of CTL lytic function by infection with HSV-1 was independent of T-cell receptor-mediated antigen recognition and did not involve virus-induced shutoff of host protein synthesis, the expression of the HSV-1 transactivation protein, ICP4, or replicating virus. Understanding the functional impairment of CTL following infection with HSV may have important implications for HSV-induced immunosuppression and the mechanism of HSV persistence in immunocompetent hosts.     

In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.     

Cytomorphologic and immunocytochemical studies of chlamydial infections in cervical smears

A total of 872 cells in 183 Papanicolaou-stained cervical smears morphologically suspected of harboring chlamydial infections were cytologically investigated in an attempt to differentiate the morphologic features of chlamydial infection from those of mucus vacuoles or bacterial infection. The observed inclusions were classified according to their morphologic appearance and their staining by a Chlamydia-specific peroxidase-antiperoxidase stain and by the periodic acid-Schiff technique. Immunoperoxidase-positive inclusions were detected in 201 cells from 13 cases; 200 (99.5%) of these cells contained "nebular" inclusions while 1 cell (0.5%) contained multiple inclusions with homogeneous central target formation. These findings suggest that nebular-type inclusions may be the key morphologic finding for the identification of chlamydial infection and that the application of an immunoperoxidase staining technique on the destained Papanicolaou preparation may be useful for the diagnosis of equivocal inclusions.     

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities.     

Interaction of herpes simplex virus type 2 with a rat glioma cell line

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.     

Herpes simplex virus type 1 strain HSV1716 grown in baby hamster kidney cells has altered tropism for nonpermissive Chinese hamster ovary cells compared to HSV1716 grown in vero cells

Chinese hamster ovary (CHO) cells are traditionally regarded as nonpermissive cells for herpes simplex virus type 1 (HSV-1) infection as they lack the specific entry receptors, and modified CHO cells have been instrumental in the identification of HSV-1 receptors in numerous studies. In this report we demonstrate that the HSV-1 strain 17+ variant HSV1716 is able to infect unmodified CHO cells but only if the virus is propagated in baby hamster kidney (BHK) cells. Infection of CHO cells by BHK-propagated HSV1716 results in expression of immediate-early, early, and late viral genes, and infectious progeny virions are produced. In normally cultured CHO cells, up to a maximum of 50% of cells were permissive for BHK-propagated HSV1716 infection, with 24 h of serum starvation increasing this to 100% of CHO cells, suggesting that the mechanism used by BHK-propagated virus to infect CHO cells was cell cycle dependent. The altered tropism of HSV1716 was also evident in another nonpermissive mouse melanoma cell line and is an exclusive property resulting from propagation of the virus using BHK cells, as viruses propagated on Vero, C8161 (a human melanoma cell line), or indeed, CHO cells were completely unable to infect either CHO or mouse melanoma cells.     

Herpes simplex virus (HSV)-specific proliferative and cytotoxic T-cell responses in humans immunized with an HSV type 2 glycoprotein subunit vaccine.

Studies were undertaken to determine whether immunization of humans with a herpes simplex virus type 2 (HSV-2) glycoprotein-subunit vaccine would result in the priming of both HSV-specific proliferating cells and cytotoxic T cells. Peripheral blood lymphocytes (PBL) from all eight vaccines studied responded by proliferating after stimulation with HSV-2, HSV-1, and glycoprotein gB-1. The PBL of five of these eight vaccines proliferated following stimulation with gD-2, whereas stimulation with gD-1 resulted in relatively low or no proliferative responses. T-cell clones were generated from HSV-2-stimulated PBL of three vaccinees who demonstrated strong proliferative responses to HSV-1 and HSV-2. Of 12 clones studied in lymphoproliferative assays, 9 were found to be cross-reactive for HSV-1 and HSV-2. Of the approximately 90 T-cell clones isolated, 14 demonstrated HSV-specific cytotoxic activity. Radioimmunoprecipitation-polyacrylamide gel electrophoresis analyses confirmed that the vaccinees had antibodies only to HSV glycoproteins, not to proteins which are absent in the subunit vaccine, indicating that these vaccinees had not become infected with HSV. Immunization of humans with an HSV-2 glycoprotein-subunit vaccine thus results in the priming of T cells that proliferate in response to stimulation with HSV and its glycoproteins and T cells that have cytotoxic activity against HSV-infected cells. Such HSV-specific memory T cells were detected as late as 2 years following the last boost with the subunit vaccine.     

Replication of herpes simplex virus in two cell systems derived from rhesus monkeys

The replication of herpes simplex virus (HSV) in two cell systems derived from rhesus monkeys (LLC-MK2 and DBS-FRhL-2) was studied. In LLC-MK2, the growth of HSV-1 was abortive or extremely limited regardless of the multiplicity of infection, while that of HSV-2 was productive only on infection at high multiplicities. DBS-FRhL-2 cells supported growth of both types of HSV, although growth was highly dependent on the age of monolayers and the infectious dose of virus inocula. Plaques were produced in DBS-FRhL-2 cell monolayers inoculated with HSV-2 but not with HSV-1, although the efficiency of their formation in the former system was much less than in a system of FL and HSV-2. On the other hand, plaques were not produced in LLC-MK2 cell monolayers by either type of HSV. The growth of adapted variants of HSV-1 was also studied. In contrast to the parental strain, these variants replicated well in LLC-MK2 even at a low multiplicity of infection and produced clear plaques in the monolayers. Furthermore, persistent infections of HSV-2 were established in DBS-FRhL-2 cell monolayers under routine culture conditions.