Biomedical subjects
Publications and source records attributed to F Rapp.
Two DNA-negative temperature-sensitive mutants of human cytomegalovirus strain AD169 were shown to be deficient in induction of several prominent viral polypeptides when grown in human foreskin fibroblasts under nonpermissive conditions. The use of a monospecific polyclonal antiserum allowed recognition of a further defect in processing of an 'early' viral polypeptide of 135 kD.
At concentrations greater than 1 mg/ml, caffeine inhibited plaque formation by herpes simplex virus type 1 (HSV-1), suppressed growth of HSV-1 at various steps of the replicative cycle, but did not inactivate virus infectivity. These effects were independent of each other and persisted through completion of virus replication, as determined by one-step growth studies. Some replication steps appeared to recover after removal of caffeine. Strong inhibition of the spread of HSV by caffeine also was demonstrated by infectious center assay. Caffeine suppressed protein synthesis, in particular that of three viral proteins.
We examined the effect of infection by other herpesviruses on the induction of Epstein-Barr virus (EBV) antigens. Raji cells were infected with a variety of viruses before superinfection with P3HR-1 virus 6 h later. Much earlier and greater synthesis of EBV antigens was observed in herpesvirus (herpes simplex virus type 1, type 2 and pseudorabies virus)-infected Raji cells than in mock-infected Raji cells prior to superinfection with P3HR-1 virus, as determined by indirect immunofluorescence tests or immunoprecipitation and PAGE. Although infection with a temperature-sensitive mutant of pseudorabies virus (tsG1) enhanced EBV antigen synthesis as well as wild-type pseudorabies virus at permissive temperature in Raji cells superinfected with P3HR-1 virus, EBV antigen synthesis was strongly inhibited at temperatures nonpermissive for tsG1. These results suggest that a herpesvirus immediate-early protein may play a role in enhancement of EBV antigen expression.
Flow cytometric detection of human immunodeficiency virus (HIV)-infected lymphoid cells at low frequencies is described. Infected cells from human T lymphoid cell lines H9 and A3.01 were detected at frequencies as low as 10(-4) following indirect immunofluorescence labeling. For labeling, cells were treated with an HIV-inactivating, permeabilizing fixative followed by binding of a monoclonal antibody specific for the HIV major core protein p24, and then by binding of fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat anti-mouse immunoglobulin antibody. We compared two fixation procedures, one using a mixture of methanol and acetone, the other a three-step fixation using methanol, paraformaldehyde and Triton X-100. The latter fixation protocol was found to be superior in its ability to resolve mixtures of infected and uninfected cells. The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. At analysis rates of several thousand cells/s, detection of HIV-infected cells as rare events was possible. Excellent agreement was obtained between flow cytometric evaluation and reverse transcriptase (RT) assay of infected H9 cells cocultured with uninfected H9 cells in various proportions for 7 days. In time course of infection experiments, cultures infected by small numbers of viral particles were positive by flow cytometry up to 3 days earlier than by RT assay.
The effect of dimethylsulfoxide (DMSO) and iododeoxyuridine (IUdR) on the growth characteristics of two established human glioblastoma cell lines (FG and HMCN-1) was studied. The FG cell line has been characterized. The HMCN-1 cell line, established in our laboratory, consisted of fibroblastoid and polygonal cells that grew without contact inhibition. Subcutaneous injection of these cells into weanling athymic nude mice induced slowly growing, solid tumors that were histologically spindly with areas that were similar to the original tumor. Chromosomal analyses revealed a human heteroploid pattern with a modal number of 69. The cells of the original human glioma contained S-100 protein and glial fibrillary acidic protein (GFA protein), whereas the established cells failed to express markers. Prolonged treatment of glioma cells with DMSO generated a more adherent, normal human fibroblastoid phenotype that grew with contact inhibition. The new phenotype and proliferative restriction of these cells was evident as late as 50 days after discontinuation of treatment. The chemical induction of cell differentiation resulted in decreased tumorigenic potential in athymic nude mice.
This report describes a latency model using human embryo lung cells that were infected with herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutants and cultivated at nonpermissive temperature (40.5 degrees C). ts mutants tsG8 (parental strain HSV-1 KOS) and tsG5 (parental strain HSV-1 13) could be maintained in a latent state at 40.5 degrees C for at least 40 days without exhibiting virus infectivity. During this time, viable virus could be reactivated by reducing the incubation temperature to the permissive level (34 degrees C). Virus replication could be detected 2 to 6 days after temperature reduction and the virus reactivated from the latent state seemed to retain the same ts phenotype as the input virus for at least 14 days.
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Previous studies have shown that herpes simplex virus (HSV) type 2 (HSV-2) can be maintained in a latent state in a limited number of cells by elevating the incubation temperature after treatment of HSV-infected human fetus lung fibroblast cells with metabolic inhibitors. Superinfection with human cytomegalovirus (HCMV) of latently infected cells maintained at the elevated temperature reactivated latent virus. In addition, superinfection with temperature-sensitive mutants indicated that reactivation of latent HSV in vitro did not require the expression of late gene function(s) of the superinfecting virus. We now report the (i) design of an in vitro HSV-2-latency system in which a higher percentage of cells contain a virus genome that can be activated; and (ii) subsequent use of this system to further characterize the virus activation process. Superinfection with a transcription-negative temperature-sensitive mutant of HSV type 1 (HSV-1) did not reactivate HSV-2-replication, suggesting that adsorption and penetration of the superinfecting virus were not sufficient for reactivation of the latent virus. Furthermore, superinfection with HSV-1 in the presence of (E)-5-(2-bromovinyl)-2'-deoxyuridine did not reactivate HSV-2 replication, suggesting that the expression of the immediate-early gene products are not sufficient for HSV-2 reactivation. Collectively, these data suggest that in addition to the expression of immediate-early gene function(s) at least a subset of early HSV-1 gene products are required for reactivation of latent HSV-2 in vitro.
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C57 mice aged 8-10 weeks in groups of 50 each received vaginal cotton pellets soaked in lysates of HEp-2 cells, either mock-infected or infected with herpes simplex virus I, herpes simplex virus 2, and highly attenuated recombinant viruses 5 times a week for 89 to 114 weeks. An untreated group was also included. The mock-infected and some of the infected cell lysates were exposed to ultraviolet light at a dose sufficient to inactivate virus. Smears of exfoliated vaginal cells collected once a month and histopathologic sections of genital organs removed at autopsy were coded and examined blind for the presence of abnormal cells indicative of malignant changes and cervical cancer, respectively. Sera collected before termination of the study were tested blind for the presence of antibody to infected cell lysates and to purified herpes simplex virus glycoprotein B. The results were as follows: Over 74% of 826 mice examined at autopsy contained tumors at non-genital sites. The tumors were randomly distributed among the various groups. Gross genital abnormalities were less common in untreated animals than in mice receiving vaginal implants. The fraction of mice which developed cervical cancer diagnosed by histopathologic examination was small (7.2%) and not significantly different among various groups. There was no correlation between the presence of abnormal exfoliated cells indicative of early invasive or invasive cancer lesions and the histopathologically proven diagnosis of micro-invasive or invasive cervical cancer. The incidence and levels of antibody were highest in animals exposed to live virus; some mice exposed to inactivated virus also developed weak or moderately high antibody levels. The presence of antibodies did not correlate with the presence of histopathologically proven cervical cancer. The results do not support the ability of herpes simplex viruses to cause genital neoplasia in mice.
Meignier et al. (1986) report the results of exposure of C57BL/6NCr mice to vaginal plugs containing live or inactivated herpes simplex virus 1 or 2 (HSV-1 or HSV-2) or recombinant viruses 5 times a week for up to 114 weeks. Genital organs showing abnormalities were transplanted into nude mice. Of 33 transplants, 13 produced subcutaneous tumors in nude mice and 12 were subsequently transplanted into C57BL/6NCr mice. We report that the DNA extracted from coded tumor tissues of nude mice and from normal viscera of the same rodents did not hybridize with HSV-1 and HSV-2 DNA probes representing the viral genomic regions shown previously to be capable of morphologically transforming cells in culture. The sensitivity of the assays was such that we could detect 0.5 copies of the HSV sequences of complexity equal to or greater than 1 Kbp per cell DNA equivalent. To control for the sensitivity of the assays in the actual hybridizations, the tumor-cell DNA was also hybridized with a beta-globin mouse DNA probe. A striking feature of these control hybridizations was the detection of beta-globin polymorphism in some nude mouse tumors. The beta-globin polymorphism allowed us to conclude that the analyzed tissues contained significant amounts of the tumor cells occurring in the C57BL/6NCr mice.
The effect of anti-mouse interferon (IFN) antibody on the course of genital herpes infection was studied in C57B1/6J mice. As we have previously described, intravaginal inoculation of C57 mice with herpes simplex virus type 2 (HSV-2) results in the development of genital lesions, subsequent encephalitis and death, with the mortality rate dependent on virus dose. In the present study, we found that intravaginal application of 1 x 10(5) units of anti-mouse (alpha/beta) IFN antibody did not significantly affect the course of infection. However, intraperitoneal or intravenous injection (the latter to a lesser extent) of anti-IFN antibody resulted in increased morbidity and mortality. In addition, intraperitoneal injection of anti-IFN antibody decreased the level of detectable endogenous IFN in peripheral blood and at the site of infection. The clear effect of anti-IFN antibody during early stages of infection points to the possible significance of early endogenous IFN production in ameliorating HSV-2 genital infection.
Human embryonic lung cells were infected with herpes simplex virus (HSV), treated with 10 micrograms/ml or more of cycloheximide for 24 h, incubated at 37 degrees C, and then shifted to 40.5 degrees C for various periods of time (0 to 40 days) without cycloheximide treatment. No infectious virus was detected after freezing and thawing of the cultures; however, infectious virus was recovered after temperature shift-down to 37 degrees C or superinfection with human cytomegalovirus (HCMV). The time course for formation of infectious centres after temperature shift-down was examined with and without HCMV superinfection during incubation at 40.5 degrees C. Two patterns of latently infected cells were identified: one pattern showed spontaneous reactivation of virus after temperature shift-down, and the second showed reactivation of HSV after superinfection with HCMV. The first pattern showed a rapid decrease in the number of infectious centres with time, whereas the second maintained a steady reactivation rate up to 40 days at 40.5 degrees C. The same tendency was observed for infectious centre formation at 37 degrees C with and without HCMV superinfection in the HSV latency system established with (E)-5-(2-bromovinyl)-2'-deoxyuridine and interferon treatment.
We previously demonstrated that herpes simplex virus type 1 (HSV-1) can be established in a latent form in vitro by the treatment of HSV-infected human cells with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in combination with human leukocyte interferon (IFN-alpha). We now report that the substitution of BVDU with 9-[(2-hydoxyethoxy)methyl]guanine (acyclovir; ACV) during a combined treatment with IFN-alpha inhibited HSV-1 replication and established in vitro virus latency that could be maintained for a longer period after inhibitor removal and a continued incubation at 37 degrees C. By contrast, the treatment of HSV-1-infected cells with combined IFN-alpha and 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a congener of ACV, failed to establish in vitro virus latency. Furthermore, none of these inhibitors used alone was sufficient to establish in vitro virus latency. The use of nucleoside analogs differing from BVDU in their modes of action has enabled us to initiate studies designed to extend in vitro virus latency.
Herpes simplex virus type 1 (HSV-1) latency was established in human embryo lung (HEL) cells by treatment with (E)-5-(2'-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and subsequent temperature increase (from 37 degrees to 40.5 degrees). This in vitro system was used to study reactivation after temperature shift-down to 37 degrees. Four methods known to induce cell DNA synthesis, superinfection with human cytomegalovirus, increased serum concentration in the medium, wound production, and superinfection with simian virus 40 (SV40), accelerated activation and replication of HSV-1. In contrast, reduced serum concentration resulted in postponement of virus reactivation and lower yield. In control experiments with mock-infected HEL cells treated with inhibitors and maintained for prolonged periods of time at 40.5 degrees, [3H]-thymidine incorporation was stimulated by increased serum concentration in the medium, wounding and SV40 superinfection, and hampered by reduced serum concentration in the medium. The data seem to indicate that cell DNA synthesis, the processes associated with it, or both, play a key role in the activation of latent HSV-1 and subsequent virus replication in this system.
Human leukocyte interferon (IFN-alpha) and acyclovir (ACV) have been used to establish human cytomegalovirus (HCMV) latency in infected human embryo lung fibroblast (HEL-F) cells. HCMV latency was maintained for a short interval (less than 9 days) after removal of inhibitors by increasing the incubation temperature. We now report a model system in which HCMV latency has been dramatically extended. HEL-F cells pretreated with IFN-alpha (200 IU/ml) and ACV (300 microM) were infected with a low MOI of HCMV, and treated for 23 days with the same inhibitor combination at 37 degrees. Infectious HCMV and virus antigens were undetectable at the time of inhibitor removal and remained undetectable during continued incubation at 40.5 degrees. A minimum of 0.4% of the cell population, however, contained a virus genome that could be reactivated at the time of inhibitor removal; this value declined to 0.0005% after 77 days at 40.5 degrees. HCMV reactivation was achieved by maintaining the infected cells at 37 degrees after inhibitor removal or by decreasing the incubation temperature to 37 degrees at any time during maintenance at 40.5 degrees. The HCMV genome was analyzed by blot hybridization in latently infected cells 23 days after inhibitor treatment at 37 degrees or 4 days after inhibitor removal at 40.5 degrees. Although many HCMV-unique DNA genomic sequences were retained in HEL-F cultures after 23 days of inhibitor treatment, a significant reduction in retained HCMV sequences occurred after inhibitor removal and temperature shift to 40.5 degrees. The XbaI HCMV DNA fragments retained in the latently infected HEL-F cultures were present at a copy number of at least 0.5 copies per haploid cell genome equivalent.
We have been actively studying herpesvirus latency and the effect of antiviral agents on latent infections in vivo and in vitro. To establish a model of recurrent herpetic genital disease, we treated latently infected C57Bl/6 mice with immunosuppressive agents. Mice were injected intraperitoneally with cyclophosphamide and antilymphocyte serum and then were examined for reactivated virus or recurrent lesions. When latently infected mice were subjected to immunosuppression, neither recurrent lesions nor virus was detected at the site of primary infection. However, when mice were selected for immunosuppressive treatment by the presence of postinfection scars, recurrent disease was induced. The results point to the significance of local factors in the pathogenicity of recurrent herpetic genital disease in mice.
The nonionic surfactant nonoxynol 9 (NP9), in combination with human alpha interferon, synergistically reduced the titer of herpes simplex virus type 2 (HSV-2) in vitro. The degree of synergy was highest at an interferon concentration of 10(3) IU/ml and an NP9 dilution of 1:1,500. We postulate that NP9 inactivates extracellular HSV-2, whereas interferon inhibits HSV-2 replication at the intracellular level.