Herpes simplex virus type 1 can either suppress or enhance human immunodeficiency virus type 1 replication in CD4-positive T lymphocytes

It was proposed recently that CEM CD4-positive T cells infected chronically by herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1) (CEM(HSV/HIV)) may be used as a model for studying HIV/HSV interactions. To ascertain whether HSV-HIV coinfection of T lymphocytes has a role in promoting progression of lentiviral infection, T cells infected chronically by either HSV-1 (CEM(HSV)) or HIV-1 (CEM(HIV)) were challenged with a superinfecting dose of HIV-1 or HSV-1. The results show a positive influence on HIV growth when CEM(HIV) cells were superinfected with HSV-1 to an extent that was dependent on the multiplicity of superinfection used. In contrast, HIV superinfection of CEM(HSV) cells resulted in a delay of HIV-1 production and in a lack of HSV-mediated LTR transactivation. These effects were due to cell growth inhibition and apoptosis, resulting from persistent HSV-1 infection. Treatment of CEM(HSV) with acyclovir inhibited completely the HSV-1 cytopathic effects and allowed efficient HIV-1 replication. These data may be relevant in clarifying the role of HIV/HSV interaction in the pathogenesis of AIDS.     

Persistent infection of BHK cells with herpes simplex virus types 1 and 2 in the absence of specific anti-herpetic antibody.

Persistent infection of BHK-21 cells with 5 strains of herpes simplex virus (HSV) type 1 and a 1 strain of HSV type 2 is described. Acute infection only was obtained with two strains of type 1 and one strain of type 2. Persistent infection could not be established in HeLa and rabbit lung cells. Persistent infection of BHK cells proceeded in the absence of specific antibody. Persistently infected cells were more resistant to superinfection with homologous or heterologous HSV as compared to infection of normal BHK cells. No interferon activity was demonstrated in persistently infected cell cultures. The results of virus titration were in accordance with the demonstration of fluorescent antigen of HSV.     

Replication of type I herpes simplex virus in primary cultures of hairy cell leukemic leukocytes.

The ability of leukemic leukocytes to support the replication of herpes simplex virus (HSV) was studied. Mononuclear leukocytes (MNL) from the peripheral blood of patients with a variety of lymphoid leukemias were isolated on Ficoll-Hypaque gradients and infected with HSV at a multiplicity of infection of 5 to 10. No virus growth was detected in cells from patients with chronic lymphocytic leukemia (9), acute lymphocytic leukemia (1), or lymphosarcoma cell leukemia (2), HSV replication did occur in hairy cell leukemic MNL from all of 4 patients studied. Maximal titers of 10(3.7) to 10(4.7) PFU/ml occurred 1 to 7 days after incubation. By electron microscopy, herpesvirus particles were seen in the nuclei of these infected cells after 3 days of culture, but none was seen in the cells not exposed to virus. Fluorescent antibody examination confirmed the presence of HSV antigens in the nuclei of infected hairy cells. No difference in the adsorption or penetration of the virus was found with the various MNL studied. Productive infection of the cells thus appeared to depend on the ability of the leukocyte ;o support a later stage of infection, either uncoating or replication of the virus.     

Persistent infection of human lymphoid and myeloid cell lines with herpes simplex virus.

Herpes simplex virus (HSV) type 1 replicated and persisted in human T, B, and myeloid cell lines with different patterns of viral replication and various effects on cell growth. T cell line CEM supported the replication of HSV for over 400 days without detectable differences in cell growth as compared with uninfected cells. HSV persisted in B cell line NC37 and myeloid cell line K562 for up to 222 and 374 days, respectively, but led to a significant decrease in the number of viable cells by 7 weeks of infection. The average number of cells producing infectious virus was very low in these cell lines (range, 0.5 to 2.7+) compared with a larger proportion of cells exhibiting HSV antigens by immunofluorescence (range, 24 to 58%). In contrast, null cell line LAZ 221 failed to replicate HSV even though the viral infection led to a cessation of cell growth.     

Multiplicity activation of herpes simplex virus in mouse neuroblastoma (C1300) cells

Summary The virus yields and number of infectious centres of HSV infected mouse neuroblastoma C1300 cells (clone 41 A₃) infected at different multiplicities of infection (MOI) were found to vary more than the differences of HSV concentrations of the virus suspensions used for infection of the cells. This suggested that a C1300 cell had to be infected with more than one HSV particle in order to produce progeny virus—multiplicity activation. The greater than expected enhancement of virus production of C1300 cell cultures receiving increasing MOI of HSV was probably not due to improved virus adsorption, nor influenced by non-virus factors in the virus inoculum stimulatory for HSV replication. A hypothesis, that the block in virus replication was promoted by an inhibitor of an HSV specified regulatory protein and could be overcome by the addition of HSV DNA copies in the infected cell, was supported by the results of two types of experiments. Presence of phosphonoformic acid, an inhibitor of the HSV specified DNA polymerase, in the culture medium of HSV infected permissive GMK cells resulted in non-linear relationships between virus yields and MOI. An HSV temperature sensitive mutant (ts B5), defective in a late structural protein, rescued wild type HSV in C1300 cells.With 4 Figures     

A persistent infection of Vero cells by egg-adapted mumps virus

Serial subculture of Vero cells infected with the chick embryo adapted Enders strain of mumps virus gives rise to a low level productive though persistent infection. Persistently infected cultures exhibit minimal cytopathology; however, widely dispersed foci of multinucleate cells are almost always present. Neither infected cell nor virus growth is temperature sensitive. Biological and biochemical evidence indicate that defective interfering particles are replicated along with infectious (nondefective) virus during the course of the persistent infection, although the plaque-purified inoculum virus stock contained only genome size RNA. With continued cell passage a heterogeneous, changing population of subgenomic sized viral RNA accumulates, suggesting that defective interfering (DI) RNA species are evolving from virion RNA, with no single DI RNA predominating. Since such factors as interferon, antibody, and temperature-sensitive mutants are absent from this system, DI particles are the likely factor modulating this persistent infection.     

Restricted replication of herpes simplex virus in human monocyte cultures: role of interferon

Exposure of fresh human monocytes in culture to high or moderate concentrations of herpes simplex virus (HSV) resulted in an abortive infection or in a highly restricted replication of the virus. Infectious progeny virus yields were obtained either by diluting the inoculum virus to multiplicities of 0.1-0.0001 PFU/cell, or by cultivation of the cells for a few days before exposure to the virus. Interferon was synthesized and released into the medium of monocyte cultures infected with the higher multiplicities, while the lower productive multiplicities or inoculations of HSV into mature macrophage cultures resulted in a production of only small amounts, if any, of interferon. Inhibition of the morphological differentiation of monocytes was seen in cultures infected with the higher multiplicities of HSV (m.o.i. 1.0-0.1 PFU/cell) and was not unlike that caused by exogenous interferon added into uninfected monocyte cultures. Antisera against the leukocyte interferon were able to enhance the production of infectious HSV in monocyte-macrophage cultures. These results suggest that interferon induced in monocytes by high multiplicities of HSV can prevent the productive replication of HSV. Apart from its possible direct antiviral effect interferon may cause this restriction of HSV replication by inhibiting the differentiation of host monocytes.     

Ultrastructural studies on the replication of herpes simplex virus in PK and XTC-2 cells

Ultrastructural changes showed the following characteristics of restricted replication of herpes simplex virus 1 (HSV 1) strains MA and HSZP in PK and XTC-2 cells: 1) minimal cytopathic changes in PK cells as compared to more pronounced alterations in XTC-2 cells; 2) formation of single nucleocapsids or their absence in the nuclei of PK cells infected with the HSZP strain; 3) lack of budding and envelopment and absence of reduplication of the nuclear membrane; 4) persistence of partially uncoated virions within the endocytic vacuoles in the cytoplasm of PK cells; and 5) formation of dense inclusion bodies in addition to the presence of defective virions in the cytoplasm of XTC-2 cells and vacuolation of their cytoplasmic membranes. The replication of HSV 1 in PK and XTC-2 cells seemed to be blocked at both early and late stages of virus replication. At low multiplicity of infection, no virus particles were formed.     

Establishment and biological characterization of an in vitro human cytomegalovirus latency model

In an attempt to develop an in vitro human cytomegalovirus (HCMV) latency model system, the growth characteristics of HCMV in a human thyroid papillary carcinoma cell line (TPC-1) were examined. When TPC-1 cultures preheated at 40.5 degrees for 48 hr were infected with HCMV and incubated at a supraoptimal temperature (40.5 degrees), the cultures could be maintained for at least 65 days without detection of infectious virus. In contrast, when the infected cultures were incubated at 37 degrees, HCMV persistently infected cultures were established. HCMV was reactivated from the latently infected cultures by decreasing the incubation temperature from 40.5 to 37 degrees, and the cultures subsequently entered into virus persistent infection. Although HCMV-specific polypeptides which comigrate with the immediate early virus polypeptides and nuclear antigens were continuously detectable in the majority (more than 95%) of the cells during the latent period, a detectable level of virus-specified DNA polymerase (one of the early virus proteins) was not induced, suggesting that the blockage of HCMV replication in the latently infected cultures occurs at the early stages of the HCMV replication cycle. Infectious center assay revealed that 0.002 to 0.2% of the cells contain an HCMV genome that can be activated during the latent period. The latently infected cells were susceptible to superinfection with homologous and heterologous strains of HCMV. In persistently infected cultures approximately 38% of the cells were lysed by reaction with HCMV immune serum and complement, whereas complement-mediated immune cytolysis could not be detected in the latently infected cultures. The data presented suggest that a temperature-sensitive cellular function(s) that controls the expression of the HCMV early functions plays an important role in maintenance of the HCMV genome in the latent state and reactivation of HCMV by decreasing the incubation temperature.     

Herpes simplex virus infection of in vitro cultured neuronal cells (mouse neuroblastoma C 1300 cells).

The nature of the restriction of herpes simplex virus replication in C 1300 neuroblastoma cells was studied. A low rate of adsorption was observed, probably due to the relatively few receptors for HSV on plasma membranes of C 1300 cells. The penetration rate of HSV to the nucleus was slow with an impaired processing of attached virus from plasma membrane to cell nucleus. Even at a high multiplicity of infection only a low percentage of the C 1300 neuroblastoma cells was permissively infected as determined by infectious centre assays. The yield of infectious HSV per virus-producing C 1300 cell was 1% of the yield from GMK control cells. The restriction in neuroblastoma cells of HSV infection could not be accounted for by sensitivity of cells to interferon or by an efficient induction of interferon. Evidence was obtained for the presence in C 1300 cells of an inhibitor of HSV replication not compatible with classical interferon. Observations on C 1300 cells maintaining many characteristics of differentiated neurons suggest that these cells may be useful as a model for studies on HSV-neuron interactions.     

Biological and biochemical characterization of a latent subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture

The present investigation describes the biological and biochemical properties of a persistent SSPE virus infection. Persistently infected cells were derived by cocultivation of infected brain cells and uninfected Vero cells, and cultures were maintained by normal subculturing methods. No infectious virus was ever released from these cultures, and all attempts to induce infectious virus release were unsuccessful. Biological assays showed that infected cells contained nucleocapsid and salt-dependent hemagglutinin antigens, whereas the normal hemagglutinin appeared not to be present. Electron microscopic examination demonstrated the presence of both intranuclear and cytoplasmic nucleocapsids together with the release of virus particles (defective?) from the cell membrane. Biochemical analysis demonstrated that approximately 90% of the intracellular genomic RNA was defective or subgenomic although a small quantity of infectious genomes was present. It is proposed that the large quantities of defective genomes in the infected cells are the major factor in the maintenance of this persistent infection.     

Evolution and properties of Aedes albopictus cell cultures persistently infected with sindbis virus

Replicate cultures of Aedes albopictus cells were infected with Sindbis virus and then maintained for long periods of time by weekly subculture. During the first week the viral titers ranged between 10⁸ and 10⁹ PFU/ml, but then gradually fell and within a few weeks stabilized at about 10⁵ to 10⁸ PFU/ml. Such cultures were followed for the appearance of temperature-sensitive (ts) virus, small-plaque virus, and for the appearance within the cells of small (12–15 S) double-stranded viral RNA (dsRNA). Cloning experiments carried out 6 months or more after the initial infection showed that persistently infected cultures gave rise to both virus-yielding and nonyielding clones. Similar results were obtained when virus-positive clones were recloned. Prolonged treatment of persistently infected cultures with anti-Sindbis virus serum resulted in curing of the virus infection. Cured cultures behaved in every way tested as normal uninfected A. albopictus cells. The ability to cure with anti-viral serum suggests that in this system extracellular virus is needed to perpetuate the long-term infection. After persistently infected cultures were subcultured, viral RNA synthesis and viral yields were maximal during the first 3–4 days. Thereafter, although the cell number continued to increase for several days, viral RNA synthesis and viral yields both decreased sharply. This result strongly suggests that A. albopictus cells have efficient means for the regulation of viral biosynthesis. Although the resistance of persistently infected cultures to superinfection could be accounted for by interfering temperature-sensitive nondefective virus, the presence in these cells of 12 S dsRNA suggests that defective viral genomes are also present.     

Herpes simplex virus infection of isolated autonomic neurons in culture: Viral replication and spread in a neuronal network

Summary Cultures of isolated neurons, derived from the superior cervical ganglion (SCG) of the newborn rat and maintained in the absence of nonneuronal cells, were infected with herpes simplex virus (HSV) type 1. By phase-contrast microscopy, including time-lapse cinematography, cytopathologic changes appeared first in neuronal cell bodies and only approximately 24 hours later were axonal abnormalities detectable. Despite low yields of viral progeny, infection spread readily within the two-dimensional network of neurons and their processes. Immuno-peroxidase staining for viral antigens confirmed the replication and spread of virus and revealed that antigen extended along axons during infection. Antiviral antibody added to the overlay medium slowed but did not prevent the spread of infection, indicating that virus passed from neuron to neuron over axonal pathways. Despite alteration of neuronal macromolecular synthesis early in infection, axonal transport is apparently preserved long enough to allow propagation of virus over interconnecting neural pathways.With 5 Figures     

Induction of an interferon-like substance in persistently infected Aedes albopictus cells

Summary The multiplication of Sindbis virus inSingh'S mosquito cell line derived from larvalA. albopictus was studied. Persistently infected cells are not able to support the growth of Sindbis virus to the same extent as cells infected for the first time. The maintenance of cell-virus equilibrium in persistently infected cells seems to be due to the presence of interferon-like antiviral substances. Mosquito cells which had been treated with media harvested from persistently infected cultures were protected from infection by Sindbis virus. The synthesis of these interferon-like substances is inhibited by actinomycin D. A possible implication of this observation is that in persistently infected mosquito cells the rate of replication of virus is controlled by the genome of the host cell.     

Role of nitric oxide in the regulation of lymphocyte apoptosis and HIV-1 replication

Nitric oxide (NO) has been implicated in certain immunopathogenetic mechanisms during the course of infection with human immunodeficiency virus (HIV). We have evaluated the levels of NO release and lymphocyte apoptosis in peripheral blood mononuclear cell (PBMC) cultures from HIV-1 infected subjects and healthy controls. We have also examined these 2 parameters in parallel cultures maintained under conditions where either NO synthesis was inhibited or high level of NO was present. Nitrite contents in culture supernatants were measured as the stable end products of the released NO. Levels of spontaneous apoptosis and activation-induced cell death (AICD) by anti-CD3 or by phytohemagglutinin were evaluated using flow cytometry. Additional experiments were also aimed at addressing a potential link between NO synthesis and HIV-1 replication in human monocyte-derived macrophages (MDMs). Acutely infected MDMs with HIV-1Bal were maintained in culture, without any additional activation signal, for a period of 14 days. Nitrites in the supernatants and mRNA accumulation of the inducible NO synthase (iNOS) in infected cells were assessed over the whole culture period. In addition, the effect of blocking NO synthesis during and after infection of MDMs, using an inhibitor of NO, was evaluated on the level of viral replication as measured by the presence of P24 antigen in the supernatants. Similarly, the effect on HIV replication of high NO levels in MDM cultures, supplied by a donor of NO during the 24 h period of infection, was also studied.We conclude that no elevation in NO release could be detected in PBMC cultures from HIV-1 infected subjects and that modulation of NO content may slightly regulate the level of spontaneous lymphocyte apoptosis but not that of AICD. Infection of MDMs with HIV-1 does not seem to induce detectable NO release or iNOS mRNA accumulation. Similarly, neither inhibition of NO synthesis nor the presence of high NO levels during the infection period could modify the outcome of virus replication in macrophages.     

T lymphocytes activated by interleukin 2 alone acquire permissiveness for replication of herpes simplex virus

Not only mitogen stimulation or mitogen stimulation in combination with interleukin 2 (IL2) was capable of causing susceptibility of human T lymphocytes to herpes simplex virus (HSV) infection, but also selective stimulation with recombinant Il2-induced permissiveness of T lymphocytes to HSV infection. Replication of HSV in such IL2-stimulated T cell cultures was shown to be restricted to a T cell subset not exceeding 5% of the total population. Furthermore, IL2 stimulation was sufficient to obtain virus replication in T cells previously infected by HSV and cultivated for several days. This could not be achieved by stimulation with mitogens such as phytohemagglutinin. The level at which virus replication was restricted in nonpermissive T cells was determined to be before immediate early gene expression as assessed by indirect immunofluorescence with monoclonal antibodies against viral proteins expressed at different stages of the viral replicative cycle.     

Production of temperature-sensitive and pathogenic virus fromAedes albopictus cells (Singh) persistently infected with chikungunya virus

Summary WhenA. albopictus, clone C6/36, cells were infected with chikungunya (CHIK) virus, high virus yield accompanied by a cytopathic effect in the acute stage of infection was followed by a relatively low yield of virus over a long period of time. Virus produced from persistently infected cultures became gradually of smaller plaque size and more temperature-sensitive; however, such virus still retained pathogenicity for suckling mice even after one year of infection.When the persistently infected cells were subcultured, a dissociation was observed between the time course of cell growth and that of virus production, suggesting some intracellular mechanisms that turn off virus production. The greater part of the interference against CHIK virus by the culture medium of the persistently infected cells appeared to be mediated by the infective virus in the medium. The infective virus was easily removed from the persistently infected cells either by subculture or by cloning in the presence of anti-CHIK serum, yielding cured cultures or virus-negative clones.With 5 Figures     

A comparison of 2 T-lymphoid cell lines persistently infected with the human immunodeficiency virus (HIV-1) with different productive capacities]

T-lymphoid cell lines (H9/CBL-4 and CEM/CBL-4) persistently infected with HIV-1 were observed simultaneously for 6.5 months. The virus activity was characterized by such parameters as the number of infected cells determined by fluorescent antibody technique, the total level of virus--specific protein synthesis determined by immune blotting method, and the capacity to infect H9 and CEM cells. A comparative analysis of the two cell lines helped define the evaluation criteria for high and low productivity cultures. It was shown that a short-term virus persistence could exist in high-productivity cultures and long-term persistence in low-productivity cultures. The cytopathic activity of virus in cultures could be judged by accumulation of virus protein p24 in cell-free supernatants, this being one of the factors defining the efficacy of infection of H9 and CEM T-lymphoid cells.