Experimental studies of acute and recurrent herpes simplex virus infections in the murine heart and dorsal root ganglia

The multiplication of HSV-1 and HSV-2 strains in the heart and the corresponding dorsal root ganglia (DRG) was examined in experimentally infected mice. Infectious HSV-1 was recovered from the heart between the second and fourth day after inoculation and 3 days later from the DRG. Both the heart and DRG yielded infectious HSV-2 from the fourth to the twenty-first day after inoculation. The HSV-2, but not the HSV-1, induced recrudescent disease in chronically infected mice up to 18 months after inoculation. These affected mice showed signs of disease similar to those observed during the acute phase of the primary infection. During the recrudescent disease, infectious HSV-2 was recovered both from the heart and DRG. The possibility of using this experimental model for investigating long-term human cardiopathies is discussed.     

Interference of the low-pH inactivated herpes simplex virus type 1 (HSV-1) strain HSZP with the early shutoff function of superinfecting HSV-1 strain KOS

In former studies, we described that the HSZP strain of herpes simplex virus type 1 (HSV-1) was defective with respect to the early shutoff of host protein synthesis but was effective at interfering with the early shutoff function of the HSV-1 strain KOS, even when heat-inactivated or neutralized by antibody. However, the HSZP strain failed to interfere when inactivated with zinc ions or purified from cells treated with 2-deoxy-D-glucose. In this study, we provide evidence that the ability of the purified low-pH inactivated (citrate buffer, pH 3.0) and gel-filtered (Sephadex G-25) HSZP virions to adsorb host cells was not significantly affected. However, their ability to induce interference with the early shutoff function of the superinfecting HSV-1 strain KOS was restricted. In comparison with native virus, up to eight times more low-pH inactivated HSZP virions were needed to interfere with the shutoff by strain KOS. The interference was not due to exclusion of strain KOS by HSZP at the level of adsorption and/or penetration. The restriction was partially overcome by treatment of the cells with polyethylene glycol after adsorption of the low-pH inactivated HSZP virions. This observation indicates that the direct fusion of the virion envelope of low-pH inactivated HSZP with the plasma cell membrane was predominantly hampered.     

In vitro acquisition of resistance against herpes simplex virus by permissive murine macrophages

Summary This study has documented that peritoneal macrophages (PM) from adult mice of strains DBA/2J, BALB/c and AKR can support productive replication of herpes simplex virus (HSV) strains Thea and Haase. Simian virus 40 transformed PM of C57 BL/6J origin yielded high titers of Thea and Haase, whereas normal PM from adult mice of the same genetic background were infected abortively. The evidence obtained suggested that multiplication of PM per se is insufficient to allow HSV replication. PM from DBA/2J mice, immune to HSV or Listeria monocytogenes lacked the capacity to support productive replication of HSV. PM from nonimmune, adult DBA/2J or suckling C57BL/6J mice acquired the potential to effectively restrict HSV replication when preincubated for 24 hours with 72 hours old MLC supernatant. These macrophages could also inhibit HSV replication if pretreated with supernatant from cultures of specific antigen stimulated, HSV or Listeria immune spleen lymphocytes (SL). Likewise, supernatant from concanavalin A stimulated SL also possessed the capacity to confer resistance against HSV replication in otherwise permissive PM. Sensitized T cells proved to be essential for the generation of active supernatants. Active supernatants had the potential to confer resistance against HSV replication within mouse but not chick or hamster embryo cells. The other characteristics of the soluble mediator included: instability at pH 2, inactivation by trypsin but not by DNase or RNase and persistence of activity when exposed to 56° C for 20 minutes.With 3 Figures     

Delayed hypersensitivity to herpes simplex virus: murine model.

Cell-mediated immunity has been shown to be clinically important in recovery from herpes simplex virus (HSV) infections. To investigate the role of delayed hypersensitivity (DH) in immunity and protection against HSV, we developed a murine model using the ear-swelling assay. Mice were infected subcutaneously with HSV-1 and ear-challenged, and the swelling was quantified. Significant ear swelling was detected by 3 to 4 days postinfection and peaked at 6 days. The kinetics of development of ear swelling were typical of DH: maximal swelling occurred 24 h post challenge and was diminished by 48 h, and the cellular infiltrate was predominantly mononuclear. Four-hour swelling, indicative of antibody-mediated, immediate-type hypersensitivity, was not detected until 15 days post immunization. The DH response was virus specific and could be transferred to normal recipients with lymph node T cells, but not with B cells or immune serum. This system will provide a useful model for evaluating the protective role of DH in HSV infection and for studying the specificity and interaction of T cells which mediate the response.     

Interactions between herpes simplex virus and murine bone marrow macrophages

Summary Macrophages from murine bone marrow (strain C3Hf Bu/Kam) were culturedin vitro in L-cell conditioned medium. After 0, 2, 4, 6, 8 and 10 days, they were infected with a clinical strain of herpes simplex virus type 1 and the outcome followed morphologically, by phagocytic index, infectious virus yields, immunofluorescence, expression of Fc receptors and major histocompatibility complex (MHC) Class II antigens. At a multiplicity of infection of 1–5, little morphological difference was apparent between infected and uninfected cultures at early stagesin vitro but marked changes occurred later with reduction in cell numbers in the infected cultures. Indirect immunofluorescence failed to detect cells expressing early viral antigens, and yields of infectious virus indicated that permissive infection was not taking place. While phagocytic index and Fc receptor expression did not change 24 hours post-infection, MHC Class II antigen expression was increased. Thus, although the bone marrow macrophages seem predominantly resistant to infection with HSV-1, they may be modified by the presence of the virus. Since macrophages may act as antigen presenting cells for the immune system, this type of mechanism may be important in the generation of local immune responses.With 5 Figures     

Impaired induction of protective immunity by highly virulent herpes simplex virus type 2 in a murine model of genital herpes

Summary.  We investigated the immune events in the vagina of mice intravaginally infected with highly virulent herpes simplex virus type 2 (HSV-2) strain 186, and compared them with those induced by HSV type 1 strain KOS, a widely known laboratory strain. Although there was no significant difference between 186 and KOS in the viral replication in the initial stage of infection, inadequate and delayed clearance of virus from the vaginal mucosa was observed in 186-challenged mice. The induction of antigen-presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ) in the vagina was slow in 186-challenged mice, and the number of T cells in the vagina in 186-challenged mice was much lower than that in KOS-challenged mice. Furthermore, the level of IL-12 as well as that of IFN-γ was significantly lower in 186-challenged mice than in KOS-challenged mice, while the level of IL-4 in 186-challenged mice was higher than that in KOS-challenged mice. On the basis of these observations, we suggest that the weak activation of epithelial cells and the delayed induction of APC by 186-infection may be involved in the inadequate activation of T cells and the ineffective virus clearance from the vaginal mucosa. Received January 5, 2000 Accepted May 22, 2000     

Efficacy of phosphonoacetic acid on herpes simplex virus infection of sensory ganglia.

Treatment of mice with phosphonoacetic acid markedly reduced the incidence of latent ganglionic infection with herpes simplex virus when administered within 24 h after viral inoculation, but had no effect on an already established ganglionic infection.     

Induction of endogenous murine type C virus by ultraviolet-irradiated herpes simplex virus: effect of metabolic inhibitors

Ultraviolet-irradiated herpes simplex virus (u.v.-HSV) induced endogenous xenotropic type C virus from AI--2 cells, derived from the BALB/c mouse, as determined by infectious centre focus-forming assay on permissive normal rat kidney (NRK) cells. The number of cells induced to release type C virus by irradiated HSV was dependent on the level of u.v. exposure received by the HSV. Optimal induction occurred when cells were infected with irradiated HSV during their exponential growth phase. Virus induction decreased under conditions of simultaneous cellular exposure to hydroxyurea or actinomycin D, inhibitors of DNA and RNA synthesis, respectively, with actinomycin D having a greater inhibitory effect. This suggests that both DNA and RNA synthesis are required for irradiated HSV induction of murine xenotropic virus. Hydroxyurea decreased induction in the first few hours after infection of A1--2 cells with irradiated HSV, suggesting that the biological events involving DNA synthesis which are required for induction by u.v.-HSV occur shortly after infection.     

Experimental immunosuppression induced by herpes simplex virus

Summary Cell-mediated immune response was studied in experimental herpetic infection in mice.Our data suggest that herpes simplex virus induced immunosuppression in mice. This depression was documented by the unresponsiveness of the lymphocytes to PHA stimulationin vitro and by the enhanced susceptibility of adult mice to Coxsackie B virus infection.Our data also suggest that levamisole, a potent immunostimulant drug, had no effect on the unresponsiveness of the lymphocytes to PHA stimulation.With 2 Figures     

Hemagglutination by herpes simplex virus type 1

Summary The McIntyre and HSZP strains as well as clinical isolate of herpes simplex virus type 1 were found to agglutinate C57B1/10su and CBA mouse red blood cells. The hemagglutinating activity was inhibited by antisera that neutralized the infectivity of the virus.     

Subacute encephalitis caused by herpes simplex virus

The authors present a case of herpes-simplex encephalitis in a 58-year-old woman. The disease had a biphasic course and lasted 87 days. The clinical picture, laboratory data and the pathologic-anatomical changes of the disease are discussed. The authors also emphasize the importance of the encephalitic process in the brain stem, which can make the clinical picture even more complicated because of the appearance of respiratory disturbances.     

Characterization of glycoprotein C of HSZP strain of herpes simplex virus 1

Sequences of UL44 genes of strains HSZP, KOS and 17 of herpes simplex virus 1 (HSV-1) were determined and the amino acid sequences of corresponding glycoproteins (gC) were deduced. In comparison with the 17 strain, the HSZP strain showed specific changes in 3 nucleotides and in 2 amino acids (aa 139 and 147, both from Arg to Trp) in the antigenic locus LII. The change at aa 147 was situated within the GAG-binding epitope. In a similar comparison, KOS strain had changes in 3 nucleotides and 3 amino acids (aa 3, 14, and 300). The UL44 genes of HSZP and KOS strains were expressed in insect Sf-21 cells by means of the baculovirus (Bac-to-Bac) expression system. As shown by immunoblot analysis, both the recombinant baculoviruses (B1-HSZP and B6-KOS) expressed a glycosylated gC, the M(r) of which (116 K) was lower than that of gC synthesized in Vero cells (129 K) infected with strains HSZP or KOS. In addition, smaller gC-specific proteins (of apparent M(r) of 50-58 K and 98 K) corresponding to a non-glycosylated precursor polypeptide and/or incomplete forms of the partially glycosylated gC were found. When Balb/c mice were immunized with Sf-21 cells expressing gC, the recombinant gC-HSZP represented a more efficient immunogen possibly due to its stronger expression in these cells. The corresponding gC-HSZP antiserum reacted in enzyme-linked immunosorbent assay (ELISA) equally well with HSZP and KOS virion antigens and neutralized HSZP strain at a low titer. Both gC-HSZP and gC-KOS antisera detected the homologous as well as the heterologous gC antigens in Vero cells regardless whether infected with strains HSZP, KOS or 17, revealing the presence of gC from 6 to 16 hrs post infection (p.i.) in the cytoplasm, on the nuclear membrane and at the cell surface.     

Viral-specific thymidine kinase in sensory ganglia of mice infected with herpes simplex virus.

Dorsal root ganglia (DRG) from herpes simplex virus (HSV)-infected mice were assayed for thymidine kinase (TK) activity at various times after infection. Polyacrylamide-gel electrophoresis revealed two peaks of activity with Rf values ranging from 0.18 to 0.24 (peak I) and 0.40 to 0.46 (peak II). In contrast, DRG from uninfected animals showed only one peak of enzyme activity with an R_f of 0.23. Incubation of infected DRG extracts with anti-HSV γ-globulin eliminated the TK activity in peak II but had no effect on the TK activity in peak I or on the TK activity found in DRG extracts from vaccinia-infected mice. The demonstration that infectious virus could be recovered from cell-free ganglionic homogenates for only 14 days, while HSV-specific TK (peak II) could be detected for up to 60 days, suggests that at least a portion of the viral genome is being continually or intermittently expressed during the chronic phase of the infection.     

Critical involvement of CD40 in protection against herpes simplex virus infection in a murine model of genital herpes

Summary.  We studied the requirement for CD40⁺ cells in the resolution of vaginal infection with avirulent herpes simplex virus type I (HSV-1) in vivo using CD40-deficient mice, which were susceptible to infection with avirulent HSV-1. Compared with wild-type mice, CD40-deficient mice could not eliminate HSV-1 virus effectively from the vaginal mucosa and produced lower amounts of interleukin-12 and interferon-γ. These results show that the induction and activation of CD40⁺ cells are important for HSV prevention, facilitating the activation of T cells to induce an efficient HSV clearance from the vaginal mucosa and to prevent lethal illness due to HSV infection. June 14, 2001 September 10, 2001     

Latent herpes simplex virus in ganglia of mice after primary infection and reinoculation at a distant site

Summary Herpes simplex virus (HSV)-infected hairless mice with evidence of latent infection in spinal ganglia did not develop latent HSV infections in trigeminal ganglia upon reinfection in the oro-facial area. HSV-infected and PAA-treated mice without evidence of latent HSV infection in spinal ganglia were resistant to reinfection in the lumbar region, but not to that performed in the oro-facial area.This is publication No. 32 from the Cooperative Antiviral Testing Group of the Antiviral Substances Program, Development and Applications Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health.     

Recovery of an unusual fusogenic herpes simplex virus type 2 strain from a clinical specimen

A highly unusual herpes simplex virus type 2 strain, strain Burr, was isolated from a female genital tract clinical specimen. This virus induced remarkably rapid and extensive syncytium formation in Vero cells involving hundreds of cells but was less fusion active in HEp-2 cells, MRC-5 cells, and mink lung cells. Virus-infected cells produced the glycoproteins gB, gC, gD and gE.     

Radioimmunoassay for herpes simplex virus

Radioimmunoassay has been used for some time in the detection of the Australia Antigen. In this experiment, radioimmunoassay with Iodine-125 (¹²⁵I) permitted rapid detection of the presence of herpes simplex virus, type I, in vitro. This is a convenient technique for the detection of virus, which now requires laborious methods involving observation for the cytopathic effect of virus upon cultured cells.