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     

In vitro antiviral activity of dermaseptins against herpes simplex virus type 1.

The in vitro antiviral activity of dermaseptins (S1-S5) against herpes simplex virus type 1 (HSV1) was investigated. These peptides were incubated with the virus and its target cells under various conditions, and their effects were examined by the cytopathic effect inhibition assay or by reduction in virus yield in Hep-2 cell cultures as well as by direct immunofluorescence. Dermaseptin S4 displayed the strongest antiviral effect against HSV1, at micromolar doses. Experiments including acyclovir as a reference antiviral agent were performed to investigate the mode of action of this dermaseptin. In contrast to acyclovir, dermaseptin S4 showed its inhibitory effect only when applied to the virus before, or during virus adsorption to the target cells. This suggested that the activity of this dermaseptin was exerted at a very early stage of the viral multiplication cycle, most likely at the virus-cell interface.     

Tumor-dependent resistance of rat peritoneal macrophages to herpes simplex virus.

By their position at sites of initial infection and their wide distribution in major organs of the body, macrophages may be decisive in determining the susceptibility or resistance of the host to virus infection. Macrophage restriction of virus replication has been shown to be closely related to virus strains or virus types and to the age of the infected host. We report the effects of the development of a solid tumor in rats on intrinsic in vitro macrophage activity against herpes simplex virus type 1. The results obtained with the infectious center assays and the analysis of single-cycle growth curves of herpes simplex virus type 1 in macrophages obtained from normal and tumor-bearing rats showed a depression of antiviral activity of macrophages from tumor-bearing rats. The possibility of immunomodulation by bacterial adjuvants on tumor-bearing rats and the effects on the antiviral activity of peritoneal macrophages were furthermore demonstrated.     

Age-dependent resistance of human alveolar macrophages to herpes simplex virus.

Studies in mice demonstrate an age-dependent susceptibility to disseminated herpesvirus infection which is mediated. at least in part, by a defect in macrophage antiviral function. We examined the growth of herpes simplex virus within human alveolar macrophages obtained by bronchopulmonary lavage from neonates, adults with a variety of immunosuppressive disorders, and healthy adult volunteers. At 24 h postinfection, mean viral titers in neonatal macrophages increased 19-fold over adsorbed virus levels, a highly significant increase when compared to either immunosuppressed or normal adult macrophages (P less than 0.0005). These findings indicate that human macrophages, like those of mice, exhibit age-dependent permissiveness for the replication of herpes simplex virus. This permissiveness may at least partially account for the clinical observation that human newborns are highly susceptible to disseminated herpes simplex virus infections, whereas adults are not.     

In vitro reactivation of latent herpes simplex virus by the demethylating compound hexamethylene-bis-acetamide

We have characterized previously a model of herpes simplex virus (HSV) infection of rat dorsal root ganglia (DRG) following cutaneous infection. During acute infection HSV can be isolated from co-cultivated rat ganglia in (mean ± S.E.M) 4.8 ± 0.33 days (d) and from latently infected ganglia in 7.8 ± 0.53 (d) (P 0.0001). We treated co-cultivated rat ganglia from acute and latent infected rats with the demethylating compound hexamethylene-bis-acetamide (HEX) to see what effect, if any, it would have on HSV infection. HEX-treated ganglia from rats with acute infection did not differ significantly from controls in the proportion of rats, skin or ganglia positive for HSV. The mean time to detect virus was not different between treated (3.6 ± 0.38 d) and control (3.1 ± 0.46 d) (P> 0.05) groups. In latent infected rats there was no difference between treated and controlled groups in the proportions of rats, skin and ganglia positive for HSV. There was a significant difference in the mean time to CPE between the HEX and control groups respectively (4.5 ± 0.72 d vs 8.92 ± 1.42 d, P < 0.01). We conclude that HEX converted latent HSV infection to a productive one.     

In vitro infection of murine macrophages with Junin virus.

Mouse peritoneal macrophages were successfully infected with two strains of Junin virus producing high titers with no apparent cell damage. Infected cultures survived longer than noninfected cultures. The pattern of virus release suggested a persistent infection. Virus replication was delayed in macrophages from mice previously immunized with Junin virus. These results support the opinion that macrophages are targets for virus replication in vivo infections.     

Blocked herpes simplex virus type 2-specific DNA synthesis in simian virus 40-transformed hamster cells permissive for herpes simplex virus type 1.

Simian virus 40-transformed hamster cells (LL-1) permissive to herpes simplex virus type 1 (HSV-1) were shown to be relatively nonpermissive to HSV-2. When LL-1 cells were infected with HSV-2, there was a 3- to 4-log reduction in infectious viral progeny at 24 h postinfection as compared with HSV-1 under identical cultured conditions. HSV-2 could be carried in the LL-1 cell line for up to 12 passages without any appreciable cytopathology. Various early functions of the replicative cycle of HSV-2 appeared to be normal. Experiments demonstrated that early enzyme activity, HSV-2 thymidine kinase, and DNA polymerase appeared at permissive levels in extracts of HSV-2-infected LL-1 cells. However, DNA analysis of HSV-2 infected LL-1 cells demonstrated a block in HSV-2-specific DNA synthesis, although HSV-2 was capable of inhibiting DNA synthesis in LL-1 cells. Furthermore, indirect immunofluorescence studies indicate that late HSV-2 structural protein synthesis was inhibited in infected LL-1 cells. Thus, the inability of HSV-2 to replicate in LL-1 cells is due to a block at or before HSV-specific DNA synthesis, resulting in a reduction of the structural protein synthesis required for viral maturation.     

Colonization of murine ganglia by a superinfecting strain of herpes simplex virus

We report on the colonization of murine trigeminal ganglia after sequential infection of mice by herpes simplex viruses (HSVs). In preliminary studies, we have established that whereas the HSV-1(F) strain efficiently colonizes ganglia when inoculated by either the ear or eye routes, the HSV-1 X HSV-2 recombinant C7D colonizes ganglia when inoculated by the eye route only. The experimental design consisted of inoculating the right eye with C7D on day 1 and with HSV-1(F) in both left and right eyes on day 26. Both right and left trigeminal ganglia were removed and analyzed independently for latent virus on day 52. Our studies indicate that HSV-1(F) viruses were recovered from all left trigeminal ganglia but from only a small number of right trigeminal ganglia. Some right trigeminal ganglia yielded no viruses, whereas others yielded both C7D and HSV-1(F) viruses identified on the basis of plaque morphology and restriction enzyme cleavage patterns of viral DNA. The results indicate that more than one virus may colonize the same ganglion and that trigeminal ganglia may be protected from colonization by a superinfecting virus by determinants acting at a local level in the absence of demonstrable virus.     

Lack of in vitro antiviral activity of fluoroquinolones against herpes simplex virus type 2

Summary The antiviral activity against herpes simplex virus type 2 (HSV-2) of five fluoroquinolones (ciprofloxacin, lomefloxacin, ofloxacin, pefloxacin, rufloxacin) was tested in vitro. Their efficacy was evaluated as reduction of the cytopathic effect (CPER) exerted by HSV-2 on Vero cells in comparison with novobiocin and acycloguanosine. Our results show a very poor antiviral effect of five quinolones (CPER₅₀=200 mg/l) that was comparable with their cytotoxicity (TCIC₅₀<200 mg/l). Novobiocin shows a lower toxicity (TCIC₅₀=400 mg/l) and a slight antiviral activity (CPER₅₀=120 mg/l). Acycloguanosine shows a TCIC₅₀ greater than 400 mg/l and a CPER₅₀ of 3.125 mg/l. The therapeutic indices gave values ranging from 0.12 to 2 for quinolones, of 3.3 for novobiocin, and greater than 128 for acycloguanosine. The antiviral efficacy of acycloguanosine was not affected by concentrations of quinolones active against bacteria (1–10 mg/l) whereas it was drastically reduced by higher doses of quinolones (>50 mg/l). Our data suggest that fluoroquinolones cannot be considered drugs able to inhibit HSV-2 replication in vitro.     

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     

In vitro antiviral activity of dermaseptin S4 and derivatives from amphibian skin against herpes simplex virus type 2

Herpes simplex virus (HSV) infections have become a public health problem worldwide. The emergence of acyclovir‐resistant viral strains and the failure of vaccination to prevent herpetic infections have prompted the search for new antiviral drugs. Accordingly, the present study was undertaken to synthesize chemically and evaluate Dermaseptin S₄ (S₄), an anti‐microbial peptide derived from amphibian skin, and its derivatives in terms of anti‐herpetic activity. The effects of biochemical modifications on their antimicrobial potential were also investigated. The peptides were incubated together with HSV‐2 on target cells under various conditions, and the antiviral effects were examined via a cell metabolic labeling method. The findings revealed that DS₄ derivatives elicited concentration‐dependent antiviral activity at micromole concentrations. The biochemical modifications of S₄ allowed for the reduction of peptide cytotoxicity without altering antiviral activity. Dermaseptins were added at different times during the viral cycle to investigate the mode of antiviral action. At the highest non‐cytotoxic concentrations, most of the tested derivatives were noted to exhibit high antiviral activity particularly when pre‐incubated with free herpes viruses prior to infection. Among these peptides, K₄K₂₀S₄ exhibited the highest antiviral activity against HSV‐2 sensitive and resistant strains. Interestingly, the antiviral activity of K₄K₂₀S₄ was effective on both acyclovir‐resistant and ‐sensitive viruses. The findings indicate that K₄K₂₀S₄ can be considered a promising candidate for future application as a therapeutic virucidal agent for the treatment of herpes viruses. J. Med. Virol. 85:272–281, 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition of herpes simplex virus production in vitro by Cyclosporin A

Summary Nontoxic concentrations of Cyclosporin A (CyA) dose-dependently inhibited herpes simplex virus (HSV) production in resting monkey kidney cells. The block was at the step of virus DNA synthesis as assessed by [³H]thymidine incorporation and by dot blot hybridization of infected cell DNA using a cloned³²P-labelled HSV DNA fragment (BamHI X) as probe. This was further supported by analysis of HSV protein synthesis in the presence of CyA as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot. A relative accumulation of HSV - (e.g., ICP 4) and ₁-proteins (e.g., ICP 6 and 8) was found, whereas HSV ₁-proteins were slightly decreased and ₂-proteins were markedly decreased by CyA. The production of thymidine kinase and DNA polymerase was decreased when CyA was added to HSV infected cells. The sensitivity to CyA was not escaped by thymidine kinase nor DNA polymerase deficient mutants. Passage of HSV in presence of CyA did not result in induction of drug resistance.     

In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant

We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection. The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion. No other known open reading frames or flanking sequences were disrupted. Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F). The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection. There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells. This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.     

Persistent herpes simplex virus infection and mechanisms of virus drug resistance

Herpes simplex virus (HSV) is susceptible to a variety of antiviral compounds, most of which are nucleoside analogues that interfere with DNA metabolism involving the virus enzymes DNA-polymerase and thymidine kinase. Single mutations in the virus genome give rise to resistant mutants following selection in vitro in the presence of a particular drug, and in this respect HSV is similar to several other viruses. Such mutants have been invaluable research tools. HSV is responsible for a variety of lesions which tend to be recurrent, owing to the special ability of the virus to remain latent in and reactivate from neural tissue. The consequences of this upon clinical resistance are discussed in the present review. In fact, clinical resistance in HSV infections has not yet become widespread but does appear to be especially important in immunocompromised patients, including those suffering from AIDS. HSV is proposed as an important model for the investigation of drug resistance in other, more complex organisms, and with respect to antiviral strategies against the human immunodeficiency virus.     

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.     

Early interactions of herpes simplex virus with mouse peritoneal macrophages.

Adsorption of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to resident peritoneal macrophages (PM) of 4-week-old Swiss albino (SA) and GR/AFib mice was studied. A significantly (P less than 0.05) higher HSV-2 adsorption rate was found with PM of SA mice than with PM of GR/AFib mice. Of added HSV-2 65% bound to the cells of SA mice over a 120-min period versus 15% to PM of GR/AFib mice. Only 15 to 20% of added HSV-1 bound to PM regardless of the mouse strain. These patterns of adsorption were found with all four HSV-1 and four HSV-2 strains tested. Pretreatment of PM with an HSV-2 mutant blocked the adsorption of added HSV-2. Thus, the receptors for HSV attachment seemed to be virus type selective. To avoid masking of adsorption by phagocytotic activity, the adsorption studies had to be performed at 4 degrees C. Transport of attached HSV-1 and HSV-2 to the nuclei of SA PM was studied with purified virus labeled with 32Pi and [3H]thymidine. In double-isotope experiments, only transport of HSV-2 was detected. The possible importance of differences in density or avidity of virus-binding receptors on the plasma membrane of PM is discussed in relation to macrophage-dependent focal liver necrosis, which was only demonstrable after intraperitoneal inoculation of HSV-2, not HSV-1, only in SA, not GR/AFib, mice.     

Protection against herpes simplex virus infection in mice by Corynebacterium parvum.

Corynebacterium parvum administered in mice prior to herpes simplex virus (HSV) infection significantly protected them against lethal encephalitis. This was seen both with a mouse strain highly susceptible to HSV and with one relatively resistant to HSV. Mice immunosuppressed by cyclophosphamide and showing an increased mortality after HSV infection were also protected by C. parvum pretreatment. However, C. parvum given simultaneously with or after HSV infection did not exert a therapeutic effect.     

In vitro repair of the preexisting nicks and gaps in herpes simplex virus DNA.

Herpes simplex virus (HSV) DNA sediments as a homogeneous band just behind T4 DNA on neutral sucrose gradients but sediments heterogeneously on alkaline sucrose gradients. If the HSV DNA is pretreated with DNA polymerase and DNA ligase, then at least half the fragmentation seen on an alkaline sucrose gradient is prevented. Thus, it is concluded that there are preexisting nicks and gaps in HSV DNA which are repairable in vitro by enzyme treatment. To determine whether the preexisting gaps are located at unique positions or are randomly located, HSV DNA was repaired in vitro using [α-³²P]dGTP as one substrate. Restriction enzyme analysis of the repaired HSV DNA suggests that the preexisting gaps are located at random on the genome.     

Role of interferon in persistent infection of macrophages with herpes simplex virus

Splenic macrophage cultures from C57BL/6 mice resistant to infection with herpes simplex virus (HSV) in vivo survived HSV infection in vitro. In contrast, macrophages from HSV-susceptible DBA/2 mice were completely lysed by the virus. During prolonged culturing, macrophages from C57BL/6 mice continued to produce infectious virus, indicating establishment of a persistent infection. At this time, interferon (IFN) was undetectable. However, as shown directly by the addition of an anti-IFN serum and indirectly by an increased activity of (2'-5')oligoadenylate synthetase, IFN was involved in the maintenance of the persistent infection. During the acute phase of virus infection, viral DNA replication was identical in macrophages from resistant or susceptible mice. Later, viral DNA content and the number of cells expressing HSV antigens decreased in macrophages from C57BL/6 mice. However, single cells remained to express viral proteins and to produce infectious particles. The results show that macrophages can be persistently infected with HSV due to their genetically controlled properties.