Ectopic expression of gamma interferon in the eye protects transgenic mice from intraocular herpes simplex virus type 1 infections.

Transgenic (rho gamma) mice provide a model for studying the influence of gamma interferon (IFN-gamma) produced in the eye on ocular and cerebral viral infection. To establish this model, we injected BALB/c- and C57BL/6-derived transgenic and nontransgenic mice of different ages intravitreally with herpes simplex virus type 1 (HSV-1) strain F. Eye and brain tissues of these mice were assessed for pathological and immunocytochemical changes. HSV-1 infection induced severe retinitis of the injected eyes and infection of the brain in all mice. In transgenic mice inoculated with HSV-1, the left, nontreated eyes were protected from retinitis, whereas nontransgenic mice developed bilateral retinitis. Additional intravitreal injection of IFN-gamma with the virus protected the noninoculated eyes of nontransgenic mice. Three-week-old nontransgenic mice died from HSV-1 infection, whereas transgenic mice of the same age and nontransgenic mice intravitreally treated with IFN-gamma survived. Ocular IFN-gamma production increased the extent of inflammation in transgenic mice but did not have a significant influence on the growth of HSV-1 until day 3 after inoculation and did not influence the neuroinvasion of this virus. Thus, the effects of IFN-gamma were not caused by an early block of viral replication. Possible mechanisms of IFN-gamma action include activation of the immune response, alteration of the properties of the virus, and direct protection of neurons.     

IL-18, but not IL-12, regulates NK cell activity following intranasal herpes simplex virus type 1 infection

Infection of the respiratory tract with HSV type 1 (HSV-1) can have severe clinical complications, yet little is known of the immune mechanisms that control the replication and spread of HSV-1 in this site. The present study investigated the protective role of IL-12 and IL-18 in host defense against intranasal HSV-1 infection. Both IL-12 and IL-18 were detected in lung fluids following intranasal infection of C57BL/6 (B6) mice. IL-18-deficient (B6.IL-18(-/-)) mice were more susceptible to HSV-1 infection than wild-type B6 mice as evidenced by exacerbated weight loss and enhanced virus growth in the lung. IL-12-deficient (B6.IL-12(-/-)) mice behaved similarly to B6 controls. Enhanced susceptibility of B6.IL-18(-/-) mice to HSV-1 infection correlated with a profound impairment in the ability of NK cells recovered from the lungs to produce IFN-gamma or to mediate cytotoxic activity ex vivo. The weak cytotoxic capacity of NK cells from the lungs of B6.IL-18(-/-) mice correlated with reduced expression of the cytolytic effector molecule granzyme B. Moreover, depletion of NK cells from B6 or B6.IL-12(-/-) mice led to enhanced viral growth in lungs by day 3 postinfection; however, this treatment had no effect on viral titers in lungs of B6.IL-18(-/-) mice. Together these studies demonstrate that IL-18, but not IL-12, plays a key role in the rapid activation of NK cells and therefore in control of early HSV-1 replication in the lung.     

Gender influences herpes simplex virus type 1 infection in normal and gamma interferon-mutant mice

Gender influences the incidence and severity of some bacterial and viral infections and autoimmune diseases in animal models and humans. To determine a gender-based difference, comparisons were made between male and female mice inoculated with herpes simplex virus type 1 (HSV-1) by the corneal route. Mortality was higher in the male mice of the three strains tested: 129/Sv//Ev wild type, gamma interferon (IFN-gamma) knockout (GKO), and IFN-gamma receptor knockout (RGKO). Similarly, in vivo HSV-1 reactivation occurred more commonly in male mice, but the male-female difference in reactivation was restricted to the two knockout strains and was not seen in the 129/Sv//Ev control. Comparison among male mice of the three strains showed a higher mortality of the RGKO mice and a higher reactivation rate of the GKO and RGKO mice than of the 129/Sv//Ev males. In contrast, female RGKO and GKO mice did not differ from female 129/Sv//Ev controls in either mortality or reactivation. HSV-1 periocular and eyelid disease was also more severe in male and dihydrotestosterone (DHT)-treated female mice than in control female mice. These results show a consistent gender difference in HSV-1 infection, with a worse outcome in male mice. In addition, the results comparing GKO and RGKO mice to controls show differences only in male mice, suggesting that some effects of IFN-gamma, a key immunoregulatory molecule, are gender specific.     

Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice

Mouse models of herpes simplex virus type 1 (HSV-1) infection provide significant insights into viral and host genes that regulate disease pathogenesis, but conventional methods to determine the full extent of viral spread and replication typically require the sacrifice of infected animals. To develop a noninvasive method for detecting HSV-1 in living mice, we used a strain KOS HSV-1 recombinant that expresses firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase reporter proteins and monitored infection with a cooled charge-coupled device camera. Viral infection in mouse footpads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. The activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes but not following the systemic delivery of substrate. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Treatment of infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase activity that correlated with changes in viral titers. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.     

Hematogenous vertical transmission of herpes simplex virus type 1 in mice

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that causes severe disease and death in newborn humans but, to date, it remains unclear how neonatal infection occurs. We show here that the vertical transmission of HSV-1 in mice is mainly hematogenous and involves the colonization of the neonate central nervous system (CNS). HSV-1 DNA was mainly detected in the blood and CNS of the offspring born to latently infected mothers; no significant differences were seen between the viral DNA concentrations in the blood of these mothers and their female progeny (either neonate or adult). The administration of acyclovir during gestation reduced or eliminated both the maternal and the neonatal viral DNA in the blood. Embryo transfer was performed to ensure (as far as possible) that only vertical hematogenous infection took place. Immunohistochemical analysis detected viral proteins in the encephalon of the offspring. Immunofluorescence studies provided immunoreactive evidence of HSV-1 proteins in the neurons of the hippocampus and showed that these viruses can molecularly reactivate after hyperthermia. Neonatal HSV-1 infection therefore appears to be mainly caused by hematogenous vertical transmission, and the viruses that colonize the offspring CNS are capable of molecular reactivation after a period of latency.     

Pathogenesis of herpes simplex virus type 2 experimental genital infection in pregnant mice

The progression of herpes simplex-2 genital infection in pregnant mice was studied by detection of viral antigens using immunoperoxidase in tissue sections, electron microscopy and virus isolation. The majority of mice (66.66%) died at 8-9 days post-inoculation. Abortions were observed in 69.23% of the infected mice along with impairment of labor and delivery. Herpes antigens were detected in most of the autonomic nerves of the uterus, including those surrounding small arterioles in the myometrium and the Auerbach and Meissner plexa of the large bowel, but not in the abortions or placentas. The infection of uterine autonomic fibers and myometrial cells could explain the delivery impairment and could have provoked a decrease in blood flow leading to abortions.     

Alteration of ribosomal protein maps in herpes simplex virus type 1 infection

At present, the effect of herpes simplex virus infection on the entire proteomes of infected cells is very poorly documented. Following several studies performed over the past few years, the modifications of a sub-cellular fraction induced by herpes simplex virus type 1 can be documented. These studies were performed in order to characterize the virally-induced modifications of a major component of the translational apparatus, the ribosomes. The very basic nature of most of the ribosomal proteins renders them very difficult to separate using isoelectric focusing (IEF). Therefore these studies were achieved using several different but related two-dimensional electrophoretic systems which allowed several two-dimensional ribosomal protein maps to be built. Comparison of the ribosomal protein maps built from non-infected cells with those built from infected cells demonstrated that infection by herpes simplex virus type 1 (HSV-1) induces important modifications of ribosomes: (i) non-reversible phosphorylation of ribosomal protein S6; (ii) unusual phosphorylation of several proteins of the small and the large subunits; and (iii) association of viral and cellular proteins to the ribosomal fraction. An overview of these published studies is presented in this review.     

Therapeutic protective effects of IL-12 combined with soluble IL-4 receptor against established infections of herpes simplex virus type 1 in thermally injured mice.

The effect of combination therapy between IL-12 and soluble IL-4R (sIL-4R) on the established infection of HSV-1 in thermally injured mice (TI mice) was investigated. All of the TI mice infected with lethal amounts of HSV-1 died when IL-12 was given therapeutically at a dose of 500 U/mouse. However, 80% of these mice treated prophylactically with IL-12 survived compared with 0% survival of the same mice treated with saline. The therapeutic administration of IL-12 to TI mice currently infected with HSV-1 caused an 80% survival of these mice when the treatment was combined with sIL-4R. Although IL-12 did not stimulate IFN-gamma production in cultures of splenic T cells from TI mice, IFN-gamma was produced by stimulation with IL-12 when the producer cells were prepared from TI mice that had been treated previously with sIL-4R. After stimulation with anti-CD3 mAb, splenic T cells from TI mice with the established infection of HSV-1 produced IL-4 into their culture fluids. However, IL-4 was not produced by splenic T cells that were prepared from the same infected mice treated with IL-12 and sIL-4R in combination. The results obtained herein indicate that the efficacies of the combination therapy against the established infection of HSV-1 may result from the IFN-gamma production stimulated by IL-12 in TI mice that are treated with sIL-4R for reducing burn-associated type 2 T cell responses.     

The immune response to ocular herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) is a prevalent microbial pathogen infecting 60% to 90% of the adult world population. The co-evolution of the virus with humans is due, in part, to adaptations that the virus has evolved to aid it in escaping immune surveillance, including the establishment of a latent infection in its human host. A latent infection allows the virus to remain in the host without inducing tissue pathology or eliciting an immune response. During the acute infection or reactivation of latent virus, the immune response is significant, which can ultimately result in corneal blindness or fatal sporadic encephalitis. In fact, HSV-1 is one of the leading causes of infectious corneal blindness in the world as a result of chronic episodes of viral reactivation leading to stromal keratitis and scarring. Significant inroads have been made in identifying key immune mediators that control ocular HSV-1 infection and potentially viral reactivation. Likewise, viral mechanisms associated with immune evasion have also been identified and will be discussed. Lastly, novel therapeutic strategies that are currently under development show promise and will be included in this review. Most investigators have taken full advantage of the murine host as a viable working in vivo model of HSV-1 due to the sensitivity and susceptibility to viral infection, ease of manipulation, and a multitude of developed probes to study changes at the cellular and molecular levels. Therefore, comments in this review will primarily be restricted to those observations pertaining to the mouse model and the assumption (however great) that similar events occur in the human condition.     

Suppression of proinflammatory cytokine expression by herpes simplex virus type 1

Viral immune evasion strategies are important for establishment and maintenance of infections. Many viruses are in possession of mechanisms to counteract the antiviral response raised by the infected host. Here we show that a herpes simplex virus type 1 (HSV-1) mutant lacking functional viral protein 16 (VP16)-a tegument protein promoting viral gene expression-induced significantly higher levels of proinflammatory cytokines than wild-type HSV-1. This was observed in several cell lines and primary murine macrophages, as well as in peritoneal cells harvested from mice infected in vivo. The enhanced ability to stimulate cytokine expression in the absence of VP16 was not mediated directly by VP16 but was dependent on the viral immediate-early genes for infected cell protein 4 (ICP4) and ICP27, which are expressed in a VP16-dependent manner during primary HSV infection. The virus appeared to target cellular factors other than interferon-induced double-stranded RNA-activated protein kinase R (PKR), since the virus mutants remained stronger inducers of cytokines in cells stably expressing a dominant-negative mutant form of PKR. Finally, mRNA stability assay revealed a significantly longer half-life for interleukin-6 mRNA after infection with the VP16 mutant than after infection with the wild-type virus. Thus, HSV is able to suppress expression of proinflammatory cytokines by decreasing the stability of mRNAs, thereby potentially impeding the antiviral host response to infection.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins. It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation. We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC). Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the Escherichia coli beta-galactosidase coding sequence were generated. Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins. The anatomic locations of beta-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus. Trigeminal ganglion neurons were positive for beta-galactosidase in adult ICP0 and ICP27 reporter transgenic mice. The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age. beta-galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody. Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for beta-galactosidase labeling in the absence of HSV-1. We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins. These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons.