Experimental Herpes Simplex Research Articles

Impact of Type I Interferon on the Safety and Immunogenicity of an Experimental Live-Attenuated Herpes Simplex Virus 1 Vaccine in Mice.

Viral fitness dictates virulence and capacity to evade host immune defenses. Understanding the biological underpinnings of such features is essential for rational vaccine development. We have previously shown that the live-attenuated herpes simplex virus 1 (HSV-1) mutant lacking the nuclear localization signal (NLS) on the ICP0 gene (0ΔNLS) is sensitive to inhibition by interferon beta (IFN-β) in vitro and functions as a highly efficacious experimental vaccine. Here, we characterize the host immune response and in vivo pathogenesis of HSV-1 0ΔNLS relative to its fully virulent parental strain in C57BL/6 mice. Additionally, we explore the role of type 1 interferon (IFN-α/β) signaling on virulence and immunogenicity of HSV-1 0ΔNLS and uncover a probable sex bias in the induction of IFN-α/β in the cornea during HSV-1 infection. Our data show that HSV-1 0ΔNLS lacks neurovirulence even in highly immunocompromised mice lacking the IFN-α/β receptor. These studies support the translational viability of the HSV-1 0ΔNLS vaccine strain by demonstrating that, while it is comparable to a virulent parental strain in terms of immunogenicity, HSV-1 0ΔNLS does not induce significant tissue pathology.IMPORTANCE HSV-1 is a common human pathogen associated with a variety of clinical presentations ranging in severity from periodic "cold sores" to lethal encephalitis. Despite the consistent failures of HSV subunit vaccines in clinical trials spanning the past 28 years, opposition to live-attenuated HSV vaccines predicated on unfounded safety concerns currently limits their widespread acceptance. Here, we demonstrate that a live-attenuated HSV-1 vaccine has great translational potential.

Protective role of CX3CR1 signalling in resident cells of the central nervous system during experimental herpes simplex virus encephalitis.

CX3CR1 is an important chemokine receptor expressed on the surface of microglia and blood leukocytes, including monocytes. Signalling through this receptor influences the immune activity of microglia and monocyte trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CX3CR1 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CX3CR1 expressed in resident cells of the CNS or peripheral monocytes in protection against HSE remains unclear. To dissect the role of CX3CR1 during HSE, we reconstituted irradiated C57BL/6 WT and CX3CR1-/- mice with CX3CR1-/- (CX3CR1-/-→WT) and WT (WT→CX3CR1-/-) bone marrow cells, respectively. Our results showed that following intranasal infection with 1.2×106 p.f.u. of HSV-1, mortality rates were significantly higher in CX3CR1-/- (61.7 %) and WT→CX3CR1-/- (66.2 %) compared to WT (16.6 %; P=0.012 and P=0.016, respectively) and CX3CR1-/-→WT animals (20 %; P=0.013 and P=0.011, respectively). Higher mortality rates in CX3CR1-/- and WT→CX3CR1-/- mice were associated with increased infectious viral titres and wider HSV dissemination in brains, as well as an overproduction of inflammatory cytokines and chemokines including IL-1β, IL-6, IFN-γ, C-C motif ligand 2 and C-C motif ligand 5. Furthermore, CX3CR1 deficiency in resident cells of the CNS resulted in excessive and sustained Ly6Chi inflammatory monocyte and neutrophil infiltration into the brain. These data suggest that CX3CR1 deficiency in resident cells of the CNS affects mouse survival, HSV-1 replication control and cerebral inflammatory response whereas its deficiency in the haematopoietic system does not appear to influence the outcome of HSE.

Both Cerebral and Hematopoietic Deficiencies in CCR2 Result in Uncontrolled Herpes Simplex Virus Infection of the Central Nervous System in Mice.

CCR2 is a chemokine receptor expressed on the surface of blood leukocytes, particularly «Ly6Chi» inflammatory monocytes and microglia. Signaling through this receptor is thought to influence the immune activity of microglia as well as monocytes egress from the bone marrow (BM) and their trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CCR2 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CCR2 expressed in cells of the CNS or peripheral monocytes in the protection against HSE remains unclear. To dissect the differential role of CCR2 during HSE, chimeric mice with receptor deficiency in the brain or blood cells were generated by transplanting wild-type (WT) C57BL/6 or CCR2-/- BM-derived cells in CCR2-/- (WT→CCR2-/-) and WT (CCR2-/-→WT) mice, respectively. Our results indicate that following intranasal infection with 1.2x106 plaque forming units of HSV-1, CCR2 deficiency in hematopoietic cells and, to a lesser extent, in CNS exacerbates the outcome of HSE. Mortality rates of CCR2-/- (71.4%) and CCR2-/-→WT (57.1%) mice were significantly higher than that of WT (15.3%; P<0.01 and P<0.05, respectively) but the difference did not reach statistical significance for WT→CCR2-/- animals (42.8%; P = 0.16). Both peripheral and CNS deficiencies in CCR2 resulted in increased infectious viral titers and wider dissemination of HSV antigens in the brain as well as an overproduction of inflammatory cytokines and chemokines including IL-1β, IL-6, CCL2, CCL3 and CCL5. Furthermore, CCR2 deficiency in the hematopoietic system altered monocytes egress from the BM and their recruitment to the CNS, which may contribute to the failure in HSV-1 containment. Collectively, these data suggest that CCR2 expressed on cells of CNS and especially on peripheral monocytes is important for the control of HSV-1 replication and inflammatory environment during experimental HSE.

Infiltration Pattern of Blood Monocytes into the Central Nervous System during Experimental Herpes Simplex Virus Encephalitis.

The kinetics and distribution of infiltrating blood monocytes into the central nervous system and their involvement in the cerebral immune response together with resident macrophages, namely microglia, were evaluated in experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE). To distinguish microglia from blood monocyte-derived macrophages, chimeras were generated by conditioning C57BL/6 recipient mice with chemotherapy regimen followed by transplantation of bone morrow-derived cells that expressed the green fluorescent protein. Mice were infected intranasally with a sub-lethal dose of HSV-1 (1.2x106 plaque forming units). Brains were harvested prior to and on days 4, 6, 8 and 10 post-infection for flow cytometry and immunohistochemistry analysis. The amounts of neutrophils (P<0.05) and «Ly6Chi» inflammatory monocytes (P<0.001) significantly increased in the CNS compared to non-infected controls on day 6 post-infection, which corresponded to more severe clinical signs of HSE. Levels decreased on day 8 for both leukocytes subpopulations (P<0.05 for inflammatory monocytes compared to non-infected controls) to reach baseline levels on day 10 following infection. The percentage of «Ly6Clow» patrolling monocytes significantly increased (P<0.01) at a later time point (day 8), which correlated with the resolution phase of HSE. Histological analysis demonstrated that blood leukocytes colonized mostly the olfactory bulb and the brainstem, which corresponded to regions where HSV-1 particles were detected. Furthermore, infiltrating cells from the monocytic lineage could differentiate into activated local tissue macrophages that express the microglia marker, ionized calcium-binding adaptor molecule 1. The lack of albumin detection in the brain parenchyma of infected mice showed that the infiltration of blood leukocytes was not necessarily related to a breakdown of the blood-brain barrier but could be the result of a functional recruitment. Thus, our findings suggest that blood monocyte-derived macrophages infiltrate the central nervous system and may contribute, with resident microglia, to the innate immune response seen during experimental HSE.

Influence of Antisense Oligonucleotides Targeting Tumor Necrosis Factor-α on Experimental Herpetic-Induced Chorioretinitis of Mouse Eye

Objective: To investigate the effect on the pathological process of topical treatment with antisense oligonucleotides (ASON) targeting tumor necrosis factor-α (TNF-α) in experimental herpes simplex virus type-1 (HSV-1)-induced chorioretinitis of mouse eye. Methods: 50 BALB/c mice were randomly divided into two different groups (25 mice in each group). The Von Szily model was induced in mice by inoculating 1×10⁵ plaque-forming units of HSV-1 (KOS strain) into the anterior chamber of the right eye. In one group, FITC-labeled ASON targeting TNF-α was injected subconjunctively into the left eye on days –1, 1, and 4, whereas phosphate buffer was used in the same way on the same days in another group. The clinical appearances were observed after infection each day and the eyes were examined histologically. The TNF-α content of retina and choroid were measured by ELISA. Results: After infection, acute inflammation appeared in the right eye of both groups. As for the non-inoculated eye, the inflammation in the ASON group was significantly decreased compared to the PBS group. The number of inflammatory cells in the ASON group was significantly lower than in the PBS group, especially in the choroid, retina and ciliary body. The TNF-α content in the choroid and retina of the ASON group was diminished. Conclusion: The results suggested that TNF-α ASON reduced the content of TNF-α in mouse eyes, and this topical treatment decreased the inflammatory reaction. It may be an effective method for treating HSV-1-induced chorioretinitis in the clinic.

Sudden Sensorineural Hearing Loss: Otolaryngologic and Audiologic Options

Sudden Sensorineural Hearing Loss: Otolaryngologic and Audiologic Options

Topical Resiquimod 0.01% Gel Decreases Herpes Simplex Virus Type 2 Genital Shedding: A Randomized, Controlled Trial

Resiquimod, an investigational immune response modifier and Toll-like receptor (TLR) 7 and 8 agonist, stimulates production of cytokines that promote an antigen-specific T helper type 1 (Th1)--acquired immune response. In animal models, induction of Th1-specific responses modifies experimental herpes simplex virus (HSV) infection. We conducted a randomized, double-blind, vehicle-controlled trial to assess the efficacy of resiquimod 0.01% gel for reducing human anogenital HSV-2 mucosal reactivation. Adults with genital HSV-2 applied resiquimod or vehicle topically to herpes lesions 2 times weekly for 3 weeks and then collected daily anogenital swabs for 60 days for HSV DNA polymerase chain reaction. Recurrences during the subsequent 7 months were treated with study gel. During the final treatment-free 60 days, participants again collected daily swabs to assess shedding. The median lesion and shedding rates were lower for resiquimod compared with vehicle recipients during the initial sampling period (10% vs. 16% [P=.03] and 10% vs. 17% [P=.08], respectively) and during the final sampling period (3% vs. 22% [P<.001] and 10% vs. 26% [P=.009], respectively). Resiquimod did not influence recurrence length. These findings suggest that the immunological control of HSV-2 reactivation and lesion clearance may differ and that TLR7 and TLR8 agonists can reduce the frequency of mucosal HSV-2 reactivation.

Delayed but Not Early Glucocorticoid Treatment Protects the Host during Experimental Herpes Simplex Virus Encephalitis in Mice

Glucocorticoids (GCs) have not been established as an evidence-based therapy for herpes simplex virus (HSV) encephalitis. We sought to evaluate the effects of early versus delayed GC administration in a model of HSV-1 encephalitis. Mice were inoculated intranasally with a clinical HSV-1 strain and had access to corticosterone (Cort; 0.2 mg/mL) in drinking water or received dexamethasone (DEX; 10 mg/kg) intraperitoneally. Cort was started immediately or 72 h after infection, whereas DEX was administrated 3 days after infection. Expression of the thymidine kinase transcript indicated widespread viral replication in the brain stem and many regions of the mediovestibular system. This neurovirulence provoked neuronal death and transcriptional activation of several immune genes. Notably, Toll-like receptors 2, 3, and 9 transcripts were strongly up-regulated. Mean life expectancy was higher in the group of mice that received delayed Cort (56%) than in the mice that received no Cort (44%) or early Cort treatments (13%) (P<.05, early vs. delayed or no Cort treatments). Delayed DEX treatment suppressed not only the expression of inflammatory genes as expected but also that of viral genes. Administration of GCs at a critical time during viral infection is associated with neuroprotection and survival in experimental HSV-1 encephalitis.

Aquaporin 4 regulation during acute and long-term experimental Herpes simplex virus encephalitis

Structural damage of the central nervous system (CNS) often leads to severely disabling residual symptoms despite effective antiviral therapy during Herpes simplex virus encephalitis (HSVE). Edematous space-occupying lesions are pathological and neuroradiological well-known phenomena for this disease. The molecular mechanisms of brain edema development in HSVE are poorly understood, the regulation of water brain-blood barrier (BBB) permeability might be disturbed. Aquaporin 4 (AQP4) is the predominant aquaporin expressed in the brain. Aquaporin 1 (AQP1) plays a role in cerebrospinal fluid modulation. Previous studies suggest that alterations of AQP expression play an important role in the development of brain edema. The mRNA expression of AQP4, AQP1, of their physiologically associated proteins Alpha-syntrophin and KIR 4.1 and of the structural glial protein glial fibrillary acid protein (GFAP) was analyzed in a well-established mice model simulating the human disease. Our data demonstrate a significant down-regulation of AQP4 in the acute phase of disease and an up-regulation of AQP4 and AQP1 in the long term. These results reveal the complex transcription pattern of AQP4, AQP1, KIR 4.1, alpha-syntrophin, and GFAP during HSVE and suggest a role for AQP4 regulation in the pathophysiology of acute and long-term HSVE. AQP4 modulation could be a potential target for brain edema treatment during HSVE.

Differential mRNA expression of neurotrophic factors GDNF, BDNF, and NT-3 in experimental herpes simplex virus encephalitis

Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNA levels were studied in the course of murine herpes simplex virus encephalitis. Induction of GNDF and NT-3 (both P < 0.05) was found during acute encephalitis. Despite absence of clinical impairment, both neurotrophic factors were overexpressed 2 months (NT-3) and 6 months (GDNF) following infection (both P < 0.05). Neurotrophic factors play an important role in neuronal survival and recovery after acute injury to the central nervous system (CNS) and may represent an additional therapeutic target for treatment of viral encephalitis.

Increased presence of matrix metalloproteinases 2 and 9 in short- and long-term experimental herpes simplex virus encephalitis

Herpes simplex virus encephalitis (HSVE) causes elevated morbidity and mortality despite antiviral treatment. Virus-independent mechanisms may perpetuate brain damage. Matrix metalloproteinases (MMPs) target extracellular matrix components. This study describes the protein and mRNA expression of MMP2 and MMP9 in experimental HSVE in the short and long term. Ten SJL-NBOM mice were infected with neurovirulent HSV-1 and compared with nine controls. The presence of MMP2 and MMP9 in brain tissue was analyzed with sodium-dodecyl-sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gelatin zymography and mRNA expression of MMP2 and MMP9 with quantitative real-time PCR at days 7, 21 and 180 post-inoculation. Infected animals had a significantly elevated gelatinolytic activity of MMP2 at all time points, and of MMP9 at days 21 and 180. Increased presence of MMP2 and MMP9 in chronic HSVE may contribute to ongoing damage. Inhibition of MMP2 and MMP9 might be a suitable target for therapeutic intervention.

Experimental herpes simplex virus encephalitis: a long-term study of interleukin-6 expression in mouse brain tissue

This study aimed to investigate the expression of interleukin-6 (IL-6) in acute and chronic herpes simplex virus encephalitis. In the brain of 15 SJL mice infected with herpes simplex virus type 1, strain F, and 14 control animals we performed a sequential quantitative analysis of expression of IL-6 mRNA with reverse transcription real-time polymerase chain reaction. The viral burden peaked in the acute disease, and then returned to a low baseline value. At day 7 following infection, IL-6 expression was significantly (2.05-fold) increased as compared with the baseline expression in uninfected animals. Twenty-one days after infection the mRNA expression still was significantly (1.78-fold) upregulated. No significant differences of IL-6 mRNA expression between infected and control mice were found after 2 and 6 months. We observed a 2.5-fold increase of IL-6 mRNA expression in control mice with increasing age of animals. We have additionally studied the clinical evolution of HSVE in IL-6 deficient mice. In experimental herpes simplex virus encephalitis IL-6, as a potent mediator of neuronal injury, is upregulated in the acute but not in the chronic disease. IL-6 deficient mice presented early and severe clinical signs of HSVE as compared to the wild-type C57/bl6 mice.

Expression of matrix metalloproteinases in experimental herpes simplex virus keratitis

To determine the distribution and activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus type-1 (HSV-1) keratitis. Keratitis was induced in BALB/c mice by inoculating the cornea with 10(5) plaque-forming units (pfu) of HSV-1 (KOS strain). Corneas were harvested at days 0, 2, 7, 14 and 28 post-infection. Expression of MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and Western blot. The enzymatic activities were analyzed by Zymography. At day 2 post-infection, MMP-2 and MMP-9 expression were increased in the epithelium as compared to the uninfected control eyes, and were detected in the superficial stroma and in inflammatory cells beneath the epithelium. Similar staining patterns were detected for TIMP-1 and TIMP-2. MMP-2 and MMP-9 epithelial staining persisted until day 28 post-infection. Necrotizing keratitis with corneal ulceration was present on days 14 and 28 post-infection. This correlated with increased expression of MMP-2 and MMP-9 within the stroma and in infiltrating inflammatory cells. MMP-2, MMP-9, TIMP-1 and TIMP-2 staining were particularly intense in the proximity of the ulcers. The neutrophils, which were abundant at the site of ulceration, were stained positive with MMP-8. Both gelatinolytic activities and caseinolytic activities were upregulated after HSV-1 corneal infection. Our data suggest that MMPs produced by resident corneal cells and by inflammatory cells possibly promote epithelial keratitis and ulcerative process after corneal HSV-1 infection. The interaction of MMPs and TIMPs may regulate the course of necrotizing HSV keratitis.

Blocking immune evasion as a novel approach for prevention and treatment of herpes simplex virus infection.

Many microorganisms encode immune evasion molecules to escape host defenses. Herpes simplex virus type 1 glycoprotein gC is an immunoevasin that inhibits complement activation by binding complement C3b. gC is expressed on the virus envelope and infected cell surface, which makes gC potentially accessible to blocking antibodies. Mice passively immunized with gC monoclonal antibodies prior to infection were protected against herpes simplex virus challenge only if the gC antibodies blocked C3b binding. Mice treated 1 or 2 days postinfection with gC monoclonal antibodies that block C3b binding had less severe disease than control mice treated with nonimmune immunoglobulin G (IgG). Mice immunized with gC protein produced antibodies that blocked C3b binding to gC. Immunized mice were significantly protected against challenge by wild-type virus, but not against a gC mutant virus lacking the C3b binding domain, suggesting that protection was mediated by antibodies that target the gC immune evasion domain. IgG and complement from subjects immunized with an experimental herpes simplex virus glycoprotein gD vaccine neutralized far more mutant virus defective in immune evasion than wild-type virus, supporting the importance of immune evasion molecules in reducing vaccine potency. These results suggest that it is possible to block immune evasion domains on herpes simplex virus and that this approach has therapeutic potential and may enhance vaccine efficacy.

Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis

To determine the distribution and activities of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus (HSV) type-1 keratitis, BALB/c mice were corneally infected with 10 5 plaque-forming units (PFU) of HSV-1 (KOS strain) and then observed for the clinical signs of keratitis. Corneas were harvested at days 0, 2, 7 and 14 post-infection (p.i.). MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and the Western blot technique. The enzymatic activities were analyzed by zymography. Epithelial HSV keratitis was present at day 2 after corneal infection and healed by day 5 p.i. While the expression and activity of MMP-2, MMP-8 and MMP-9 increased in the corneas at day 2 p.i., it was reduced at day 7 p.i. TIMP-1 and -2 were expressed in the corneas before and seven days after infection. Necrotizing stromal keratitis with corneal ulceration and dense polymorphonuclear leukocyte (PMN) infiltration was present at day 14 p.i. This correlated with increased expression of MMP-2, MMP-8 and MMP-9 in the corneas. MMP-8, MMP-9 and MMP-2 staining was particularly intense in the proximity of the ulcers and in areas of PMN infiltration. At day 14 p.i., MMP-2, -8 and -9 activities were upregulated, and TIMP-2 was expressed. These data suggest that MMPs produced by resident corneal cells and PMNs may possibly play a role in early epithelial keratitis and in the ulcerative process in the late phase after corneal HSV-1 infection. The ratio of MMPs to TIMPs may be important for the course of necrotizing HSV keratitis. TIMPs might participate in the repair process.

A novel permissive role for glucocorticoids in induction of febrile and behavioral signs of experimental herpes simplex virus encephalitis

Herpes simplex virus type 1 (HSV-1) encephalitis may present with fever and behavioral changes, to the extent of a psychotic state and psychomotor agitation. We developed a clinically relevant experimental model of HSV-1 encephalitis and investigated host brain responses associated with its clinical signs and whether these responses depend on the presence of circulating glucocorticoids. Intracerebral inoculation of HSV-1 in rats induced fever, motor hyperactivity and aggressive behavior. In adrenalectomized rats HSV-1 failed to induce these signs, although mortality rate was identical to sham-operated rats. Hypophysectomy or blocking glucocorticoid receptors also prevented HSV-1-induced fever. Dexamethasone replacement therapy to adrenalectomized rats restored the HSV-1-induced fever and behavioral abnormalities. HSV-1 inoculation produced hyperproduction of prostaglandin E 2 by brain slices. This effect was abolished in adrenalectomized rats and was restored by dexamethasone treatment. In intact rats HSV-1 induced brain interleukin-1β (IL-1β) gene expression. Adrenalectomy alone caused brain IL-1β expression, which did not increase after HSV-1 infection. Similarly, HSV-1 induced IL-1β expression in astrocyte cultures. Removal of cortisol from the culture medium caused basal IL-1β mRNA expression which was not increased by infection. In conclusion, fever, motor hyperactivity and aggressive behavior during experimental HSV-1 encephalitis are dependent on brain responses, including prostaglandin E 2 and IL-1β synthesis. Circulating glucocorticoids play an essential permissive role in the induction of these host brain responses.

Experimental herpes simplex virus encephalitis: inhibition of the expression of inducible nitric oxide synthase in mouse brain tissue

In the brain tissue of 36 mice infected with herpes simplex virus type 1, strain F, we determined the expression of inducible nitric oxide synthase (iNOS) with semiquantitative reverse transcription polymerase chain reaction. The viral burden was quantitated by polymerase chain reaction. Nitric oxide, induced by iNOS, may contribute to neuronal cell damage following virus infection. As the experimental therapeutic strategy in herpes simplex virus encephalitis (HSVE), we used: N-nitro- l-arginin (L-NA), a selective inhibitor of iNOS; and combination therapies of either methylprednisolone/acyclovir or L-NA/acyclovir. The viral burden peaked in acute disease, and then returned to a low baseline value, except in untreated controls. The expression of iNOS mRNA was suppressed by L-NA and by acyclovir/corticosteroids. INOS inhibition may provide an additional therapeutic strategy targeted specifically to suppress iNOS expression as a potential secondary mechanism of tissue damage in acute and chronic HSVE.

Therapy of idiopathic sudden sensorineural hearing loss: antiviral treatment of experimental herpes simplex virus infection of the inner ear.

Experimental herpes simplex virus type 1 (HSV-1) labyrinthitis provides a model of idiopathic sudden sensorineural hearing loss (ISSHL). Corticosteroids improve the prognosis for hearing recovery in ISSHL, but the effects of acyclovir are unknown. To establish the therapeutic efficacy of acyclovir (Zovirax) and prednisolone in experimental HSV-1 viral labyrinthitis, we induced HSV-1 labyrinthitis in 12 guinea pigs. Three animals received no treatment, 3 received prednisolone, 3 received acyclovir, and 3 received both. Four other animals served as controls, receiving culture medium only. Hearing, HSV-1 antibody titers, and cochlear damage were evaluated. The HSV-1 labyrinthitis caused hearing loss within 24 hours. Combination treatment consisting of prednisolone and acyclovir resulted in earlier hearing recovery and less extensive cochlear destruction compared to prednisolone or acyclovir as a monotherapy. The beneficial effect of this treatment modality remains to be demonstrated in ISSHL.

Herpes simplex virus encephalitis: cranial magnetic resonance imaging and neuropathology in a mouse model

We performed a long-term magnetic resonance imaging (MRI) study in a mouse model of herpes simplex virus encephalitis. Mice were infected with herpes simplex virus type 1 (HSV-1) strain F. A 1.5-T cranial MRI scanner with standard spin-echo sequences was used. Neuropathological studies included immunohistochemistry. The presence of HSV DNA in brain tissue was determined with a polymerase chain reaction assay. Clinical assessment was performed daily: within the first 2 weeks the animals were severely affected and recovered thereafter. MRI and histopathological abnormalities corresponded well. HSV DNA was detectable initially and at 6 months. Extent and severity of structural abnormalities increased at 6 months. MRI offers a new in vivo approach for the detection of structural changes in the disease course of experimental herpes simplex virus encephalitis.

Herpes simplex virus encephalitis: long-term comparative study of viral load and the expression of immunologic nitric oxide synthase in mouse brain tissue

In the brain tissue of 21 mice infected with herpes simplex virus type 1 (HSV-1) strain F we determined the expression of immunologic nitric oxide synthase (iNOS) as a potential mediator of neuronal injury with a semiquantitative reverse transcription polymerase chain reaction. Viral burden in brain tissue was quantitated with a dilutional polymerase chain reaction assay. Viral burden and iNOS-expression peaked at day 7 following infection. Thereafter viral burden declined to a low baseline value at 6 months following infection, whereas iNOS-expression was still 4-fold increased compared to baseline levels. In experimental herpes simplex virus encephalitis iNOS, as one potent mediator of neuronal injury, is upregulated in the acute and chronic disease. In future, in addition to antiviral treatment, inhibitors of iNOS might offer new therapeutic strategies in herpes simplex virus encephalitis.