The dual role of CD8+ T lymphocytes in the development of stress-induced herpes simplex encephalitis

Despite the generally restrictive nature of the blood–brain barrier (BBB), circulating lymphocytes can infiltrate into the central nervous system (CNS) during a variety of disease states. Although the contributions of these lymphocytes to CNS-associated disease have been identified in some viral models, the factors which govern this infiltration following herpes simplex virus (HSV) infection remain to be elucidated. We have developed a murine model of HSV encephalitis (HSE) to define the relationship among psychological stress, the recruitment of HSV-specific T cells into the CNS, and the development of HSE. Naive mice, as well as mice that had been vaccinated with a recombinant vaccinia virus (rVVESgB498–505) that elicits the generation of HSV-1 gB498–505-specific CD8⁺ T cells, were infected intranasally (i.n.) with HSV-1 McIntyre. Beginning one day prior to HSV-1 infection and continuing for a total of 9 days, naive and vaccinated mice were exposed to a well-established stressor, restraint stress. Naive, stressed mice exhibited increased symptoms of HSE and HSE-associated mortality as compared to non-stressed controls. A concomitant increase in CD4⁺ and CD8⁺ T cells in the brain was observed throughout the infection, with CD8⁺ T cells outnumbering CD4⁺ T cells. The development of HSE in these naive, stressed mice was accompanied by a delayed infiltration of gB498–505-specific CD8⁺ T cells after HSV spread into the brain. In contrast, both stressed and non-stressed rVVESgB498–505-vaccinated mice possessed gB498–505-specific CD8⁺ T cells prior to HSV challenge and were protected against HSE despite having detectable HSV-1 DNA in the brain. Together, these findings suggest that a delayed infiltration of CD8⁺ T cells into the brain may promote HSE in naive mice, while the presence of HSV-specific CD8⁺ T cells in the brain prior to HSV challenge is protective, possibly by limiting HSV replication and spread within the CNS.     

A polymerase chain reaction assay of cerebrospinal fluid in patients with suspected herpes simplex encephalitis.

A polymerase chain reaction (PCR) was used to detect herpes simplex virus (HSV) deoxyribonucleic acid (DNA) in CSF of 109 patients with possible herpes simplex encephalitis. HSV DNA was found in 20/109 patients. In 14 of these patients the diagnosis was confirmed by a rise in CSF antibodies, isolation of HSV from the brain, or both. In 3 patients CSF antibodies did not rise and 3 patients did not have a follow up lumbar puncture or a brain biopsy. In 19/20 patients HSV DNA was present in the first CSF specimen. The virus was identified as HSV I in 15 patients and HSV II in 4; the virus was not typed in the other patient. A possible diagnosis of herpes simplex encephalitis was not confirmed in the 89 PCR-negative patients. HSV DNA was present in CSF of 3 patients who had meningitis with herpetic genital infections but it was not found in 24 patients with other neurological diseases. The results suggest that the detection of HSV DNA in CSF using a PCR assay will be an accurate method of early diagnosis of herpes simplex encephalitis.     

Innate and adaptive host response during the initial phase of herpes simplex virus encephalitis in the neonatal mouse

To study early events of neonatal herpes simplex virus (HSV) encephalitis and its sequelae, the authors induced a controlled infection in the brains of mice using HSVgH, a genetically modified Disabled Infective Single Cycle virus. Neonatal Balb/C mice were infected with various amounts of HSVgH- virus by intracerebral injection. Results showed that the survival of infected mice was dependent on the amount of virus injected. Infection with 200,000 plaque forming units (pfu) of HSVgH-, virus resulted in 0% survival, whereas 25,000 pfu resulted in 75% survival. If the mice died, 98% of the deaths occurred between 3 and 7 days after infection. Replication competent virus was recovered from 20% of mice brains infected with 25,000 pfu of HSVgH-. Neutralizing antibodies were not detected 6 weeks post infection in sera of mice, which survived infection with 25,000 pfu of HSVgH-. LacZ histochemistry and immunoperoxidase staining using anti-HSV and anti- beta-galactosidase antibodies revealed that the infection was limited to the site of injection. Tissue destruction was observed at the site of inoculation 3 days post infection using cresyl violet staining. At 3 days post infection adjacent sections showed positive cells for viral antigens and apoptotic cells in the infected area. Immunoperoxidase staining using antibodies to surface markers showed microglial activation beginning on day 1 and astrocyte proliferation beginning on day 3 post infection. B and T lymphocytes were not detected on day 1 through 7 post infection. This controlled experimental HSV infection suggests a limited non-specific early host response in the neonate to HSV encephalitis.     

Herpes-simplex-related antigen in human demyelinative disease and encephalitis

Summary Using immunohistochemical methods optimized to detect herpes simplex virus type 2 (HSV-2) antigen, paraffin sections from human central nervous system tissues from 31 cases pathologically diagnosed as multiple sclerosis (MS), 34 cases of other neurological diseases, 4 adult cases of HSV encephalitis, and mouse brains infected with various HSV strains were examined. Two distinct patterns of immunoreactivity with HSV antisera were seen. In typical acute human and experimental encephalitis, antigen was readily detected using high dilutions of antisera to both HSV types –1 and –2, and was found nonselectiviely in both neurons and glia. Lesions were destructive, with necrosis of all neural cell types, and inflammation was a mixture of polymorphonuclear and mononuclear cells. By contrast, immunoreactivity in lesions in each of three MS cases and in one case of brain stem encephalitis was found only with HSV-2 antisera, and relatively high antiserum concentrations were required to detect it. Reactivity appeared to be largely restricted to glial cell nuclei within and near lesions that were selectively demyelinated. Only mononuclear inflammation was present. These experiments suggest that HSV-related antigen may be found in a broader spectrum of human CNS lesions than has previously been recognized, and that HSV or a related agent may be associated with a selective infection of glial cells and with CNS demyelination.     

Intranasal herpes simplex virus type 2 inoculation causes a profound thymidine kinase dependent cerebral inflammatory response in the mouse hindbrain

The herpes simplex virus (HSV) has the ability to replicate in the central nervous system (CNS), which may cause fatal encephalitis. The present study investigated the activity of the nuclear factor kappa B (NF-kappa B) and the pattern of cytokine/chemokine gene expression across the brain of HSV-infected mice and the role of the viral thymidine kinase (TK) in mediating these effects. Mice were killed 1-8 days after intranasal inoculation with either HSV-2 TK-competent or TK-deficient clinical isolates. Animals infected with the TK-competent virus exhibited first signs of infection at day 5 postinoculation, whereas severe signs of sickness were observed between day 6 and 8. A robust hybridization signal was found in the brain of these animals for the gene encoding the inhibitory factor kappa B alpha (I kappa B alpha, index of NF-kappa B activity), toll-like receptor 2 (TLR2), tumour necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) in numerous regions of the pons and medulla. The levels of expression of these genes increased 4 days after the inoculation and peaked at day 6 within the endothelium of the brain capillaries and cells of myeloid origin. A robust signal for the TK gene and its encoding protein was detected selectively within the regions that exhibited expression of the immune molecules. In contrast, animals that received the TK-deficient virus did not show any signs of sickness or cerebral inflammation or HSV replication within the cerebral tissue. The present data provide clear evidence that HSV-2 has the ability to trigger a profound inflammatory response in a pattern that follows the viral TK-dependent HSV replication in neurons. Such neurovirulence occurring in the hindbrain is proposed here to be directly responsible for neurodegeneration and to lead to the cerebral innate immune response, which in turn could play a key role in fatal HSV-2-induced encephalitis.     

Herpes simplex virus infection in capsaicin-treated mice

Following inoculation into the snout herpes simplex virus (HSV) spread to neurons in mouse trigeminal ganglion and subsequently to the brain. Capsaicin treatment of neonatal mice, which causes a loss of unmyelinated sensory neurons, some of which contain substance P, reduced the mortality rate of HSV-infected mice. Moreover, a lower percentage of mice survived the infection with reactivatable virus. There was also an extensive infection of glial cells proximal to the transitional zone in the trigeminal root between the peripheral and central nervous system. Distal to this zone there was an accumulation of substance P immunoreactivity in centrally directed fibres. This amplified degenerative effect on central branches of the substance P containing sensory nerves by glial infection may contribute to the deafferentiation pain syndrome following HSV infection.     

Pathogenesis of Semliki Forest virus encephalitis

This article provides a review of the pathogenesis of Semliki Forest virus (SFV) encephalitis. In mice, outcome of infection varies according to age of the mouse and strain of the virus and can include acute encephalitis, subacute demyelinating meningoencephalomyelitis, and persistent subclinical central nervous system (CNS) infection. All strains of virus are virulent in mice infected <12 days of age. The L10 strain is also virulent in mice >14 days age, whereas the A7(74) strain is avirulent. The genetic difference between these strains maps to the nsp3 gene. For A7(74) virus, age-related virulence correlates with ability of CNS neurons to replicate virus and undergo apoptotic cell death. Immature developing neurons support complete virus replication but as neuronal populations and circuits mature in the postnatal brain, virus infection becomes progressively restricted and nonproductive. This restricted replication can be overcome by gold I compounds, which may function by inducing neuronal dedifferentiation to a state permissive for virus replication. Biochemical pathways associated with membrane biogenesis may be an important determinant of this effect. Infection of some developing neuronal populations results in apoptosis, whereas infection of mature neurons results in persistent infection. An active type-I interferon system prevents virus spread in extraneural tissues. An initial high-titer plasma viremia is controlled by immunoglobulin M (IgM) antibodies. Virus enters the brain across cerebral endothelial cells and initiates scattered foci of perivascular infection. The blood-brain barrier is disrupted. Neurons and oligodendrocytes are the cell types most frequently infected. Infectivity in the brain can be eliminated by IgG antibodies, though an active T-cell response is required for virus elimination. Lesions of inflammatory demyelination require the presence of CD8(+) T lymphocytes and probably result from destruction by these cells of virally infected oligodendrocytes.     

The immune response to picornavirus infection and the effect of immune manipulation on acute seizures

Viral infection of the central nervous system can result in encephalitis. About 20% of individuals who develop viral encephalitis go on to develop epilepsy. We have established an experimental model where virus infection of mice with Theiler’s murine encephalomyelitis virus (TMEV) leads to acute seizures, followed by a latent period (no seizures/epileptogenesis phase) and then spontaneous recurrent seizures—epilepsy. Infiltrating macrophages (CD11b⁺CD45^hi) present in the brain at day 3 post-infection are an important source of interleukin-6, which contributes to the development of acute seizures in the TMEV-induced seizure model. Time course analysis of viral infection and inflammatory [CD11b⁺CD45^hiLy-6C^hi] and patrolling [CD11b⁺CD45^hiLy-6C^low] monocyte and T cell infiltration into the brains of TMEV-infected C57BL/6J mice over the entire course of the acute viral infection was performed to elucidate the role of virus and the immune response to virus in seizures and viral clearance. The infiltrating inflammatory macrophages were present early following infection but declined over the course of acute viral infection, supporting a role in seizure development, while the lymphocyte infiltration increased rapidly and plateaued, advocating that they play a role in viral clearance. In addition, we showed for the first time that, while TMEV infection of RAG1^?/? mice did not alter the number of mice experiencing acute seizures, TMEV infection of C57BL/6J mice depleted of macrophages resulted in a significant decrease in the number of mice experiencing seizures, again supporting a role for infiltrating macrophages in the development of acute seizures in the TMEV-induced seizure model.     

A critical role for IL-10 in limiting inflammation during toxoplasmic encephalitis

IL-10 plays a vital role in controlling the inflammatory response during acute Toxoplasma gondii infection, however the production of IL-10 during the chronic phase of toxoplasmosis has been associated with parasite persistence. To address this paradox, the production and effect of IL-10 in the brain during toxoplasmic encephalitis (TE) was investigated. Analysis of brain mononuclear cells (BMNC) from chronically infected mice revealed that infiltrating macrophages and CD4(+) T cells were the major sources of IL-10. Endogenous levels of IL-10 inhibited the production of IL-12, IFN-gamma, TNF-alpha, and IL-6 from both hematopoetic and non-hematopoetic cells in the brain, as well as anti-microbial activity of astrocytes. Furthermore, IL-10-/- mice that progressed to the chronic phase of infection had equivalent parasite burden to WT mice but developed a lethal inflammatory response within the brain characterized by increased numbers of CD4(+) T cells and macrophages, and elevated production of inflammatory cytokines. Finally, partial depletion of CD4(+) T cells decreased the severity of the inflammation in the brain and allowed IL-10-/- mice to survive infection. Together these results point to a vital role for IL-10 in the control of CD4(+) T cell mediated inflammation in the brain during TE.     

Human Hendra virus infection causes acute and relapsing encephalitis

Aim : To study the pathology of two cases of human Hendra virus infection, one with no clinical encephalitis and one with relapsing encephalitis. Methods : Autopsy tissues were investigated by light microscopy, immunohistochemistry and in situ hybridization. Results : In the patient with acute pulmonary syndrome but not clinical acute encephalitis, vasculitis was found in the brain, lung, heart and kidney. Occasionally, viral antigens were demonstrated in vascular walls but multinucleated endothelial syncytia were absent. In the lung, there was severe inflammation, necrosis and viral antigens in type II pneumocytes and macrophages. The rare kidney glomerulus showed inflammation and viral antigens in capillary walls and podocytes. Discrete necrotic/vacuolar plaques in the brain parenchyma were associated with antigens and viral RNA. Brain inflammation was mild although CD68⁺ microglia/macrophages were significantly increased. Cytoplasmic viral inclusions and antigens and viral RNA in neurones and ependyma suggested viral replication. In the case of relapsing encephalitis, there was severe widespread meningoencephalitis characterized by neuronal loss, macrophages and other inflammatory cells, reactive blood vessels and perivascular cuffing. Antigens and viral RNA were mainly found in neurones. Vasculitis was absent in all the tissues examined. Conclusions : The case of acute Hendra virus infection demonstrated evidence of systemic infection and acute encephalitis. The case of relapsing Hendra virus encephalitis showed no signs of extraneural infection but in the brain, extensive inflammation and infected neurones were observed. Hendra virus can cause acute and relapsing encephalitis and the findings suggest that the pathology and pathogenesis are similar to Nipah virus infection.     

A case of neonatal herpes simplex virus encephalitis with calcifications of thalamus and basal ganglia

A male infant developed left-sided myoclonus associated with low grade fever at 21 days of age. The course of pregnancy and delivery had been uneventful. Birth weight was 2,240g at 38 weeks of gestation. Physical examination at 26 days of age revealed hyperirritability and incomplete Moro reflex. EEG showed periodic discharges originating in the right central region. Initial brain CT at 26 days of age showed no apparent abnormality. In lumbar CSF protein was 173 mg/dl, and nucleated cells 328/mm3 (polynuclear cells 6 and mononuclear cells 322). Cultures of CSF for bacteria and viruses were negative. Although, the serum titers of CF antibody to herpes simplex virus (HSV) were 1:4 to 1:8 during hospitalization and those in CSF were not increased significantly, ELISA titers for IgG and IgM in serum and CSF rose significantly. The diagnosis of HSV encephalitis was made at 41 days of age. His myoclonus was well controlled with phenobarbital and clonazepam. He was treated with acyclovir and CSF findings returned to normal. On 2nd brain CT (39 days of age), calcification at thalamus, basal ganglia and right frontal lobe, which is extremely rare CT findings for HSV encephalitis, was noticed. His DQ at discharge was about 90. It is emphasized that the early diagnosis of HSV encephalitis can be made by ELISA and serial brain CT examinations, and that HSV encephalitis should be considered when calcification of thalamus and basal ganglia are detected on brain CT.     

Toxoplasma encephalitis of immunocompetent and nude mice: immunohistochemical characterisation of Toxoplasma antigen, infiltrates and major histocompatibility complex gene products

The experimental infection of immunocompetent and immunodeficient athymic mice with an avirulent encephalitogenic Toxoplasma strain (DX strain) was employed to study the ensuing encephalitic process by use of histological and immunocytochemical methods. In the acute phase of the infection Toxoplasma cysts and tachyzoites were accompanied by an infiltrate composed of macrophages, CD4+ and CD8+ T cells. In the chronic stage a granulomatous encephalitis developed. In contrast to immunocompetent NMRI mice, athymic nude NMRI mice died 3 weeks post-infection because of a generalized toxoplasmosis with predominant involvement of the brain. A salient feature of murine Toxoplasma encephalitis was up-regulation of class I and II major histocompatibility complex (MHC) gene products. Class I antigen was widely expressed on microglial cells and astrocytes. Class II antigen was only expressed on microglial cells despite a considerable astrogliosis. Our results indicate a differential expression of MHC-determined antigens on brain cells in acute and chronic murine Toxoplasma encephalitis.     

Specific IgG subclass reactivity in herpes simplex encephalitis

Summary Serum and cerebrospinal fluid (CSF) samples from 19 patients with a previous diagnosis of herpes simplex virus encephalitis (HSVE), from 14 patients with a previous diagnosis of non HSVE encephalitis and from 21 healthy subjects were examined to detect IgG subclasses 1–4 reactive with herpes simplex virus (HSV), cytomegalovirus (CMV) and varicella zoster virus (VZV). Antibodies to HSV were detected in CSF and serum from the 14 HSVE-patients with a reactivated HSV infection and from 3 of the 5 patients with a primary HSV infection. The predominant subclass pattern was an early HSV-specific IgG1 rise, followed by IgG3 and, more seldom, IgG4; HSV IgG2 was rarely seen. In HSVE patients, HSV IgG3 was absent in early samples and usually appeared 10–20 days after onset of disease. In 14 out of 16 seropositive healthy controls, on the other hand, HSV IgG3 was present in the CSF. Rising VZV IgG levels in serum and CSF were found in 11 HSVE patients. Eight of them showed signs of intrathecal VZV IgG1 synthesis. The VZV IgG reactivity was restricted to IgG1 in 7 of these whereas the HSV IgG subclass response also included IgG3 or 4. The appearance of several HSV IgG subclasses appeared to serve as a marker of HSV infection in spite of the serological VZV reaction, usually restricted to VZV IgG1. Intrathecal synthesis of the quantitatively minor HSV IgG3 and 4 subclasses was detected earlier than intrathecal synthesis of total HSV IgG, dominated by IgG1 in 4 patients with HSVE.     

Relapse of herpes simplex encephalitis

This report describes a child with herpes simplex virus (HSV) encephalitis who improved dramatically while being treated with acyclovir but subsequently had neurological deterioration and died. A severe necrotizing process was present in the brain at autopsy but there were no focal areas of demyelination and poor inflammatory response. HSV was not cultured from brain biopsy during relapse or autopsy. Fourteen previous cases of relapsing herpes encephalitis are reviewed and treatment regime and mechanisms of relapse are discussed.     

Herpes simplex encephalitis: Immunohistological demonstration of spread of virus via olfactory pathways in mice

Six-week-old Balb/c mice were inoculated intranasally with a suspension of HSV1 virus and the distribution of viral antigen in the brain 3–7 days later was surveyed using the immunoperoxidase technique. Virus was first detectable in the brain 4 days later at 2 distinct sites: the trigeminal root entry zone in the brain stem and the olfactory bulbs. On succeeding days virus spread from the trigeminal focus to many other brain stem nuclei and, in some mice, to the thalamus and the cerebellum. From the olfactory bulbs, in a proportion of mice, virus spread to anterior olfactory nucleus, lateral olfactory tract, septal nuclei, temporal lobe, hippocampus and cingulate cortex. Infection of olfactory bulbs was found to occur following intracorneal as well as intranasal inoculation of virus. The relevance of this model to human herpes simplex encephalitis is discussed.     

Relapse of herpes encephalitis after acyclovir therapy: report of two new cases and review of the literature

Relapse of herpes simplex virus (HSV) encephalitis following acyclovir therapy has been reported infrequently in children beyond the neonatal period. The pathogenic mechanism of the recurrence is not fully understood. We report two new cases that support a mechanism of latent HSV infection with reactivation of the disease. Our patients were 2 years (#1) and 8 months (#2) old at initial infection. Both presented with fever, lethargy, focal seizures, and focal motor abnormalities. Serum HSV antibodies (Abs) were negative. The patients were treated with acyclovir for 14 and 21 days, respectively. They were readmitted at 1 month, and 4 days after discharge, respectively, with recurrent lethargy, seizures, and choreo-athetoid movements. Serum and CSF HSV Abs were significantly increased. CSF PCR was positive. In patient # 2 acyclovir-sensitive HSV was isolated from a brain biopsy. Both patients were re-treated with acyclovir, but progressed to a neurovegetative state. In our cases, latent HSV infection and reactivation is the most likely explanation for recurrent encephalitis. The immuno-pathogenic mechanisms of the infection recurrence are discussed. Based on the reported cases in the literature, patients younger than 2 years of age and with lower total dose of acyclovir treatments have a higher risk of recurrence.     

Psychological stress exacerbates primary vaginal herpes simplex virus type 1 (HSV-1) infection by impairing both innate and adaptive immune responses

Chronic psychological stress is generally immunosuppressive and contributes to an increase in herpes simplex virus (HSV) pathogenicity. We have previously shown that mice experiencing stress at the time of intranasal HSV infection have increased levels of infectious virus in their nasal cavity, as compared to control mice that were not subjected to stress. We have extended our studies to determine the effects of stress at another clinically-relevant mucosal site by examining the immune response to and pathogenesis of vaginal HSV infection. Mice experiencing psychological stress during vaginal HSV infection exhibited an increase in both vaginal viral titers and the pathology associated with this HSV infection. We demonstrate that these observations result from the failure of both the innate and HSV-specific adaptive immune responses. At 2 days post-infection, NK cell numbers were significantly decreased in mice experiencing restraint stress. Studies examining the adaptive immune response revealed a decrease in the number of HSV-specific CD8⁺ T cells in not only the vaginal tissue itself but also the draining iliac lymph nodes (ILN). Furthermore, the number of functional cells, in terms of both their degranulation and interferon-γ production, in the ILN of stressed mice was decreased as compared to non-stressed mice. We conclude that psychological stress, through its suppression of both innate and adaptive immune responses, may be an important factor in the ability to control vaginal HSV infection.     

Herpes simplex virus encephalitis. Neuroanatomical and neurochemical selectivity

Intracerebral infection of mice with HSV-1 was found to produce a 2-3-fold increase in dopamine and serotonin metabolism in cortex, striatum, diencephalon and brain stem. Neurochemical markers of GABA and acetylcholine neurones, and neurotransmitter receptor binding sites were unchanged. The immunohistochemical distribution of virus antigen revealed high levels of immunoreactivity in s. nigra, ventral tegmental area, locus coeruleus and dorsal raphe nucleus, whilst other areas of brain stem were clear of virus antigen. The changes in monoamine metabolism observed in experimental HSV encephalitis may be related to the concentration of virus in monoamine neurones.     

A medical overview of encephalitis

Encephalitis is uncommon but is a neurological emergency which must be considered in a patient presenting with altered consciousness. Encephalitis is a diffuse inflammatory process of the brain parenchyma associated with evidence of brain dysfunction. The presentation of encephalitis can be acute or chronic. The aetiology of encephalitis can be broadly divided into two major subtypes. (1) Infection-related encephalitis which is a direct consequence of pathogenic viral, bacterial or parasitic agents. Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are the most common cause of acute infectious encephalitis. (2) Autoimmune-mediated encephalitis which is mediated by an aberrant immune response. This can be triggered by a recent viral infection or vaccination. An example of this would be acute disseminated encephalitis (ADEM). This article will focus on the medical management of acute encephalitis. This will involve an extensive overview of the literature reviewing the diagnosis, investigation and treatment of acute viral encephalitis, ADEM and acute haemorrhagic leukoencephalopathy (AHLE). Encephalitis can also present chronically, and some of the different types of chronic encephalitis will be discussed.     

Overlapping Guillain–Barré syndrome and Bickerstaff's brainstem encephalitis associated with anti-GQ1b IgG antibody after herpes simplex virus infection

Herpes simplex virus (HSV) is a rare, antecedent infectious agent in Guillain-Barré syndrome (GBS). We report a patient with overlapping GBS and Bickerstaff's brainstem encephalitis (BBE). The patient had a vesicular lesion on her nose. Antecedent HSV type 1 (HSV-1) infection was confirmed by isolation of the virus and detection of the presence of serum anti-HSV-1 IgM antibody during the acute phase. Her serum IgG had high anti-GQ1b antibody titer. External ophthalmoplegia has been noted in 2 of 4 reported cases of HSV-associated GBS. Herpetic brainstem encephalitis cases of poor prognosis are known, but only 2 cases of benign brainstem encephalitis secondary to HSV infection, in which there was acute ophthalmoplegia and clinical features consistent with those of BBE have been reported.