Demyelinating strain of mouse hepatitis virus infection bridging innate and adaptive immune response in the induction of demyelination.

The presence of immunoglobulin oligoclonal bands in the cerebrospinal fluid of Multiple Sclerosis (MS) patients supports the hypothesis of an infectious etiology, although the antigenic targets remain elusive. Neurotropic mouse hepatitis virus (MHV) infection in mice provides a useful tool for studying mechanisms of demyelination in a virus-induced experimental model of MS. This study uses Affymetrix microarray analysis to compare differential spinal cord mRNA levels between mice infected with demyelinating and non-demyelinating strains of MHV to identify host immune genes expressed in this demyelinating disease model. The study reveals that during the acute stage of infection, both strains induce inflammatory innate immune response genes, whereas upregulation of several immunoglobulin genes during chronic stage infection is unique to infection with the demyelinating strain. Results suggest that the demyelinating strain induced an innate-immune response during acute infection that may promote switching of Ig isotype genes during chronic infection, potentially playing a role in antibody-mediated progressive demyelination even after viral clearance.

Differential regulation of matrix metalloproteinases in varicella zoster virus-infected human brain vascular adventitial fibroblasts.

Upon reactivation, varicella zoster virus (VZV) spreads transaxonally, infects cerebral arteries and causes ischemic or hemorrhagic stroke, as well as aneurysms. The mechanism(s) of VZV-induced aneurysm formation is unknown. However, matrix metalloproteinases (MMPs), which digest extracellular structural proteins in the artery wall, play a role in cerebral and aortic artery aneurysm formation and rupture. Here, we examined the effect of VZV infection on expression of MMP-1, -2, -3, and -9 in primary human brain vascular adventitial fibroblasts (BRAFS). At 6 days post-infection, VZV- and mock-infected BRAFs were analyzed for mRNA levels of MMP-1, -2, -3 and -9 by RT-PCR and for corresponding total intra- and extracellular protein levels by multiplex ELISA. The activity of MMP-1 was also measured in a substrate cleavage assay. Compared to mock-infected BRAFs, MMP-1, MMP-3 and MMP-9 transcripts, cell lysate protein and conditioned supernatant protein were all increased in VZV-infected BRAFs, whereas MMP-2 transcripts, cell lysate protein and conditioned supernatant protein were decreased. MMP-1 from the conditioned supernatant of VZV-infected BRAFs showed increased cleavage activity on an MMP-1-specific substrate compared to mock-infected BRAFs. Differential regulation of MMPs in VZV-infected BRAFs may contribute to aneurysm formation in VZV vasculopathy.

Burning mouth syndrome due to herpes simplex virus type 1.

Burning mouth syndrome is characterised by chronic orofacial burning pain. No dental or medical cause has been found. We present a case of burning mouth syndrome of 6 months duration in a healthy 65-year-old woman, which was associated with high copy numbers of herpes simplex virus type 1 (HSV-1) DNA in the saliva. Her pain resolved completely after antiviral treatment with a corresponding absence of salivary HSV-1 DNA 4 weeks and 6 months later.

Prevalence and distribution of VZV in temporal arteries of patients with giant cell arteritis.

OBJECTIVE: Varicella-zoster virus (VZV) infection may trigger the inflammatory cascade that characterizes giant cell arteritis (GCA). METHODS: Formalin-fixed, paraffin-embedded GCA-positive temporal artery (TA) biopsies (50 sections/TA) including adjacent skeletal muscle and normal TAs obtained postmortem from subjects >50 years of age were examined by immunohistochemistry for presence and distribution of VZV antigen and by ultrastructural examination for virions. Adjacent regions were examined by hematoxylin & eosin staining. VZV antigen-positive slides were analyzed by PCR for VZV DNA. RESULTS: VZV antigen was found in 61/82 (74%) GCA-positive TAs compared with 1/13 (8%) normal TAs (p < 0.0001, relative risk 9.67, 95% confidence interval 1.46, 63.69). Most GCA-positive TAs contained viral antigen in skip areas. VZV antigen was present mostly in adventitia, followed by media and intima. VZV antigen was found in 12/32 (38%) skeletal muscles adjacent to VZV antigen-positive TAs. Despite formalin fixation, VZV DNA was detected in 18/45 (40%) GCA-positive VZV antigen-positive TAs, in 6/10 (60%) VZV antigen-positive skeletal muscles, and in one VZV antigen-positive normal TA. Varicella-zoster virions were found in a GCA-positive TA. In sections adjacent to those containing VZV, GCA pathology was seen in 89% of GCA-positive TAs but in none of 18 adjacent sections from normal TAs. CONCLUSIONS: Most GCA-positive TAs contained VZV in skip areas that correlated with adjacent GCA pathology, supporting the hypothesis that VZV triggers GCA immunopathology. Antiviral treatment may confer additional benefit to patients with GCA treated with corticosteroids, although the optimal antiviral regimen remains to be determined.

Mouse hepatitis virus infection upregulates genes involved in innate immune responses.

Neurotropic recombinant strain of Mouse Hepatitis Virus, RSA59, induces meningo-encephalitis, myelitis and demyelination following intracranial inoculation. RSA59 induced neuropathology is partially caused by activation of CNS resident microglia, as demonstrated by changes in cellular morphology and increased expression of a microglia/macrophage specific calcium ion binding factor, Iba1. Affymetrix Microarray analysis for mRNA expression data reveals expression of inflammatory mediators that are known to be released by activated microglia. Microglia-specific cell surface molecules, including CD11b, CD74, CD52 and CD68, are significantly upregulated in contrast to CD4, CD8 and CD19. Protein analysis of spinal cord extracts taken from mice 6 days post-inoculation, the time of peak inflammation, reveals robust expression of IFN-γ, IL-12 and mKC. Data suggest that activated microglia and inflammatory mediators contribute to a local CNS microenvironment that regulates viral replication and IFN-γ production during the acute phase of infection, which in turn can cause phagolysosome maturation and phagocytosis of the myelin sheath, leading to demyelination.

Inhibition of phosphorylated-STAT1 nuclear translocation and antiviral protein expression in human brain vascular adventitial fibroblasts infected with varicella-zoster virus.

In varicella-zoster virus (VZV)-infected primary human brain vascular adventitial fibroblasts (BRAFs), levels of beta interferon (IFN-ß,) STAT1, and STAT2 transcripts as well as STAT1 and STAT2 protein were decreased. IFN-α transcript levels were increased but not secreted IFN-α protein levels. Compared to IFN-α-treated control results, in VZV-infected BRAFs, phosphorylated STAT1 did not translocate to the nucleus, resulting in impaired downstream expression of interferon-inducible antiviral Mx1. Overall, VZV interference with the type I interferon pathway may promote virus persistence in cerebral arteries.

Biopsy-negative, varicella zoster virus (VZV)-positive giant cell arteritis, zoster, VZV encephalitis and ischemic optic neuropathy, all in one.

A 72-year-old man developed clinical features of giant cell arteritis (GCA) and ipsilateral ophthalmic-distribution zoster, followed within 2 weeks by VZV encephalitis and 2 months later by ischemic optic neuropathy. Temporal artery biopsy was histopathologically negative for GCA, but contained VZV antigen and VZV DNA in multiple non-contiguous (skip) areas. The collective clinical and laboratory findings revealed a remarkably close temporal association of zoster, multifocal VZV vasculopathy with temporal artery infection, biopsy-negative VZV-positive GCA and VZV encephalitis.

Frequency and abundance of alphaherpesvirus DNA in human thoracic sympathetic ganglia.

Alphaherpesvirus reactivation from thoracic sympathetic ganglia (TSG) and transaxonal spread to target organs cause human visceral disease. Yet alphaherpesvirus latency in TSG has not been well characterized. In this study, quantitative PCR detected varicella-zoster virus (VZV), herpes simplex virus 1 (HSV-1), and HSV-2 DNA in 117 fresh TSG obtained postmortem from 15 subjects. VZV DNA was found in 76 (65%) ganglia from all subjects, HSV-1 DNA was found in 5 (4%) ganglia from 3 subjects, and no HSV-2 was found.

Varicella zoster virus (VZV) in cerebral arteries of subjects at high risk for VZV reactivation.

With a decline in varicella zoster virus (VZV)-specific cell-mediated immunity, VZV can reactivate, infect cerebral arteries and cause stroke. Previous studies of cerebral arteries from subjects without a history of transient ischemic attacks or stroke revealed no VZV DNA or VZV antigen; however, VZV DNA and VZV antigen were found in the cerebral arteries of a subject with diabetes, a known risk factor for VZV reactivation and zoster. The present study analyzed an additional 55 cerebral arteries from 18 subjects with co-morbidities that may increase risk of VZV reactivation: a history of alcohol abuse, tricyclic antidepressant intoxication, cocaine abuse, HIV or being over age 70 years. VZV antigen was detected in 24 (44%) cerebral arteries from 14 (78%) subjects.

Varicella zoster virus in the temporal artery of a patient with giant cell arteritis.

We recently detected varicella zoster virus (VZV) in the temporal arteries (TA) of 5/24 patients with clinically suspect giant cell arteritis (GCA) whose TAs were GCA-negative pathologically; in those GCA-negative, VZV+TAs, virus antigen predominated in the arterial adventitia, but without medial necrosis and multinucleated giant cells. During our continuing search for VZV antigen in GCA-negative TAs, in the TA of one subject, we found abundant VZV antigen, as well as VZV DNA, in multiple regions (skip areas) of the TA spanning 350 µm, as well as in skeletal muscle adjacent to the infected TA. Additional pathological analysis of sections adjacent to those containing viral antigen revealed inflammation involving the arterial media and abundant multinucleated giant cells characteristic of GCA. Detection of VZV in areas of the TA with pathological features of GCA warrants further correlative pathological-virological analysis of VZV in GCA.

GeXPS multiplex PCR analysis of the simian varicella virus transcriptome in productively infected cells in culture and acutely infected ganglia.

Simian varicella zoster virus (SVV) infection of non-human primates serves as a model to study varicella zoster virus (VZV) infection and pathogenesis in humans. While macroarray analysis detected all 69 predicted unique open reading frames (ORFs) in SVV-infected cells in culture, it lacked the sensitivity to detect the low-abundance transcripts expressed in latently infected monkey ganglia. Recently, a multiplex RT-PCR assay using the GenomeLab Genetic Analysis System (GeXPS) identified 10 VZV transcripts in latently-infected human ganglia. GeXPS was used to analyze the SVV transcriptome in SVV-infected monkey cells in culture as well as in acutely infected ganglia from African green monkeys. Oligonucleotide primers containing both SVV ORF- and cell-specific sequences linked to universal DNA sequences were used in RT-PCR to produce products of predetermined sizes. Amplification products were resolved by capillary gel electrophoresis and detected by fluorescence spectrophotometry. Transcripts corresponding to all 69 predicted SVV ORFs, in addition to transcripts within the leftward end region and ORF 61 antisense transcripts were detected in virus-infected cells in culture. Except for two transcripts (ORFs 14 and 35), all transcripts found in infected tissue culture cells were also found in acutely infected monkey ganglia.

Multifocal VZV vasculopathy with temporal artery infection mimics giant cell arteritis.

OBJECTIVE: To address the incidence of varicella-zoster virus (VZV) infection in patients with biopsy-negative giant cell arteritis (GCA), we examined archived biopsy-negative temporal arteries from subjects with clinically suspected GCA for the presence of VZV antigen. METHODS: Formalin-fixed, paraffin-embedded temporal arteries that were pathologically negative for GCA and normal temporal arteries were analyzed immunohistochemically for VZV and herpes simplex virus-1 (HSV-1) antigen. RESULTS: Five (21%) of 24 temporal arteries from patients who were clinically suspect but biopsy negative for GCA revealed VZV but not HSV-1 by immunohistochemical analysis. Thirteen normal temporal arteries did not contain VZV or HSV-1 antigen. All 5 subjects whose temporal arteries contained VZV antigen presented with clinical and laboratory features of GCA and early visual disturbances. CONCLUSION: Multifocal VZV vasculopathy can present with the full spectrum of clinical features and laboratory abnormalities characteristically seen in GCA.

Search for varicella zoster virus and herpes simplex virus-1 in normal human cerebral arteries.

Virological confirmation of varicella zoster virus (VZV) vasculopathy is provided by presence of virus in the cerebral arteries, frequently associated with inflammation. Yet, cerebral arteries from normal subjects have never been studied for VZV DNA or antigen. We analyzed 63 human cerebral arteries from 45 subjects for VZV DNA and antigen, control herpes simplex virus (HSV)-1 DNA and antigen, and leukocyte-specific CD45 antigen. No cerebral arteries contained VZV or HSV-1 DNA or antigen; eight arteries from seven subjects contained leukocytes expressing CD45. Thus, the presence of VZV antigen in cerebral arteries of patients with stroke is likely to be clinically significant.

VZV multifocal vasculopathy with ischemic optic neuropathy, acute retinal necrosis and temporal artery infection in the absence of zoster rash.

We describe a 54-year-old diabetic woman who developed ischemic optic neuropathy followed by acute retinal necrosis and multiple areas of focal venous beading. Vitreous fluid contained amplifiable VZV DNA but not HSV-1, CMV or toxoplasma DNA. The clinical presentation was remarkable for jaw claudication and intermittent scalp pain, prompting a temporal artery biopsy that was pathologically negative for giant cell arteritis, but notable for VZV antigen. The current case adds to the clinical spectrum of multifocal VZV vasculopathy. The development of acute VZV retinal necrosis after ischemic optic neuropathy supports the notion that vasculitis is an important additional mechanism in the development of VZV retinal injury.

Varicella-zoster virus vasculopathy: immune characteristics of virus-infected arteries.

OBJECTIVE: Pathologic changes in varicella-zoster virus (VZV)-infected arteries include inflammation, thickened intima, and paucity of smooth muscle cells. Since no criteria have been established for early vs late VZV vasculopathy, we examined inflammatory cells and their distribution in 6 normal arteries, and 2 VZV-infected arteries 3 days after onset of disease (early) and 10 months after protracted neurologic disease (late). METHODS: VZV-infected temporal artery obtained 3 days after onset of ischemic optic neuropathy from an 80-year-old man, VZV-infected middle cerebral artery (MCA) obtained 10 months after protracted disease from a 73-year-old man, and 5 MCAs and 1 temporal artery from normal subjects, age 22-60 years, were examined histologically and immunohistochemically using antibodies against VZV and inflammatory cell subsets. RESULTS: In both early and late VZV vasculopathy, T cells, activated macrophages, and rare B cells were found in adventitia and intima. In adventitia of early VZV vasculopathy, neutrophils and VZV antigen were abundant and a thickened intima was associated with inflammatory cells in vaso vasorum vessels. In media of late VZV vasculopathy, viral antigen, but not leukocytes, was found. VZV was not seen in inflammatory cells. Inflammatory cells were absent in control arteries. CONCLUSIONS: Both VZV and neutrophils exclusively in adventitia in early VZV vasculopathy indicate that disease begins there. Late VZV vasculopathy is distinguished by viral antigen without inflammation in media, revealing a human virus in an immunoprivileged arterial media. Association of thickened intima and inflammation in vaso vasorum vessels in early VZV vasculopathy support the role of virus-induced inflammation in vessel wall remodeling.

Restricted varicella-zoster virus transcription in human trigeminal ganglia obtained soon after death.

We analyzed the varicella-zoster virus (VZV) transcriptome in 43 latently infected human trigeminal ganglia (TG) with postmortem intervals (PMIs) ranging from 3.7 to 24 h. Multiplex reverse transcriptase PCR (RT-PCR) revealed no VZV transcripts with a PMI of <9 h. Real-time PCR indicated a significant increase (P = 0.02) in VZV ORF63 transcript levels but not the virus DNA burden with longer PMI. Overall, both the breadth of the VZV transcriptome and the VZV ORF63 transcript levels in human cadaver TG increased with longer PMI.

Reactivation of type 1 herpes simplex virus and varicella zoster virus in an immunosuppressed patient with acute peripheral facial weakness.

We describe a 26-year-old man treated with azathioprine for myasthenia gravis who developed acute left-sided peripheral facial weakness. Brain magnetic resonance imaging (MRI) revealed enhancement in the left geniculate ganglion and in the intracanalicular and tympanic segments of the facial nerve. Analysis of cerebrospinal fluid (CSF) and serum revealed intrathecal synthesis of anti-varicella zoster virus (VZV) IgG antibody. Although previous analyses of saliva, blood mononuclear cells, serum antibodies, middle ear fluid, and auricular and geniculate zone skin scrapings have shown that a small but definite proportion of patients with idiopathic peripheral facial palsy ("Bell's palsy") have the Ramsay Hunt syndrome zoster sine herpete (RHS ZSH), this is the first confirmation of RHS ZSH by intrathecal synthesis of anti-VZV IgG antibody. In addition, herpes simplex virus (HSV)-1 DNA was found in saliva of the patient on 3 consecutive days. Simultaneous reactivation of two alphaherpesviruses (HSV-1 and VZV) in our immunosuppressed patient underscores the need to consider opportunistic infection as a cause of facial weakness.

Persistence of varicella zoster virus DNA in saliva after herpes zoster.

Analysis of saliva samples from individuals aged ≥ 60 years who had a history of zoster (group 1), zoster and postherpetic neuralgia (PHN; group 2), or no history of zoster (group 3) revealed varicella zoster virus (VZV) DNA in saliva samples from 11 of 17 individuals in group 1, 10 of 15 individuals in group 2, and 2 of 17 individuals in group 3. The frequency of VZV DNA detection was significantly higher (P = .001) in saliva of subjects with a history of zoster, with or without PHN (21 [67%] of 32 subjects in groups 1 and 2), than in saliva of age-matched subjects with no zoster history (2 [12%] of 17 subjects in group 3). Thus, persistence of VZV DNA in saliva is the outcome of zoster, independent of PHN. Because VZV infection can produce neurological and ocular disease without zoster rash, future studies are needed to establish whether VZV DNA can be detected in the saliva of such patients.

Varicella-zoster virus transcriptome in latently infected human ganglia.

We recently developed a novel multiplex reverse transcription (RT)-PCR assay that allows rapid and sensitive detection of transcripts corresponding to all 68 unique varicella-zoster virus (VZV) open reading frames (ORFs) in only five amplification reactions (M. A. Nagel, D. Gilden, T. Shade, B. Gao, and R. J. Cohrs, J. Virol. Methods 157:62-68, 2009). Herein, we applied multiplex RT-PCR analysis to mRNA extracted from 26 trigeminal ganglia latently infected with VZV and one control trigeminal ganglion negative for VZV DNA that were removed from 14 men and women, 16 to 84 years of age, within 24 h after death. Analysis identified VZV transcripts mapping to VZV ORFs 29, 62, and 63, previously detected and sequence verified; VZV ORFs 4 and 40, previously detected by in situ hybridization; and VZV ORFs 11, 41, 43, 57, and 68, not previously detected. VZV ORF 63 transcripts were the most prevalent. Comparison of the 10 VZV ORFs transcribed during latency to their herpes simplex virus type 1 homologues reveals that the latently transcribed VZV genes encode immediate-early, early, and late transcripts.