Simian varicella virus induces apoptosis in monkey kidney cells by the intrinsic pathway and involves downregulation of bcl-2 expression.

Simian varicella virus (SVV) causes varicella in primates, becomes latent in ganglionic neurons, and reactivates to produce zoster. SVV produces a cytopathic effect in monkey kidney cells in tissue culture. To study the mechanism by which SVV-infected cells die, we examined markers of apoptosis 24 to 64 h postinfection (hpi). Western blot analysis of virus-infected cell lysates revealed a significant increase in the levels of the cleaved active form of caspase-3, accompanied by a parallel increase in caspase-3 activity at 40 to 64 hpi. Caspase-9, a marker for the intrinsic pathway, was activated significantly in SVV-infected cells at all time points, whereas trace levels of the active form of caspase-8, an extrinsic pathway marker, was detected only at 64 hpi. Bcl-2 expression at the mRNA and protein levels was decreased by 50 to 70% throughout the course of virus infection. Release of cytochrome c, an activator of caspase-9, from mitochondria into the cytoplasm was increased by 200% at 64 hpi. Analysis of Vero cells infected with SVV expressing green fluorescent protein (SVV-GFP) at 64 hpi revealed colocalization of the active forms of caspase-3 and caspase-9 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with GFP. A significant decrease in the bcl-2 mRNA levels along with an abundance of mRNA specific for SVV genes 63, 40, and 21 was seen in the fraction of Vero cells that were infected with SVV-GFP. Together, these findings indicate that SVV induces apoptosis in cultured Vero cells through the intrinsic pathway in which Bcl-2 is downregulated.

Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia.

Analysis of cells infected by a wide range of herpesviruses has identified numerous virally encoded microRNAs (miRNAs), and several reports suggest that these viral miRNAs are likely to play key roles in several aspects of the herpesvirus life cycle. Here we report the first analysis of human ganglia for the presence of virally encoded miRNAs. Deep sequencing of human trigeminal ganglia latently infected with two pathogenic alphaherpesviruses, herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV), confirmed the expression of five HSV-1 miRNAs, miR-H2 through miR-H6, which had previously been observed in mice latently infected with HSV-1. In addition, two novel HSV-1 miRNAs, termed miR-H7 and miR-H8, were also identified. Like four of the previously reported HSV-1 miRNAs, miR-H7 and miR-H8 are encoded within the second exon of the HSV-1 latency-associated transcript. Although VZV genomic DNA was readily detectable in the three human trigeminal ganglia analyzed, we failed to detect any VZV miRNAs, suggesting that VZV, unlike other herpesviruses examined so far, may not express viral miRNAs in latently infected cells.

CSF IgG heavy-chain bias in patients at the time of a clinically isolated syndrome.

Using FACS and single cell reverse transcriptase polymerase chain reaction, we examined the cerebrospinal fluid (CSF) IgG VH repertoires from 10 subjects with a clinically isolated demyelinating syndrome (CIS). B and plasma cell repertoires from individual subjects showed similar VH family germline usage, nearly identical levels of post-germinal center somatic hypermutation, and significant overlap in their clonal populations. Repertoires from 7 of 10 CIS subjects demonstrated a biased usage of VH4 and/or VH2 family gene segments in their plasma or B cell repertoires. V-regionbias, however, was not observed in the corresponding peripheral blood CD19+ B cell repertoires from 2 CIS subjects or in normal healthy adults. Clinically, subjects with VH4 or VH2 CSF IgG repertoire bias rapidly progressed to definite MS, whereas individuals without repertoire bias did not develop MS after a minimum of 2 years of follow-up (p=0.01).

Varicella zoster virus infection: clinical features, molecular pathogenesis of disease, and latency.

Varicella zoster virus (VZV) is an exclusively human neurotropic alphaherpesvirus. Primary infection causes varicella (chickenpox), after which virus becomes latent in cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia along the entire neuraxis. Years later, in association with a decline in cell-mediated immunity in elderly and immunocompromised individuals, VZV reactivates and causes a wide range of neurologic disease. This article discusses the clinical manifestations, treatment, and prevention of VZV infection and reactivation; pathogenesis of VZV infection; and current research focusing on VZV latency, reactivation, and animal models.

Characterization of phage peptide interaction with antibody using phage mediated immuno-PCR.

Real-time immuno-PCR (RT-IPCR) is a powerful technique that combines ELISA with the specificity and sensitivity of PCR. RT-IPCR of phage-displayed peptides exploits the unique physical associations between phenotype (the displayed peptide) and genotype (the encoding DNA) within the same phage particle. Previously, we identified phage peptides specific for recombinant antibodies (rAbs) prepared from clonally expanded plasma cells in multiple sclerosis (MS) cerebrospinal fluid (CSF) and subacute sclerosing panencephalitis (SSPE) brain. Herein, we applied phage-mediated RT-IPCR to study reactivity of these specific phage peptides for the rAbs. Compared to standard ELISA, which required greater than 10(4) or 10(5) phage particles to detect binding to rAbs, RT-IPCR detected binding with as few as 100 phage particles. RT-IPCR was also superior to ELISA in determining relative affinities of rAbs for phage peptides and was effective in screening MS CSF for IgG reactivity to phage peptides. Phage-mediated RT-IPCR is a rapid, high-throughput technology that avoids the requirement for synthetic peptides and will facilitate the identification of candidate peptides that react with the IgG in MS CSF.

Analysis of multiple sclerosis cerebrospinal fluid reveals a continuum of clonally related antibody-secreting cells that are predominantly plasma blasts.

Fluorescence-activated cell sorting (FACS) analysis of B cell subtypes in 17 CSF samples from 15 patients with clinically-definite MS revealed that CD19+ B cells accounted for 2 to 11% (mean 5%) and CD138+ cells constituted 0 to 5% (mean 2%) of total CSF lymphocytes. Further stratification of CD138+ cells based on expression levels of CD19 showed that CD138+19+ plasma blasts constituted 89+/-2% (mean+/-SE) of the CD138+ cell population (P<0.00001), with more mature plasma cells (CD138+19-) constituting the remaining 11+/-2%. Sequence analysis of immunoglobulin variable regions in single CD138+19+ and CD138+19- cells sorted from MS CSF identified many of the same clonal populations in both populations, indicating a continuum of clonally related plasma cell subtypes of which CD138+19+ plasma blasts are most abundant.

Herpesvirus infections of the nervous system.

There are eight human herpesviruses (HHVs). Primary infection by any of the eight viruses, usually occurring in childhood, is either asymptomatic or produces fever and rash of skin or mucous membranes; other organs might be involved on rare occasions. After primary infection, the virus becomes latent in ganglia or lymphoid tissue. With the exception of HHV-8, which causes Kaposi's sarcoma in patients with AIDS, reactivation of HHVs can produce one or more of the following complications: meningitis, encephalitis, myelitis, vasculopathy, ganglioneuritis, retinal necrosis and optic neuritis. Disease can be monophasic, recurrent or chronic. Infection with each herpesvirus produces distinctive clinical features and imaging abnormalities. This Review highlights the patterns of neurological symptoms and signs, along with the typical imaging abnormalities, produced by each of the HHVs. Optimal virological studies of blood, cerebrospinal fluid and affected tissue for confirmation of diagnosis are discussed; this is particularly important because some HHV infections of the nervous system can be treated successfully with antiviral agents.

The protean neurologic manifestations of varicella-zoster virus infection.

Multiple neurologic complications may follow the reactivation of varicella-zoster virus (VZV), including herpes zoster (also known as zoster or shingles), postherpetic neuralgia, vasculopathy, myelitis, necrotizing retinitis, and zoster sine herpete (pain without rash). These conditions can be difficult to recognize, especially as several can occur without rash.

Improvement of postherpetic neuralgia after treatment with intravenous acyclovir followed by oral valacyclovir.

BACKGROUND: Postherpetic neuralgia (PHN) is a complication of shingles (herpes zoster), a painful rash due to varicella-zoster virus reactivation. Studies of patients with PHN and zoster sine herpete (radicular pain without rash) support the notion that low-grade viral ganglionitis contributes to pain. If chronic pain reflects active infection, then antiviral therapy may help patients with PHN. OBJECTIVE: To determine whether antiviral treatment helps reduce PHN-associated pain. DESIGN: Prospective, open-label phase I/II clinical trial. SETTING: Tertiary care university hospital. PATIENTS: Fifteen patients with moderate to severe PHN. INTERVENTIONS: Intravenous acyclovir at a dosage of 10 mg/kg every 8 hours for 14 days followed by oral valacyclovir at a dosage of 1000 mg 3 times per day for 1 month. MAIN OUTCOME MEASURE: Numeric Rating Scale for Pain score. RESULTS: As defined by a decrease of 2 or more points on the Numeric Rating Scale for Pain, 8 (53%) of 15 patients reported improvement. CONCLUSION: Clinical improvement reported by most of our patients warrants further investigation in a larger, randomized, double-blind, placebo-controlled trial.

Rapid and efficient identification of epitopes/mimotopes from random peptide libraries.

Phage-displayed random peptide libraries are important tools in identifying novel epitopes/mimotopes that may lead to the determination of antigen specificity. In this approach, high-affinity phage peptides are enriched by affinity selection (panning) on a monoclonal antibody. To facilitate identification of all potential phage peptides specific for recombinant monoclonal antibodies (rAbs) previously generated from clonally expanded plasma cells from the cerebrospinal fluid of patients with multiple sclerosis (MS), we developed a high-throughput method to determine phage specificity. In contrast to the 8-9 days needed in the standard large-scale method of amplifying phage clones for ELISA, the high-throughput method takes only 1 day. ELISA using phage clones amplified directly in 96-well plates avoids large-scale phage purification and enables rapid identification of specific epitopes/mimotopes. This technique will expedite identification of MS-specific peptides that can be used to discover the corresponding protein antigens.

Specificity of recombinant antibodies generated from multiple sclerosis cerebrospinal fluid probed with a random peptide library.

We generated recombinant antibodies (rAbs) from over-represented IgG sequences expressed by single plasma cells from multiple sclerosis (MS) cerebrospinal fluid (CSF). Panning of a phage-displayed random peptide library with the rAbs revealed several specific peptide sequences. Inhibition assays confirmed specific binding of the peptides to the antigen-binding site of the antibody. The native IgG of MS CSF from which the recombinant antibody was cloned also recognized these peptides. Our data demonstrate that MS rAb reflects the specificity of IgG in the CSF. Thus, the epitopes/mimotopes identified by MS rAb may provide clues to disease-relevant antigens.

The B cell response in multiple sclerosis.

Multiple sclerosis (MS) plaques and CSF contain increased amounts of intrathecally synthesized IgG, manifest as oligoclonal bands (OCBs) after protein electrophoresis. OCBs are not unique to MS and are also produced in infectious diseases of the CNS, in which the oligoclonal IgG has been shown to be antibody directed against the disease-causing agent. Thus, analysis of antibody specificity may identify the causative agent/antigen in MS. This review discusses recent studies that have analyzed the phenotypes of B cells in MS which infiltrate the CNS and the molecular features of their antigen-binding regions. Together with histologic studies showing the presence of ectopic lymphoid follicles in the meninges of some MS patients, this data supports the notion of a targeted and compartmentalized humoral response in MS.

Infectious causes of multiple sclerosis.

Multiple sclerosis (MS) is a serious chronic neurological disorder in which demyelination and inflammation occur in the white matter of the CNS. The findings of many epidemiological studies and a discordance of MS in monozygotic twins suggest that the disorder is acquired. The most likely cause is a virus because more than 90% of patients with MS have high concentrations of IgG, manifest as oligoclonal bands, in the brain and CSF. Most chronic inflammatory CNS disorders are infectious. More indirect evidence that MS is caused by a virus is the association of several viruses with demyelinating encephalomyelitis in human beings, and the induction of demyelination in animals infected with viruses in research. Nevertheless, no virus has been isolated from the brains of patients who had MS. Molecular analysis of IgG gene specificity in the brain and CSF of those with MS has shown features of an antigen-driven response: clonal amplification and extensive somatic mutations. A viral antigen against which the IgG in MS brain and CSF is directed might be identified.

Laser capture microdissection and single-cell RT-PCR without RNA purification.

Chronic infectious diseases of the central nervous system (CNS) are characterized by intrathecal synthesis of increased amounts of immunoglobulin G (IgG) directed against the agent that causes disease. In other inflammatory CNS diseases such as multiple sclerosis and CNS sarcoid, the targets of the humoral immune response are uncertain. To identify the IgGs expressed by individual CD38(+) plasma cells seen in human brain sections, we merged the techniques of laser capture microdissection (LCM) and single-cell RT-PCR. Frozen brain sections from a patient who died of subacute sclerosing panencephalitis (SSPE), were rapidly immunostained and examined by LCM to dissect individual CD38(+) cells. After cell lysis, we developed two techniques for reverse-transcription (RT) of unpurified total RNA in the cell lysates. The first method performed repeated and rapid freeze-thawing, followed by centrifugation of the cell lysate into tubes for subsequent RT. The second, more successful method performed RT in situ on detergent-solubilized cells directly on the cap surface; subsequent nested PCR identified heavy and light chain sequences expressed by two-thirds of individually isolated plasma cells. These techniques will streamline the identification of gene expression products in single cells from complex tissues and have the potential to identify IgGs expressed in the CNS of inflammatory diseases of unknown etiology.

Multifocal vasculopathy due to Varicella-Zoster Virus (VZV): serial analysis of VZV DNA and intrathecal synthesis of VZV antibody in cerebrospinal fluid.

Recognition of multifocal vasculopathy due to varicella-zoster virus (VZV) is often problematic. We describe a human immunodeficiency virus-infected patient who had progressive central nervous system disease for >3 months. Both VZV DNA and antibody were detected in cerebrospinal fluid (CSF) specimens; serial polymerase chain reaction analyses confirmed the diagnosis and guided the duration of therapy. Reduced ratios of VZV antibody in serum to that in CSF were also demonstrated.

Surgical induction of zoster in a contralateral homologous dermatomal distribution.

Herpes zoster occurs most often in elderly and immunocompromised individuals, and rarely after surgery. We report 2 cases in which zoster developed in the contralateral dermatome distribution homologous to the surgical site. The mechanism by which unilateral surgery might affect homologous ganglia is likely to involve spinal cord pathways above the dermatomal level of surgical trauma.

Multifocal varicella-zoster virus vasculopathy without rash.

A 51-year-old woman with CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) developed stepwise progressive focal neurological deficits without zoster rash. Multifocal ischemic infarcts were seen on magnetic resonance imaging, and cerebral angiography revealed focal stenosis of arteries affecting the intracranial circulation. A brain biopsy was nondiagnostic. Virological etiology of the disease was verified by the detection of varicella-zoster virus antibody in cerebrospinal fluid and by reduced serum-cerebrospinal fluid varicella-zoster virus IgG ratios (compared with normally high ratios of total IgG and albumin). Treatment with intravenous acyclovir stabilized but did not significantly improve her neurological deficits.

Varicella zoster virus latency, neurological disease and experimental models: an update.

Varicella zoster virus (VZV), a ubiquitous neurotropic human herpesvirus, causes chickenpox (varicella) and then remains latent for decades in cranial nerve, dorsal root and autonomic nervous system ganglia along the entire neuraxis. Virus reactivation, most often after age 60, produces shingles (zoster), characterized by pain and rash usually restricted to 1-3 dermatomes. In elderly individuals, zoster is frequently complicated by postherpetic neuralgia (PHN), pain that persists for months to years after the resolution of rash. Virus may also spread beyond ganglia to the spinal cord to cause myelitis, as well as to blood vessels of the brain, producing a unifocal or multifocal vasculopathy. The increased incidence of zoster in the elderly and immunocompromised individuals appears to be due to a VZV-specific host immunodeficiency. Recent studies indicate that PHN may be due to a chronic active VZV ganglionitis, and that VZV vasculopathy is caused by a productive virus infection in cerebral arteries. Since neurological disease produced by VZV is due to reactivation from ganglia, the physical state of viral nucleic acid and expression during latency as well as the possible mechanisms by which VZV latency is maintained and reactivates are discussed. Finally, VZV is an exclusively human herpesvirus, and experimental infection of animals with VZV does not produce disease nor does VZV reactivate from ganglia. Two varicella models in primates have proven useful: one that mimics varicella latency in humans, and one that can be used to study the efficacy of antiviral agent in driving varicella virus back to a latent state.

B cells in multiple sclerosis.

The most common laboratory abnormality in multiple sclerosis (MS) is an increased amount of cerebrospinal fluid IgG and the presence of oligoclonal bands. Despite studies of the humoral response that suggest the involvement of an infectious agent or autoantigen in disease, the major targets of the oligoclonal response are still unknown. Identification of these targets will reveal valuable insights into the cause and pathogenesis of MS and is likely to lead to effective treatment.