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.

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.

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.

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).

Asymptomatic reactivation and shed of infectious varicella zoster virus in astronauts.

Varicella zoster virus (VZV) causes varicella (chickenpox), after which virus becomes latent in ganglia along the entire neuraxis. Virus reactivation produces zoster (shingles). Infectious VZV is found in vesicles of patients with zoster and varicella, but virus shed in the absence of disease has not been documented. VZV DNA was previously detected in saliva of astronauts during and after spaceflight, a uniquely stressful environment in which cell mediated immunity (CMI) is temporally dampened. The decline in CMI to VZV associated with zoster led to the hypothesis that infectious VZV would also be present in the saliva of astronauts subjected to stress of spaceflight. Herein, not only was the detection of salivary VZV DNA associated with spaceflight validated, but also infectious virus was detected in saliva from 2 of 3 astronauts. This is the first demonstration of shed of infectious VZV in the absence of disease.

Varicella-zoster virus in the saliva of patients with herpes zoster.

Fifty-four patients with herpes zoster were treated with valacyclovir. On treatment days 1, 8, and 15, pain was scored and saliva examined for varicella-zoster virus (VZV) DNA. VZV DNA was found in every patient the day treatment was started and later disappeared in 82%. There was a positive correlation between the presence of VZV DNA and pain and between VZV DNA copy number and pain (P <.0005). VZV DNA was present in 1 patient before rash and in 4 after pain resolved and was not present in any of 6 subjects with chronic pain or in 14 healthy subjects. Analysis of human saliva has potential usefulness in the diagnosis of neurological disease produced by VZV without rash.

Construction of recombinant mouse IgG1 antibody directed against varicella zoster virus immediate early protein 63.

Five varicella zoster virus (VZV) genes are known to be transcribed in latently infected human ganglia. Transcripts from VZV gene 63, which encodes an immediate early (IE) protein, are the most prevalent and abundant. To obtain a reagent that might facilitate studies of the role of the IE63 protein in latency and reactivation, we selected an IE63-specific Fab fragment from a phage library and used it to prepare a recombinant mouse IgG1 antibody that detects IE63 and functions in Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assays.

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.

VH4 gene segments dominate the intrathecal humoral immune response in multiple sclerosis.

A characteristic feature of the CNS inflammatory response in multiple sclerosis (MS) is the intrathecal synthesis of IgG and the presence of oligoclonal bands. A strong correlation between CD138(+) plasma blast numbers in MS cerebrospinal fluid (CeSF) and intrathecal IgG synthesis suggests that these cells are the major Ab-secreting cell type in MS CeSF. Sequencing of V regions from CD138(+) cells in MS CeSF has revealed somatically mutated and expanded IgG clonotypes consistent with an Ag-targeted response. In the present study, single-cell RT-PCR analysis of CD138(+) cells from 11 MS patients representing differing clinical courses and stages of disease identified expansion of CD138(+) cells with functionally rearranged V(H)4 gene segments as an overriding feature of MS CeSF repertoires. V(H)4 dominance was attributed to the preferential selection of specific V(H)4 genes, particularly gene segment V(H)4-39, which displayed a significant enrichment in CeSF compared with MS peripheral blood B cells. A modest increase in V(H)4 prevalence among MS peripheral blood IgG memory cells was also noted, suggesting that factors shaping the CD138 repertoire in CeSF might also influence the peripheral IgG memory cell pool. These results indicate a highly restricted B cell response in MS. Identifying the targets of CeSF plasma cells may yield insights into disease pathogenesis.

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.

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.

Simian varicella virus reactivation in cynomolgus monkeys.

SVV infection of primates closely resembles VZV infection of humans. Like VZV, SVV becomes latent in ganglionic neurons. We used this model to study the effect of immunosuppression on varicella reactivation. Cynomolgus monkeys latently infected with SVV were irradiated and treated with tacrolimus and prednisone. Of four latently infected monkeys that were immunosuppressed and subjected to the stress of transportation and isolation, one developed zoster, and three others developed features of subclinical reactivation. Another non-immunosuppressed latently infected monkey that was subjected to the same stress of travel and isolation showed features of subclinical reactivation. Virus reactivation was confirmed not only by the occurrence of zoster in one monkey, but also by the presence of late SVV RNA in ganglia, and the detection of SVV DNA in non-ganglionic tissue, and SVV antigens in skin, ganglia and lung.

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.

Disseminated simian varicella virus infection in an irradiated rhesus macaque (Macaca mulatta).

We describe correlative clinicopathological/virological findings from a simian varicella virus (SVV)-seronegative monkey that developed disseminated varicella 105 days after gamma-irradiation. Twelve other monkeys in the colony were also irradiated, none of which developed varicella. Before irradiation, sera from the monkey that developed disseminated infection and one asymptomatic monkey were available. Analysis indicated that subclinical reactivation of latent SVV from an asymptomatic irradiated monkey likely led to disseminated varicella in the seronegative irradiated monkey. These findings parallel those from humans with disseminated varicella infection and support the usefulness of SVV infection as a model for human varicella-zoster virus infection, particularly virus reactivation after gamma-irradiation.

Prevalence and abundance of latently transcribed varicella-zoster virus genes in human ganglia.

In human ganglia latently infected with varicella-zoster virus (VZV), sequence analysis has revealed that five viral genes (VZV genes 21, 29, 62, 63, and 66) are transcribed. However, their comparative prevalence and abundance are unknown. Here, using real-time PCR, we analyzed 28 trigeminal ganglia from 14 humans for RNA corresponding to the five virus genes known to be transcribed in latently infected human ganglia. The most prevalent transcript found was VZV gene 63 (78%), followed by gene 66 (43%), gene 62 (36%), and gene 29 (21%). No gene 21 transcripts were detected in any of the 28 ganglia. VZV gene 63 RNA was also the most abundant (3,710 +/- 6,895 copies per 1 microg of mRNA) transcript detected in latently infected human ganglia, followed by VZV gene 29 (491 +/- 594), VZV gene 66 (117 +/- 85), and VZV gene 62 (64 +/- 38). Thus, the repeated detection and high abundance of VZV gene 63 transcripts in latently infected ganglia suggests that VZV gene 63 may be more important for the maintenance of virus latency than the less abundantly transcribed and randomly detected VZV genes 21, 29, 62, and 66.