Through a glass, darkly: cerebrospinal fluid viral load measurements and the pathogenesis of human immunodeficiency virus infection of the central nervous system.
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Central Nervous System↗
Biomedical subjects
Kenneth L Tyler
Publications and source records attributed to Kenneth L Tyler.
Quantitative CSF PCR in Epstein-Barr virus infections of the central nervous system.
Acute Epstein-Barr virus (EBV) infection of the central nervous system (CNS) is associated with meningoencephalitis and other neurological syndromes and with CNS lymphomas (CNSLs). Diagnosis is based on serological studies and more recently on detection of EBV DNA in cerebrospinal fluid (CSF) by polymerase chain reaction (PCR). We measured EBV DNA by quantitative PCR and EBV mRNA by RT-PCR in the CSF in patients with EBV-associated neurological disorders. EBV was identified as the cause of CNS infection in 28 patients: 14 with CNSL, 10 with encephalitis, and 4 with postinfectious neurological complications. CSF analysis showed that patients with CNSL had high EBV load (mean +/- standard error of 4.8 +/- 0.2 log(10) DNA copies/ml) and low leukocyte counts (22 +/- 7 cells/microl); encephalitis was characterized by high EBV load (4.2 +/- 0.3 log(10) DNA copies/ml) and high leukocyte counts (143 +/- 62 cells/microl); and patients with postinfectious complications showed low EBV load (3.0 +/- 0.2 log(10) DNA copies/ml) with high leukocyte counts (88 +/- 57 cells/microl). Lytic cycle EBV mRNA, a marker of viral replication, was identified in 10 CSF samples from patients with CNSL and encephalitis. These studies demonstrate the utility of quantitative CSF PCR and establish the presence of lytic cycle EBV mRNA in CSF of patients with EBV-associated neurological disease.
Adult↗
Use of PCR for the diagnosis of herpesvirus infections of the central nervous system.
Polymerase chain reaction (PCR) analysis of cerebrospinal fluid (CSF) has revolutionized the diagnosis of nervous system viral infections, particularly those caused by human herpesviruses (HHV). The PCR technique allows the detection of minute quantities of DNA or RNA in body fluids and tissues. Both fresh-frozen and formalin-fixed tissues may be utilized for PCR assays, with the latter making archival studies possible. CSF PCR has now replaced brain biopsy as the gold standard for the diagnosis of herpes simplex virus (HSV) encephalitis. PCR analysis of both CSF and nervous system tissues has also broadened our understanding of the spectrum of disease caused by HSV-1 and -2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV) and HHV-6. PCR results obtained from tissue specimens must be interpreted cautiously, since this highly sensitive technique may detect portions of viral genomic material that may be present even in the absence of active viral infection. Tissue PCR results in particular must be corroborated with clinical and neuropathologic evidence of central nervous system (CNS) infection. In several neurological diseases, negative PCR results have provided evidence against a role for herpesviruses as the causative agents. This review summarizes the role of CSF PCR in the diagnosis and therapeutic management of herpesvirus infections of the nervous system, particularly those caused by HSV and VZV.
Central Nervous System Viral Diseases↗
Reovirus-induced neuronal apoptosis is mediated by caspase 3 and is associated with the activation of death receptors.
Reovirus infection of the central nervous system (CNS) is an important experimental system for understanding the pathogenesis of neurotropic viral infection. Infection of neonatal mice with T3 reoviruses causes lethal encephalitis in which injury results from virus-induced apoptosis. We now show that this apoptosis in vivo is associated with activation of caspase 3, and use neuroblastoma and primary neuronal cultures to identify the cellular pathways involved. Reovirus-induced apoptosis in neuronal cultures is initiated by activation of the tumor necrosis factor (TNF) receptor superfamily death receptors and is inhibited by treatment with soluble death receptors (DRs). The DR-associated initiator caspase, caspase 8, is activated following infection, this activation is inhibited by a cell-permeable peptide inhibitor (IETD-CHO). In contrast to our previous findings in non-neuronal cell lines, reovirus-induced neuronal apoptosis is not accompanied by significant release of cytochrome c from the mitochondria or with caspase 9 activation following infection. This suggests that in neuronal cells, unlike their non-neuronal counterparts, the mitochondria-mediated apoptotic pathway associated with cytochrome c release and caspase 9 activation does not play a significant role in augmenting reovirus-induced apoptosis. Consistent with these results, peptide caspase inhibitors show a hierarchy of efficacy in inhibiting reovirus-induced apoptosis, with inhibitors of caspase 3 > caspase 8 >>> caspase 9. These studies provide a comprehensive profile of the pattern of virus-induced apoptotic pathway activation in neuronal culture.
Animals↗
Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels.
Many viruses belonging to diverse viral families with differing structure and replication strategies induce apoptosis both in cultured cells in vitro and in tissues in vivo. Despite this fact, little is known about the specific cellular apoptotic pathways induced during viral infection. We have previously shown that reovirus-induced apoptosis of HEK cells is initiated by death receptor activation but requires augmentation by mitochondrial apoptotic pathways for its maximal expression. We now show that reovirus infection of HEK cells is associated with selective cytosolic release of the mitochondrial proapoptotic factors cytochrome c and Smac/DIABLO, but not the release of apoptosis-inducing factor. Release of these factors is not associated with loss of mitochondrial transmembrane potential and is blocked by overexpression of Bcl-2. Stable expression of caspase-9b, a dominant-negative form of caspase-9, blocks reovirus-induced caspase-9 activation but fails to significantly reduce activation of the key effector caspase, caspase-3. Smac/DIABLO enhances apoptosis through its action on cellular inhibitor of apoptosis proteins (IAPs). Reovirus infection is associated with selective down-regulation of cellular IAPs, including c-IAP1, XIAP, and survivin, effects that are blocked by Bcl-2 expression, establishing the dependence of IAP down-regulation on mitochondrial events. Taken together, these results are consistent with a model in which Smac/DIABLO-mediated inhibition of IAPs, rather than cytochrome c-mediated activation of caspase-9, is the key event responsible for mitochondrial augmentation of reovirus-induced apoptosis. These studies provide the first evidence for the association of Smac/DIABLO with virus-induced apoptosis.
Apoptosis↗
Reovirus-induced alterations in gene expression related to cell cycle regulation.
Mammalian reovirus infection results in perturbation of host cell cycle progression. Since reovirus infection is known to activate cellular transcription factors, we investigated alterations in cell cycle-related gene expression following HEK293 cell infection by using the Affymetrix U95A microarray. Serotype 3 reovirus infection results in differential expression of 10 genes classified as encoding proteins that function at the G(1)-to-S transition, 11 genes classified as encoding proteins that function at G(2)-to-M transition, and 4 genes classified as encoding proteins that function at the mitotic spindle checkpoint. Serotype 1 reovirus infection results in differential expression of four genes classified as encoding proteins that function at the G(1)-to-S transition and three genes classified as encoding proteins that function at G(2)-to-M transition but does not alter any genes classified as encoding proteins that function at the mitotic spindle checkpoint. We have previously shown that serotype 3, but not serotype 1, reovirus infection induces a G(2)-to-M transition arrest resulting from an inhibition of cdc2 kinase activity. Of the differentially expressed genes encoding proteins regulating the G(2)-to-M transition, chk1, wee1, and GADD45 are known to inhibit cdc2 kinase activity. A hypothetical model describing serotype 3 reovirus-induced inhibition of cdc2 kinase is presented, and reovirus-induced perturbations of the G(1)-to-S, G(2)-to-M, and mitotic spindle checkpoints are discussed.
Cell Cycle↗