[Relationship between variation of coxsackievirus B3 VP1 sequence from cerebrospinal fluid of children and severity of damage to central nervous system].

OBJECTIVE: To investigate the possible relationship between variation of coxsackievirus B3 (CoxB3) VP1 sequence from cerebrospinal fluid of children with severe and mild central nervous system (CNS) infection and damage to CNS in children from Shandong province. METHODS: The enteroviruses were detected using VP1 typing and sequencing primer for enteroviruses from 73 enterovirus-infected cases confirmed by detection of cerebrospinal fluid by enteroviruses common primer. VP1 sequences (450 nucleotides) were determined and analyzed for 21 CoxB3 enteroviruses strains isolated in Qingdao and Binzhou, and were compared with that of BLAST search procedures from GeneBank in NCBI. The variation of VP1 gene and amino acids sequence of CoxB3 enteroviruses was analyzed for severe and mild CNS infection. RESULTS: The nucleotide homogeneity of these CoxB3 appeared to be 97% - 99%, however, the homogeneity among different genotypes were 83% - 76%. Replacement of glutamine by histidine at amino acid locus 856 of VP1 CoxB3 was found in 4 cases with severe encephalitis. There were different variation in VP1 nucleotide sequence of CoxB3 in 3 cases with mild encephalitis and 14 cases with meningitis, but amino acids sequences had no regular variation. The modified Glasgow's coma score was below 7 in all the 4 cases with severe encephalitis. Of these 4 cases, 3 had consciousness disturbance for less than 3 days. Lethargy, restlessness and psychiatric symptoms were major manifestations, of whom 3 also had dysphagia, 1 had encephalatrophy obviously, Glasgow's coma score was 3, deep coma lasted for 9 days, and had concomitant fatal epileptic attacks. Of these 4 cases, 2 completely recovered, 1 had high muscle tone, 1 remained under anti-epileptic drug treatment at follow-up 6 months later. CONCLUSION: There were a small epidemic of CoxB3 CNS infection in children in 2005 in this area. The amino acid variation of CoxB3 VP1 possibly caused increased viral virulence and caused damage to CNS.

Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR.

Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/microl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10-100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100-500 copies/10(5) PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/10(5) cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50-10 ng DNA.

Presence of JC virus-specific CTL in the cerebrospinal fluid of PML patients: rationale for immune-based therapeutic strategies.

OBJECTIVES: There is urgent need of a treatment for progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). To evaluate the rationale for immunotherapy of PML, we explored whether JCV-specific cytotoxic T lymphocytes (CTL) can penetrate the central nervous system (CNS). In addition, we studied the breadth of their T-cell receptor (TCR) repertoire, and sought to establish a reliable method to expand these cells in vitro. DESIGN AND METHODS: We enrolled 18 patients in this study, including 16 with proven or possible PML (15 HIV-positive and one HIV-negative), and two HIV-positive patients with other neurological diseases. Detection of JCV-specific CTL in the blood and the cerebrospinal fluid was performed by Cr release and tetramer staining assays in 15 patients. RESULTS: Of 11 PML patients with analyzable cerebrospinal fluid (CSF), two had no detectable JCV-specific CTL in the blood and CSF and died 3.7 and 7.2 months later. The nine remaining patients had an inactive course of PML and detectable JCV-specific CTL in the blood. In addition, four of them (44%) also had detectable JCV-specific CTL in the CSF. Both HIV-positive patients with OND had detectable JCV-specific CTL in the blood and one in the CSF. Using tetramer technology, we obtained highly enriched JCV-specific CTL lines that were able to kill target cells presenting JCV peptides. The breadth of the TCR repertoire was CTL epitope dependent. CONCLUSIONS: These results indicate that JCV-specific CTL are present in the CNS of PML patients and pave the way for an immune-based therapeutic approach.

HIV-1 promotes quiescence in human neural progenitor cells.

The exact mechanism by which human immunodeficiency virus type 1 (HIV-1) produces dementia remains obscure. We have recently found that chemokines can inhibit neural progenitor cell proliferation. We hypothesized that HIV-1 could also inhibit neural progenitor cell proliferation by chemokine receptor signaling. We found that HIV-1 coat proteins that used C-C chemokine receptor 3 or C-X-C chemokine receptor 4 as coreceptors inhibited proliferation of neural progenitor cells in isolated cultures, as well as in hippocampal slices. The cerebrospinal fluid from patients with dementia also inhibited neural progenitor cell proliferation in these culture systems. To obtain an in vivo correlation, we examined hippocampus tissue obtained from patients with dementia at autopsy and found reduced numbers of neural progenitor cells in patients with dementia, compared with patients without dementia. Apolipoprotein E3, but not E4, antagonized the effects of coat proteins. We found reduced phosphorylation of extracellular signal-regulated kinase in neural progenitor cells treated with coat proteins, which may explain the protein's mechanism of action. We conclude that HIV-1 inhibits neural progenitor cell proliferation, which may result in impaired ability to form new memories and learn new tasks.