[Show abstract][Hide abstract]ABSTRACT: Characterization of a novel electrochemiluminescece assay for detection of HHV-6 using monoclonal anti-HHV-6 antibodies. (A) HHV-6B lysate (Z29 infected SupT-1 cells) reactivity with HHV-6 specific p41 monoclonal antibody. Reactivity is measured as photon emission. (B) HHV-6B lysate (Z29 infected SupT-1 cells) reactivity with larger panel of HHV-6 specific monoclonal antibodies except for the HHV-6A specific anti-p41/38 monoclonal antibody. (C) Lysate of uninfected SupT-1 cells reactive with the panel of HHV-6 specific antibodies. (D) Capture-detection assay using HHV-6 variant B lysate with the indicated monoclonal antibodies. (E) Capture-detection assay using HHV-6 variant A lysate with the indicated monoclonal antibodies. (F) Reactivity of 14 healthy control sera (dotted lines) against HHV-6B lysate. Solid black line illustrates the average antibody reactivity against HHV-6 in 14 healthy controls. (G) Sera from two natalizumab-treated patients TY20 and TY11 were tested for antibody reactivity against HHV-6B antigens at various dilutions as indicated. For panels D-G, reactivity is measured as photon emission on virus infected lysate minus photon emission on uninfected cells.
(1.55 MB TIF)
[Show abstract][Hide abstract]ABSTRACT: The alpha(4) integrin antagonist natalizumab was shown to be effective in patients with immune-mediated disorders but was unexpectedly associated with JC polyomavirus associated progressive multifocal leukoencephalopathy (PML) in two multiple sclerosis (MS) and one Crohn's disease patients. Impaired immune surveillance due to natalizumab treatment may have contributed to the JCV reactivation. As HHV-6 has been suggested to play a role in MS, we asked whether this virus could also have been reactivated during natalizumab therapy. Matched sera and CSF from a limited set of MS patients treated with and without natalizumab were examined for evidence of HHV-6. In addition, we also superinfected a persistent JC virus infected glial cell with HHV-6A to determine if JC virus can be increased. Elevated serum HHV6 IgG and HHV-6A DNA was detected in the CSF of a subset of patients but not controls. We confirmed that superinfection with HHV-6 of a JC virus infected glial cells increased expression of JCV. These results support the hypothesis that treatment with natalizumab may be associated with reduced immune surveillance resulting in reactivation of viruses associated with MS pathogenesis.
Full-text available · Article · Feb 2008 · PLoS ONE
[Show abstract][Hide abstract]ABSTRACT: Human herpesvirus-6 (HHV-6) is a beta-herpesvirus with 90% seroprevalence that infects and establishes latency in the central nervous system. Two HHV-6 variants are known: HHV-6A and HHV-6B. Active infection or reactivation of HHV-6 in the brain is associated with neurological disorders, including epilepsy, encephalitis, and multiple sclerosis. In a preliminary study, we found HHV-6B DNA in resected brain tissue from patients with mesial temporal lobe epilepsy (MTLE) and have localized viral antigen to glial fibrillary acidic protein (GFAP)-positive glia in the same brain sections. We sought, first, to determine the extent of HHV-6 infection in brain material resected from MTLE and non-MTLE patients; and second, to establish in vitro primary astrocyte cultures from freshly resected brain material and determine expression of glutamate transporters.
HHV-6B infection in astrocytes and brain specimens was investigated in resected brain material from MTLE and non-MTLE patients using PCR and immunofluorescence. HHV-6B viral DNA was detected by TaqMan PCR in brain resections from 11 of 16 (69%) additional patients with MTLE and from zero of seven (0%) additional patients without MTLE. All brain regions that tested positive by HHV-6B variant-specific TaqMan PCR were positive for viral DNA by nested PCR. Primary astrocytes were isolated and cultured from seven epilepsy brain resections and astrocyte purity was defined by GFAP reactivity. HHV-6 gp116/54/64 antigen was detected in primary cultured GFAP-positive astrocytes from resected tissue that was HHV-6 DNA positive-the first demonstration of an ex vivo HHV-6-infected astrocyte culture isolated from HHV-6-positive brain material. Previous work has shown that MTLE is related to glutamate transporter dysfunction. We infected astrocyte cultures in vitro with HHV-6 and found a marked decrease in glutamate transporter EAAT-2 expression.
Overall, we have now detected HHV-6B in 15 of 24 patients with mesial temporal sclerosis/MTLE, in contrast to zero of 14 with other syndromes. Our results suggest a potential etiology and pathogenic mechanism for MTLE.
Full-text available · Article · Jun 2007 · PLoS Medicine
[Show abstract][Hide abstract]ABSTRACT: Primary Astrocytes Isolated and Cultured from HHV-6B–Positive MTLE Brain Resections Express Viral DNA and Antigen
Primary astrocytes were isolated from fresh brain material obtained during epilepsy brain resection.
(A) Cells were cultured for 3–4 wk and stained for GFAP (green), DAPI (blue; nuclei), and the nonvariant specific HHV-6 antigen gp116/54/64 (red). Representative immunofluorescence images show primary astrocyte cultures from four epilepsy brain resections (patients 14, 15, 5, and 6). All images were acquired with a 20× objective.
(B) Cells were scraped from fixed slides (patients 5 and 6), DNA was extracted, and DNA for HHV-6 U57 (major capsid protein) was detected by nested PCR. Negative and positive controls used were uninfected SupT1 T cells and HHV-6B (strain Z29)–infected SupT1 T cells, respectively.
(36 KB JPG)
[Show abstract][Hide abstract]ABSTRACT: One-half of bone-marrow transplant (BMT) and stem-cell transplant recipients have reactivation of latent human herpesvirus
(HHV)-6 2–4 weeks after transplant. Although the detection of viral DNA, RNA, and antigen in brain material confirmed active
HHV-6 variant B infection, peak viral loads in cerebrospinal fluid (CSF) and serum occurred 2–4 weeks before death and decreased
to low levels before or at autopsy. All autopsy samples consistently demonstrated HHV-6 active infection in the hippocampus.
Astrocytic cells positive for viral antigen provided support for an HHV-6-specific tropism for hippocampal astrocytes. HHV-6
DNA in CSF and serum may not reflect the level of active viral infection in the brain after BMT.
Full-text available · Article · Mar 2007 · The Journal of Infectious Diseases