Detection of human herpesvirus-6 in mesial temporal lobe epilepsy surgical brain resections

Abstract

Background: Human herpesvirus-6 (HHV-6), a ubiquitous beta-herpesvirus, is the causative agent of roseola infantum and has been associated with a number of neurologic disorders including seizures, encephalitis/meningitis, and multiple sclerosis. Although the role of HHV-6 in human CNS disease remains to be fully defined, a number of studies have suggested that the CNS can be a site for persistent HHV-6 infection.

Objective: To characterize the extent and distribution of HHV-6 in human glial cells from surgical brain resections of patients with mesial temporal lobe epilepsy (MTLE).

Method: Brain samples from eight patients with MTLE and seven patients with neocortical epilepsy (NE) undergoing surgical resection were quantitatively analyzed for the presence of HHV-6 DNA using a virus-specific real-time PCR assay. HHV-6 expression was also characterized by western blot analysis and in situ immunohistochemistry (IHC). In addition, HHV-6-reactive cells were analyzed for expression of glial fibrillary acidic protein (GFAP) by double immunofluorescence.

Results: DNA obtained from four of eight patients with MTLE had significantly elevated levels of HHV-6 as quantified by real-time PCR. HHV-6 was not amplified in any of the seven patients with NE undergoing surgery. The highest levels of HHV-6 were demonstrated in hippocampal sections (up to 23,079 copies/10(6) cells) and subtyped as HHV-6B. Expression of HHV-6 was confirmed by western blot analysis and IHC. HHV-6 was co-localized to GFAP-positive cells that morphologically appeared to be astrocytes.

Conclusions: HHV-6B is present in brain specimens from a subset of patients with MTLE and localized to astrocytes in the absence of inflammation. The amplification of HHV-6 from hippocampal and temporal lobe astrocytes of MTLE warrants further investigation into the possible role of HHV-6 in the development of MTLE.

Figure 1

Quantitative detection of human herpesvirus-6 variant B (HHV-6B) DNA in samples from normal donors and from brain surgery patients. PBMC ND = peripheral blood mononuclear cells (PBMC) from normal donors; PBMC Epilepsy patients = available PBMC from all brain surgery patients examined (mesial temporal lobe epilepsy [MTLE] and neocortical epilepsy [NE] patients); MTLE Brain resections = fresh brain tissue samples from surgical resections of MTLE patients; NE Brain resections = fresh brain tissue samples from surgical resections of NE patients; circles = PBMC; squares = hippocampus samples; triangles = lateral temporal lobe samples; rectangles = frontal lobe samples. Samples above the cutoff threshold (dotted line) were considered positive. ■ ▲ = Patient 2; = Patient 3; ▨ = Patient 6; = Patient 15.

Figure 2

Immunoreactivity to human herpesvirus-6 (HHV-6) p41 antigen protein extracts from frozen samples from Patient 2. Lane 1 = uninfected SupT-1 cells; lanes 2 to 4 = lateral temporal lobe, 5, 10, and 20 μL; lanes 5 to 7 = hippocampus, 5, 10, and 20 μL; lane 8 = protein ladder; lane 9 = Z29-infected SupT-1 cells. Respective band molecular weights are shown on the right.

Figure 3

In situ immunohistochemistry staining and localization of human herpesvirus-6 (HHV-6) gp116/54/64 in formalin-fixed paraffin-embedded tissue from patients with mesial temporal lobe epilepsy and control subjects. Samples are from hippocampus (A) (32×) and lateral temporal lobe (B) (40×) from Patient 2; hippocampus (C) (40×) and lateral temporal lobe (D) (32×) from Patient 3; and hippocampus (E) (32×) and lateral temporal lobe (F) (32×) from Patient 1. Positively stained cells of tissue samples from Patients 2 and 3 morphologically appear to be astrocytes (filled arrows), whereas oligodendrocytes and a proportion of astrocyte-appearing cells (open arrows) are negatively stained.

Figure 4

Co-localization of anti-human herpesvirus 6 (anti-HHV-6) gp116/54/64 reactivity in glial fibrillary acidic protein (GFAP)–reactive cells using double-immunofluorescence assay on formalin-fixed paraffin-embedded brain tissue. Positive staining for GFAP (green) but not for HHV-6 gp116/54/64 (red) of astrocytic cell line U251 was visualized with both fluorescein isothiocyanate and rhodamine filters (A). Positive staining for HHV-6 gp116/54/64 but not for GFAP of HHV-6B (strain Z29)–infected SupT-1 cell line (B) was seen after simultaneous incubation with both sets of antibodies. Lateral temporal lobe of Patient 2 shows presence of GFAP-reactive cells (green) (C, arrows) and of HHV-6 gp116/54/64-positive cells (red; D, arrows) incubated with both anti-GFAP and anti-gp116/54/64 primary antibodies. Juxtaposition of the combined images shows co-localization of GFAP and HHV-6 gp116 reactivity (yellow) (E, arrows) (20× magnification). All fields were counterstained with 4,6-diamidino-2-phenylindole (blue) to visualize nuclei.