Association of human herpesvirus-6B with mesial temporal lobe epilepsy

Abstract

Background: 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.

Methods and findings: 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.

Conclusions: 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.

Figure 1. High Levels of HHV-6 DNA Are Detected in Brain Resections from Patients with MTLE

HHV-6B DNA was quantitated in PBMCs, serum, and fresh brain (hipp, hippocampus; LTL, lateral temporal lobe) from epilepsy brain resections by TaqMan PCR. Viral load was normalized as DNA copies/10⁶ cells (brain and PBMCs) or DNA copies/ml (serum). Data from brain, serum, and PBMCs are represented graphically from patients with and without MTLE. Means are shown by black bars.

Figure 2. Primary Astrocytes Isolated and Cultured from HHV-6B–Positive MTLE Brain Resections Express Viral Antigen

Primary astrocytes were isolated from fresh brain material obtained during epilepsy brain resection. Cells were cultured for 3–4 wk and costained for the nonvariant specific HHV-6 gp116 surface glycoprotein and GFAP as a marker for astrocytes (A–C), the neuronal marker Tuj1 (A–B), or the microglial marker CD68 (D). (A) Epilepsy patient 2a: GFAP = blue; HHV-6 = green; Tuj1 = red, 20×. (B) Epilepsy patient 2a: GFAP = blue; HHV-6 = green; Tuj1 = red, 32×. (C) Epilepsy patient 15; GFAP = green, HHV-6 = red; DAPI = blue, 40×. (D) Epilepsy patient 15; GFAP = green; CD68 = red; DAPI = blue, 40×.

Figure 3. Longitudinal Characterization of HHV-6 Infection in One Patient with Multiple Brain Resections Followed by Hemispherectomy

(A) HHV-6B viral load was quantitated from fresh tissue obtained from three consecutive brain resections (13 February 2004, 18 March 2004, and 15 June 2004) followed by hemispherectomy (19 January 2005) using variant-specific TaqMan PCR. Primary astrocytes were cultured from tissue obtained at hemispherectomy from frontal/parietal and temporal lobes. HHV-6B viral load was quantitated by Taqman in these astrocyte cultures (1 March 2005) approximately 6 wk after surgery. All viral loads are represented as DNA copies/10⁶ cells. (B) Expression of viral RNA was determined by RT-PCR using primers for three HHV-6 genes (from top to bottom: U86, immediate early [primers specific for variants A and B], U67, late; U100, late). Lane 1, patient 16 frontal/parietal lobe astrocytes; lane 2, patient 16 temporal lobe astrocytes; lane 3, negative control temporal lobe; lane 4, patient 16 frontal/parietal lobe; lane 5, patient 16 temporal lobe; lane 6, patient 16 occipital lobe; lane 7, patient 16 frontal lobe; lane 8, uninfected JJahn (negative control); lane 9, U1102-infected JJahn (positive control).

Figure 4. Decreased Expression of the Glutamate Transporter EAAT-2 in HHV-6–Infected Astrocytes

(A) EAAT-2 mRNA was detected by RT-PCR in fresh brain from patient 16, in astrocytes cultured from patient 15, and in astrocytes cultured from patient 20, and infected with HHV-6 in vitro. Samples from the two different patients were run in two separate PCR reactions (on different days) using the same set of EAAT-2 primers but different actin primers. The products of these PCR reactions were run on the same gel. Lane 1, patient 16 frontal/parietal lobe; lane 2, patient 16 temporal lobe; lane 3, patient 16 occipital lobe; lane 4, patient 16 frontal lobe; lane 5, patient 16 frontal/parietal lobe astrocytes; lane 6, patient 16 temporal lobe astrocytes; lane 7, uninfected astrocytes cultured from patient 20; lane 8, HHV-6A (strain U1102)–infected astrocytes cultured from patient 20; lane 9, HHV-6B (strain Z29)–infected astrocytes cultured from patient 20; lane 10, negative control (JJhan T cells); lane 11, positive control (U251 astrocytes). (B) EAAT-2 mRNA was detected by quantitative TaqMan and normalized to expression of HPRT from astrocytes cultured from frontal/parietal (F/P) and temporal (TL) lobes of patient 16. Astrocytes from patient 20 were mock-infected or were infected with HHV-6A (strain U1102) or HHV-6B (strain Z29).