Detection of human herpesvirus-6 in cerebrospinal fluid of patients with encephalitis.

OBJECTIVE: Virus infections are the most common causes of encephalitis, a syndrome characterized by acute inflammation of the brain. More than 150 different viruses have been implicated in the pathogenesis of encephalitis; however, because of limitations with diagnostic testing, causative factors of more than half of the cases remain unknown. METHODS: To investigate whether human herpesvirus-6 (HHV-6) is a causative agent of encephalitis, we examined for evidence of virus infection by determining the presence of viral sequence using polymerase chain reaction and assessed HHV-6 antibody reactivity in the cerebrospinal fluid of encephalitis patients with unknown cause. In a cohort study, we compared virus-specific antibody levels in cerebrospinal fluid samples of patients with encephalitis, relapsing-remitting multiple sclerosis, and other neurological diseases. RESULTS: Our results demonstrated increased levels of HHV-6 IgG, as well as IgM levels, in a subset of encephalitis patients compared with other neurological diseases. Moreover, cell-free viral DNA that is indicative of active infection was detected in 40% (14/35) of encephalitis patients, whereas no amplifiable viral sequence was found in either relapsing-remitting MS or other neurological diseases patients. In addition, a significant correlation between polymerase chain reaction detection and anti-HHV-6 antibody response was also demonstrated. INTERPRETATION: Collectively, these results suggested HHV-6 as a possible pathogen in a subset of encephalitis cases.

Reactivation of human herpesvirus-6 in natalizumab treated multiple sclerosis patients.

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.

Human herpesvirus 6 (HHV-6) induces dysregulation of glutamate uptake and transporter expression in astrocytes.

Human herpesvirus 6 (HHV-6) infects and establishes latency in the central nervous system (CNS). Reactivation of latent HHV-6 has been associated with neurologic diseases including epilepsy and multiple sclerosis (MS). In vivo, HHV-6 has been localized to astrocytes and can infect human astrocytes in vitro, suggesting that this virus may have a tropism for glial cells and may affect glial cell function. An essential role of astrocytes in the CNS is active maintenance of the excitatory neurotransmitter glutamate. Dysregulation of glutamate has been implicated as a potential mechanism of disease in both epilepsy and MS. Both disorders have demonstrated elevated glutamate in CSF and may be associated with dysregulation of glutamate signaling, uptake, and metabolism. This study demonstrates dysregulation of glutamate uptake in human astrocytes infected with both variants of HHV-6, A and B, with differential effects of HHV-6 in acute and persistently infected cells. Whereas astrocytes acutely infected with HHV-6 demonstrated increased glutamate uptake, cells persistently infected with HHV-6A and HHV-6B demonstrated impaired glutamate uptake. Functional dysregulation of glutamate uptake was associated with early increases in mRNA and protein expression of the glial glutamate transporter EAAT-2 followed by a sustained decrease in mRNA expression in astrocytes infected with both HHV-6A and HHV-6B. Dysregulated glutamate uptake and transporter expression suggests a mechanism for dysregulation of glutamate levels in vivo and a potential mechanism for virus-associated neurologic disease.

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

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.

Detection of active human herpesvirus-6 infection in the brain: correlation with polymerase chain reaction detection in cerebrospinal fluid.

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.

Complete replication cycle and acquisition of tegument in nucleus of human herpesvirus 6A in astrocytes and in T-cells.

The ultrastructural replication cycle of human herpesvirus 6A and 6B, both T-lymphotropic viruses, with tropism for the central nervous system, was compared by electron microscopy in the same cells, that is, in the T-lymphoblastoid cell line SupT-1 and in human astrocytes. Both HHV-6A and HHV-6B replicated efficiently in SupT-1 and formed viral particles. The tegument is the least characterized structure of the herpesviral particle and both variants were able to form intranuclear membrane compartments called tegusomes in SupT-1 where tegumentation occurred. Also, tegumentation occurred in HHV-6A infected cells in the nucleoplasm without the presence of a tegusome. This suggests that there is more than one possible route of tegumentation. Differences in the replication cycles between HHV-6A and HHV-6B were also observed in the cytoplasm. One such difference was that prominent annulate lamellae were only found in the cytoplasm of HHV-6A infected cells. In astrocytes a successful formation of viral particles was only seen with the HHV-6A variant. The HHV-6A virus life cycle in astrocytes resembled the life cycle in the T-cell line SupT-1, except that no annulate lamellae were found. Complete viral particles were found extracellularly around the astrocytes and the supernatant of infected astrocytes were able to re-infect SupT-1 cells. This suggests that HHV-6A infection in astrocytes can generate complete, viable, and infectious viral particles. The HHV-6 variants behave differently in the same type of cells and have different tropisms for astrocytes, supporting the notion that the variants might induce different diseases.

Efficacy of antiviral compounds in human herpesvirus-6-infected glial cells.

The beta-herpesvirus human herpesvirus-6 (HHV-6) is becoming increasingly recognized as an important pathogen in immunocompromised patients, particularly in post bone marrow transplant (BMT). Reactivation of latent HHV-6 resulting in encephalitis has been reported in BMT and stem cell transplant (SCT) patients. The development of HHV-6 encephalitis can be a fatal complication, the frequency of which is increasing likely due to improved diagnosis with quantitative polymerase chain reaction (PCR) of cerebrospinal fluid. There are currently no antiviral compounds approved for HHV-6, nor have any controlled clinical trials been conducted. The frequency and severity of HHV-6 encephalitis in both immunocompetent and immunocompromised patients necessitates studies on the usefulness of currently available anti-viral compounds. The authors compared the antiviral efficacy of four drugs currently used for cytomegalovirus (CMV) infection, a beta-herpesvirus sharing homology with HHV-6. In HHV-6A- and HHV-6B-infected T cells, acyclovir, ganciclovir, foscarnet, and cidofovir exhibited antiviral activity consistent with that published in other studies. In HHV-6-infected human astrocytes (U251), however, only foscarnet and cidofovir exhibited antiviral activity and this effect was restricted to infection with HHV-6 variant A. In pathological brain sections from patients with neurological disorders such as multiple sclerosis and epilepsy, HHV-6 has been localized to glial cells. Determination of antiviral activity in human glial fibrillary acidic protein (GFAP)-positive astrocytes of currently used antiviral compounds is essential for potential treatment of HHV-6 and neurological disorders. Our data highlight the necessity for further study of antiviral compound in HHV-6-infected glial cells as well as the development of more selective compounds for HHV-6.

Differential tropism of human herpesvirus 6 (HHV-6) variants and induction of latency by HHV-6A in oligodendrocytes.

Human herpesvirus 6 (HHV-6) is a ubiquitous beta -herpesvirus associated with a number of clinical disorders. Two closely but biologically distinct variants have been described. HHV-6 variant B causes the common childhood disease exhanthem subitum, and although the pathologic characteristics for HHV-6 variant A are less well defined, HHV-6A has been suggested to be more neurotropic. We studied the effect of both HHV-6 variants in an oligodendrocyte cell line (MO3.13). Infection of M03.13 was monitored by cytopathic effect (CPE), quantitative TaqMan PCR for viral DNA in cells and supernatant, reverse transcriptase-polymerase chain reaction (RT-PCR) to detect viral RNA, and indirect immunofluorescence (IFA) to detect viral protein expression. HHV-6A infection induced significantly more CPE than infection with HHV-6B. HHV-6B induced an abortive infection associated with a decrease of the initial viral DNA load over time, early RNA expression, and no expression of viral antigen. In contrast, infection with HHV-6A DNA persisted in cells for at least 62 days. During the acute phase of infection with HHV-6A, intracellular and extracellular viral load increased and cells expressed the viral protein IE-2 and gp116/54/64. No HHV-6A RNA or protein was expressed after 30 days post infection, suggesting that HHV-6A formed a latent infection. These studies provide in vitro support to the hypothesis that HHV-6 can actively infect oligodendrocytes. Our results suggest that HHV-6A and HHV-6B have different tropism in MO3.13 cells and that an initially active HHV-6A infection can develop latency. Differences between HHV-6A and -6B infection in different neural cell types may be associated with different neurological diseases.