Tissue specific signature of HHV-6 infection in ME/CFS

Abstract

First exposure to various human herpesviruses (HHVs) including HHV-6, HCMV and EBV does not cause a life-threatening disease. In fact, most individuals are frequently unaware of their first exposure to such pathogens. These herpesviruses acquire lifelong latency in the human body where they show minimal genomic activity required for their survival. We hypothesized that it is not the latency itself but a timely, regionally restricted viral reactivation in a sub-set of host cells that plays a key role in disease development. HHV-6 (HHV-6A and HHV-6B) and HHV-7 are unique HHVs that acquire latency by integration of the viral genome into sub-telomeric region of human chromosomes. HHV-6 reactivation has been linked to Alzheimer's Disease, Chronic Fatigue Syndrome, and many other diseases. However, lack of viral activity in commonly tested biological materials including blood or serum strongly suggests tissue specific localization of active HHV-6 genome. Here in this paper, we attempted to analyze active HHV-6 transcripts in postmortem tissue biopsies from a small cohort of ME/CFS patients and matched controls by fluorescence in situ hybridization using a probe against HHV-6 microRNA (miRNA), miR-aU14. Our results show abundant viral miRNA in various regions of the human brain and associated neuronal tissues including the spinal cord that is only detected in ME/CFS patients and not in controls. Our findings provide evidence of tissue-specific active HHV-6 and EBV infection in ME/CFS, which along with recent work demonstrating a possible relationship between herpesvirus infection and ME/CFS, provide grounds for renewed discussion on the role of herpesviruses in ME/CFS.

Keywords: EBV; HHV-6; ME/CFS; epstein-barr virus; herpesvirus; viral pathology.

FIGURE 1

Representative fluorescence microscopy images of different human brain tissue types of ME/CFS patients. Tissues were co-stained with antibodies against HHV-6 specific proteins and/or neuronal tissue-specific marker and counter-stained for DAPI. (A) Human Choroid plexus tissues co-stained for HHV-6B U94 or OHV3, respectively. For each Panel images from the left to the right show markers for HHV-6 infection (red) and cell nuclei stained by DAPI (blue) and an overlay of the images. (B) Human anterior Hippocampus and Amygdala tissues co-stained for Iba1, HHV-6B U94 or OHV3. For each Panel images from the left to the right show astrocytes stained with Glial fibrillary acidic protein (GFAP) (red), markers for HHV-6 infection (green), cell nuclei stained by DAPI (blue) and an overlay of the images. (C) Human anterior Hippocampus and Amygdala tissues co-stained for Iba1 and HHV-6 miR-aU14. For each Panel images from the left to the right show astrocytes stained with GFAP (red), markers for HHV-6 infection (green), cell nuclei stained by DAPI (blue) and an overlay of the images. The scale bars represent 100 μm.

FIGURE 2

Representative fluorescence microscopy images of different human neuronal tissue types from two different ME/CFS patients and a control individual. For each panel images from the left to the right show HHV-6 p41 (red), a marker for active HHV-6A infection, miR-aU14 (green), cell nuclei stained with DAPI (blue) and an overlay of the images. (A) Human Dorsal root ganglia tissues of a ME/CFS patient. (B) Nerve, Lumbosacral nerve root, lumber nerve root and cervical nerve root tissues of a ME/CFS patient. (C) Lumbosacral nerve root tissue of a ME/CFS patient. The scale bars represent 100 μm.

FIGURE 3

Absence of HHV-6 infection in cerebellum of ME/CFS patients. Representative images showing Immuno fluorescence analysis for HHV-6B U94 in cerebellum samples. Cerebellum samples were stained using antibody against HHV-6B U94 together with GFAP (marker for astrocytes). DAPI was used to counterstain DNA. Each panel represents a different ME/CFS patient or a non-ME/CFS control. The scale bars represent 100 μm.

FIGURE 4

Co-infection of Epstein-Barr virus (EBV) in ME/CFS. Representative images showing Immuno fluorescence analysis for EBV dUTPase in multiple tissue samples from different ME/CFS patients and a non-ME/CFS control. Tissue samples were stained using a rabbit polyclonal antibody against EBV dUTPase. DAPI was used to counterstain DNA. The scale bars represent 100 μm.