CHRONIC ENTEROVIRUS INFECTION IN A PATIENT WITH MYALGIC ENCEPHALOMYELITIS/ CHRONIC FATIGUE SYNDROME (ME/CFS) – CLINICAL, VIROLOGIC AND PATHOLOGICAL ANALYSIS

John Chia, David Wang, Andrew Chia, Rabiha El-Habbal. 2015

Source: 19th International Picornavirus Meeting, 2016

OBJECTIVES: A 23 y/o Caucasian male developed prolonged, recurrent gastrointestinal symptoms, followed by onset of severe ME/CFS (CDC criteria, ICC). At initial evaluation, Echovirus 11 antibody titer was ≥1:640 (normal <1:10); IgG and IgM antibody for EBV and HHV6 were negative, CMV IgG was positive. He failed to respond to a combination of α and γ interferon; and debilitating symptoms of the stomach and central nervous system were minimally alleviated by SSRI, benzodiazepine and acid-suppressants. Repeated MRI scans of brain and spinal cords showed normal results. The patient committed suicide 6 years after the onset of symptoms. Brain was harvested and frozen within 24 hours of death for evaluation of chronic viral infection.

METHOD: Using 5D8/1 and J2 monoclonal antibody, stomach and colon biopsies obtained 5 months after onset of illness were stained for viral capsid protein (VP1) and dsRNA, respectively, by immunoperoxidase technique. Blood drawn in Paxgene tube 3 years after illness was screened for enterovirus RNA by RT-PCR. 1 cm3 sample was taken from the ponto-medullary junction (PM), medial temporal lobe (MT), frontal lobe (FL), occipital lobe (OL), cerebellum (CL) and midbrain/hypothalamus area (MB) of brain. The brain samples were homogenized in 10 ml of serum-free medium. Aliquots were processed for viral cultures. Trizol-LS reagent was used for RNA and protein extraction, as well as other lysis agents. Protein samples were separated with Tris-Glycine and MES gels, wet and semi-dry transfer, then western blot was performed with Ibind (Life technology) using EV-, CMV- and HHV6-speci c mAbs, patient’s own serum and control serum samples. Viral culture was performed in WI-38 and BGMK-DAF cell lines. RT-PCR for conserved highly-conserved sequences of 5’ end and 3D polymerase sequence were performed on extracted RNA.

RESULTS: Stomach and colon biopsies stained positive for EV VP1 soon after initial infection documenting the initial viral infection; dsRNA was detected in the stomach biopsies. EV RNA was not detected in blood 3 years after illness. Initial culture of brain samples did not grow virus; 5’ EV RNA sequence was not detected by RT-PCR. Using 5 D8/1 mAb, western blot revealed 37K protein band in the brain samples, which corresponded to viral protein extracted from infected stomach biopsies and enterovirus culture, but not in brain biopsy samples taking from patients with brain tuberculoma and lymphoma. 3D pol gene was ampli ed from the DNase-treated RNA extracted from PM, MT and FL.

CONCLUSION: The analysis of the second brain specimen taken from a ME/CFS patient replicated the British ndings published in 1994 (Ann. IM). The nding of viral protein and RNA in the brain specimens 6 years after documented acute enterovirus infection of the gastrointestinal tract is consistent with a chronic, persistent infection of the brain causing debilitating symptoms. EV is clearly one of the causes of ME/CFS, and antiviral therapy should be developed for chronic EV infection.