Bulgarian Participation in European Network on Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Human Herpesvirus Infection Associated Biomarkers of ME/CFS – Optimization of PCR-Based Detection

Infections of Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus-6 and -7 (HHV-6, HHV-7) are suspected as etiological agents of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). The aim of this study was to select and optimize PCR-based systems for detection of EBV, CMV and HHV-6 in biological samples from ME/CFS patients and controls. Whole blood and plasma samples obtained from 5 individuals, and DNA samples containing the entire viral genome of EBV, CMV and HHV-6 were used in this study. Conventional and nested PCR systems were assessed for their efficiency in virus DNA amplification. For PCR optimization modifications in reaction volume, concentration of primers and MgCl2, and in annealing temperatures were assessed. Sensitivity of reactions was estimated by serial dilutions of viral DNA. PCR systems were tested with HSV, VZV, EBV, CMV and HHV-6 for cross-reactivity. EBV, CMV and HHV6 DNA were not detected in blood and serum samples by conventional PCR and the first round of nested PCR. EBV, CMV and HHV-6 positive samples were only detected in the second round of nested PCR in blood samples (two samples were EBV-positive, CMV and HHV-6 were detected in one sample each). The PCR conditions for each virus in both steps of amplification were optimized. The sensitivity of nested PCR was determined to be not less than 200 copies/mL of sample. No cross-reactivity was detected with other herpes viruses. The optimized nested PCR assays are highly sensitive and specific and allow detection of EBV, CMV and HHV-6 in clinical specimens to study the potential role of these viruses in ME/CFS.