title: Enteroviruses and chronic fatigue syndrome
Creators: name: Nairn, Carron
Publisher: name: University of Glasgow
Year: 1999

description: A prospective study was initiated to follow a group of patients with chronic fatigue syndrome (CFS) and examine their serum for evidence of enteroviral sequences over time and to compare any positive PCR products by sequence analysis. Additionally, a questionnaire was developed to determine the clinical nature of the illness and to try to correlate this with the enteroviral status of the patient at a particular time. Initial phylogenetic analysis of sequence derived from twenty serum positive samples showed that the sequences derived from CFS patients grouped apart from the known enteroviruses and from sequences derived from other clinical cases on a phylogenetic tree (Galbraith et al., 1995). The sequences were approximately 69-84% similar to the closest sequence of a coxsackievirus B3. However, the sequences could have been from known enteroviruses with no sequence data available, from known enteroviruses with variant 5' NTRs or from previously unknown enteroviruses. Further analysis at a later date with other available sequence data showed that the sequences grouped within the coxsackie/echovirus group although some were still 10-13% different. The variation observed was of the order of that seen between clinical isolates of the same serotype, thus the CFS patient sequences may have represented known enteroviruses with variant 5' NTRs. Without corroboration from other regions this tentative group could not be described further. The capsid region was chosen as an alternative target region since it is relatively conserved among the enteroviruses, containing the neutralizing regions which form the basis of the serotypes. Semi-nested priming could amplify the majority of the known enteroviruses to a sensitivity of 1 TCID50, which was 100-fold less than the standard PCR. Testing of 55 samples previously positive by the standard PCR did not generate any positives. The use of other techniques (inverse PCR, long PCR and production of a cDNA library) to acquire additional sequence were also unsuccessful. Either the assays were not sensitive enough to detect the low levels of RNA present in the serum (as assessed by the standard PCR) or the genome is so different or deleted (perhaps due to the presence of defective-interfering particles) that amplification was not possible. Indeed, the discovery that 85% of enteroviral RNA samples lacked a poly-A tail may also point to there being atypical sequence present. Limited sample volume prevented this from being pursued further. Persistence was examined by analyzing the 5' NTR sequence of two sequential samples obtained from sixteen patients. Many sequences, however, were more similar to those isolated in the same year than to their respective pair. Additionally, correlation with clinical data did not suggest a role for enteroviral persistence in the maintenance of the syndrome, although a positive correlation between severity of symptoms (based on ability to work and time spent walking for example) and the presence of enteroviral sequence was noted on analysis of first questionnaires from a cohort of 130 patients. This was not present on analysis of the second questionnaire from each patient. The average duration of fatigue for this group of patients was 3.9 years and thus questionnaire analysis proved difficult in terms of patients recalling features prior to their fatigue. In a preliminary study, a group of patients who had a history of fatigue of approximately six months was described. Enteroviral sequences were detected in the serum of 42% of CFS patients compared to 27% of acutely-ill patients and 2% of healthy control individuals. Additionally, a number of positives in the CFS patient group were detected after one round of PCR only, perhaps indicating a higher titre of enterovirus in these samples. While this study was being carried out there was an outbreak of echovirus 4 and this suggested therefore that the virus played a direct role in triggering the syndrome. Sequence analysis of PCR positives was not performed at this time to confirm this theory. Thus it may be useful to test patients shortly after the onset of fatigue where there is a history of a flu-like illness, when there is a greater chance of recovering a possible triggering agent, even though a formal diagnosis has not been made. Consistently, a higher proportion of CFS patients than comparison individuals were positive for enteroviral sequences throughout this study (except in 1997) and thus this work does not rule out the enteroviruses as being involved in CFS. However, the methods employed did not elucidate any further sequence information nor provide a definitive answer regarding the role of these viruses in individuals with a long history of illness.