List of enterovirus infection studies

Dr John Chia uses the 5-D8/1 monoclonal antibody to reveal enterovirus VP1 protein in the stomach biopsy tissues of ME/CFS patients (the brown stains in images A and C), and uses the J2 monoclonal antibody to reveal enterovirus dsRNA in the stomach tissues (brown stains in images B and D). 100X magnification. ©︎ Frontiers 8(6), 2020.

Enteroviruses such as Coxsackie B virus have long been associated with myalgic encephalomyelitis, with nearly thirty studies finding evidence of chronic enterovirus infection in ME/CFS patients. The enterovirus theory of ME/CFS is the oldest and probably most researched.

Early studies in the 1970s and 1980s focused on the elevated coxsackievirus B antibody titers often found in ME/CFS, which suggested persistent enterovirus infections in these patients, but did not provide direct evidence of the virus. Once molecular testing methods such as PCR became available towards the end of the 1980s, then studies were able to demonstrate direct evidence of enterovirus infection, by detecting enteroviral RNA in the tissues of ME/CFS patients.

The late 1980s also saw the introduction of the 5-D8/1 monoclonal antibody, developed by Professor James Mowbray and his team, for detecting enterovirus VP1 protein in tissue biopsies and in the blood (the antibody binds to VP1 and stains the tissues brown when enterovirus is present). This again provided direct evidence of persistent enterovirus infection.

Throughout the 1990s, using molecular methods such as PCR, a series of British studies detected enterovirus RNA much more frequently in the muscle tissues of ME/CFS patients compared to healthy controls. And one important study by Cunningham et al uncovered the first evidence that the persistent infections found in ME/CFS were due to a mutated enterovirus; this mutated virus is now reasonably well understood and referred to as non-cytolytic enterovirus. 

These British findings of enterovirus infection in the tissues of ME/CFS patients were not replicated in the United States until Dr John Chia's 2008 study, which found both enterovirus RNA and enterovirus VP1 protein much more frequently in the stomach tissues of ME/CFS patients compared to controls. 

There have been over 30 ME/CFS enterovirus studies in total, and the majority have been positive (finding enterovirus much more commonly in ME/CFS patients than healthy controls); in the list below, positive studies are indicated by a + symbol. However, there were also 4 negative studies (finding no difference between patients and healthy controls) and these are indicated by the − symbol. Note that two of these negative studies had small cohorts of only around 30 patients. Note also that some studies used PCR primers targeting the 5′UTR region of the enteroviral genome — a region known to contain deletions in chronic enterovirus infections; thus these PCR primers may not be appropriate for detecting chronic enterovirus, which might explain some negative study results.

Early ME/CFS enterovirus research

This early period of enterovirus research from 1970 to 2003 (comprises mainly British studies).

	Study Title	Date	Details
+	Encephalomyelitis Resembling Benign Myalgic Encephalomyelitis Innes SGB	1970	This study examined 4 cases of ME. In the cerebrospinal fluid they found coxsackievirus B2 in one case and echovirus 3 in another. In the sera of the two other cases they found elevated coxsackievirus B2 and elevated coxsackievirus B5 titers via antibody testing.
+	Sporadic myalgic encephalomyelitis in a rural practice Keighley BD, Bell EJ	1983	This study found elevated coxsackievirus B titers in 16 of of 20 patients in an ME/CFS outbreak in Ayrshire, UK.
+	Myalgic encephalomyelitis — report of an epidemic Fegan KG, Behan PO, Bell EJ	1983	This study found elevated coxsackievirus B titers in 18 out of 20 patients in an ME/CFS outbreak in Ayrshire, UK
+	Coxsackie B infection in a Scottish general practice Calder BD, Warnock PJ	1984	This study found high antibody titers to coxsackievirus B in 38 out of 81 patients who experienced a syndrome with many of the features of myalgic encephalomyelitis.
	Some long-term sequelae of Coxsackie B virus infection Gray JA	1984	Discussion of coxsackievirus B and echovirus in relation to ME/CFS.
+	A study of Coxsackie B virus infections, 1972-1983 Bell EJ, McCartney RA	1984	Found that in well-documented cases of ME/CFS, 41% of patients had elevated neutralizing antibody titers, compared to 4% of healthy controls.
+	Coxsackie B viruses and the post-viral syndrome: a prospective study in general practice. Calder BD, Warnock PJ, McCartney RA, Bell EJ	1987	This study found significant neutralizing antibodies to Coxsackie B viruses in the blood sera of 46% of patients with symptoms of post-viral syndrome, compared to 25% of controls. Of the patients testing posting for Coxsackie B antibodies 55% were "still unwell after one year and high antibody titres persisted in all but two of the patients". Recovery was not found to correlate with a fall in antibody level, but was more rapid in patients whose presenting symptoms were paraesthesiae, anorexia or dyspnoea".
+	Chronic enterovirus infection in patients with postviral fatigue syndrome Yousef GE, Bell EJ, Mann GF, Murugesan V, Smith DG, McCartney RA, Mowbray JF	1988	This study on 87 ME/CFS patients found 51% were positive for the enterovirus VP1 antigen in the serum. None of the 30 healthy controls were positive for the VP1 antigen. SIGNIFICANCE: One of the first studies to find direct evidence of persistent enterovirus in the blood.
+	Postviral fatigue syndrome: persistence of enterovirus RNA in muscle and elevated creatine kinase Archard, LC, Bowles, NE, Behan PO, Bell EJ, Doyle D	1988	Using molecular hybridization testing to detect RNA from coxsackievirus B, they found enteroviral RNA in the quadriceps muscles of 20 out of 96 ME/CFS patients, but in none of the 4 controls. Enteroviral RNA was found even in ME/CFS patients who had had this illness for 20 years, indicating a very long-term persistent infection. SIGNIFICANCE: One of the first studies to find direct evidence for persistent enterovirus infection in the skeletal muscles of ME/CFS patients.
+	Coxsackie B viruses and myalgic encephalomyelitis Bell EJ, McCartney RA, Riding MH	1988	This study on 290 adults and 47 children with ME/CFS found 37% and 38% respectively were IgM positive for coxsackievirus B, compared to 9% in 500 healthy adult controls.
+	VP-1 antigen in chronic postviral fatigue syndrome Halpin D, Wessely S	1989	This study on 38 ME/CFS patients found 30% of the blood samples were positive for the enterovirus VP1 antigen, compared to 12% out of 43 controls who had other non-ME/CFS neurological illnesses.
+	Persistence of enteroviral RNA in chronic fatigue syndrome is associated with the abnormal production of equal amounts of positive and negative strands of enteroviral RNA Cunningham L, Bowles NE, Lane RJ, Dubowitz V, Archard LC	1990	This study found enterovirus RNA in the skeletal muscles of 4 out out 8 ME/CFS patients, and in none of the 4 controls. But moreover they found that in ME/CFS patients' muscles, there were roughly equal amounts of enteroviral positive strand RNA and negative strand RNA (ratio of 1:1), whereas in normal acute lytic enterovirus infection, the positive strand is around 100 times more common than the negative strand (ratio of 100:1). The authors thus suggested a mutant defective enterovirus may be responsible for the persistent infections found in ME/CFS. SIGNIFICANCE: This study was the first to demonstrate evidence for a mutated defective enterovirus in ME/CFS, which we now refer to as non-cytolytic enterovirus (or non-cytopathic enterovirus). The same mutated non-cytolytic enterovirus has also been found in several other chronic diseases.
+	Myalgic encephalomyelitis--a persistent enteroviral infection? Dowsett EG, Ramsay AM, McCartney RA, Bell EJ	1990	This study found that out of 420 cases of ME/CFS, 205 had significant titers to coxsackievirus B.
-	Antibody to coxsackie B virus in diagnosing postviral fatigue syndrome Miller NA, Carmichael HA, Calder BD, Behan PO, Bell EJ, McCartney RA, Hall FC	1991	In Dunbartonshire, Scotland, 243 patients with postviral fatigue syndrome and an equal number of matched controls had serological tests to detect coxsackievirus B IgM antibodies by ELISA, and IgG antibodies by the micrometabolic inhibition method. No significant difference was observed in the titer levels of patients and controls. However, Dr John Chia found that the ELISA, IFA and CFT antibody test methods are not sufficiently sensitive to detect chronic enterovirus infections in ME/CFS patients; he found only the gold-standard plaque reduction neutralization method was sensitive enough to detect chronic enterovirus.Note that this study did not test for echovirus, another enterovirus associated with ME/CFS.
+	Enteroviral RNA sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome Gow JW, Behan WM, Clements GB, Woodall C, Riding M, Behan PO	1991	This study of 60 ME/CFS patients found 20% had high titers to coxsackievirus B, compared to 14% of healthy controls. Furthermore, 53% of these ME/CFS patients had enteroviral RNA sequences in their muscles, compared to 15% for healthy controls.
+	Chronic fatigue syndrome: clinical condition associated with immune activation Landay AL, Jessop C, Lennette ET, Levy JA	1991	This study of found a higher of coxsackievirus B antibody titers in ME/CFS patients compared to healthy controls.
+	Amplification and identification of enteroviral sequences in the postviral fatigue syndrome Gow JW1, Behan WM	1991	This study of 60 ME/CFS patients found that ME/CFS patients were 6.7 times more likely to have enteroviral RNA in their muscle tissue, compared to healthy controls.
+	Persistent virus infection of muscle in postviral fatigue syndrome Cunningham L, Bowles NE, Archard LC	1991	This study of 140 ME/CFS patients found enteroviral RNA in 24% of their muscle biopsy samples, and Epstein-Barr virus DNA in a further 9% of these biopsy samples (though no ME/CFS patient had both enterovirus and EBV in their muscle biopsies). No enterovirus RNA was detected in any of the 152 control samples of human muscle.
+	Interferon-α therapy for patients with chronic fatigue syndrome Brook MG, Bannister BA, Weir WRC	1993	18 patients who had ME/CFS were given interferon alpha therapy for 12 weeks, after which patients were observed for a further 3 months. Patients were divided into two groups of 9, one group were treated immediately, and the second group had their treatment delayed for 3 months; none of the second group recovered significantly while waiting for treatment. At the end of the study, 3 out of 18 patients recovered completely, and 2 of these 3 remained well 12 months later. A further 2 patients were improved at the end of the 3 month follow-up period and remained so up to 8 months later. Out of the 5 patients who recovered or improved, 4 had coxsackievirus B IgM antibodies in their serum; but only 1 of the patients who did not improve had coxsackievirus B antibodies. SIGNIFICANCE: the study concluded that if persisting coxsackievirus B antibodies reflect chronic enterovirus infection, interferon alpha may ameliorate ME/CFS by suppressing enterovirus replication; but the study gave an alternative explanation that the known immunostimulatory properties of interferon alpha may account for the treatment response. However, the fact clinical response only occurred in CVB patients tends to argue against the alternative explanation.
+	Persistence of enterovirus RNA in muscle biopsy samples suggests that some cases of chronic fatigue syndrome result from a previous, inflammatory viral myopathy Bowles NE, Bayston TA, Zhang HY, Doyle D, Lane RJ, Cunningham L, Archard LC	1993	This study of 158 ME/CFS patients found enteroviral RNA in 26% of the patients' muscle biopsy samples, compared to only 1% in healthy controls.
+	Studies on enterovirus in patients with chronic fatigue syndrome Gow JW, Behan WM, Simpson K, McGarry F, Keir S, Behan PO	1994	This study of 121 patients with ME/CFS found enteroviral RNA in 26.4% of the patients' muscle biopsy samples, and found enteroviral RNA in 19.8% the muscle biopsies of patients other neuromuscular disorders. From these results the authors concluded that "it is unlikely that persistent enterovirus infection plays a pathogenetic role in CFS, although an effect in initiating the disease process cannot be excluded." However, this conclusion may not be sound, firstly because persistent enterovirus is associated with a wide range of diseases, including chronic inflammatory myopathy, and thus might be playing a role in these other neuromuscular disorders as well; and secondly because persistent enterovirus infections are also found in other organs in ME/CFS patients, such as the brain and stomach, and it is possible ME/CFS might in fact be caused by an enterovirus brain infection, rather than (or in addition to) a muscle infection. Dr John Chia also points out that patients with chronic neuromuscular diseases are not as active as normal people, and the highly sensitive but qualitative RNA PCR test used in this study could not compare quantitatively the amounts of viral RNA in tissue specimens of the two groups.
+	Enterovirus in the chronic fatigue syndrome McGarry F, Gow J, Behan PO	1994	This study details an autopsy of a deceased ME/CFS patient. Enteroviral RNA was found in the heart, muscles, hypothalamus and brainstem of this patient (this brain infection shown in red in the image), and this RNA showed an 83% similarity to coxsackievirus B3. Control tissue samples taken from four patients who died of cerebrovascular diseases, and another four who had depression and committed suicide, showed no evidence of enteroviral RNA. SIGNIFICANCE: This was the first of three ME/CFS autopsies which found enterovirus infection in the brain. See also: post-mortem brain studies.
-	Enteroviruses and the chronic fatigue syndrome Swanink CM, Melchers WJ, van der Meer JW, Vercoulen JH, Bleijenberg G, Fennis JF, Galama JM	1994	This study, conducted by some of the psychologists at the Nijmegen Group in the Netherlands did not find any difference between the blood and stool samples of 76 ME/CFS patients and 76 healthy controls. The study used IgG, IgM, and IgA antibody ELISA testing (not very sensitive for chronic enterovirus), antibody complement fixation testing (insensitive for chronic enterovirus), Prof Mowbray's VP1 test on the serum, isolation of virus from stool specimens, and nested PCR on stool samples. In the case of the stool testing, almost all patients and controls were negative. Dr John Chia found the ELISA, IFA and CFT antibody test methods are not sufficiently sensitive to reliably detect chronic enterovirus infections in ME/CFS patients; Dr Chia found only the gold-standard plaque reduction neutralization method was sufficiently sensitive to detect chronic enterovirus.
+	Detection of enterovirus-specific RNA in serum: the relationship to chronic fatigue Clements GB, McGarry F, Nairn C, Galbraith DN	1995	This study of 88 ME/CFS patients found enteroviral RNA in the serum of 41% of patients, compared to 2% of healthy controls.
+	Phylogenetic analysis of short enteroviral sequences from patients with chronic fatigue syndrome Galbraith DN, Nairn C, Clements GB	1995	This study examined the 5′ region of the genome of enteroviruses taken from ME/CFS patients and found that patients nearly always have viruses with a different genetic makeup compared to the enteroviruses found in acute, self-limiting infections in healthy controls. SIGNIFICANCE: We now know that most ME/CFS patients are infected with non-cytolytic enterovirus, an enterovirus that due to acquired mutations in the 5′ region transmutes into a form capable of causing persistent infection. This study was the first to provide genomic evidence that ME/CFS patients are infected with a mutated enterovirus.
-	Investigation by polymerase chain reaction of enteroviral infection in patients with chronic fatigue syndrome McArdle A, McArdle F, Jackson MJ, Page SF, Fahal I, Edwards RH	1996	This study examined 34 muscle biopsies from ME/CFS patients and 10 muscle biopsies from healthy controls, but did not detect enterovirus RNA in the patient or control muscle tissues. But commenting on the differences between their negative and the positive results found in Gow 1991, the authors say: "The difference in findings between our study and that of Gow et al. may be due to the population of CFS patients studied. Although the diagnostic criteria of both groups was broadly similar, the patients used in the study of Gow et al. all reported that the illness had an acute onset after a feverish illness, whereas only 58% of patients in our study reported a viral infection before the onset of their illness. Another possible explanation comes from the work of Galbraith et al. Direct sequencing of PCR products from enterovirus-positive serum samples of patients with CFS by these authors has suggested the presence of distinct novel enteroviruses. These viruses are thus unclassified and their site of replication is unknown. Thus, the different findings of different studies may reflect the nature of the samples examined. "
-	No findings of enteroviruses in Swedish patients with chronic fatigue syndrome Lindh G, Samuelson A, Hedlund KO, Evengård B, Lindquist L, Ehrnst A	1996	This Swedish study examined 29 muscle biopsies from ME/CFS patients, but could not detect enterovirus RNA in the tissues.
	Evidence for enteroviral persistence in humans Galbraith DN, Nairn C, Clements GB	1997	8 ME/CFS patients were tested for enterovirus by PCR via 2 serum samples taken at least 5 months apart. 4 patients had virtually identical nucleotide sequences in the 5′ region of the enteroviral genome in both samples, and the sequence pairs also each had a unique shared pattern indicating that the virus had persisted.
+	Viral Isolation from Brain in Myalgic Encephalomyelitis Richardson J	2001	This study examined the brain of an ME patient who died through suicide, and found enteroviral VP1 protein in the fibroblasts of small blood vessels in the cerebral cortex, plus some patchy distribution of enteroviral VP1 protein in a small fraction of glial cells (this brain infection shown in red in the image). Note that most glial cells are astrocytes, and one study found coxsackie B virus can create a persistent infection in human astrocyte cells lines, and another study showed this virus can infect the astrocytes in murine brains. See also: post-mortem brain studies.
+	Enterovirus related metabolic myopathy: a postviral fatigue syndrome Lane RJ, Soteriou BA, Zhang H, Archard LC	2003	This study of 48 patients with ME/CFS found enteroviral sequences by reverse transcription nested polymerase chain reaction (RT-NPCR) in 20.8% of the patients' muscle biopsy samples, while all the 29 control samples were negative for such sequences. SIGNIFICANCE: This could be the first study demonstrating an an association between enterovirus and post-exertional malaise (PEM). While this 2003 study makes no mention of PEM, the conclusion suggests an association between enterovirus RNA in muscle and abnormal lactate response to exercise. In 2019, studies began appearing showing how abnormal blood lactate corresponds with PEM severity in ME/CFS.
+	Detection of enterovirus in human skeletal muscle from patients with chronic inflammatory muscle disease or fibromyalgia and healthy subjects Douche-Aourik F, Berlier W, Féasson L, Bourlet T, Harrath R, Omar S, Grattard F, Denis C, Pozzetto B	2003	This study used RT-PCR to test the skeletal muscle biopsies of 30 patients with fibromyalgia/chronic fatigue syndrome for enterovirus: 13% of patients were found positive. They also tested the muscles of 15 patients with chronic inflammatory muscle diseases: 20% of patients were positive. None of the 29 healthy control muscle biopsies were found positive.

Dr John Chia's ME/CFS enterovirus research

John Chia in California: his research papers on enterovirus in ME/CFS from 2005 to present.

	Study Title	Date	Details
+	The role of enterovirus in chronic fatigue syndrome Chia J	2005	A review paper summarizing experimental and clinical evidence supporting the role of enterovirus in chronic fatigue syndrome. This paper also details some experimental antiviral treatments of ME/CFS. The first experiment used ribavirin plus interferon alpha therapy, resulting in improvements in symptoms, the elimination of enteroviral RNA from peripheral blood mononuclear cells (PBMC), and a fourfold reduction in coxsackievirus B antibody titers. However, after therapy was complete, relapse typically occurred 4 to 5 months later, along with antibody titers increasing to pretreatment values and the enteroviral RNA returning to the PBMC. In another experiment, interferon alpha plus interferon delta were used in combination to treat severe bedbound ME/CFS patients: 6 out of 11 treated patients were able to return to full or part-time work as a result, but again relapse occurred some month later. Another study also found interferon alpha therapy improves only enterovirus-associated ME/CFS.  SIGNIFICANCE: although interferon therapy is usually not a long-term solution to ME/CFS, its temporary major efficacy provides supporting evidence for the theory that enterovirus can cause and maintain the ME/CFS condition. For further info on interferon for ME/CFS, see: interferon.
+	Chronic fatigue syndrome is associated with chronic enterovirus infection of the stomach Chia JK, Chia AY	2008	This study of 165 ME/CFS patients found enterovirus VP1 protein in 82% of stomach tissue samples from patients, compared to 20% in healthy controls. 37% of the ME/CFS patient stomach tissue samples also tested positive for enterovirus RNA, compared to less than 1% for healthy controls. The persistent presence of enteroviral VP1 and RNA in the tissues is a signature of non-cytolytic enterovirus infection. SIGNIFICANCE: This was the first US study to replicate the original British research. It also introduced a significant innovation: previous British studies had used muscle tissue biopsies in order to test for enterovirus infections in ME/CFS. But muscle biopsies are painful and leave a scar, and so are not ideal for routine clinical use. Dr John Chia realized that in ME/CFS, enterovirus also usually infects stomach tissues, and that stomach tissue biopsies are painless and easier to perform. Testing the stomach tissues is thus a very useful proxy for less accessible tissues such as the muscle and of course the brain.
+	Acute enterovirus infection followed by myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and viral persistence Chia J, Chia A, Voeller M, Lee T, Chang R	2010	This study followed patients who were hospitalized for acute enterovirus infections, and found that in the next few years subsequent to the acute infections, symptoms consistent with ME/CFS emerged in 3 patients.
+	Functional Dyspepsia and Chronic Gastritis Associated with Enteroviruses Chia JK, Chia AY, Wang D, El-Habbal R	2015	In this study of 416 ME/CFS patients with functional dyspepsia (FD), and 66 patients with functional dyspepsia but without ME/CFS, who underwent stomach tissue biopsies between 2006 and 2012, enterovirus VP1 protein was found in stomach tissue samples of 82% of the ME/CFS + FD patients, and 83% of the FD only patients. Enterovirus double-stranded RNA (dsRNA) was found in in the stomach tissues of 64% and 63% of these two patient cohorts respectively. SIGNIFICANCE: This larger study confirms the result of Dr John Chia's previous 2008 study. This study also extended the earlier results by finding enteroviral dsRNA in the stomach tissues of ME/CFS patients, which is a characteristic feature of persistent non-cytolytic enterovirus infection.
+	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	This brain autopsy on a deceased ME/CFS patient found evidence of enterovirus infection in various areas of the brain: the pontomedullary junction and midbrain (both are in the brainstem), medial temporal lobe, lateral frontal cortex, occipital lobe and cerebellum (these various infected brains areas shown in red in the image). See post-mortem brain studies for more details about this study.

Other investigations by John Chia

Dr Chia took more than 2500 blood samples from more than 510 ME/CFS patients, and in their peripheral blood lymphocyte (PBL) cells, 35% of these patients tested positive for enterovirus RNA, using a very sensitive test: the Chemicon RT-PCR EAI 3 primer test, with Qiagen 1-step RT-PCR enzyme.

Interestingly, for the more severe bedridden patients, Dr Chia found enterovirus RNA in 70% of the PBL cells. But for the less ill patients, enterovirus RNA was found in only 12%.

The sensitivity of the test was high, around 80 to 800 RNA copies per ml of blood, yet typically the same patient would not always test positive using this test (they would be positive on some occasions and negative on others), so Chia says it is clear that the enteroviral RNA present in the blood in ME/CFS is at very low levels. Dr Chia says any PCR tests which have insufficient sensitivity (above 1000 copies of RNA per ml of blood) will almost always give negative results in ME/CFS patients.

See also

• Enterovirus
• Non-cytolytic enterovirus
• Coxsackie B virus
• Echovirus
• Post-mortem brain studies
• John Chia
• List of herpesvirus infection studies

Learn more

• Dr John Chia, International Symposium on Viruses In CFS 2008, The Role of Enterovirus in ME/CFS
• Dr John Chia, State of Knowledge Workshop on ME/CFS Research 2011 (Day 1) Part 1
• Dr John Chia, State of Knowledge Workshop on ME/CFS Research 2011 (Day 1) Part 2

• Invest in ME International ME Conference, London 2009: Diagnosis and Treatment of ME/CFS Associated With Chronic Enterovirus Infection

• Invest in ME International ME Conference, London 2010: Enterovirus Infection in ME/CFS

• Invest in ME International ME Conference, London 2011: Clinical and Research Experience of Enteroviral Involvement in ME/CFS
• Invest in ME International ME Conference, London 2015: Enteroviruses and ME/CFS: An Update on Pathogenesis
• Chronic viral infections in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), Enterovirus Section.
• The Enterovirus Theory of Disease Etiology in ME/CFS: A Critical Review, paper by Adam O'Neal and Maureen R. Hanson, 2021.
• The viral origin of myalgic encephalomyelitis/chronic fatigue syndrome, paper by Maureen R. Hanson, 2023.