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Non-cytolytic enterovirus
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== Detection of non-cytolytic infections == Non-cytolytic infection replicates at too low a level for detection by cytopathic effect, but non-cytolytic enterovirus can be detected in tissues samples by sensitive reverse transcription-PCR (RT-PCR) and by immunohistochemistry.<ref name="Chapman2012">{{Cite web|url=https://www.jdrfnpod.org/wordpress/wp-content/uploads/2013/08/1a-chapman.pdf | title = Replication Defective Enterovirus Infections:Implications for Type I Diabetes. Slides from a presentation by Prof Nora M. Chapman given at the 2012 Annual Meeting of the Network for Pancreatic Organ Donors with Diabetes, Page 22 | last = Chapman | first = Nora | date = 2012 | website = jdrfnpod|archive-url=|archive-date=|url-status=|access-date=}}</ref> In their 1999 study Tam and Messner<ref name="Tam1999" /> used RT-PCR to detect the non-cytolytic enteroviral RNA in the tissues, with different primers to distinguish positive from negative viral ssRNA. In order to detect the dsRNA, they first used the RNase I enzyme to destroy the viral ssRNA in the tissue, after which a positive result from RT-PCR testing indicates the presence of dsRNA. [[File:Enterovirus VP1 & dsRNA in stomach, Chia 2015. 400px.png|thumb|400x400px|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.]] For the immunohistochemistry detection of non-cytolytic enterovirus, Dr John Chia uses the 5-D8/1 monoclonal antibody stain for highlighting enterovirus VP1 protein, and the J2 monoclonal antibody stain to reveal the dsRNA component of non-cytolytic infection.<ref name="Chia2015" /> Chia employs such tissue staining to detect non-cytolytic enterovirus infections in ME/CFS patients' stomach tissue biopsies. In a study, Chia found enterovirus VP1 protein in 82% of ME/CFS patients' stomach biopsies, compared to 20% of the controls.<ref name="ChiaChia2008" /> Indirect evidence of chronic enterovirus infection is suggested by the persistently elevated coxsackievirus B or echovirus antibody titers often observed in ME/CFS patients with these infections. However these raised antibody titers are usually only detected when sensitive techniques such as plaque reduction neutralization testing or micro-neutralization testing are employed. Complement fixation testing (CFT) is not sensitive enough to detect antibody titers in chronic enterovirus infections (CFT is only useful in the first few months of initial infection).<ref>{{Cite journal | last = Christensen | first = M. L. | last2 = Pachman | first2 = L.M. | last3 = Schneiderman | first3 = R. | last4 = Patel | first4 = D.C. | last5 = Friedman | first5 = J.M. | date = Nov 1986 | title = Prevalence of Coxsackie B virus antibodies in patients with juvenile dermatomyositis|url=https://www.ncbi.nlm.nih.gov/pubmed/3022759|journal=Arthritis and Rheumatism|volume=29|issue=11|pages=1365–1370|issn=0004-3591|pmid=3022759|quote=Patient and control sera were tested by 2 main serologic methods that primarily reflect IgG function: the complement-fixation (CF) test and the neutralization test. The CF test measures antibody that, usually, can be detected for only a few months after the onset of a viral infection, whereas neutralizing antibody can be detected for years after the initial infection.|via=}}</ref><ref>{{Cite web|url=http://www.investinme.org/IIMEC4.shtml | title = Diagnosis and Treatment of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome Associated with Chronic Enterovirus Infection. Presentation at the Invest in ME International ME Conference, London 2009 (available on DVD). Timecode: 27:51. | last=Chia | first = John | date = 2009 | website = [[Invest in ME Research]]|archive-url=|archive-date=|url-status=|access-date=|quote=The typical antibody that the laboratory would do is called the complement fixation test, which is neither sensitive nor specific. That means if you get a positive test, it's worthless. And if you get a negative test, it's worthless. Well that's wonderful.}}</ref> Dr Chia measured the antibody titers of 200 consecutive ME/CFS patients and 150 controls, using the [http://ltd.aruplab.com/tests/pub/0060055 ARUP Lab] CVB and echovirus antibody micro-neutralization tests, and found the patients' titers were often substantially higher than the mean titers of the controls.<ref>{{Cite web|url=https://www.youtube.com/watch?v=Xz7CaxWtCUU&t=10m34s | title = The Role of Enteroviruses in Myalgic Encephalomyelitis / Chronic Fatigue Syndrome. Enterovirus Session, International Symposium on Viruses in CFS & Post-viral Fatigue, Maryland, US, June 2008. Timecode: 10:34 . | last=Chia | first = John | date = 2008 | website = YouTube | archive-url=|archive-date=|url-status=|access-date=}}</ref><ref>{{Cite journal | last = Chia | first = J K S | date = Nov 2005 | title = The role of enterovirus in chronic fatigue syndrome|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1770761/|journal=Journal of Clinical Pathology|volume=58|issue=11|pages=1126–1132|doi=10.1136/jcp.2004.020255|issn=0021-9746|pmc=1770761|pmid=16254097|quote=During our study to determine the infectious aetiology in the first 200 patients with severe fatigue following flu-like illnesses, ... Significantly raised neutralising antibody titres against six coxsackieviruses or five echoviruses were found in more than half of those patients who had initial respiratory and/or gastrointestinal tract infections, compared with 150 control subjects|via=}}</ref> Highly elevated CVB titers may also be seen in a small subset of dilated cardiomyopathy patients.<ref>{{Cite journal | last = Cambridge | first = G. | last2 = MacArthur | first2 = C.G. | last3 = Waterson | first3 = A.P. | last4 = Goodwin | first4 = J.F. | last5 = Oakley | first5 = C.M. | date = Jun 1979 | title = Antibodies to Coxsackie B viruses in congestive cardiomyopathy|url=https://www.ncbi.nlm.nih.gov/pubmed/223612/|journal=British Heart Journal|volume=41|issue=6 | pages = 692–696|issn=0007-0769|pmid=223612|quote=High neutralisation titres (≥1024) to Coxsackie B viruses were more common among the patients than among the controls. ... A micro-neutralisation test system was used for the Coxsackie B antibody studies|via=}}</ref> PCR blood testing is not sufficiently sensitive for the chronic enteroviral infections found in ME/CFS, because enteroviral RNA is only present at low levels in the blood, Dr Chia discovered. Chia determined that with special techniques and repeated testing, enterovirus RNA can be detected in around 30% of whole blood samples taken from chronically infected enterovirus-associated ME/CFS patients.<ref>{{Cite web|url=https://www.enterovirusfoundation.org/treatments-1/ | title = Enterovirus Foundation | last = Chia | first = John | date = |website=Enterovirus Foundation|archive-url=|archive-date=|url-status=|access-date=}}</ref> This finding emerged from Chia's lab work in which over a 5 year period he tested more than 2,500 blood samples from over 510 ME/CFS patients by PCR. Chia observed that only 35% of patients tested positive twice or more for enteroviral RNA in their peripheral blood lymphocytes (PBL), using a highly sensitive semi-nested RT-PCR test kit from Chemicon (with an assay sensitivity of 80 to 800 copies of RNA per ml of blood). In the same RT-PCR test 4% of the controls tested positive. Interestingly, in these RT-PCR blood tests, Chia found enterovirus RNA in 70% of the severe bedridden patients, but only in 12% of the less ill.<ref>{{Cite web|url=https://www.youtube.com/watch?v=Xz7CaxWtCUU&t=15m22s | title = The Role of Enteroviruses in Myalgic Encephalomyelitis / Chronic Fatigue Syndrome. Enterovirus Session, International Symposium on Viruses in CFS & Post-viral Fatigue, Maryland, US, June 2008. Timecode: 15:22. | last=Chia | first = John | date = 2008 | website = YouTube | archive-url=|archive-date=|url-status=|access-date=}}</ref><ref>{{Cite web|url=https://www.youtube.com/watch?v=obHtCwhg3-0&t=9m25s | title = The Pathogenic Role of Enteroviruses in Myalgic Encephalomyelitis / Chronic Fatigue Syndrome. Presentation at the State of Knowledge Workshop on ME/CFS, National Institutes of Health, April 2011, Day 1, Part 1. Timecode: 9:25. | last=Chia | first = John | date = 2011 | website = YouTube | archive-url=|archive-date=|url-status=|access-date=}}</ref> Furthermore, when 20 of the sickest patients' PBL cells were retested every 3 to 6 months for 2 years, Chia found that patients who had tested positive might be negative on the next test, and then might be positive again on a subsequent test; he observed it was rare for a patient with a positive PCR result to consistently test positive. Chia concluded it was clear that the enteroviral RNA present in the blood is at very low levels.<ref>{{Cite web|url=https://www.youtube.com/watch?v=Xz7CaxWtCUU&t=16m04s | title = The Role of Enteroviruses in Myalgic Encephalomyelitis / Chronic Fatigue Syndrome. Enterovirus Session, International Symposium on Viruses in CFS & Post-viral Fatigue, Maryland, US, June 2008. Timecode: 16:04. | last=Chia | first = John | date = 2008 | website = YouTube | archive-url=|archive-date=|url-status=|access-date=}}</ref> Dr Chia says any PCR blood tests which have insufficient sensitivity (of above 1000 copies of RNA per ml of blood) will almost always give negative results in ME/CFS patients.<ref>{{Cite web|url=http://www.investinme.org/IIMEC10.shtml | title = Dr John Chia: Enteroviruses and Myalgic Encephalomyelitis / Chronic Fatigue Syndrome: An Update on Pathogenesis. Presentation at the Invest in ME International ME Conference, London 2015 (available on DVD). Timecode: 12:00. | last=Chia | first = John | date = 2015 | website = Invest in ME Research|archive-url=|archive-date=|url-status=|access-date=}}</ref> Note that when Dr Chia was aiming to detect non-cytolytic enterovirus infection in the brain tissue of a deceased ME/CFS patient, on RT-PCR testing he initially got a negative result. Dr Chia then remembered a phenomenon whereby viral RNA may bind to chromosomal DNA, which prevents the RT-PCR from detecting the viral RNA. So Dr Chia then used the DNase enzyme to digest the chromosomal DNA in the brain tissue, and after doing this, he was able to find enterovirus RNA in various brain areas.<ref>{{Cite web|url=https://www.me-pedia.org/wiki/Autopsy_in_Myalgic_Encephalomyelitis|title=Autopsy in Myalgic Encephalomyelitis|at=See the section "Chia 2015 study"}}</ref>
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