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Non-cytolytic enterovirus
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== Some studies in non-cytolytic enterovirus research == Within a few years of their discovery in the late 1940s, the Coxsackie B viruses were shown to be involved in myocarditis,<ref name="Chapman1997">{{Cite book | last = Chapman | first = N.M. | last2 = Ramsingh | first2 = A.I. | last3 = Tracy | first3 = S. | date = 1997 | title=Genetics of Coxsackievirus Virulence|url=https://link.springer.com/chapter/10.1007/978-3-642-60687-8_11|language=en|location=Berlin, Heidelberg|publisher=Springer Berlin Heidelberg |pages=227–258 |doi=10.1007/978-3-642-60687-8_11|isbn=9783642645075}}</ref> but direct evidence that enterovirus was able to maintain a persistent infective presence in the heart muscle tissues would have to wait until molecular testing techniques became available in the late 1980s which could detect the virus in the tissues by its RNA. Once these molecular techniques were employed, they not only demonstrated that enteroviral RNA is chronically present in the tissues, but they led to an intriguing further discovery: that in persistent enterovirus tissue infections, the ratio of positive to negative strand enteroviral RNA was greatly reduced compared to acute enterovirus infection. This prompted the speculation that persistent enterovirus infections might involve a virus with a mutated defective genome. Fifteen years later, these genomic defects were first detected by Nora Chapman, Steven Tracy, Kyung-Soo Kim, William Tapprich and colleagues in a landmark 2005 paper.<ref name="Kim2005b" /> The defects were in the form of deletions in the 5′ region of the genome. In this same paper, Chapman identified the precise molecular mechanism by which these acquired genome defects were able to transform lytic enterovirus (which normally only causes acute infections) into a virus capable of very long-term viral persistence in the tissues. Later studies confirmed the presence of these deletions in the 5′ region in other chronic enterovirus human heart infections, thus providing supporting evidence for Chapman's proposed mechanism. * '''Archard''' et al 1987: <ref name="Archard1987" /> using molecular hybridization testing detected Coxsackie B virus RNA in 56% of the heart tissue biopsies from myocarditis or dilated cardiomyopathy, whereas biopsies from controls with non-viral heart diseases were all negative. The authors also found viral RNA in the skeletal muscles of patients with chronic myositis (juvenile dermatomyositis and adult polymyositis). This may have been one of the first studies to provide solid evidence of the persistent presence of enterovirus in the tissues. * '''Archard''' et al 1988: <ref name="Archard1988">{{Cite journal | last = Archard | first = L. C. | last2 = Bowles | first2 = N.E. | last3 = Behan | first3 = P.O. | last4 = Bell | first4 = E.J. | last5 = Doyle | first5 = D. | date = Jun 1988 | title = Postviral fatigue syndrome: persistence of enterovirus RNA in muscle and elevated creatine kinase|url=https://www.ncbi.nlm.nih.gov/pubmed/3404526|journal=Journal of the Royal Society of Medicine|volume=81|issue=6|pages=326–329|doi=10.1177/014107688808100608|issn=0141-0768|pmc=1291623|pmid=3404526}}</ref> may have been the first to find evidence for persistent enterovirus infection in the skeletal muscles of ME/CFS patients, using molecular hybridization testing to detect enteroviral RNA. They found enteroviral RNA even in ME/CFS patients who had had this illness for 20 years. * '''Righthand''' et al 1989: <ref name="Righthand1989">{{Cite journal | last = Righthand | first = V. F. | last2 = Blackburn | first2 = R.V. | date = Dec 1989 | title = Steady-state infection by echovirus 6 associated with nonlytic viral RNA and an unprocessed capsid polypeptide|url=https://www.ncbi.nlm.nih.gov/pubmed/2585604/|journal=Journal of Virology|volume=63|issue=12 | pages = 5268–5275|issn=0022-538X|pmid=2585604}}</ref> demonstrated that an echovirus 6 infection could be maintained for over 6 years in a cell line, and that this infection was and was not cytolytic (showed no cytopathic effect) when transfected into uninfected susceptible cells. * '''Cunningham''' et al 1990: <ref name="Cunningham1990">{{Cite journal | last = Cunningham | first = L. | last2 = Bowles | first2 = N.E. | last3 = Lane | first3 = R.J. | last4 = Dubowitz | first4 = V. | last5 = Archard | first5 = L.C. | date = Jun 1990 | title = 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|url=https://www.ncbi.nlm.nih.gov/pubmed/2161907|journal=The Journal of General Virology|volume=71|issue = Pt 6|pages=1399–1402|doi=10.1099/0022-1317-71-6-1399|issn=0022-1317|pmid=2161907}}</ref> was possibly the first indication that there was something odd about persistent enterovirus infections. In normal lytic enterovirus infections, there is around 100 times more positive strand RNA than negative strand RNA in infected cells. But in the enterovirus-infected muscles of ME/CFS patients, Cunningham et al found roughly equal amounts of positive and negative strand RNA. Thus they found instead of the normal approximately 100:1 positive to negative strand ratio, in ME/CFS the ratio is closer to 1:1. The authors concluded that this finding of near equal amounts, together with the failure to isolate and culture infectious lytic virus or detect virus-specific antigens, suggested that a mutant defective enterovirus may be responsible for these persistent muscle infections. * '''Archard''' et al 1991: <ref name="| last = Archard1991">{{Cite journal | last = Archard | first = L. C. | last2 = Bowles | first2 = N.E. | last3 = Cunningham | first3 = L. | last4 = Freeke | first4 = C.A. | last5 = Olsen | first5 = E.G. |last6 = Rose | first6 = M.L. | last7 = Meany | first7 = B. | last8 = Why | first8 = H.J. | last9 = Richardson | first9 = P.J. | date = Aug 1991 | title = Molecular probes for detection of persisting enterovirus infection of human heart and their prognostic value|url=https://www.ncbi.nlm.nih.gov/pubmed/1655452|journal=European Heart Journal|volume=12|issue=Suppl D | pages = 56–59|issn=0195-668X|pmid=1655452}}</ref> found a reduced positive to negative strand RNA ratio of around 1:1 in the heart muscle in dilated cardiomyopathy, and in myocarditis after the acute inflammation had cleared up. This is just as Cunningham had found in the skeletal muscles of ME/CFS patients. The authors concluded that the persisting enterovirus infection of the myocardium is probably underpinned by defective virus mutants which do not complete a productive replication cycle. * '''Klingel''' et al 1992: <ref name="Klingel1992">{{Cite journal | last = Klingel | first = K. | last2 = Hohenadl | first2 = C. | last3 = Canu | first3 = A. | last4 = Albrecht | first4 = M. | last5 = Seemann | first5 = M. | last6 = Mall | first6 = G. | last7 = Kandolf | first7 = R. | date = 1992-01-01 | title = Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation | url =https://www.ncbi.nlm.nih.gov/pubmed/1309611/|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=89|issue=1|pages=314–318|issn=0027-8424|pmid=1309611}}</ref> found the same reduced positive to negative strand ratio of around 1:1 in an immunocompetent mouse model of chronic myocarditis. They also noted a strong correlation, both spatial and temporal, between viral replication and formation of lesions in both the acute and chronic phase of infection. * '''Tam and Messner''' 1999: <ref name="Tam1999" /> in a chronic CVB myositis mouse model, detected a decreased positive to negative strand RNA ratio of about 1:1 in the muscle tissues, mirroring what Cunningham had found in ME/CFS patients. They also demonstrated that enteroviral persistence in the muscle is characterized by the formation of enteroviral dsRNA. The authors pointed out that intracellular ssRNA decays within hours, so they postulated that the persistence of non-cytolytic enterovirus may instead arise from the dsRNA component of this infection, suggesting that the stability of dsRNA prevents its elimination by the immune system. They also suggested this dsRNA may contribute to pathogenicity of non-cytolytic enterovirus infection. So in the Tam and Messner model of non-cytolytic enterovirus, the viral dsRNA is hypothesized to be central to sustaining this form of infection. * '''Feuer''' et al 2002: <ref name="Feuer2002">{{Cite journal | last = Feuer | first = Ralph | last2 = Mena | first2 = Ignacio | last3 = Pagarigan | first3 = Robb | last4 = Slifka | first4 = Mark K. | last5 = Whitton | first5 = J. Lindsay | date = May 2002 | title = Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro|url=https://www.ncbi.nlm.nih.gov/pubmed/11932410/|journal=Journal of Virology|volume=76|issue=9 | pages = 4430–4440|issn=0022-538X|pmid=11932410}}</ref> proposed that persistent coxsackievirus B infection may be based on infection of quiescent cells, and observed that in quiescent cells, CVB appears to be able to persist in a form similar to viral latency. * '''Kim''' et al 2005: <ref name="Kim2005b" /> in this landmark study, Prof Nora Chapman identified the mechanism which underpins enteroviral RNA persistence. They isolated persistent coxsackievirus B3 from the hearts of mice that had been infected with this enterovirus one year earlier, and discovered mutations in this virus: deletions in the 5′ region at the end of the viral genome. They point out that such deletions are observed to inhibit lytic virus replication by decreasing positive strand viral RNA production, and propose that this lytic virus inhibition provides the opportunity for mutated virus populations to dominate the cell, thereby giving birth to persistent non-cytolytic infection. In the Chapman theory of non-cytolytic enterovirus, the deletions in the viral genome are central to creating and sustaining this persistent form of enterovirus infection. * '''Chapman''' et al 2008: <ref name="Chapman2008a">{{Cite journal | last = Chapman | first = Nora M. | last2 = Kim | first2 = Kyung-Soo | last3 = Drescher | first3 = Kristen M. | last4 = Oka | first4 = Kuniyuki | last5 = Tracy | first5 = Steven | date = 2008-06-05 | title = 5' terminal deletions in the genome of a coxsackievirus B2 strain occurred naturally in human heart|url=https://www.ncbi.nlm.nih.gov/pubmed/18378272/|journal=Virology|volume=375|issue=2 | pages = 480–491|doi=10.1016/j.virol.2008.02.030|issn=0042-6822|pmc=2440640|pmid=18378272}}</ref> found for the first time non-cytolytic enterovirus with deletions in the genomic 5′ region in human coxsackievirus B2 myocarditis. Previously non-cytolytic enterovirus with such deletions had only been detected in mouse models. * '''Chia and Chia''' 2008: <ref name="ChiaChia2008">{{Cite journal | last = Chia | first = J.K. S. | last2 = Chia | first2 = A.Y. | date = Jan 2008 | title = Chronic fatigue syndrome is associated with chronic enterovirus infection of the stomach|url=https://www.ncbi.nlm.nih.gov/pubmed/17872383|journal=Journal of Clinical Pathology|volume=61|issue=1 | pages = 43–48|doi=10.1136/jcp.2007.050054|issn=1472-4146|pmid=17872383|quote=No significant cytopathic effect was shown in the EV RNA-positive cultures, whereas wild type strains of enteroviruses would cause major cytopathic changes within one week.|via=}}</ref> found enteroviral VP1 protein in stomach tissues of 82% of ME/CFS patients, compared to 20% of the controls, and found enteroviral RNA in 37% of patient tissues compared to 5% of controls. They discovered that in the stomach tissues of ME/CFS patients, lytic virus is not always found, despite the presence of enteroviral RNA. This is a signature of non-cytolytic infection. * '''Feuer''' et al 2009: <ref name="Feuer2009">{{Cite journal | last = Feuer | first = Ralph | last2 = Ruller | first2 = Chelsea M. | last3 = An | first3 = Naili | last4 = Tabor-Godwin | first4 = Jenna M. | last5 = Rhoades | first5 = Ross E. | last6 = Maciejewski | first6 = Sonia | last7 = Pagarigan | first7 = Robb R. | last8 = Cornell | first8 = Christopher T. | last9 = Crocker | first9 = Stephen J. | date = Sep 2009 | title = Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period | url =https://www.ncbi.nlm.nih.gov/pubmed/19570873/|journal=Journal of Virology|volume=83|issue=18 | pages = 9356–9369|doi=10.1128/JVI.02382-07|issn=1098-5514|pmc=2738251|pmid=19570873}}</ref> showed that coxsackievirus B3 may persist as a chronic low-level non-cytolytic infection in the central nervous system of mice. Three case studies have also found chronic enterovirus infection in the brains of ME/CFS patients; see [[post-mortem brain studies]]. * '''Lévêque''' et al 2012: <ref>{{Cite journal | last = Lévêque | first = Nicolas | last2 = Renois | first2 = Fanny | last3 = Talmud | first3 = Déborah | last4 = Nguyen | first4 = Yohan | last5 = Lesaffre | first5 = François | last6 = Boulagnon | first6 = Camille | last7 = Bruneval | first7 = Patrick | last8 = Fornes | first8 = Paul | last9 = Andréoletti | first9 = Laurent | date = Oct 2012 | title = Quantitative genomic and antigenomic enterovirus RNA detection in explanted heart tissue samples from patients with end-stage idiopathic dilated cardiomyopathy|url=https://www.ncbi.nlm.nih.gov/pubmed/22837323/|journal=Journal of Clinical Microbiology|volume=50|issue=10|pages=3378–3380|doi=10.1128/JCM.01612-12|issn=1098-660X|pmc=3457437|pmid=22837323}}</ref> found for the first time non-cytolytic enterovirus in the heart tissues in human idiopathic dilated cardiomyopathy: they found enterovirus genomes with deletions in the 5′ region, and they found a reduced positive to negative enteroviral ssRNA ratio. * '''Tracy''' et al 2015: <ref name="Tracy2015" /> found non-cytolytic coxsackievirus B with deletions in the 5′ region can persist in the pancreas of mice. This has implications in the study of type 1 diabetes, which has long been linked to CVB. The fact that non-cytolytic CVB can form long-term infection in the pancreas raises the possibility that this infection might be a cause of TID, at least in a subset of patients. Pre-existing insulitis in murine pancreatic islets makes the islets more susceptible to CVB infection.<ref>{{Cite journal | last = Drescher | first = Kristen M. | last2 = Kono | first2 = Ken | last3 = Bopegamage | first3 = Shubhada | last4 = Carson | first4 = Steven D. | last5 = Tracy | first5 = Steven | date = 2004-11-24 | title = Coxsackievirus B3 infection and type 1 diabetes development in NOD mice: insulitis determines susceptibility of pancreatic islets to virus infection | url =https://www.ncbi.nlm.nih.gov/pubmed/15518817|journal=Virology|volume=329|issue=2|pages=381–394|doi=10.1016/j.virol.2004.06.049|issn=0042-6822|pmid=15518817}}</ref><ref name="Tracy2014-2">{{Cite journal | last = Tracy | first = Steven | date = 2014 | title = Human enteroviruses and type 1 diabetes|url=http://www.investinme.org/Documents/Journals/Journal%20of%20IiME%20Vol%208%20Issue%201.pdf|journal=Journal of IiME|volume=8|issue=1|pages=27-33|via=}}</ref> * '''Chia''' et al 2015: <ref name="Chia2015">{{Cite journal | last = Chia | first = John K. | authorlink = John Chia | last2 = Chia | first2 = Andrew Y. | authorlink2 = Andrew Chia | last3 = Wang | first3 = David | author-link3 = David Wang | last4 = El-Habbal | first4 = Rabiha | date = 2015 | title=Functional Dyspepsia and Chronic Gastritis Associated with Enteroviruses|url=http://file.scirp.org/Html/2-1900264_55465.htm|journal=Open Journal of Gastroenterology|language=en|volume=05|issue=04|pages=21–27|doi=10.4236/ojgas.2015.54005|issn=2163-9450}}</ref> using VP1 and dsRNA staining found both enteroviral VP1 protein and enteroviral dsRNA in the stomach tissues of functional dyspepsia and chronic gastritis patients both with and without an ME/CFS diagnosis. The dsRNA is postulated to be a mechanism of non-cytolytic enteroviral persistence. * '''Smithee''' 2015: <ref name="Smithee2015">{{Cite journal | last = Smithee | first = S. | last2 = Tracy | first2 = S. | last3 = Chapman | first3 = N.M. | date = Dec 2015 | title = Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5'-Terminal Genomic Deletions|url=https://www.ncbi.nlm.nih.gov/pubmed/26355088/|journal=Journal of Virology|volume=89|issue=23|pages=11761–11772|doi=10.1128/JVI.01308-15|issn=1098-5514|pmc=4645312|pmid=26355088|quote=CVB3-CKO replicated to a level approximately 5 log units lower than that for wt CVB3|via=}}</ref> demonstrated that artificially engineered non-cytolytic CVB populations in vitro displayed impaired replication, with RNA levels 100,000-fold lower than those of lytic (wild-type) virus. * '''Bouin''' et al 2016: <ref name="Bouin2016">{{Cite journal | last = Bouin | first = Alexis | last2 = Nguyen | first2 = Yohan | last3 = Wehbe | first3 = Michel | last4 = Renois | first4 = Fanny | last5 = Fornes | first5 = Paul | last6 = Bani-Sadr | first6 = Firouze | last7 = Metz | first7 = Damien | last8 = Andreoletti | first8 = Laurent | date = Aug 2016 | title = Major Persistent 5' Terminally Deleted Coxsackievirus B3 Populations in Human Endomyocardial Tissues|url=https://www.ncbi.nlm.nih.gov/pubmed/27434549/|journal=Emerging Infectious Diseases|volume=22|issue=8|pages=1488–1490|doi=10.3201/eid2208.160186|issn=1080-6059|pmc=4982168|pmid=27434549|quote=Our results estimated a major proportion (84.8%) of reads presenting with a terminal 48-nt deletion associated with minor proportions of reads deleted of 15 nt (14.3%) and nondeleted (0.9%).|via=}}</ref> found a mix of non-cytolytic (terminally deleted) and complete coxsackievirus B3 genomes in the endomyocardial tissues of a patient with dilated cardiomyopathy, along with a low viral RNA load, and a decreased positive to negative strand RNA ratio of 4.8:1. They found the majority of the CVB genomes in the tissues were terminally deleted (with deletions varying from 15 to 48 nucleotides is length), but a small minority (0.9%) of these genomes were found to be the complete genomes of the lytic (wild-type) virus. * '''Lévêque''' et al 2017: <ref name="Leveque2017a">{{Cite journal | last = Lévêque | first = Nicolas | last2 = Garcia | first2 = Magali | last3 = Bouin | first3 = Alexis | last4 = Nguyen | first4 = Joseph H.C. | last5 = Tran | first5 = Genevieve P. | last6 = Andreoletti | first6 = Laurent | last7 = Semler | first7 = Bert L. | date = 2017-08-15 | title = Functional Consequences of RNA 5'-Terminal Deletions on Coxsackievirus B3 RNA Replication and Ribonucleoprotein Complex Formation | url =https://www.ncbi.nlm.nih.gov/pubmed/28539455|journal=Journal of Virology|volume=91|issue=16|pages=|doi=10.1128/JVI.00423-17|issn=1098-5514|pmc=5533909|pmid=28539455|quote=Taken together, our cell culture reporter transfection experiments and in vitro assays demonstrated the ability of TD RNAs harboring deletions of up to 49 nucleotides to synthesize viral proteins. CVB protein expression could, in part, be involved in DCM pathophysiology due to the expression of the viral 2A proteinase in infected cardiomyocytes. ... It should be noted that overall viral protein expression by TD replicons remained lower than that of wild-type RNAs. ... Our in vitro replication experiments did not show detectable levels of RNA synthesis for deletions larger than 21 nucleotides. ... The wild-type virus could also play the role of helper virus, allowing TD viruses to replicate|via=}}</ref> in a series of in vitro experiments on non-cytolytic coxsackievirus B3 with artificially engineered 5′ terminal deletions, found that this terminally deleted CVB3 RNA synthesized viral proteins in similar quantities to lytic CVB3 infection, although non-cytolytic CVB3 RNA with the largest deletions generated less protein (see [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533909/figure/F4/ Fig 4]). They were unable to detect RNA synthesis in CVB3 with deletions larger than 21 nucleotides, though point out that other studies have detected very low levels of RNA replication in such terminally deleted enteroviruses in vitro. They suggest this discrepancy is most likely be due to their testing methods being not sufficiently sensitive to detect the very low levels of RNA present. They also suggest another possibility for this discrepancy: that the small amount of lytic virus which Bouin et al found in vivo mixed in with the terminally deleted virus could act as a helper virus, facilitating terminally deleted viruses to replicate. In this model, non-cytolytic (terminally deleted) viruses must coexist with a small amount of lytic virus in order to facilitate their replication (or facilitate more efficient replication). * '''Flynn''' et al 2017: <ref>{{Cite journal | last = Flynn | first = Claudia T. | last2 = Kimura | first2 = Taishi | last3 = Frimpong-Boateng | first3 = Kwesi | last4 = Harkins | first4 = Stephanie | last5 = Whitton | first5 = J. Lindsay | date = Dec 2017 | title = Immunological and pathological consequences of coxsackievirus RNA persistence in the heart|url=https://www.ncbi.nlm.nih.gov/pubmed/28950225|journal=Virology|volume=512|pages=104–112|doi=10.1016/j.virol.2017.09.017|issn=1096-0341|pmc=5653433|pmid=28950225|via=|quote=Following inoculation into mice, these variants could be identified in the heart, where they persisted but – mirroring their behavior in tissue culture – they did not trigger any detectable pathology. ... These TD viruses are non-cytopathic, and their inoculation in vivo does not initiate detectable disease. However, even if unable to kill infected cells, or to trigger disease de novo, in principle these TD viruses could exacerbate existing disease, e.g. by prolonging immunopathogenic responses to the original infection.}}</ref> argue that the persistent enteroviral RNA found in myocarditis and dilated cardiomyopathy are probably not the cause of these diseases. They point out that in the experiments of Nora Chapman and colleagues inoculating mice with cultured non-cytolytic infection, this infection made its way to the heart, where it persisted, but it did not trigger any detectable pathology. However, Nora Chapman says it likely requires a high level of acute infection to seed a sufficient number of quiescent cells with enterovirus before a non-cytolytic infection emerges.<ref>{{Cite web|url=https://www.youtube.com/watch?v=3Ro7UlhSD-w&t=13m53s | title = How Does a Lytic Enterovirus Persist and Cause Chronic Disease? Enterovirus Session, International Symposium on Viruses in CFS & Post-viral Fatigue, Maryland, US, June 2008. Timecode: 13:53. | last=Chapman | first = Nora | date = 2008 | website = YouTube | archive-url=|archive-date=|url-status=|access-date=}}</ref>
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