Non-cytolytic enterovirus

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Introduction
Non-cytolytic enterovirus is an aberrant form of viral infection that enterovirus B species such as coxsackievirus B can transmute into within the host. Evidence for the existence of this form of infection dates back to at least 1990, but an understanding of the molecular mechanism of non-cytolytic enterovirus has only been obtained in recent years.

The transformation of normal lytic enterovirus into the non-cytolytic form is underpinned by deletions in the genome the virus acquires in the host. This altered form of enterovirus may then cause persistent intracellular infections which replicate very slowly without killing the cells. Non-cytolytic enterovirus infections have been shown to lead to cellular dysfunction, and may affect immune signaling, induce autoimmunity, or elicit a pro-inflammatory immune response. Non-cytolytic enterovirus is difficult for the immune system to eradicate, so it can persist for very long periods, and is associated with a range of chronic diseases.

Persistent enteroviral infections have been found in: The persistent enterovirus infections found in the above diseases are in several cases (those shown in italics) explicitly demonstrated to be non-cytolytic. Several researchers including Prof Nora Chapman, Prof Steven Tracy and Dr John Chia theorize that it is specifically the non-cytolytic form of enterovirus infection may cause ME/CFS. Persistent enterovirus is also being investigated as a possible cause for the other above-listed diseases as well.
 * The brain, muscle and stomach tissues of myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) patients; see enterovirus infection studies.
 * The heart muscle in murine and human coxsackievirus B (CVB) chronic myocarditis.
 * The heart muscle in dilated cardiomyopathy (a sequelae of myocarditis).
 * The skeletal muscles in murine and human chronic myositis.
 * The cerebrospinal fluid and spinal cord tissue of amyotrophic lateral sclerosis (a motor neuron disease).
 * The brainstem in Parkinson's disease.
 * The salivary gland epithelial cells and lymphocyte infiltrates in primary Sjögren's syndrome.
 * The cerebrospinal fluid in post-polio syndrome.
 * The murine pancreas and human pancreas, which has implications for type 1 diabetes.

Note: non-cytolytic enterovirus is also referred to as: non-cytopathic enterovirus, defective enterovirus, and terminally deleted enterovirus.

The nature of non-cytolytic infection
When enterovirus is initially contracted by a host, it begins as a normal acute lytic infection. Lytic infection involves the virus entering into host cells and replicating rapidly, producing tens of thousands of new viral particles (virions) in each infected cell, then killing the cell through lysis, allowing the new virions to escape and infect more cells.

But during such acute infections, enterovirus B serotypes such as coxsackievirus B are capable of transforming inside infected host cells into the aberrant non-cytolytic form. Non-cytolytic infections are not a different species of enterovirus; they arise from regular enterovirus B species in common circulation, but which get transformed in the host into a different quasispecies as a result of deletions that develop in the viral genome. Viral quasispecies are viruses which within the host have acquired small mutations (such as deletions) in their genome, but which are still closely related to their parent virus. In the case of non-cytolytic enterovirus, these mutations lead to a dramatically altered virus lifecycle.

The lifecycle of a regular lytic enterovirus centers on the virion, but once this is transformed to a non-cytolytic infection, the virus then exists in a very different configuration: as strands of naked viral RNA that reside within host cells creating a persistent intracellular infection. This viral RNA is self-replicating and self-sustaining, and is thought can survive independently of any help from the lytic infection. Furthermore, whereas the lytic virus destroys by lysis the cells it infects, non-cytolytic enterovirus does not typically kill the host cells it inhabits, allowing the non-cytolytic infection to reside in these cells on a long-term basis.

The self-sustaining RNA infection of non-cytolytic enterovirus consists of positive and negative single-stranded viral RNA (ssRNA), as well as double-stranded viral RNA (dsRNA). The latter is formed when the positive ssRNA and negative ssRNA in the cell join to create dsRNA. The genome deletions of the non-cytolytic viral RNA differentiate it from the lytic virus RNA, which has a complete genome.

Non-cytolytic infections very rarely produce lytic virions (infectious virions with a complete genome), neither in chronic myocarditis or dilated cardiomyopathy, nor myositis, nor ME/CFS. Non-cytolytic infection can nevertheless propagate by packing its defective genomes into capsids (viral shells) to create virions; this is one way the defective virus can spread.

Note that the non-cytolytic state of enterovirus is distinct from viral latency: in the latent state, viruses are typically dormant for long periods and do not engage in viral replication or viral propagation; whereas a non-cytolytic infection actively replicates and propagates, albeit very slowly.

RNA viruses such as enterovirus are generally not capable of latency (usually only DNA viruses are able to go into latency). So latency is not a mechanism that enterovirus could use to create a persistent presence in the host. But it is now clear enterovirus can form chronic low-level intracellular infection as a non-cytolytic virus and can persist even in immunocompetent hosts for very long periods.

Non-cytolytic enterovirus is resistant to immune elimination
Non-cytolytic infection can persist for very long periods in the host, and its mutated enteroviral RNA is not cleared by the immune system. The reason why this infection can evade the immune response is not clear.

One theory is that the dsRNA component of this infection confers resistance to immune eradication. The immune enzyme RNase L, released inside the cell as part of the type 1 interferon response to viral infection, is able to destroy (cleave) ssRNA, but dsRNA is resistant to destruction by this enzyme.

Dr John Chia likens dsRNA to a seed, which is hardy and allows the non-cytolytic infection to survive through periods of immune attack. Chia says that when the pressure of the immune response abates, it is probable that the dsRNA can dissociate back into ssRNA and then recommence replication. In this way, the dsRNA may constantly reseed the non-cytolytic infection. (Self-replicating strands of RNA or DNA are well studied, and are known as replicons).

Another theory suggested by Lévêque et al is that the defects found at the terminal end of the non-cytolytic enterovirus genome might facilitate immune evasion: alphaviruses are known to utilize such mutations within their genome to evade the type 1 interferon-induced immune response, and non-cytolytic enterovirus has defects in the same area of the genome as alphavirus. If the genomic mutations of enterovirus also confer resistance to the interferon-induced immune response, that may explain their resistance to immune clearance.

Non-cytolytic enterovirus may cause pathogenic effects leading to disease
In the cells it infects, non-cytolytic enterovirus synthesizes the full range of viral proteins, and these viral proteins can interfere with cellular function. For example, enterovirus 2A protein from non-cytolytic enterovirus decreases the contractility of heart cardiomyocyte cells in dilated cardiomyopathy, as well as increasing cell membrane permeability; and 2A can also promote virus spread to adjacent cells.

Furthermore, viral dsRNA is a potent inducer of type 1 interferons, so even the low levels of dsRNA found in non-cytolytic infections may be sufficient to chronically induce interferon, which might then lead to adverse effects. Although it remains to be seen if the low level of dsRNA in non-cytolytic infections is sufficient to stimulate type I interferon secretion.

Non-cytolytic enterovirus infection in the heart muscle in chronic CVB myocarditis and dilated cardiomyopathy has also been shown in some studies to induce autoantibodies which target mitochondria and thus substantially inhibit energy metabolism. Article here.

How does lytic enterovirus transmute into the non-cytolytic form?
The mechanism by which lytic enterovirus can get transformed into a non-cytolytic virus in the host was identified in a landmark 2005 study by Prof Nora Chapman and her colleagues.

Prof Nora Chapman says that the conversion of acute lytic enterovirus into a non-cytolytic infection can only occur in specific cell types, namely in non-dividing quiescent cells (such as muscle cells), but cannot occur in dividing cells (like liver cells).

A certain amount of non-cytolytic enterovirus is always generated during lytic replication, through random mutations from replication errors; but in dividing cells, because production of lytic virus is efficient, lytic virus populations dominate over non-cytolytic populations, and the cell thus is co-opted for lytic replication. By contrast, in quiescent cells, due to specific cellular conditions, lytic virus manufacture is very inefficient, and this provides an opportunity for non-cytolytic populations in quiescent cells to grow and prevail. Once non-cytolytic virus establishes itself as the dominant species in the cell, it co-opts the cell for non-cytolytic replication.

This is the essence of how non-cytolytic enterovirus infection is born, but we now explain in detail how this transmutation from lytic to non-cytolytic virus occurs.

In the mechanics of lytic enterovirus infection, viral negative ssRNA is employed as a template to create numerous positive ssRNA copies (like a photographic negative producing lots of prints). The positive ssRNA copies are then packed into capsids (viral shells) to make new enterovirus virions. Enteroviruses are positive single-stranded RNA viruses, meaning their genome comprises positive ssRNA; so in order to create thousands of new enterovirus virions, thousands of copies of the positive ssRNA are required.

When enterovirus enters a rapidly dividing cell, it efficiently produces tens of thousands of copies of positive ssRNA with the help of an important factor present in the cell known as hnRNP C (heterogeneous nuclear ribonucleoprotein C). This factor works by binding to the negative ssRNA, where it has the effect of accelerating production of positive ssRNA.

With the aid of hnRNP C, each strand of enterovirus negative ssRNA is able to efficiently produce around 100 strands of positive ssRNA. So in rapidly dividing cells, a 100-fold excess of positive strands over negative strands is observed.

However, in a quiescent cell, the factor hnRNP C is not available to the virus, as this factor is confined to the cell nucleus during quiescence. Only when a cell starts dividing (mitosis) does hnRNP C move into the cytoplasm (the region between the nucleus and the cell membrane), where enterovirus replicates. Without the assistance of hnRNP C, positive ssRNA production is severely impacted, as now each negative ssRNA template is only able to manufacture in the order of 1 strand of positive ssRNA. In consequence, roughly equal numbers of positive and negative ssRNA strands are observed in quiescent cells.

Because of the great shortfall of positive ssRNA in quiescent cells, very few lytic virions are created. As a result, cellular lysis — the destruction of the cell as the virions are released — does not occur. Instead the cell survives, and is populated with roughly equal amounts of enteroviral positive and negative ssRNA.

With these conditions found in quiescent cells, a non-cytolytic enterovirus infection can emerge. The emergence pivots on natural defects appearing in the viral genome: in viral replication, during the synthesis of viral RNA, reproduction errors naturally arise. These errors will often be in the form of deletions in the 5′ (pronounced "five primed") region at the terminal end of the genome (5′ is an untranslated region of the genome). These deletions result from premature termination of the process of genome transcription.

Now it just so happens that these deletions in the 5′ region are located in precisely the part of the genome that hnRNP C binds to on the negative ssRNA template. Thus once the deletions occur in the virus, this genomic defect permanently prevents efficient production of positive ssRNA.

So now, not only do we have a cell which being quiescent lacks cytoplasmic hnRNP C, but this situation is compounded by the deletions in the viral genome, which prevent hnRNP C binding to the negative strand template. Under these conditions, lytic virus creation greatly inhibited, and the replication process starts duplicating the genomes containing deletions, ie, duplicating the non-cytolytic virus. These circumstances favor the evolution and domination of non-cytolytic enterovirus.



Deletions in the 5′ terminal end of the viral genome in persistent low-level coxsackievirus B infections were first detected by Nora Chapman and colleagues in her 2005 study, and have since been demonstrated in other studies. The deletions range from 7 to 49 nucleotides in length.

Lévêque et al 2017 also speculate that these genome deletions might even facilitate immune evasion, thereby making non-cytolytic enterovirus largely invulnerable to immune clearance.

This transmutation of a lytic infection into non-cytolytic one by means of genome deletions cannot occur in a dividing cell, because although viral replication in dividing cells produces the same genomic deletions, these deleted genomes are outnumbered by the high amounts of lytic virions efficiently generated, thanks to hnRNP C. And furthermore, during lytic virus production in a dividing cell, the process rapidly kills the cell by lysis, so the deleted genome RNA infection has nowhere to live. Thus conversion to the non-cytolytic form is thought possible only in quiescent cells.

So these are the conditions under which non-cytolytic enterovirus infection is born, and once this intracellular infection is created, it is observed that the immune system cannot easily clear it.

Factors that promote the creation of a non-cytolytic infection
The question arises: why do these persistent non-cytolytic infections not always occur after acute coxsackievirus B infection? What circumstances lead to acute lytic enterovirus transforming into a chronic non-cytolytic infection within the host?

Prof Nora Chapman suggests that it probably requires a high level of acute infection to seed quiescent cells with enterovirus, such that a non-cytolytic infection has a chance to emerge.

Interestingly, Dr Chia found that when corticosteroids are inadvertently given to a patient during the acute stage of an enterovirus infection, this is almost a recipe for triggering ME/CFS. Corticosteroids suppress the antiviral immune response, and may thus cause a higher level of acute infection.

The spread of non-cytolytic enterovirus infection in the host
Non-cytolytic enterovirus, though defective due to deletions in its genome, appears capable of propagating itself within the host. This is demonstrated when cultured non-cytolytic enterovirus is injected intraperitoneally into mice: the infection travels to the heart, and then produces a persistent non-cytolytic infection in the heart tissues.

Non-cytolytic enterovirus is able to propagate because although the virus is defective, this virus is still packaged into capsids to make virions which can transport the non-cytolytic infection to other cells. Evidence for this virion mode of propagation comes from the fact that when cultured non-cytolytic coxsackievirus B3 is introduced to a cell line, infection of those cells can be prevented by CVB3 neutralizing antibody.

Interestingly, when non-cytolytic enteroviral RNA is packed into virions, as well as virions containing the normal positive ssRNA genome, negative ssRNA is also encapsulated in almost as many virions. Thus in non-cytolytic infection, virions containing the negative ssRNA anti-genome are found, which is very unusual.

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.

In their 1999 study Tam and Messner used RT-PCR to detect the non-cytolytic enteroviral RNA in the tissues, with different primers to separately detect both positive and negative viral ssRNA. In order to detect dsRNA, they 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. For the immunohistochemistry detection of non-cytolytic enterovirus on stomach biopsy tissues, Dr John Chia uses the 5-D8/1 monoclonal antibody stain to highlight enterovirus VP1 protein, and J2 monoclonal antibody stain to reveal the dsRNA component of non-cytolytic infection. Dr Chia employs such tissue staining to detect non-cytolytic enterovirus infection in ME/CFS patients in research routine clinical contexts. In a study, Chia found enterovirus in 82% of ME/CFS patients' stomach tissue biopsies, compared to 20% of the controls.

Indirect evidence of chronic enterovirus infection is suggested by the persistently elevated coxsackievirus B antibody titers often detected in patients with these infections. However these raised antibody titers 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).

When Dr Chia measured the antibody titers of 200 consecutive ME/CFS patients and 150 controls, using the ARUP Lab CVB and echovirus antibody micro-neutralization tests, he found the patients' titers were often substantially higher than the mean titers of the controls. Raised coxsackievirus B antibody titers have also been reported in association with myositis (both dermatomyositis and polymyositis), and highly elevated CVB titers may be seen in a subset of dilated cardiomyopathy patients.

In ME/CFS patients, Dr Chia found enteroviral RNA is only present at low levels in the blood, and discovered blood PCR testing is thus not sensitive for chronic enteroviral infections. He found 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. This is based on Dr Chia's work taking more than 2500 blood samples from over 510 ME/CFS patients and testing with PCR. He found only 35% of patients tested positive 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 a 800 copies of RNA per ml of blood). Interestingly, for the severe bedridden patients, Chia found enterovirus RNA in 70%, but enterovirus RNA was only found in 12% of the less ill patients. Furthermore, when 20 of the sickest patients' PBL cells were retested every 3 to 6 months for 2 years, for any given patient, Chia found sometimes they tested positive, sometimes negative; it was rare for any patient to always test positive. Chia concluded it is clear the enteroviral RNA present in the blood is at very low levels. Dr Chia says any PCR 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.

Treatment of non-cytolytic infections
Interferon alpha therapy has been found temporarily effective in enterovirus-associated ME/CFS in several studies,  with treatment often resulting in large improvement in symptoms, elimination of enteroviral RNA from peripheral blood mononuclear cells (PBMC), and a fourfold reduction in coxsackievirus B antibody titers. However, typically these improvements only last for 2 to 4 months after the therapy is complete, then relapse occurs, along with antibody titers increasing to pretreatment values and the enteroviral RNA returning to the PBMC.

Dr Chia says that there is an urgent priority to develop inhibitors for viral RNA replicase, the main mechanism for RNA replication, which allows the persistence of the viral genome in infected cells.

Summary characteristics of non-cytolytic enterovirus infection
Non-cytolytic enterovirus infection:

Is a self-sustaining intracellular infection of naked viral RNA possessing genomic deletions which are responsible for transmuting the original lytic virus into the non-cytolytic form.

Is born in non-dividing (quiescent) cells such as muscle cells, where cellular conditions promote the evolutionary selection of non-cytolytic virus defective genomes in preference to intact lytic virus genomes.

Is made of positive and negative single-stranded viral RNA as well as double-stranded viral RNA, which replicate very slowly and at a low level. The dsRNA may contribute to pathogenicity of non-cytolytic infection.

Is likely capable of replicating independently of the lytic enterovirus infection; tissues infected with non-cytolytic enterovirus are often largely devoid of lytic virus (the absence of lytic virus is a characteristic feature of non-cytolytic infection).

Is resistant to the immune response, and can thus reside inside host cells for very long periods. Its double-stranded RNA form is postulated to confer resistance to immune clearance; other theories suggest the genomic deletions furnish the non-cytolytic virus with immune resistance.

Is not cytolytic or cytopathic: there is no significant destruction of cells (a marker of lytic infection) when non-cytolytic infections are cultured in vitro; non-cytolytic infections do not readily kill the cells they inhabit.

Is able to synthesize all the normal viral proteins in the host cell, which can alter cellular functioning and cause dysfunction.

Is able to encapsulate its defective genome into capsids to create virions, enabling non-cytolytic virus to spread. Just as many negative strand anti-genomes are encapsulated as positive strand genomes.

Is characterized by a reduced positive to negative single-stranded RNA ratio: the ratio in acute lytic infection is around 1:100, but in persistent non-cytolytic infection the ratio is closer to 1:1.

Learn more
How Does a Lytic Enterovirus Persist and Cause Chronic Disease? Presentation by Prof Nora M. Chapman.

Human Enteroviruses and Chronic Infectious Disease. Steven Tracy & Nora Chapman. Also as pdf (p.23-31).

The role of enterovirus in chronic fatigue syndrome. Dr John Chia.

Replication Defective Enterovirus Infections: Implications for Type I Diabetes. Presentation slides by Nora M. Chapman.

Human Enteroviruses and Type 1 Diabetes (p.27-33). Prof Steven Tracy. Also as pdf (p.27-33).

Revealing enterovirus infection in chronic human disorders: An integrated diagnostic approach.

Enterovirus Persistence as a Mechanism in the Pathogenesis of Type 1 Diabetes.