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

[halcyon]

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

Enteroviruses (EVs) causing persisting infection are characterized by minimal replication and genetic changes. Typing of these agents may complement disease assessment and shed light on pathogenesis. Here we report an integrated approach for EV detection in human samples that is based on pre-enrichment of virus in cell culture before search for the viral genome and viral antigens. Cases of post-polio syndrome, type 1 diabetes, and chronic cardiomyopathy were investigated. As tissue-based approaches require invasive procedures, information was mainly gleaned from virus in blood. Molecular assays targeting conserved genome regions of all EV types (5′UTR, 2 C, 3Dpol) were employed. As compared to direct assays of plasma or leukocytes, the EV detection rate was significantly enhanced by co-culture of leukocytes with cell lines prior to molecular and immunologic tests. Results of RT-PCR and sequencing were confirmed by staining cell cultures with a panel of EV-specific antibodies. Sequence and phylogenetic analysis showed that EVs of the C species (polioviruses) were associated with the post-polio syndrome, while members of the B species were found in type 1 diabetes and cardiomyopathy. The procedure may be used for investigating the possible association of different EVs with a variety of chronic neurologic, endocrine, and cardiac disorders.

Interesting study with implications for future ME pathogen research. These are the sorts of methods that need to be attempted going forward I believe if researchers insist on using blood instead of tissues for pathogen detection in ME.

@Janet Dafoe (Rose49) I hope you can share this study with Ron as it has several important notes regarding difficulties with detection and identification of enteroviruses in chronic disease.

The 5′UTR region comprises the targets of current commercial diagnostic assays for EV infections. These tests do not recognize the closely related human Parechoviruses (PeVs)1. Since low EV levels are produced in persistent infection14, 28 and the causative agents may carry genetic changes, commercial assays rarely give positive results in chronic disorders.

First, identification of EV isolates could not be obtained via sequencing the capsid-coding regions (mainly the VP1 gene) according to published procedures39, 42–44, nor using poliovirus-specific PV1 primers. In prospect, identification of defective EV types may be achieved using sequencing tools of enhanced sensitivity48, or via sequencing cDNA fragments subjected to preliminary enrichment with primer sets covering the entire genome of EV groups/species43. In addition, novel cell culture methods allowing serial passage of persistent EVs at sufficiently high titers may also favor sequencing.

 

[drob31]

@Hip

 

[anciendaze]

This is very interesting, but I fear we will be going through another cycle in which these procedures will be attacked for producing false positives, when molecular biology does not support clinical diagnosis. An example of that took place with culture tests for borrelia burghdorferi. We still don't have a gold-standard test for that infection.

If doctors don't want to see a chronic infection they will not, unless the patient is close to dying.

It is possible this will be different, due to the results on type-1 diabetes, for which there is little diagnostic uncertainty. Could we finally be on the path to prevention?

 

[Hip]

This is very interesting, but I fear we will be going through another cycle in which these procedures will be attacked for producing false positives, when molecular biology does not support clinical diagnosis. An example of that took place with culture tests for borrelia burghdorferi. We still don't have a gold-standard test for that infection.

There are always going to be some skeptical opinions until the matter is decided beyond all doubt, but were are fortunate that chronic non-cytolytic enterovirus infections are associated with a number of diseases, as the following paragraphs from the paper indicate:

Though most enterovirus infections appear to be self-resolving within a few weeks, prolonged fecal shedding of non-polio EVs and attenuated strains used in live poliovirus vaccine has been documented in normal children. Persistent EV infections are frequently encountered in immunodeficiency, but long-term infection of immunocompetent hosts is still a matter of debate.

some studies indicate that subclinical EV infection may be associated with lifelong disorders such as the post-polio syndrome (PPS), myasthenia gravis, autoimmune thyroiditis, type 1 diabetes (T1D), chronic viral cardiomyopathy (CVC).

They don't mention ME/CFS, but of course that too is linked to chronic non-cytolytic enterovirus infections. And chronic enterovirus is linked to Crohn's disease.

Thus because several diseases are associated with chronic non-cytolytic enteroviruses, the clinical significance of these infections is quite high, and hopefully that fact will spur on research faster than if there were only one disease involved.

 

[bel canto]

Might be interesting to see how many people here had family members with one of those disorders noted above. I had a parent with post-polio syndrome. Subclinical doesn't necessarily mean non-transmissible, I think.

 

[anciendaze]

@Hip

My comment above was meant to refer to diagnostic ambiguity for several of the listed illnesses: post-polio syndrome, myasthenia gravis, autoimmune thyroiditis and chronic viral cardiomyopathy. In practice, there are large gray areas, not black and white distinctions. You can easily find doctors who consider "chronic viral cardiomyopathy" an oxymoron. They expect viral cardiomyopathy to be rapidly life-threatening as in the well-know case of singer Randy Travis.

There is much less ambiguity about type-1 diabetes (T1D), and one would hope this will spur general research into chronic enterovirus infections.

Incidentally, there is evidence treating T1D does not stop the pathological process. This is probably because cytotoxic T-cells remain activated and continue to kill islet cells. We need to stop the underlying enterovirus infection, and that is a problem if you can't even detect virions in the blood.

This relates to two other papers I've mentioned: one on the role of cytotoxic T-cells in inflammatory bowel diseases and diabetes, and another on persistence of immune cells with defective viral infections. If, as I'm thinking, this method works when others do not because these enteroviruses are defective, specific detection will be more difficult because not all such viruses are defective in the same way.

Current methods of reducing autoimmune activity are like taking a hammer to the problem. We need to find and treat the specific subset of immune cells with persistent infections, not stop or damage most immune activity.

 

[Pyrrhus]

Antonio Toniolo is the author of this study. Unfortunately, no one has tried to replicate Toniolo's technique described in this article.

Furthermore, Toniolo is now retired and has recently remarked that his students can not get funding to continue this line of research.

 

[Pyrrhus]

Related discussion:

The Enterovirus Theory of Disease Etiology in ME/CFS: A Critical Review (O'Neal and Hanson, 2021)
https://forums.phoenixrising.me/thr...-critical-review-oneal-and-hanson-2021.84191/

Specifically, this post which discusses methodological problems in enterovirus detection:
https://forums.phoenixrising.me/thr...eal-and-hanson-2021.84191/page-6#post-2354208