Interview with Dr. Diane Griffin: Mechanisms of viral RNA persistence: Amy Proal interviews Diane Griffin

[Pyrrhus]

Dr Diane Griffin's most recent paper:

Griffin DE. Why does viral RNA sometimes persist after recovery from acute infections? PLoS Biol. 2022 Jun 1;20(6):e3001687
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9191737/

But the virus in the brain is still susceptible to the tissue-resident immune system of the brain. The immune cells of the brain can destroy viruses floating around in the extracellular space, and can also kill some infected cells where the virus is hiding. There is one notable exception however: viruses hiding inside infected neurons. The immune system of the brain can identify and surround infected neurons, but they can not kill infected neurons, with very few exceptions. (This is probably an evolutionary adaptation to avoid massive brain damage from otherwise harmless neurotropic infections. Even if the brain were capable of rapidly regenerating the lost neurons, any cognitive memory associated with the lost neurons will also be lost.)

Dr. Diane Griffin also describes this concept in her paper:

However, adaptive immune-mediated virus clearance is not always cytolytic (Box 1). For essential cells that are not easily replaced, such as neurons, noncytolytic control is advantageous for the host [97,98]. Antibodies that recognize alphavirus surface glycoproteins are required for clearance of infectious virions from the brains of infected mice and act by inducing antiviral signaling cascades that suppress production of viral RNA and infectious virions and inhibit virus release without harming the infected neurons [96,99–103]. Thus, the infected neuron survives with viral RNA still present. [...] The adaptive immune response can employ several noncytolytic mechanisms for clearance of infectious virus that allow survival of cells that still harbor viral RNA (Fig 3).
[...]
Viruses infecting long-lived cells in immune-privileged tissues may be particularly likely to survive and retain persistent RNA after infection [11,19,21,50,109–113]. Several early studies of progressive tick-borne and western equine viral encephalitis conducted prior to the availability of sensitive methods for detecting viral RNA provided clinical and pathological evidence of RNA persistence and ongoing inflammation in the absence of infectious virus in the CNS [17,20,114–116].

 

[SNT Gatchaman]

Dr Amy Proal is part of PolyBio. Along with collaborators they have been awarded 1.3m to investigate virus/viral protein persistence in the gut in long Covid.

(Boston, MA – July 18, 2022) – PolyBio Research Foundation has been gifted $1.3 million to spearhead a collaborative LongCovid research study with scientists from the Icahn School of Medicine at Mount Sinai in New York City and the J. Craig Venter Institute in San Diego. The funding from Balvi, a scientific investment and direct gifting fund for COVID projects, will allow the collaborative team to use innovative technologies to determine if SARS-CoV-2 or its proteins can persist for long periods of time in the gastrointestinal (GI) tract of patients with LongCovid. The study sets the stage for the possible use of antiviral treatments in LongCovid.

https://polybio.org/portfolio/intestinal-tissue/

 

[Pyrrhus]

The viruses also use convoluted membrane structures as part of its replication machinery. These membrane structures can encircle and enclose the dsRNA and associated viral proteins inside "intracellular vesicles", where intracellular enzymes can not attack or degrade them. These "intracellular vesicles" can then be exported out of the cell as "extracellular vesicles", for transport to other cells. (For more information on this process, see Viral Extracellular Vesicles known as ‘Stealth Spheres’)

Dr. Diane Griffin also alludes to the concept of viruses spreading from cell-to-cell via extracellular vesicles in her paper:

In the measles virus-induced late disease SSPE, virion proteins required for particle assembly at the plasma membrane (hemagglutinin, fusion, and matrix) have acquired changes that prevent cell surface expression and virion assembly but promote cell-to-cell ribonucleoprotein transfer to uninfected cells, thereby allowing continued spread of viral RNA without producing infectious virions [121–124]. Similar mutations have been observed in the viral RNAs from persistent CNS infections due to mumps and mouse hepatitis viruses [113,125].

 

[Pyrrhus]

Many people have wondered why the immunity from most vaccines wane over time, but not from the measles vaccine. The measles vaccine, which uses a live - but attenuated - measles virus, apparently results in life-long immunity.

Dr. Diane Griffin explains that the reason for this life-long immunity is likely due to persistence of the vaccine virus in lymph nodes:

Macaques infected with measles virus have persistent RNA in lymphocytes and myeloid cells for months after resolution of the acute rash disease. Pathologic examination of their lymph nodes shows a progressive increase in germinal centers with proliferating B cells accompanied by continued appearance of virus-specific peripheral follicular helper CD4+ T cells and antibody-secreting cells in circulation and affinity maturation of antiviral antibody [41]. This contrasts with the short-lived immunity induced by SARS-CoV-2 and many other respiratory viruses potentially due to a failure to establish the persistence of RNA in lymphoid tissue required for prolonged synthesis of viral antigens for immune stimulation [157–161].

 

[Marylib]

I'm just happy that enough of you retain enough cognitive function to analyze all this. Thanks for sharing. And true, @Pyrrhus live attenuated vaccines might solve some problems. I've gotten to the point that immunizations are so tricky and so ongoing these days, that I'd probably be happy to have those as long as I could rest up and have someone take care of me for a year or so. Just lucky I already had the measles and the polio and smallpox vaccine - the usual childhood vaccines. I think the sugar-cube polio one was live attenuated. Fortunately I avoided polio while many of my contemporaries did not. They have to deal with post-polio now. Formerly thought to 'not exist.' They have plenty of energy though - and plenty of braces and pain and spinal fusions and dead muscles, sadly. Depends on which muscles were attacked. They were told to 'build up their muscles,' which damaged them even more.

 

[Marylib]

Dr Amy Proal is part of PolyBio. Along with collaborators they have been awarded 1.3m to investigate virus/viral protein persistence in the gut in long Covid.



https://polybio.org/portfolio/intestinal-tissue/

I can pretty much guarantee you it is in there. And plenty of other places, too. Why someone doesn't just provide aggressive, monitored and prompt treatment is beyond me. It's cruel. My head may explode if I hear one more statement like 'that's not how science works.' It should work better and more doctors need to be able to take chances. The list of obituaries grows all the time.
http://www.drvallings.co.nz/obituaries.html

 

[CSMLSM]

Many people have wondered why the immunity from most vaccines wane over time, but not from the measles vaccine. The measles vaccine, which uses a live - but attenuated - measles virus, apparently results in life-long immunity.

Dr. Diane Griffin explains that the reason for this life-long immunity is likely due to persistence of the vaccine virus in lymph nodes:

The vaccines for covid 19 are less effective than real infection, for protection against reinfection due to a broader antibody production against many targets of the virus from the immune system when the full virus is encountered, rather than from just the spike protein in the vaccine. More targets to connect an antibody to.
This is what I have come to understand. What do you think Pyrrhus? I am sure you are more well informed about this than I.

 

[Pyrrhus]

The vaccines for covid 19 are less effective than real infection, for protection against reinfection due to a broader antibody production against many targets of the virus from the immune system when the full virus is encountered, rather than from just the spike protein in the vaccine. More targets to connect an antibody to.
This is what I have come to understand. What do you think Pyrrhus?

There has been a lot of talk about protection from coronavirus vaccines versus protection from coronavirus infection. A lot of data has been interpreted in different ways.

Generally speaking, what you say is true regarding protection from vaccines vs. protection from infection. But every infection is different, new variants are constantly emerging, and the immune system in one person can react differently from the immune system in another person, so it can be very difficult to generalize.

I hope this helps.

 

[hapl808]

Generally speaking, what you say is true regarding protection from vaccines vs. protection from infection. But every infection is different, new variants are constantly emerging, and the immune system in one person can react differently from the immune system in another person, so it can be very difficult to generalize.

I think this is the most accurate thing. Generalizing is very difficult, yet I see it done all the time no matter the viewpoint. Either vaccines are always better, or natural immunity is always better, or unvaccinated will always have worse symptoms, or unvaccinated will always recover faster. As we know, the immune systems for different people vary wildly, and it's almost impossible to predict with our current level of knowledge.

 

[CSMLSM]

I think this is the most accurate thing. Generalizing is very difficult, yet I see it done all the time no matter the viewpoint. Either vaccines are always better, or natural immunity is always better, or unvaccinated will always have worse symptoms, or unvaccinated will always recover faster. As we know, the immune systems for different people vary wildly, and it's almost impossible to predict with our current level of knowledge.

Some research I am aware of shows night and day between vaccine and real infection on longevity of protection and broader antibody coverage of the real infection, but I totally understand and know of the variability of the variables you speak of.
If you are immune compromised, have previous inflammatory condition or have genetic alterations to immune functions that are required yes I can see the ungeneralisable point.

 

[Pyrrhus]

I also found this surprising tidbit about Borna disease virus in Dr. Diane Griffin's paper:

For Borna disease virus that replicates in the nucleus, persistently infected cells retain genomic RNA in aggregates of viral ribonucleoproteins tethered to host chromosomes with host nuclear proteins that are maintained in daughter cells through the cell cycle.

This behavior is extremely unusual for an RNA virus. This behavior means that when the infected cell divides into two daughter cells, the virus may be expected to survive in both daughter cells!

Now imagine if that infected cell is a stem cell - could viral replication and spread be a simple result of normal cell division?

 

[Pyrrhus]

This persistent RNA state is called a non-cytolytic infection.

Dr. Diane Griffin might not think that it's accurate to equate a persistent infection with a non-cytolytic infection:

Although viruses frequently lyse cells in tissue culture, primary cells and cells infected in vivo are often resistant to induction of cell death.

This happens to coincide with what has been observed with enteroviruses:

Although enteroviruses can generate cytolytic infections in tissue culture, they mostly appear to generate non-cytolytic infections in human beings, whether in the acute or persistent phase of the infection.

Specifically, the best-studied enterovirus - poliovirus - readily generates cytolytic infections in tissue culture, but there is very little evidence that poliovirus actually generates cytolytic infections in a typical human infection. The permanent paralysis sometimes seen in acute poliovirus infection is actually caused by a particularly aggressive immune response to infected neurons, but the neurons are not actually lysed by the poliovirus itself...

 

[Hip]

Although viruses frequently lyse cells in tissue culture, primary cells and cells infected in vivo are often resistant to induction of cell death.

Presumably an acute virally infected cell which avoids either apoptosis or lysis does so because the immune response in the cell fights off the virus. I believe immune system-initiated apoptosis is a last resort, if the virus in the cell cannot be eliminated by the intracellular immune response.

But I suspect that acute infection would be cleared, and would not become chronic.

 

[Pyrrhus]

Presumably an acute virally infected cell which avoids either apoptosis or lysis does so because the immune response in the cell fights off the virus.

Unfortunately, it appears that that would be an incorrect assumption for an in vivo infection. Remember that most RNA viruses have multiple means to avoid the intracellular immune response, and it is in the virus's self-interest to find a cell where it can remain indefinitely, and to re-program that cell for viral replication.

It is not in the virus's self-interest to destroy this cell. (at least not before it has had a chance to spread to a new host!) In this case, the in vivo infection will always be "non-cytolytic" whether or not the cell is in the acute phase of the infection or in the persistent phase of the infection.

I hope this is clear.

 

[Hip]

It is not in the virus's self-interest to destroy this cell.

It is generally in the interests of a virus to destroy the cell by lysis, since in an acute infection, that's usually how the viral particles newly-created within the cell escape from that cell, and then go off and infect further cells. Entering cells, making thousands of copies of itself, and then bursting out of the cell via lyse is the typical viral lifecycle.

On exception is in the case of DNA viruses, which may choose not to destroy the cell, and instead go into latency inside the cell. That is the "run away and live to fight another day" strategy of viruses. In this case, the virus becomes persistent in the host. But we do not usually refer to the latent state as non-cytopathic or non-cytolytic.


But RNA viruses have no latency mechanism. So that means many RNA viruses can only cause acute infections, not chronic ones.

However, we know that RNA viruses like enterovirus can sometimes undergo mutations to its genome while in the host, which then in effect turns it into a different virus. This mutated virus can then live inside cells on a long term chronic basis, without killing the cell. That is the chronic non-cytopathic / non-cytolytic enterovirus found in ME/CFS patients. As soon as this mutated virus appears, that is the beginning of the chronic phase of the enterovirus infection.

Alphaviruses do a very similar thing to enterovirus: they can also undergo genome mutations in the host, and then form chronic non-cytolytic infections. One alphavirus which is linked to causing ME/CFS is Ross River virus.

Hepatitis C virus and dengue virus are a further two RNA viruses which are known to cause chronic infections (and also fatiguing ME/CFS-like illnesses), but I am not sure of the mechanics of this, and whether these viruses also undergo genome mutations in order to acquire the ability to cause a chronic infection.


I just Googled a bit, and found that lymphocytic choriomeningitis virus (LCMV) is an RNA virus which is non-cytopathic, in that it propagates from cell to cell without lysis (without destroying the cell).

So this may be an example of what you are referring to: a virus which during its acute phase does not kill the cells it infects, and is thus non-cytopathic during both acute and chronic phases.

 

[Pyrrhus]

Related discussions:

Neuroinflammation in Long Covid
https://forums.phoenixrising.me/threads/neuroinflammation-in-long-covid.81396/#post-2298672

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/

 

[Shanti1]

Another potential mechanism for SARS-CoV2 RNA viral spread. While only observed in a cell study, it gives a mechanism for SARS-CoV2 entry in to cell where the expression of the viral entry port (ACE2 receptors) is low, ie neurons and brain tissue in general.

Pepe A, et al. Tunneling nanotubes provide a route for SARS-CoV-2 spreading. Sci Adv. 2022 Jul 22;8(29):eabo0171.
https://www.science.org/doi/10.1126/sciadv.abo0171

Abstract
Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection represent a major issue in long coronavirus disease. How SARS-CoV-2 gains access to the brain and how infection leads to neurological symptoms are not clear because the principal means of viral entry by endocytosis, the angiotensin-converting enzyme 2 receptor, are barely detectable in the brain.

We report that human neuronal cells, nonpermissive to infection through the endocytic pathway, can be infected when cocultured with permissive infected epithelial cells. SARS-CoV-2 induces the formation of tunneling nanotubes (TNTs) and exploits this route to spread to uninfected cells. In cellulo correlative fluorescence and cryo–electron tomography reveal that SARS-CoV-2 is associated with TNTs between permissive cells.

Furthermore, multiple vesicular structures such as double-membrane vesicles, sites of viral replication, are observed inside TNTs between permissive and nonpermissive cells. Our data highlight a previously unknown mechanism of SARS-CoV-2 spreading, likely used as a route to invade nonpermissive cells and potentiate infection in permissive cells.

Tunneling nanotube bridge provides a conduit for SARS-COV-2 to cross from one cell to another. A closeup is shown below with viruses in transit. Credit: Anna Pepe/Institut Pasteur