Human Herpesvirus-6 Reactivation, Mitochondrial Fragmentation, and the Coordination of Antiviral and Metabolic Phenotypes in ME/CFS - 2020 - Schreiner

[wigglethemouse]

There's a bit about "structured illumination microscopy (SIM)" i.e. techniques used by Bupesh Prusty to measure mitochondrial length ---.

New optical microscopes have become powerful enough to observe mitochondria in real time in 3D and that is bringing new insights. In the past cells had to be fixed to slides and put in an electron microscope to get a good picture, and that process changes the cells.
e.g. https://www.sciencedaily.com/releases/2019/08/190808100336.htm

Light microscopy is the only way in which we can look inside a living cell, or living tissues, in three dimensions. An electron microscope only gives a two-dimensional view, and the organic sample would quickly burn up due to the extreme heat of the electron beam, and therefore cannot be observed alive. Moreover, by marking the biomolecules of the structure we are interested in with a specially designed fluorescent molecule, we can distinguish it from the surroundings: this is fluorescence microscopy.

Until the mid-1990s fluorescence microscopy was hampered by basic physics: due to the diffraction limit, any features on the sample closer together than about 250 nanometres would be blurred together. Viruses and individual proteins are much smaller than this, so they could not be studied this way. But in around 1994, in a wonderful lesson teaching us that we must take care when applying fundamental physical principles, Stefan Hell discovered Stimulated Emission Depletion (STED) microscopy, which is now one of several optical microscopy approaches that achieve "super-resolution," resolution beyond the diffraction limit. He received the Nobel Prize in Chemistry in 2014 "for the development of super-resolved fluorescence microscopy," together with Eric Betzig and William Moerner.

 

[wigglethemouse]

Can't remember if I posted this or not. What's intriguing about the HSV-1 and H1N1 virus challenges in this paper is that renowned virus hunter Dr. Ian Lipkin and team are heading down a similar experimental path in their ME studies.

In Dr. Lipkins talk at the CDC last September he presented the Tru-Culture system from the Institut Pasteur, Paris, that could monitor a virus/bacterial challenge on a patient sample by depressing a plunger at certain time intervals


This paper explains the method and how the Tru-culture system compares to conventional PBMC methods
Standardized whole blood stimulation improves immunomonitoring of induced immune responses in multi-center study.

Whole blood and PBMCs from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies.

 

[Andy]

 

[wigglethemouse]

 

[Milo]

do you mean this one?
Mitochondrial abnormalities in the postviral fatigue syndrome
W. M. H. Behan

sci-hub.tw/10.1007/bf00294431

Interesting that such study has been done. My muscle biopsy performed within 3 years of onset showed slow-twitch fibre atrophy, which is type 1 and not type 2 as the quoted study found.

Though muscle biopsy is invasive and not that pleasant, in my view we would gain knowledge by studying a cohort of volunteer patients.

 

[wigglethemouse]

A user on PR made a very interesting point I think it's worth repeating.

By using a model cell line of A549 cells which are grown in patient or HC serum means means replication of the work is possible. I remember @Jonathan Edwards discussing on the nanoneedle thread after that paper was released that there was no way to replicate that work as the nanoneedle is proprietary. This is very different and it's not complex.

It also allows other researchers to join the hunt for the "something in the blood" molecules, and using model cell lines they can pick apart and analyse what is happening extremely closely.

More researchers can join the hunt which was not possible with the nanoneedle!

 

[sebaaa]

A user on PR made a very interesting point I think it's worth repeating.

By using a model cell line of A549 cells which are grown in patient or HC serum means means replication of the work is possible. I remember @Jonathan Edwards discussing on the nanoneedle thread after that paper was released that there was no way to replicate that work as the nanoneedle is proprietary. This is very different and it's not complex.

It also allows other researchers to join the hunt for the "something in the blood" molecules, and using model cell lines they can pick apart and analyse what is happening extremely closely.

More researchers can join the hunt which was not possible with the nanoneedle!

That's a really good point!

I was thinking that you could avoid the issue of needing the nanoneedle to find the "something in the blood" by replicating Fluge & Mella's study where "cultured human muscle cells indicated that exposure to ME/CFS serum led to increased rates of mitochondrial respiration." Using this you could isolate the "factor" in the serum that causes the the increased mitochondrial respiration and lactate production. I think they used Seahorse equipment to measure the changes. What you provided is definitely another really good approach especially if you want to look into HHV6.

 

[ZeroGravitas]

A user on PR made a very interesting point

I did a good...?!

Cort has also supplied the press release for the study.

I'm guessing that he has liased with Bhupesh Prusty & Robert Naviaux over the study.

When I commented asking on his HR blog post, Cort said it was just Naviaux he talked to for that post. Kinda surprised me, as this study seems like Prusty's baby and he's communicative (on Twitter at least). Not sure exactly what aspects Bob contributed to the study? Other than advice on his CDR theory framework...?

cell danger response

I've not really seen detailed discussion of this. (Maybe elsewhere in dedicated threads...?) It seems so revelatory and essential to be thinking about the behavioural states of cells in this kind of way, hammering it out, even if (or especially if) Naviaux hasn't got it all right yet.

I mean, I see a lot of fairly detailed talk (much more than I know or can retain) of what might be directly affecting mitochondria in specific ways and looking closely at them in various contexts. But it seems a little like Lilliputians focusing intently upon a giant's foot, trying to predict how it will move, while failing to think on a grander scale, of the the fact it's attached to a person, who may be seated, walking to the kitchen or laid out dead. Sure if something totally sweeps their feet out from under them, that'll dominate the behaviour. But mostly a top-down theory is essential in systems with so much regulated feedback control. Can't always build up a novel new picture by purely working around the solid edges of the jigsaw puzzle, with only solidly grounded double blind controlled experiments. Although I'll admit the overstated conclusions in the HHV-6 paper did jump out at me, too. Anyway...

The new paper reported seeing SOD2 expression reduced, and smaller mitochondria, so super-oxide (and other ROS) production should be increased, as per Naviaux's Oxidative Shielding [2012]... Yes? And as per point "3)" in section 3 of his [2013] paper on CDR's features.

New paper also effectively reports seeing "5)" in "strongly induced [...] 1-carbon metabolism" (aka 'methylation cycle', right?), so making methyl groups (that can silence genes?; Lipkin mentions seeing hypermethylation in above video, I think). dUTPase and Thymidylate synthase up too, do these promote making DNA instead of RNA, preventing RNA viruses (right?). But I'm totally unclear of wider implications (and mechanistic details)...

Anyway. Prusty describes "M1-proinflammatory form of mitochondria" in this paper, which implies a CDR1 state, from Naviaux's [2018] paper...:



...But Naviaux had ME/CFS as CDR3 illness, there. I wondered if that's why they've not talked about the CDR sub-divisions at all - calling that order into question. Or if that's more because the cancer cells aren't in a normal healthy M2 state to start with? More like M0, with aerobic glycolysis for cell growth...?

Was rambling about this on PR; might they have seen a more notable difference in ATP levels between an M2 and M1 state, than the modest shift actually observed in the cancer cell's hypometabolic shift?

 

[wigglethemouse]

I did a good...?!

You did very good sir! I don't think rules here allow posters to name users on other forums so I kept it anonymous. Until you revealed yourself.

Feel free to include your post on how you interpreted the paper here, if you want to that is, perhaps it will generate further discussion.

And welcome to s4me @ZeroGravitas . This forum was set up to debate and pick apart the science, so while it can seem a bit negative, it's done with good intentions to try to get a better understanding.

 

[ZeroGravitas]

Feel free to include your post on how you interpreted the paper here

Well, I sneakily linked it in that quote already [PhoenixRising]. I still have some refinements and additions to edit in, based on (your) comments, etc. May quote paste it here after.

I meant it more as an educational summary, that I didn't think most here would benefit from... Lurked here last year, love the pure science angle, leaving the personal treatments aspect aside, but too intimidated by the obvious bio/medical expertise of most posters. (As a failed physicist & systems engineer with a sieve brain for metabolite names and terminology.) Didn't want to post screens worth of half-knoweledgable speculation/nonsense, as I did with Phair [PhoenixRising]. Or be tempted into a futurism rant about how big data, cheap metabolomics, AI, genetic engineer, etc, will make medicine an exponentially booming information science extension of computing during this decade, finally overhauling our outdated medial practices...

pick apart the science

... So yeah:

(1) Anyone any thoughts on CDR1 vs CDR3. Have I failed to understand them or trying to read too much into it, above?

(2) Another question I had in mind for Prusty: do these experiments implicitly give a mechanism for the delay inherent in PEM (24h + ish)?

They cultured the OS-U2 cells for a couple days, to collect the X-factor(s) in the medium. Then cultured the A549 in that medium for a couple more days. Are these time scales *purely* necessitated by the technical requirements of the various analysis they then perform on the cells? Or would it intrinsically take about a day (or few) for a secreted signal to accumulate in the blood to a sufficient concentration? Are sustained levels needed for other cells to react to that. Is there a threshold for cells switching state, rather than an analogue influence (continuos function)?

Naviaux himself has said [2018]:

Mitochondria change their function rapidly under stress. Within minutes, the normal anti-inflammatory M2 form of mitochondria that is specialized to meet the metabolic needs of the differentiated cell, is polarized toward pro-inflammatory, M1 mitochondria

So, initial reactions can be very fast, but what about the propagated signal...?

 

[Trish]

Lurked here last year, love the pure science angle, leaving the personal treatments aspect aside, but too intimidated by the obvious bio/medical expertise of most posters.

Welcome aboard, @ZeroGravitas. Glad you took the plunge and joined. Most of us don't have in depth scientific knowledge, though I'm glad some do. Please don't feel intimidated to post. We all learn by trying to understand each others' posts and asking questions, and I'm grateful when others pick apart my ideas (when I manage to have any) and explain to me when I've got things wrong. No one has all the answers with ME - I wish someone did!

 

[ZeroGravitas]

....Aaand, I killed the thread. May as well stick some nails in the coffin:

Cort just published a nice write-up on a paper from November 2019 by lead author Nuno Sepúlveda [SimmaronResearch]. Full paper text here [NCBI].

"ME/CFS as a Hyper-Regulated Immune System Driven by an Interplay Between Regulatory T Cells and Chronic Human Herpesvirus Infections"

This seems really promising to me, in terms of potential tie-in with this work from Prusty's team. Would it theoretically give a mechanism (and site) for sustained transactivation of HHV-6 (like in their U2-OS cell model)?

Although I see from the previous discussion of this work, in this thread [S4ME], that the supporting clinical data is tenuous (or absent)...?:

How solid is the statement that an increased density and percentage of Tregs is observed in ME/CFS?

My information is that the Biobank team have found some differences in mucosa-associated invariant T cells (MAIT) but not T regs.

I've not read it through, yet (not sure I'll be able to), but this table of information seems quite relevant, here, in describing which cells they think HHV-6 (and the other viruses) infect and then are latent in. Mostly immune cells for HHV-6. Do we think that's vaguely accurate? Given that we know from other work on HHV-6 that it's turned up all over the body, in autopsies, etc:

 

[ZeroGravitas]

OK, so here's my paper summary previously posted here on the other forum (a couple of tweaks noted):


I think this is the most exciting study since I first came across the hypometabolism (aka dauer) metabolomic studies and Naviaux's Oxidative Shielding, CDR (Cell Danger Response) and Healing Cycle framework, upon which this paper builds!

I'm a decades long gradual onset case, with no obvious acute/infectious events and I've not had colds or flu in many years (hmmm ). So viruses have largely been outside of my self-research interest. Hence I've now been reading up on (and around) this HHV-6 topic since Prusty posted the paper. Minus a few days of frustrating energy crashes, of course. I'll try to summarise my understanding (please correct and question), then do my usual of posting a big list of over-excited questions, later on...

To oversimplify (and and probably overstate) the findings:

ME/CFS is caused by *partial* reactivation of human herpes virus 6 DNA in *some* of our cells, which then send a mystery signalling molecule(s) through our blood that shuts down the rest of our cells.


In more detail:

Prusty et al. demonstrated that human cells (U2-OS), cultured in a lab dish and treated to undergo partial HHV-6 reactivation, secreted a substance into the surrounding medium (supernatant) that, when transferred to a second (responder) culture of separate cells (A549), inhibited cellular energy production.

Serum from each of 10 ME/CFS patients produced the exact same effects! Both also gave the second culture of cells an innate immune resistance to infection from influenza-A (H1N1) and HSV-1 viruses.

They say HHV-6, Human Herpes virus, is found in 90-100% of all humans by age 3. So virtually everyone has it and HHV-7 is very similar. Herpes viruses insert their RNA into the nucleus of human cells, becoming a 'latent' infection, without need to retain viral particles anywhere. But HHV-6 is unique in making its code 'chromosomally integrated', directly part of our own DNA strands. In fact, up to 1% of people are born with inherited HHV-6! Having it in every cell.

They used a drug called TSA (trichostatin-A) that's well known to 'reactivate' HHV-6 in cells they had infected previously. They describe the drug as modelling "genetic and environmental stressors". I think this might equate to the initial acute infectious events, etc, that initiate ME/CFS.

This reactivation didn't go as far as making actual functional virus particles, hence they termed it "transactivation". Only non-structural helper proteins are being made inside of the cells, which are causing metabolic changes and mitochondria to splitting into smaller structures (fission). They found that the most indicative of these proteins is U14, which was also detected in the blood of 40% (8 of 20) patient samples.

They still haven't been able to separate the active (fatigue factor) substance(s) from the transferred cell culture liquid (or ME/CFS serum), in order to identify it. But that seems extremely close at hand, now! Prusty claimed in January that they had a candidate and a couple tests in mind already [previous thread].


Some more notes related to all this:

► The laboratory cells used in the experiments are from cancers. These cell lines are effectively immortal, convenient for standardising lab-work. But they behave a little differently to regular healthy cells, including a strong tenancy towards growth, etc, so exact results aren't proof of behaviour of patient's cell in the body. I.e. ME/CFS patients are not immune to infections, line Covid-19 [Twitter]! The cell lines are:

► U2-OS - Human Bone Osteosarcoma Epithelial Cell, 2T line, established 1964 [microscopyu].
• Their custom U2-OS cells were developed for this 2018 paper [Nature], with latent (chromosomally integrated) ciHHV-6 in the DNA.
• GFP (green fluorescence protein) was integrated into the mitochondrial DNA (for clear visualisation under microscope) of a second batch of U2-OS cells without ciHHV-6 DNA. These were studied after transfer of supernatant from the ciHHV-6 cells, also.

► A549 - Adenocarcinomic human alveolar basal epithelial cells, established 1972 [Wikipedia].
• Chosen to be able to support infection by the test viruses.

[Note: like the majority of human cells, neither cell line can support complete production of HHV-6 virus particles, so are good for studying early stages of reactivation.]


► TSA - histone deacetylase inhibitor trichostatin-A [Wikipedia] - the reactivation trigger which "models Genetic and environmental stressors".
• An antifungal and antibiotic with possible anti-cancer action (via encouraging apoptosis).
• Inhibits the class I and II mammalian histone deacetylase (HDAC) enzyme family [Wikipedia]. Interfering with the removal of acetyl groups, altering gene expression.


► Viral immunity experiment - Comparing effect on (A549) responder cells of being cultured in liquid from (1) trans activated U2-OS cells, (2) ME/CFS patient serum, (3) non-activated U2-OS cells, (4) Control subject serum:




► HHV-6 transactivation's altered enzymes levels in U2-OS cells, (+) = Increased factors, (-) = Decreased, from the paper's abstract:

(+) 1-carbon metabolism - AKA methylation cycle.
(+) dUTPase [Wikipedia] - produces dUMP (precursor of thymidine), decreases dUTP (avoiding uracil's accidental use in DNA in place of thymine) [PhosphoSite]. dUTPases (Udeoxyuridine triphosphate nucleotidohydrolases) are key modulators of innate and adaptive immune responses [NCBI].
(+) Thymidylate synthase - dUMP to dTMP (thymidine monophosphate - DNA nucleotide) [Wikipedia]...

(-) SOD2 (superoxide dismutase 2) - clears reactive oxygen species from mitochonrial energy production. Protects against cell death and inflamatory cytokines.
(-) PDH (pyruvate dehydrogenase) and other proteins required for mitochondrial oxidation of fatty acid, amino acid, and glucose metabolism.

Expressed levels of many mitochondria associated proteins (enzymes) where changed, as measured by pSILAC experiment (pulsed stable isotope labeling by amino acids in cell culture) some significant ones labelled in [Fig1 of paper]:



Note: I think it's only implied that all of these changes in the transactivated cells are transferred via the factor in the cell medium to the second set of cells... Also, I'm not sure how much we should keep in mind that these were cancer cells, with a quite different metabolic resting state that may be affected differently to healthy cells.


► sncRNA-U14 - small noncoding RNA from HHV-6, potent biomarker of recent reactivation, found in 8 of 20 ME/CFS pateint serum (0 of 5 controls). I don't know if it might be present in all ME/CFS cases at below detectable levels [?], but I feel that host cell signals are more likely the main factor (like Naviaux talks about with ATP secretion). List of factors currently being investigated, below...


► Other HHV-6 Viral proteins [Nature] :
• U3-U7 - latency associated.
• U14 - transactivation (early stage - which has been found in many other viruses and bacteria).
• U11 - late stage, needed for making viron (virus particles).
• [Incomplete, could not find a clear list and hard to extract from research papers...]


► Beyond the immediate findings, the setup used is itself a big deal! Giving an alternative to the nano-needle test for detecting the serum factor. Far less practical in an eventual clinical setting, as is, but allowing reproduction in other experimental laboratories. It's also a model for *creating* fatigue factor molecules, in a well known cell line that they can pick apart and analyse extremely closely while its doing it:

"Our mitochondrial reporter-based cell system will provide an opportunity to develop a diagnostic test for ME/CFS as well as provide a platform for further identification of potential factors that define ME/CFS pathophysiology."


► Herpes viruses (all types) establish lifelong infection via many tricks [Wikipedia]. E.g.:

• Producing protein mimicking human interleukin 10 (hIL-10) = cmvIL-10.
Inhibits pro-inflamatory cytokines (IFN-γ, IL-1α, GM-CSF, IL-6 and TNF-α).
• Downregulating MHC I and II (major histocompatibility complex):
- Prevents them presenting viral antigen proteins from inside to cell surface.
- This stops cytotoxic T lymphocytes identifying the infected cell.
• HLA-G upregulated to suppress natural killer cells which usually attack cells not presenting MHCs.


► Roseola [NHS | Wikipedia] body rash following fever is how *initial* HHV-6 (A and B) and HHV-7 infections present in young childhood. Although sometimes asymptomatic, these infections may account for up to ~30% of ER visits for young children with sudden high fever (for up to 5 days).


► Receptor binding for cell entry of virus (and other details), CD = "Cluster of differentiation":
[Note: Prusty's said that the receptors are expressed everywhere (to some extent) so the viruses could access all cells.]

• HHV-6A => CD46 [Wikipedia]
Inhibitory complement receptor (system that enhances antibodies and phagocytic cells).
Also exploited by a strain of measles and group B adenoviruses.

• HHV-6B => CD134 [PubMed | Wikipedia]
AKA "OX40". Part of Tumor necrosis factor receptor superfamily, member 4 (TNFRSF4).
Mostly on CD4 T-Cells.
Activation increases cytokine production.
Associated with pathologic cytokine storm in e.g. H5N1 bird flu.
Activation critical for sustained immune response (past first few days).

• HHV-7 => CD4 (and some cell surface glyoproteins) [Wikipedia]
Enters CD4+ T cells (and macrophages, dendritic cells).
Downregulates CD4 a week after infection (so may interfere with HIV, but reactivaties HHV-6).
95% of adults infected by age 2-5 (generally after HHV-6).
20% show virus in blood, 98% in mouth.


► ME/CFS and HHV-6 on [HHV-6 foundation]: Diagnosis & antiviral treatment; Chromosomally integrated HHV-6 or CIHHV-6; Infecting the brain via the Olfactory (nose) Nerve (affecting Limbic system, Hippocampus); reactivated in transplant patients & under stress; resident in sensory ganglia (per VanElzakker's hypothesis), list of key papers (from researchers e.g. Montoya, Learner, Komaroff, etc).


► Fatigue factor molecule types Prusty has talked about looking for specifically [YouTube]:
• Mitochondrial metabolites (analysing in conjunction with Naviaux).
• Exosomes containing small non-coding RNAs or proteins. Are these the "cryptic peptides" [Twitter]?
• RNA - Cellular or pathogenic (including U14).
• Antibodies - auto (against self), against viruses.
• Calcium flux - altered inside cells so also outside.


List of questions (for researchers or whomever) to follow...

 

[ZeroGravitas]

Questions asked and replied to on this tweet. Prusty's responses in quotes:

(1) Does time frame of transfer effect suggest mechanism for patient's 1-2 day delayed PEM, crashes, etc? Naviaux's previously said [2018] that "Mitochondria change their function rapidly under stress. Within minutes[...]". But you cultured the HHV-6 transactivated cells for 2 days and then the responder cells for 2 days in the transferred supernatant.

(a) Were these durations necessary for the phenotype transfer to work? (I.e. to accumulate or respond to the molecular factor.) Did you try shorter times to find a minimum?

(b) Or was the duration part of technical requirements of the analysis methods?

Ql: PEM related crashes and fatigue is a complex physiological process and can involve many factors. We are in the process of understanding this complex phenomenon. Mitochondrial fragmentation is a very quick process under experimental conditions. With a sufficient stimulus, one can induce mitochondrial fragmentations within 2-3 h. But this is not how it works in nature. The CDR response is initiated from a limited number of cells and it requires sufficient time to reach every cell and completely shut down every cell. This is usually in the range of 2-4 days depending upon the number of original events. Yes, we can see effects starting to show up within 24 h. But the statistically significant effect that we want to show takes a longer time. In short, the metabolically and energetically crunch time is the hardest time that the cell or the body faces, when every other alternative is exhausted. [Twitter]

(2) Did this paper show cells in a CDR1 state? "M1" mitochondria and anti-viral protection are characteristic of Naviaux's CDR1 state. But he categorises ME/CFS as being a CDR3 disease [2019, Fig.2]:


(a) Was he possibly mistaken about ME/CFS being CDR3 associated?

(b) Or are the majority of our cells stuck in CDR3 as a result of a smaller population of cells in CDR1 (e.g. with HHV-6 transactivation) preventing completion of the healing cycle?

Q2: This is what I also asked Bob when I had my results and he explained me very nicely, which is not possible to do it here. Just to tell you that M1 mitochondria is the shape of mitochondria when they are under stress like PEM. But if the mitochondria remain in M1 form forever, the cells will simply die (mitophagy). This possibly happens in neurons causing neuronal damage in long run. The M2 form is probably preferred under resting conditions. But unfortunately, we cannot test this in the body. [Twitter]

(c) Or are the your observations not sufficient to draw conclusions on this because they are in cancer cells?

These cells are treated with both control & patient serum. So cell line does not make any difference here. [Twitter]

Q2: Our work is done in cancer cell lines as it is necessary for creating the HHV-6 reactivation system. All the other work are in cancer cell lines because we need to have a uniform system to work. [Twitter]

(d) Are the lab cancer cells naturally in a proliferative CDR2 state...?


(3) Was Naviaux's contribution to the paper purely advice and/or interpretation (no lab work)?

Q3: Bob has helped in me in making a lot of sense out of my work. He has not done any actual experiment for this paper. But we are continuing on some of the cool stuffs now [Twitter]

(4) Which cells in patients are (or aren't) affected?:

(a) Are initial HHV-6A infections mostly limited to cells with higher CD46 expression (chart below)? And HHV-6B to cells with CD134, primarily CD4 T-Cells, etc?

Q4: CD46 is expressed in every nucleated human cell. Hence HHV-6 can infect virtually every nucleated human cell. Same is also true for HHV-6B. T-cells only allow productive virus infection, which is clinically not that relevant in my personal opinion. [Twitter]

(b) Was HHV-6A virus chosen, instead of 6B or 7, too be able to infect U2-OS and A549

We wanted to have a unique fluorophore-based system for our study. As HHV-6A is the only virus out of the three (HHV-6A, HHV-6B and HHV-7), whose genome is available in the form of a BAC, we chose this one for our work. We will continue working with the rest of the two in future. The cell type in which the virus is integrated and where (chromosomal site) it is integrated, might influence the rate of reactivation and hence the severity of the disease. [Twitter]

(c) Could the extent of the initial (latent) infection determine the severity of ME/CFS, after the reactivation triggering event has passed?

(d) Or is infection and reactivation in specific locations more likely to be key? E.g. You've found infections all over the brain and brainstem [YouTube].

(e) Could worsening of ME/CFS severity come from a spread of HHV-6 infection to more cells?

I do not think that these viral reactivation spreads from cell to cell as there is literally no virus particle formation. Virus particle formation would be the simplest infection scenario possible as the host immune system would then recognize it and will eliminate it. [Twitter]

(5) Why do most people *not* get ME/CFS after acute infections, surgery, etc? If everyone has HHV-6 (and other viruses) latent in their bodies. (Some factors in 3, genetic and/or metabolic status?)

Q5: People do reactivate these viruses from time to time. But most of the times, our own immune system takes care of it. We do not even realize it. But in some of us, there must be a genetic component that makes us susceptible to conditions like ME/CFS. We are trying to understand that. [Twitter]

(6) How does HHV-6 transactivation become chronic in patients?

(a) The U2-OS and A549 cells are incapable of late stage viral replication. Is this also true for some cells in the human body (that can be infected)?

(b) Does the virus deliberately prevent itself from complete replication? What's the evolutionary advantage, if so? Is it related to blocking competing viruses from getting its host cell destroy by the immune system...?

(c) Is another mechanism needed for chronic HHV-6 activation? e.g. another infection, accumulation of toxic elements or other researcher's hypothesis (below)?

Q6: In our body, most of the cell types does not allow productive HHV-6 infection. Only CD4+ cells allow productive HHV-6 infection. HHV-6 would never want to get fully eliminated from the cell. So, it would prefer to stop itself being detected as soon as it starts reactivating. The Immediate early (IE) viral RNA would immediately be detected in cell cytoplasm by host innate immune machinery. Hence it starts producing small non-coding RNAs first, which helps in preparing the stage for further virus reactivation. It is a constant battle between the cells and the virus. Sometimes the cell wins and sometimes the virus.

An ideal cellular environment is the most important requirement for fully productive virus infection. [Twitter]

(7) Do you suspect interactions with any other proposed mechanisms? I.e. as them causing sustained HHV-6 transactivation, or vice versa? Specifically:

(a) Robert Phair's IDO2 mutation tryptophan trap [2019]?

(b) Nuno Sepúlveda's "Hyper-Regulated Immune System" [2019]?

(c) Michael VanElzakker's "vagus nerve infection" hypothesis [2013]?

(d) Any others you have an eye on...? (E.g. research on ROS/oxidative stress.)



(8) Any hints of patient symptom phenotypes being separable by something you've measured...?:

(a) Detectable U14 (in 40% of sampled patients)?

(b) Inherited ciHHV-6?

(c) What about the never sick patients verses those who catch everything (or at least have repeatedly infection symptops).



(9) ATP production:

(a) Was this shown halved in the U2-OS cells, upon transactivation or with transfered supernatant. But a much more marginal reduction in the A549 cells (am I'm reading the paper right)?

(b) Does [Fig.2D] show halved ATP production purely due to application of TSA? (If so, is that a big distorting factor on results?)

Q9: The figure 2D is the ATP content in U2-OS cells after adoptive transfer of the culture supernatant. The results are more or less the same in A549 cells. TSA do have some effect, but it is very clear that virus reactivation itself has a stronger effect. If one normalizes the data to the TSA added sample, the difference still stands clear and significant.

The A549 ATP assay that you see in figure 5 is from patient serum treated cells. I guess we need to grow the cells for longer time to see a more significant effect on cellular ATP content. But I do not think that we can see a dramatic decrease in ATP content under any condition. Minor changes in Physiological levels of ATP can have a drastic effect on cells. [Twitter]

(c) Are the measured ATP concentrations indicate a matching scaling of flux (in production rate)?

No, we did not measure the production rate of ATP. [Twitter]

(d) Would you expect to see a similar contrast in effect between different tissues within a patient? Or between patients? Or are these results not indicative at all, because they are cancer cells?

Again, cancer cells have a different physiology. But until we have another system, we have to depend upon these cells for our work. In future, we will try utilizing primary cells for such work.

Of course, different tissues or cells will show different levels of effect. [Twitter]

(e) Why is there a factor of 10 difference between ATP concentrations graphed in fig.2d verses fig.1c...? (An error?)

Good observation. This depends upon the number of cells being used for the study. We did not show ATP content per cell. We take similar number of cells per experiment. The figure 1C was from experiments done a long time back. Figure 2D was done during the revision. As we have used more cells for 2D, we have almost 10 times more ATP content. [Twitter]

(10) Issue with the paper - Does the last sentence of your abstract seems to claim too much? You clearly showed strong similarities but no direct evidence of causation by HHV-6 in patients; it was an in vitro study. Is there unpublished work that bridges this gap?

Q10: I do not think that it will ever be possible to show HHV-6 infection in ME/CFS patients causing the disease. It is not a localized disease like cancer, and it is not an overnight disease. So, it is up to you to decide whether we claim too much or not. We do not claim that we solved the mystery. [Twitter]

(11) Serum factor...:

(a) Can you give us any more hints about what molecules you are currently honing in on, or the nature of the tests you've mentioned?

Q11: We are trying everything that we can think about. I am not in a position to tell you anything. It is a very competitive field. If I tell you anything, someone will come out and tell that it is too early, and we do not have enough data to support. So let us work and come to stronger results. [Twitter]

(b) Could the U14 protein be the factor? Seeing as it turns up in so many pathogens. But you only found it in 40% patient serum, so would that make it one of multiple factors? Or could it be pressent in all, but below your detection threshold?

No, I do not think that U14 protein is a factor. I guess you are talking about U14 small non-coding RNA. It is very much possible that other viruses have similar RNA. But our probes are extremely stringent and do not detect anything having more than one nucleotide difference. So, we are definitely not detecting any similar RNA from other herpesviruses. It needs tons of money to test all the other herpesviruses. We will do it systematically as and when possible. [Twitter]

(c) Have you been able to rule out any of the types of molecule you mentioned previously [YouTube]? I.e. Mitochondrial metabolites, exosomes containing small non-coding RNAs or proteins, cellular RNA, antibodies, calcium flux...? And what about purinergic signalling (ATP, etc)?

Yes, we are ruling out some of them. But once again, I cannot tell anything about it at this moment. [Twitter]

(12) Personal relevance:

(a) Gradual onset - can this work on latent virus reactivation fit with (very) gradual onset of ME/CFS?

Q12: Yes, I think so. Virus reactivation and its consequential effects are slow process. It matches well with the slow onset process of the disease. [Twitter]

(b) Predisposing factors - could deleterious SNPs of SOD2 (or methylation enzymes, upregulated COMT, etc) make viral activation more likely? Or its effects more pronounced?

Yes, it can have consequences. [Twitter]

(c) Delayed sleep - Could the CDR state (or viral proteins) directly slow down the 24h clock gene expression pattern? Specifically, the astrocytes in the SCN seem to be the body's master time keepers. Because circadian rhythm is so commonly delayed in the illness - probably more of a high level neurological issue (but it was my first symptom, worsening with very gradual onset).

The link to circadian clock is fascinating. Some of the ME/CFS patients have spoken to me on this. I am very much fascinated with the idea and the literature do support an association between circadian rhythm and mitochondrial morphology. I think hormonal changes, metabolism and other factor that has a link to circadian rhythm can make the situation more interestink. I would love to test this in future if time and finances permits. [Twitter]

 

[ZeroGravitas]

(13) General curiosities:

(a) Can HHV-6 DNA show up sometimes in whole genome sequencing? I assume this is deliberately filtered out of the final data, even if its chromosomally integrated...?

Yes, one can see HHV-6 in whole genome data. But an extremely high sequencing depth is required to see these sequences. Yes, most of the times these sequences are discarded during initial data filtration. [Twitter]

(b) How close are we to having a working CRISPR system that could remove HHV-6 from humans (in vivo)? Would have to do this by providing immunity, blacklisting the key viral proteins, or something?

Some of my colleagues are working on CRISPR technology to remove integrated HHV-6 from the genome. But I guess we are still very far away from any successful results. [Twitter]

(c) Are there any exciting new laboratory technologies or equipment you expect to have access to in the near future?

We keep developing exciting technologies and also keep looking for new ones developed by others. It is hard to say here what we really want. [Twitter]

(14) So, following on for question 6, incomplete (HHV-6) reactivation is actually the more common, by cell type. Even amongst nucleated cells, the interior is more limiting of viral replication than the surface proteins needed for virus ingress...

(a) Do most cells lack certain molecular machinery/enzymes/gene expression? What is it?

(b) Or are some/most cell types just better at actively suppressing complete replication?

Q14: Still not fully understood. It is hard to say whether it is virus or the host who decides the fate of virus reactivation. I would say it's the host cell. [Twitter]

(15) How do anti-virals suppress transactivation (or its effect)? If indeed they do? (Sorry, I've no idea of their mechanisms of action.)

Q15: Most of the antivirals against herpesviruses inhibit virus DNA replication. [Twitter]

(16) Do you think it could be possible to (quickly) confirm the finding of reduced PDH, SOD2, etc, activity directly in cells taken from patients?

(a) What cell type(s) might be suitable for this, if any? (Keeping in mind the lab methods required, too.)

Q16: Yes, it should be possible to test PDH and SOD2 levels in patients. Blood cells would be ideal as it will be difficult to get any other cells. But two factors are key to success. First, A simple western blot or mass spec would not tell you anything as it measures everything in the cells. One has to pSlLAC like experiments that we did where you measure the proteins as they are synthesized avoiding the proteins that have a longer half like and they are there in cell without much of changes. We plan to follow another very fascinating approach to check this in future. [Twitter]

(b) Or is there little value in this? As you've said, above, that the serum/supernatant factor is all important?

It is hard to say what is important and what is not. Every result at this stage is valuable. We will see how to utilize the data at a later time. [Twitter]

 

[wigglethemouse]

Inhibits the class I and II mammalian histone deacetylase (HDAC) enzyme family [Wikipedia]. Interfering with the removal of acetyl groups, altering gene expression.

McGregor talked about HDAC at the Australia conference in 2019. I believe he also mentioned it as a topic for further research here
https://www.s4me.info/threads/post-...-2019-mcgregor-et-al.10260/page-2#post-181942

I see a new study is going to be looking at gene expression of HDAC
https://www.s4me.info/threads/epige...t-yet-open-for-recruitment.15047/#post-258995

 

[Ravn]

Don't think this has been posted yet. I found it very informative.

Note: I'm pretty sure this talk was recorded before Prusty's paper was published so may not include the very latest results but this video helped me understand Prusty's thinking around HHV6/HHV7, mitochondria and ME much better.

Further points of interest:

He's planning a collaboration with other researchers to see if EBV causes similar findings - he hypothesises that all herpes viruses could have the same effect.

He's testing some existing drugs that may prevent the mito fragmentation; says we currently can't fight the actual virus (unless it's actively replicating which mostly it isn't in ME) but treating downstream effects of virus may help symptoms.

He has started analysing donated tissue samples from 6 (?) bodies to see if there are "hidden" herpes infections in e.g. brain tissues - the sort of thing you can't get at when people are alive.

Note: at the start Prusty says a couple of sentences in German but the actual talk is in English. The slides are in German but reasonably self-explanatory. Questions at the end are also in German but with English answers that mostly make sense even without having understood the question.