Human Herpesvirus-6 Reactivation, Mitochondrial Fragmentation, Paper Pub. 4/1/20 - Dr. Prusty

[junkcrap50]

I've had fantastic results with Andrographis, it's helped treat part of my ME I have no idea what. Could be lyme bacteria, could be anything.
....
I only stopped taking this about a month ago as I left the winter season and entered spring. I always feel good for a few weeks after stopping Andrographis and then all my symptoms return. Maybe it's due in part to the DRP1 content?

Could you make a new thread/post about Andrographis, your experience, and any research you've come across?

 

[raghav]

@sebaaa The factor identified by OMF and the factor that Prusty has identified need not be the same. Maybe because of its small size it is able to penetrate cell membranes and cause a mess inside the cells. Just guessing.

 

[Marylib]

As I've said before and elsewhere, I often wonder if the antiretorivirals I take are why I'm 'healthier' then the majority of ME people I know.

I have heard of a couple of other people who do well on ARV's too. Not easy to get, from what I can tell, or to pay for? One takes antivirals along with an ARV and says she never thinks about giving any of them up, because they really improve her quality of life.

 

[raghav]

@godlovesatrier What brand , dosage and after how long did you get benefits ? Was it in a few days or more ?

 

[raghav]

https://twitter.com/i/web/status/1255825866103365636

Got one more paper accepted today showing reactivation of donor-derived inherited HHV-6A after kidney transplantation in a recipient leading to fatal consequences. It's high time to screen both donor & recipient for HHV-6 before transplantation. Team work between France & Germany

 

[godlovesatrier]

@raghav it's a lot to explain in a single reply. Too much really. Feel free to PM me if you want more info though.

 

[Slushiefan]

Respectfully, I cannot agree. If I sleep less, I feel much more energetic and clear-headed. So my working hypothesis is that the "something in my blood" is created while I sleep. If there is an agent that can stop this substance from being produced by my body, then I expect it would have a major beneficial effect on how I feel.

I experienced the same re: sleep. I'll add that I have also had good results from methotrexate and to a lesser degree other DMARDS.

All in all to me there seems to be an autoimmune condition here that results in something being released in the later stages of sleep.

If that is the something in the blood, it may be that which creates mito fission.

 

[Rufous McKinney]

It's high time to screen both donor & recipient for HHV-6 before transplantation.

How do we reconcile the most individuals are reportedly exposed to HHV-6? (same with EBV).

"Human Herpesvirus 6 (HHV-6) is a set of two closely related herpes viruses known as HHV-6A and HHV-6B. HHV-6B infects nearly 100% of human beings, typically before the age of three" HHV-6 Foundation...

Clearly there is sometjhing about all this I don't understand.

 

[godlovesatrier]

How do we reconcile the most individuals are reportedly exposed to HHV-6? (same with EBV).

"Human Herpesvirus 6 (HHV-6) is a set of two closely related herpes viruses known as HHV-6A and HHV-6B. HHV-6B infects nearly 100% of human beings, typically before the age of three" HHV-6 Foundation...

Clearly there is sometjhing about all this I don't understand.

I thought that too. If everyone has it, like ebv and only way 5% get ME then something else has to be causing the ME?

 

[sb4]

I think it's that something causes transactivation of HHV6 chronically. I read that it's theorised that when you become infected with a different pathogen, HHV6 transactivates in order to protect itself from another pathogen getting in its turf; this causes mito fragmentation acutely but it switches off after the infection is over in healthy people however in us it does not for whatever reason.

 

[ZeroGravitas]

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 [I may add to and edit these]:

► 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.​

• U2-OS cells apparently can't support complete production of HHV-6 virus particles, so are ideal for studying early stages of reactivation.​

• 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.​

​

► 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).
• 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":

• 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, which has been found in many other viruses and bacteria).
• Antibodies - auto (against self), against viruses.
• Calcium flux - altered inside cells so also outside.


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

 

[raghav]

In one of the slides Prusty uses the term scrambled RNA any idea what it means ?

 

[raghav]

https://www.researchgate.net/public...ndependent_inhibitor_of_mitochondrial_fission

Sulforaphane is a Nrf2-independent inhibitor of mitochondrial fission
The KEAP1-Nrf2-ARE antioxidant system is a principal means by which cells respond to oxidative and xenobiotic stresses. Sulforaphane (SFN), an electrophilic isothiocyanate derived from cruciferous vegetables, activates the KEAP1-Nrf2-ARE pathway and has become a molecule-of-interest in the treatment of diseases in which chronic oxidative stress plays a major etiological role. We demonstrate here that the mitochondria of cultured, human retinal pigment epithelial (RPE-1) cells treated with SFN undergo hyperfusion that is independent of both Nrf2 and its cytoplasmic inhibitor KEAP1. Mitochondrial fusion has been reported to be cytoprotective by inhibiting pore formation in mitochondria during apoptosis, and consistent with this, we show Nrf2-independent, cytoprotection of SFN-treated cells exposed to the apoptosis-inducer, staurosporine. Mechanistically, SFN mitigates the recruitment and/or retention of the soluble fission factor Drp1 to mitochondria and to peroxisomes but does not affect overall Drp1 abundance. These data demonstrate that the beneficial properties of SFN extend beyond activation of the KEAP1-Nrf2-ARE system and warrant further interrogation given the current use of this agent in multiple clinical trials.

 

[Badpack]

@raghav so where to get it now?
edit: nevermind, its just broccoli haha.

 

[raghav]

@raghav so where to get it now?
edit: nevermind, its just broccoli haha.

Excellent question. It is available on Amazon as Broccoli extract of sulforaphane. You can get pure sulforaphane also. I think we may have to try a combination of mito fragmentation inhibitors to push start our systems. Just guessing.

 

[Badpack]

@raghav yeah, yesterday i made an amazon haul like the cool kids these days. I got
BodyBio/E-Lyte - Sodium Butyrate 600 mg 100 caps and Andrographis (Kalmegh) (250 g), now Vitalfuel® - Sulforaphan 60 caps. And still waiting for my kuding tea. My mitochodria dont know whats coming for them.

 

[raghav]

@Badpack Andrographis is a powerful anti glycemic, so be careful with the starting dose and keep checking your sugar levels. Sulforaphane is also known to help with diabetes. Being a diabetic it will be useful for me. But most of the forum members are hypoglycaemic so they should keep an eye on their sugar levels.

 

[wigglethemouse]

This Reactivation didn't go as far as making actual functional virus particles,

I think I read either in the paper or elsewhere, or perhaps both, that U2-OS cells don't support HHV6 virus replication and were specifically used for this purpose.

sncRNA-U14 - small noncoding RNA of HHV-6, potent biomarker of recent reactivation, found in 8 of 20 ME/CFS pateint serum (0 controls).

Worth pointing out only 5 out of the 10 controls had the FISH analysis for sncRNA-U14 done (table S1 in the supplemental section, second to last column).

@ZeroGravitas In case you haven't seen it here is Dr. Prusty's CMRC2020 talk from March this year where he describes the work in the paper and some extra bits as to thing he is looking for that might be "the factor". He mentions looking for HHV6 in ME autopsies.

► Beyond the immediate findings, the setup used is itself a big deal. Giving an alternative to the nano-needle test (albeit far less practical for now, I think). But also a model for *creating* a fatigue factor molecules, in a well known cell line that they can pick apart and analyse extremely closely while its doing it:

Thank you for pointing this out. Having a model cell line means replication of the work should be easier too. More people can join the hunt which was not possible with the nanoneedle.

 

[Learner1]

assumption right now is that the mitochodria are in a state of infinite fission because of, yet unknown (possible small non-coding RNA U14), components in the blood. So the approach of "just make new ones" wont bring us far. Because the mitochondria arent defect.

The problem is mitochondrial fragmentation, isn't it? It is doubtful that every mitochondrion in the body is fragmented. If over about half are bad, it is pretty difficult to recover, at least that's what I learned in the doctor sessions at the mitochondria conference, along with learning about his they can be repaired as they recycle. Seems that if you can stop what's damaging them, and supply what's needed, they have a good of recycling. I believe I posted last year's Mitochondrial Correction article by Thomas Seyfried, Garth Nicolson, Dom D'Agostino, etc. discussing how mitos can be repaired.

Respectfully, I cannot agree. If I sleep less, I feel much more energetic and clear-headed. So my working hypothesis is that the "something in my blood" is created while I sleep. If there is an agent that can stop this substance from being produced by my body, then I expect it would have a major beneficial effect on how I feel.

Again, by the time we've had this disease for awhile, it's highly likely a cascade of damage and deranged metabolic pathways has occurred. So, applying one treatment is unlikely to fix us. Thoughtfully applying a sequence if treatments us more likely to gave a positive impact.

I don't think it's too difficult to get them on the market without FDA approval. They're readily available from Sigma-Aldrich and Alibaba. Just about anything can be sold as a research chemical.

That sounds a little scary. And illegal.

no FDA approval, no patent, no money. So dont expect anything below 5-10 years without patients going out of there way to play the guinea pig.

Agreed.

Which is literally what most ME/CFS patients are doing. How many times have you, among others, mentioned wanting to get your hands on ss-31?

There are many tolls that are available today that can be used.

yea, its pretty sad that this is our only way. Just like the Ampligen study, which needs the participants to pay for the Ampligen to get it done.
I already paid 8k for rituximab and other treatments by now that all did absolutely nothing.

Rituximab has helped a few of us. Having had it, and knowing what it costs, it's doubtful $8k would do much. $60k worth is a fair try and some patients have had results with more.
But, it is helping in a totally different way.

There are many potentially positive treatments. Understanding the mechanisms of what has gone wrong, and applying solutions to each step, in sequence may have a cumulative positive impact.

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.

I don't think all our cells are really shut down, do you? Maybe a certain number aren't so perky, but we are pretty alive with organ systems functioning.

HHV-6 transactivation's cellular effects observed, (+) = Increased factors, (-) = Decreased:

(+) 1-carbon metabolism - AKA methylation cycle.
(+) dUTPase [Wikipedia] - which decreases dUTP, so uracil can't be used for making 3/4 of DNA bases [?]. dUTPases (Udeoxyuridine triphosphate nucleotidohydrolases) are key modulators of innate and adaptive immune responses [NCBI].
(+) Thymidylate synthase - dUMP to dTMP [Wikipedia]...

(-) SOD2 (superoxide dismutase 2) - clears reactive oxygen sepcies 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.

This is helpful to know. So, in addition to stopping the HHV6, maybe we need to work out way down this list and correct what's out of whack. How do we right the methylation cycle, increase SOD2 and PDH, and correct uracil and thymidine?

https://www.researchgate.net/public...ndependent_inhibitor_of_mitochondrial_fission

Sulforaphane is a Nrf2-independent inhibitor of mitochondrial fission

I think we may have to try a combination of mito fragmentation inhibitors to push start our systems. Just guessing

Now this looks useful!! Sulforophane also has snyi cancer properties.

Thank you for pointing this out. Having a model cell line means replication of the work should be easier too.

Cell lines are a funny topic. I've always found it odd that we test things in very abnormal cancer cells.

 

[ZeroGravitas]

that U2-OS cells don't support HHV6 virus replication and were specifically used for this purpose.

Hmmm, you mean this line?:

The possibilities of productive viral life cycle in U2-OS cells was eliminated by quantifying P41 and U94 mRNAs (Fig. 1D) in the same mRNA preparations.

I think its just saying that they eliminated the possibility that the virus reached the later stages of replication by confirming that there was no late stage proteins (P41 and U84)... Right? (Or a different bit?)

only 5 out of the 10 controls had the FISH analysis for sncRNA-U14

Errm... ah yes, ok. Edited in. So you're saying it could easily be fluke that no u14 was seen in blood of controls. Hence this study doesn't indicate it as a potential distinctive serum marker for HHV-6 transactivation (or ME/CFS).

Dr. Prusty's CMRC2020 talk

Yes, thank you. (Although I've forgotten half the things I've seen and most the things I've read in the last week or so...)

I'll make a note of the serum factors he talks about looking at [YouTube]:

► 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, which has been found in many other viruses and bacteria).
• Antibodies - auto (against self), against viruses.
• Calcium flux - altered inside cells so also outside.

In one of the slides Prusty uses the term scrambled RNA any idea what it means ?

In the paper, that slide (shown from 10:55 in the video) includes the description: "Representative images showing FISH analysis for HHV-6 sncRNA-U14, human U6 snRNA (positive control), and a scrambled RNA (negative control)."
So it's literally just a random nonsense sequence of RNA that they search for with the FISH process, to confirm that it's returning negative results correctly, as it should never find that as a hit. U6 it should always find in all human samples, confirms positive results are working. (I'm inferring - I have almost no idea what FISH is or how it works.)