As the other co-author, (of the peer reviewed work, not this article or the others on this substack, which are all Dan's work. And, in fact, he really did most of the work on that peer reviewed article, too, I just acted like an encouraging major professor with a student who picked his own project and whose work and energy did the work, with minor supervision.) I am hoping to engage with the readers. Dan was ahead of me, in spite of, and maybe because of my formal training as a microbiologist, as I worked with bacteriophage T4, not prone to being a quasi species. Or, staring in the 1970s. Loved this above piece, but perhaps I can add something by being available as a sort of "ask the professor" thread. After all, I did teach Molecular Virology at the University level, before designing and implementing internationally used genetic databases.
I will NEVER criticize or even critique answers. All I ask is for those to whom the material I present is well known, and there will be some, that I not be criticized for that.
So, when I say I did not see swarms, in my graduate work, it was because we wanted pure genomes which we could get by diluting phage solutions and spreading them on a lawn of bacterial hosts, in which one phage would eat a hole, leaving gobs of copies of itself.
This was the DNA phage T4 which recombines a lot, but whose genome is normally pretty stable.
The questions are:
1. What is different from T4 in flu and coronavirus?
2. Would a plaque (or equivalent) represent one genome, for these other viruses, of not, why not?
3. What experiments, or procedures should be done to measure swarms in RNA viruses?
This is the level of understanding the world needs of this virus, and which has been lacking to date. The entire field of microbiology has lacked a theorist who can make the scales fall from their eyes. and this is the price we pay.
Thanks so much, glad it helped you wrap your head around things, and yeah it is past time for a bit of a revolution but it doesn't look like anyone is interested unfortunately.
Well, although I have no idea what really goes on behind closed doors with whom, don't forget scientific inertia as well as the quest for fame as additional forces.
Is it your theory that people in Wuhan were given these LAVs as part of a human experiment and somehow they were infected by the man made virus and it spread so quickly because it reverted to the original highly pathogenic virus?
I'm new to all this language but fascinated beyond my imagination at what is going on. Can you first please tell me what exactly the furin cleavage site is and what is does ? This paper says that it occurs naturally in coronaviruses but I thought it was not a natural occurrence instead it is n insertion.
It's explained in this paper, which is linked at the top, it sounds like you're going to want to spend some more time familiarizing yourself and reading some more because there's a lot to learn!!
Mr. Sirotkin...This is simply incredible work. I'm going to have to read this about 12 more times for it to absorb into the limited bandwidth I have between the ears. A few questions, but let me know if I'm being a pest. Incredibly presciently, you published this article and discussed the issue of the Vero cells vs. the Calu-3 LONG BEFORE this seems to have become a defining issue of BA.2.86; it shuns Vero cells, but loves Calu-3 cells. And I imagine that is not good. Nice crystal ball you have there.
Were the folks at Wuhan actively trying to produce an FCS from the starting point of a bat virus?
Is the final deattenuated virus a bat virus that is adapted for human transmission?
Amazing hubris to be playing with this stuff. Thanks for the education. I'm just blown away by all this.
The credit for the cell-line stuff belongs to my dad, in our first peer-reviewed paper linked at the top, this is his observation:
"It might be more than a coincidence that the Vero cells often used in serial passage are derived from kidney epithelial cells extracted from African green monkeys, which have ACE2 receptors very similar to those found in humans and would be shared by the humanized mice that are also used for serial passage research."
So that put the phenomenon on the radar for me, since at the time I had no idea WTF a Vero cell was, and then once reports came out the FCS appeared to fall-off in Vero cells but reappear in Calu-3 cells, I was reminded of the quasispecies stuff around minority elements of the swarm being able to quickly reach fixation and seem to appear from the murk, and figured things out.
I don’t think the folks in Wuhan were trying to make an FCS necessarily, although they likely expected one to appear based on what happens with influenza, they were looking to make a highly-pathogenic Frankenvirus to use as a template for attenuation, which explains what Baric was trying to do at UNC, splicing around SARS-like viruses to see what would work. They thought they removed the FCS during attenuation of the LAV, but it was still there hiding in the quasispecies swarm, possibly as a result of industrial slippage – maybe not even sample had a hidden FCS, but it would only take a few.
And the original parent virus will be something like SARS or MERS in lethality, and very adapted to humans. When you get a chance don’t forget to check out this longer piece on the quasispecies stuff:
Wow...this is deep. I'm a bit of a dolt, but can you clarify something for me?
This phrase caught my attention "...long way from surgically splicing precise nucleotides in-and-out, which led to the cryptic emergence of the FCS in small minority subpopulations"
Are you saying that the FCS was not specifically inserted in the Wuhan lab, but had originated earlier in what amounted to 'gain of function' experiments in other labs as the swarm mutated haphazardly encouraged by human manipulation? Thanks for any clarification you can provide.
Yup sounds like you're getting it, not sure if you're done with the article but I later use the 2012 serial passage GoF experiments as a parallel, keep on reading if you're not done yet but happy to answer more questions!!
“Each and everyone one of these arrogant old hacks was drawn into the siren song of multi-billion dollar defense and pharmaceutical contracts long ago, and they’re going to remain pushing for a fascist and entirely ineffective vaccination program because they’re rotten, filthy, diseased whores, and that is exactly what they are being paid to do.”
Just dropping by a year later to say “thanks”. Vital and prescient read, and perhaps even more relevant today than it was a year ago. Thanks again.
Nope, this is simply what happens when a virus that was created as a highly pathogenic chimera on humanized mice is able to gatekeep back closer to its original form and gain its original host back
It's now airborne among mice because it was trained on mice and is very well acclimated to their immune systems now that it is closer to its original strongest form.
So serial passage was used to select less pathogenic genomes, but when the artificial selection constraints from serial passage are removed by infections in the wild, that sequence of mutations will revert over time to its more virulent form?
Presumably there could be new mutation pathways as well, leading to genomes with more or less virulence, depending on selection pressure? E.g. crowding selecting for faster growth/more virulence.
Yup, ferrets are used to attenuate it down into a vaccine. And no, OPV demonstrates the gatekeeping that always happens to an original form - didn't you read Golden Silkworms?
OPV reversion to one of its original forms makes sense to me, because they were all optimized by selection pressure in the wild, so are known to have excellent fitness. The OPV also changes viral selection pressure with its widespread use. It's interesting that OPV seems to cause high rates of paralysis from non-OPV viral strains (e.g. India sees high rates of NPAFP following OPV drops -- https://www.scidev.net/asia-pacific/news/oral-polio-drops-linked-to-paralysis-in-india/). Maybe there is some epistatic effect of wild-type virus and OPV...
I'm not seeing how this applies to lab-created virulence. In the case of a cross-species chimera, or lab selection for virulence from high-mutation-rate passages, the original virulent genome has selection pressure from the lab environment. Not seeing how that implies a fitness peak in the wild, and seems like an LAV could evolve back towards the original lab genome, or something as virulent but with better fitness in the wild.
I'm having a hard time believing you've read my articles, you seem to just be here to voice your opinion, OPV was not built from a wild type it was built from a chimera that was constructed in the lab.
i'm not going to respond unless you have questions about my text because otherwise it's become clear you're just talking at me and have no interest in actually engaging with the science.
I don't understand this response. I'm trying to understand how this works, using what I think is valid, plus what I've learned from you and related research papers.
I read the OPV article, it says that vaccine was a chimera of three viral genomes. I said "OPV reversion to one of its original forms", meaning reversion to one of the three original genomes. How is that not consistent with your article? How is trying to understand the selection pressures on OPV viruses not consistent?
I understand all this in terms of evolutionary fitness (aka selection pressure). If an LAV viral genome is going to evolve back to some original more-virulent form, and become widely distributed, that genome has to have better fitness in the wild...
I've been re-reading this and the Golden Silkworms articles. Is my understanding of this correct?
* viral mutation rates are greatly increased because of the quasispecies nature of an infection. Whenever virons with two different RNA sequences infect the same cell, they can recombine per https://www.nature.com/articles/nrmicro2614
* could an mRNA vaccine combined with an active infection allow cross-combining vaccine-mRNA spike sequences with active viral sequences? Since the vaccine uses psuedo-uridine, which has lower copy accuracy, this would increase mutation rates in the spike protein
* using drugs such as molnupiravir that increase viral mutation rates are ill-advised
Not really, it's not recombination and no that wouldn't work.
The virus is going to get back to its original strongest form, there's no way to do what you're talking about safely or affectively, public places need more ventilation, that is an easy fix that no one wants to talk about because of commercial real estate, doctors and scientists are not going to be able to play God and come up with some magical cure that gets everybody out of this.
We seem to be talking at cross-purposes, sorry I wasn't more clear.
I completely agree that better ventilation would solve things, and I've been in that camp since the pandemic was declared in early 2020.
Re my question, I'm not sure of the official terminology, so does this work? Say a quasispecies swarm is composed of individual virons, each with its own enveloped RNA replication pattern called a "variant". A swam will have high-percentage variants and lower-percentage variants. The same variant (aka RNA replication pattern) could have different envelopes as well, but not relevant to what I was asking.
I'm asking about how a viral quasispecies swarm mutates (e.g. creates additional variants), and how this relates to current public health measures of mRNA vaccination and molnupiravir.
It looks like new variants can be created either by copy errors or by recombination. Is this correct, do they both work, and are there any other ways? If so, then the recombination events will presumably create new mutations at a faster rate than copy errors, because the recombination can blend multiple changes from each piece, and each piece is part of a viable variant so the recombination is more likely to be viable.
I understand that there are a wide variety of variants in a swarm, and it is even possible that it directly includes small-numbers of copies of the original strongest form that will emerge over repeated passage. But ongoing recombination, mutations and selection pressure seem like they have to be relevant.
I'm not sure why you keep on bringing up recombination. Please read this article it either answers your questions or has links to academic papers that do:
Also keep in mind I'm standing behind my work publicly, I'm basing all of this very soundly on actual science and not conjecture, and that I think it's beyond abhorrent to downplay the effect and impact of a virus WHEN IT IS STILL JUST BARELY GETTING STARTED WITH US.
I can barely fucking fathom the lengths at which sophomoric assholes have fallen all over themselves to act like an expert, when all they're really doing is telling people the happy bullshit they want to hear - in the process endangering their lives.
Hi Dan, could you explain why The Ethical Skeptic's nucleotide entropy approach for dating SARS-COV-2 back to 2018 doesn't make sense? It's what his whole argument is based off of
As the other co-author, (of the peer reviewed work, not this article or the others on this substack, which are all Dan's work. And, in fact, he really did most of the work on that peer reviewed article, too, I just acted like an encouraging major professor with a student who picked his own project and whose work and energy did the work, with minor supervision.) I am hoping to engage with the readers. Dan was ahead of me, in spite of, and maybe because of my formal training as a microbiologist, as I worked with bacteriophage T4, not prone to being a quasi species. Or, staring in the 1970s. Loved this above piece, but perhaps I can add something by being available as a sort of "ask the professor" thread. After all, I did teach Molecular Virology at the University level, before designing and implementing internationally used genetic databases.
I will NEVER criticize or even critique answers. All I ask is for those to whom the material I present is well known, and there will be some, that I not be criticized for that.
So, when I say I did not see swarms, in my graduate work, it was because we wanted pure genomes which we could get by diluting phage solutions and spreading them on a lawn of bacterial hosts, in which one phage would eat a hole, leaving gobs of copies of itself.
This was the DNA phage T4 which recombines a lot, but whose genome is normally pretty stable.
The questions are:
1. What is different from T4 in flu and coronavirus?
2. Would a plaque (or equivalent) represent one genome, for these other viruses, of not, why not?
3. What experiments, or procedures should be done to measure swarms in RNA viruses?
(Happy to discuss all this.)
Hints:
https://en.wikipedia.org/wiki/Whole_genome_sequencing
https://en.wikipedia.org/wiki/Polymerase_chain_reaction
For those more familiar with these technology, what role would primer choice make?
This is the level of understanding the world needs of this virus, and which has been lacking to date. The entire field of microbiology has lacked a theorist who can make the scales fall from their eyes. and this is the price we pay.
Thanks so much, glad it helped you wrap your head around things, and yeah it is past time for a bit of a revolution but it doesn't look like anyone is interested unfortunately.
Yikes, could they (CDC,NIH,NIAID,Gates, Fauci, Daszak, etc) all be in on it just for the money? I can't wrap my head around that...
Well, although I have no idea what really goes on behind closed doors with whom, don't forget scientific inertia as well as the quest for fame as additional forces.
Is it your theory that people in Wuhan were given these LAVs as part of a human experiment and somehow they were infected by the man made virus and it spread so quickly because it reverted to the original highly pathogenic virus?
It's explained in part two of golden silkworms.
Pretty much: https://harvard2thebighouse.substack.com/p/understanding-covid-19-and-seasonal
Awesome, thanks!!
I'm new to all this language but fascinated beyond my imagination at what is going on. Can you first please tell me what exactly the furin cleavage site is and what is does ? This paper says that it occurs naturally in coronaviruses but I thought it was not a natural occurrence instead it is n insertion.
It's explained in this paper, which is linked at the top, it sounds like you're going to want to spend some more time familiarizing yourself and reading some more because there's a lot to learn!!
https://onlinelibrary.wiley.com/doi/10.1002/bies.202000091
Mr. Sirotkin...This is simply incredible work. I'm going to have to read this about 12 more times for it to absorb into the limited bandwidth I have between the ears. A few questions, but let me know if I'm being a pest. Incredibly presciently, you published this article and discussed the issue of the Vero cells vs. the Calu-3 LONG BEFORE this seems to have become a defining issue of BA.2.86; it shuns Vero cells, but loves Calu-3 cells. And I imagine that is not good. Nice crystal ball you have there.
Were the folks at Wuhan actively trying to produce an FCS from the starting point of a bat virus?
Is the final deattenuated virus a bat virus that is adapted for human transmission?
Amazing hubris to be playing with this stuff. Thanks for the education. I'm just blown away by all this.
The credit for the cell-line stuff belongs to my dad, in our first peer-reviewed paper linked at the top, this is his observation:
"It might be more than a coincidence that the Vero cells often used in serial passage are derived from kidney epithelial cells extracted from African green monkeys, which have ACE2 receptors very similar to those found in humans and would be shared by the humanized mice that are also used for serial passage research."
So that put the phenomenon on the radar for me, since at the time I had no idea WTF a Vero cell was, and then once reports came out the FCS appeared to fall-off in Vero cells but reappear in Calu-3 cells, I was reminded of the quasispecies stuff around minority elements of the swarm being able to quickly reach fixation and seem to appear from the murk, and figured things out.
I don’t think the folks in Wuhan were trying to make an FCS necessarily, although they likely expected one to appear based on what happens with influenza, they were looking to make a highly-pathogenic Frankenvirus to use as a template for attenuation, which explains what Baric was trying to do at UNC, splicing around SARS-like viruses to see what would work. They thought they removed the FCS during attenuation of the LAV, but it was still there hiding in the quasispecies swarm, possibly as a result of industrial slippage – maybe not even sample had a hidden FCS, but it would only take a few.
And the original parent virus will be something like SARS or MERS in lethality, and very adapted to humans. When you get a chance don’t forget to check out this longer piece on the quasispecies stuff:
https://harvard2thebighouse.substack.com/p/understanding-covid-19-and-seasonal
Wow...this is deep. I'm a bit of a dolt, but can you clarify something for me?
This phrase caught my attention "...long way from surgically splicing precise nucleotides in-and-out, which led to the cryptic emergence of the FCS in small minority subpopulations"
Are you saying that the FCS was not specifically inserted in the Wuhan lab, but had originated earlier in what amounted to 'gain of function' experiments in other labs as the swarm mutated haphazardly encouraged by human manipulation? Thanks for any clarification you can provide.
Yup sounds like you're getting it, not sure if you're done with the article but I later use the 2012 serial passage GoF experiments as a parallel, keep on reading if you're not done yet but happy to answer more questions!!
Thank you SO MUCH for responding. This is quite the revelation to me and changes my preconceived notions.
I'm going to have to chew on that article in multiple sessions as my brain was screaming "Overload, Danger Will Robinson"
The imagery of the quantum viral swarm is sobering.
Sure thing, and yeah the whole quasispecies phenomenon is a completely legit scientific theory, just one that's been buried.
And if you REALLY want to go down the rabbit-hole, this article is my original discussion of it:
https://harvard2thebighouse.substack.com/p/understanding-covid-19-and-seasonal
“Each and everyone one of these arrogant old hacks was drawn into the siren song of multi-billion dollar defense and pharmaceutical contracts long ago, and they’re going to remain pushing for a fascist and entirely ineffective vaccination program because they’re rotten, filthy, diseased whores, and that is exactly what they are being paid to do.”
Just dropping by a year later to say “thanks”. Vital and prescient read, and perhaps even more relevant today than it was a year ago. Thanks again.
Sure thing, glad someone's out there reading and hopefully benefitting!!
These researchers say it looks like SARS-Cov-2 jumped to mice, then back to people. Seems compelling to me, but I'm not an expert.
December 14th, 2021
Evidence for a mouse origin of the SARS-CoV-2 Omicron variant
https://www.biorxiv.org/content/10.1101/2021.12.14.472632v1.full.pdf
Nope, this is simply what happens when a virus that was created as a highly pathogenic chimera on humanized mice is able to gatekeep back closer to its original form and gain its original host back
It's now airborne among mice because it was trained on mice and is very well acclimated to their immune systems now that it is closer to its original strongest form.
So serial passage was used to select less pathogenic genomes, but when the artificial selection constraints from serial passage are removed by infections in the wild, that sequence of mutations will revert over time to its more virulent form?
Presumably there could be new mutation pathways as well, leading to genomes with more or less virulence, depending on selection pressure? E.g. crowding selecting for faster growth/more virulence.
Yup, ferrets are used to attenuate it down into a vaccine. And no, OPV demonstrates the gatekeeping that always happens to an original form - didn't you read Golden Silkworms?
OPV reversion to one of its original forms makes sense to me, because they were all optimized by selection pressure in the wild, so are known to have excellent fitness. The OPV also changes viral selection pressure with its widespread use. It's interesting that OPV seems to cause high rates of paralysis from non-OPV viral strains (e.g. India sees high rates of NPAFP following OPV drops -- https://www.scidev.net/asia-pacific/news/oral-polio-drops-linked-to-paralysis-in-india/). Maybe there is some epistatic effect of wild-type virus and OPV...
I'm not seeing how this applies to lab-created virulence. In the case of a cross-species chimera, or lab selection for virulence from high-mutation-rate passages, the original virulent genome has selection pressure from the lab environment. Not seeing how that implies a fitness peak in the wild, and seems like an LAV could evolve back towards the original lab genome, or something as virulent but with better fitness in the wild.
I'm having a hard time believing you've read my articles, you seem to just be here to voice your opinion, OPV was not built from a wild type it was built from a chimera that was constructed in the lab.
i'm not going to respond unless you have questions about my text because otherwise it's become clear you're just talking at me and have no interest in actually engaging with the science.
I don't understand this response. I'm trying to understand how this works, using what I think is valid, plus what I've learned from you and related research papers.
I read the OPV article, it says that vaccine was a chimera of three viral genomes. I said "OPV reversion to one of its original forms", meaning reversion to one of the three original genomes. How is that not consistent with your article? How is trying to understand the selection pressures on OPV viruses not consistent?
I understand all this in terms of evolutionary fitness (aka selection pressure). If an LAV viral genome is going to evolve back to some original more-virulent form, and become widely distributed, that genome has to have better fitness in the wild...
I've been re-reading this and the Golden Silkworms articles. Is my understanding of this correct?
* viral mutation rates are greatly increased because of the quasispecies nature of an infection. Whenever virons with two different RNA sequences infect the same cell, they can recombine per https://www.nature.com/articles/nrmicro2614
* could an mRNA vaccine combined with an active infection allow cross-combining vaccine-mRNA spike sequences with active viral sequences? Since the vaccine uses psuedo-uridine, which has lower copy accuracy, this would increase mutation rates in the spike protein
* using drugs such as molnupiravir that increase viral mutation rates are ill-advised
Not really, it's not recombination and no that wouldn't work.
The virus is going to get back to its original strongest form, there's no way to do what you're talking about safely or affectively, public places need more ventilation, that is an easy fix that no one wants to talk about because of commercial real estate, doctors and scientists are not going to be able to play God and come up with some magical cure that gets everybody out of this.
We seem to be talking at cross-purposes, sorry I wasn't more clear.
I completely agree that better ventilation would solve things, and I've been in that camp since the pandemic was declared in early 2020.
Re my question, I'm not sure of the official terminology, so does this work? Say a quasispecies swarm is composed of individual virons, each with its own enveloped RNA replication pattern called a "variant". A swam will have high-percentage variants and lower-percentage variants. The same variant (aka RNA replication pattern) could have different envelopes as well, but not relevant to what I was asking.
I'm asking about how a viral quasispecies swarm mutates (e.g. creates additional variants), and how this relates to current public health measures of mRNA vaccination and molnupiravir.
It looks like new variants can be created either by copy errors or by recombination. Is this correct, do they both work, and are there any other ways? If so, then the recombination events will presumably create new mutations at a faster rate than copy errors, because the recombination can blend multiple changes from each piece, and each piece is part of a viable variant so the recombination is more likely to be viable.
I understand that there are a wide variety of variants in a swarm, and it is even possible that it directly includes small-numbers of copies of the original strongest form that will emerge over repeated passage. But ongoing recombination, mutations and selection pressure seem like they have to be relevant.
I'm not sure why you keep on bringing up recombination. Please read this article it either answers your questions or has links to academic papers that do:
https://harvard2thebighouse.substack.com/p/understanding-covid-19-and-seasonal
Just trying to understand how things work, what the mechanisms are
It's a different model altogether, it's not linear it is a much more Baysian dynamic - what actually goes on with viral infections in a community.
I can't explain to you why some thing that isn't vaguely scientific and has no basis in science, isn't scientific man.
he is hand waving and making shit up, there is a reason there's nothing published peer reviewed and he is trying to invent something out of his ass.
If you can't see that I am sorry I can't help you
It seems like this aligns pretty well with the Ethical Skeptic's work: https://theethicalskeptic.com/2021/11/15/chinas-ccp-concealed-sars-cov-2-presence-in-china-as-far-back-as-march-2018/
Does that make sense?
Nope not really, you should read this: https://harvard2thebighouse.substack.com/p/understanding-covid-19-and-seasonal
Also keep in mind I'm standing behind my work publicly, I'm basing all of this very soundly on actual science and not conjecture, and that I think it's beyond abhorrent to downplay the effect and impact of a virus WHEN IT IS STILL JUST BARELY GETTING STARTED WITH US.
I can barely fucking fathom the lengths at which sophomoric assholes have fallen all over themselves to act like an expert, when all they're really doing is telling people the happy bullshit they want to hear - in the process endangering their lives.
Hi Dan, could you explain why The Ethical Skeptic's nucleotide entropy approach for dating SARS-COV-2 back to 2018 doesn't make sense? It's what his whole argument is based off of