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The Virus Lab Grown Whom Should we Trust and Follow?



With many lockdowns and  viral deaths growing and

senseless measures being enforced

 the Question asked is there anyone to

blame?

 

Maybe some mad scientists led by Doctor Fauci himself?

We came across this article in Medium; to me it is incrminating

Lab-Made? SARS-CoV-2 Genealogy Through the Lens of Gain-of-Function Research

Yuri Deigin

Yuri Deigin

Apr 22, 2020·64 min read

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Staff celebrating the physical completion of the laboratory in 2015, Wuhan, China (Source)

How I Learned to Start Worrying

Oh, come on. Lab-made? Nonsense! Back in January, that was my knee-jerk reaction when ideas that Covid-19 is caused by a laboratory leak had just surfaced. Bioweapon? Well, that is just Flat Earth crazies territory. Thus, whenever I kept hearing anything about non-natural origins of SARS-CoV-2, I brushed it aside under similar sentiments. So what if there is a virology institute in Wuhan? Who knows how many of those are sprinkled throughout China.

Biology

Ok, let’s start from the basics. What’s a furin site, an RBM, or a spike protein? Bear with me: once you wade through the jungle of terminology, conceptually, everything is pretty straightforward. For example, spike proteins are those red things sticking out of a virus particle — the very reason for which these viruses got “crowned”:

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Overall structure of 2019-nCoV RBD bound with ACE2.
(a) Overall topology of 2019-nCoV spike monomer. NTD, N-terminal domain. RBD, receptor-binding domain. RBM, receptor-binding motif. SD1, subdomain 1. SD2, subdomain 2. FP, fusion peptide. HR1, heptad repeat 1. HR2, heptad repeat 2. TM, transmembrane region. IC, intracellular domain.
(b) Sequence and secondary structures of 2019-nCoV RBD. The RBM is colored red.
© Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.
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Pangolins

Next, pangolins appeared on the scene: in February, another group of Chinese scientists discovered a peculiar strain of pangolin coronavirus in their possession, which, while generally being only 90% similar to CoV2, in the RBM region was almost identical to it, with only a single amino acid difference (see the upper two sequences, dots indicate a match with the top sequence):

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Royal Genealogy

In order to better understand CoV2 origins, let’s take a look at spike protein sequences of our Unholy Trinity: CoV2, RaTG13 and MP789 (pangolin-2019). Let’s compare the pairwise differences between them (identical amino acids are marked with dots, red letters denote differences, and dashes indicate deleted/inserted amino acids):

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(Image Source)

A Killer Intro

It is impossible to ignore the introduction of a PRRA insert between S1 and S2: it sticks out like a splinter. This insert creates the furin cleavage site, which I mentioned at the very beginning. Let me explain what a furin site is. Remember the structure of our spike protein? Here is a detailed diagram:

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Virus replication in the lung tissues of hamsters infected with either WT or Del-mut-1 SARS-CoV-2 virus. Virus titration by plaque assay of lung and tracheal tissues collected on day 2 and 4 post-infection
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Beijing 2019

But the most recent work of this kind that I came across was an October 2019 paper from several Beijing labs, where the new furin site RRKR was inserted into not just some pseudovirus, but into an actual live chicken coronavirus, infectious bronchitis virus (IBV):

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Where Did RaTG13 Come From?

RaTG13 is a very unusual strain. Odd to see that Shi Zhengli’s group was silent about it for all these years. After all, it is very different from its SARS-like siblings, especially in the spike protein, which is precisely what determines which types of cells (and in which animals) this virus can infect. Here is a genome similarity graph of CoV2 compared to other bat coronaviruses (panel B):

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UPD: Is RaTG13 the same as RaBtCoV/4991?

[UPDATED] After I had published this post, I was pointed to this preprint that alleges that RaTG13 is, in fact, RaBtCoV/4991 (KP876546), which Shi Zhengli had previously reported discovering in an abandoned mineshaft in Yunnan in 2013. There indeed are several reasons to think so. First and foremost, the only published sequence for RaBtCoV/4991 is 100% identical to that of RaTG13 at the nucleotide level, albeit being just a 370-bp stretch of the RdRp gene:

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“Wuhan-1”

In that paper, Shi Zhengli’s group also reported that by culturing the isolated samples in monkey Vero cells, they managed to isolate a live virus that was almost identical to the Rs3367 strain. The authors named their creation WIV1 (where WIV stands for Wuhan Institute of Virology):

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Other Yunnan Strains

In 2011, other researchers had also found samples of coronaviruses from the Yunnan Rhinolophus affinis. The strain LYRa11 seemed to me the most interesting:

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1999: First Chimeric Coronavirus

If you think that all of the gain-of-function coronavirus research into what exactly allows coronaviruses to jump from one species to another began in response to the first SARS outbreak in 2002, you’d be mistaken. Virologists experimented with chimeric coronaviruses long before that. Here, for example, is a 1999 paper from the Dutch group of Peter Rottier from Utrecht University with a revealing title Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier:

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(F) Schematic of single and double mutation emergence in MERS-CoV spike over different passages.
(G) Location of mutations within MERS-CoV spike.

Ralph “Trailblazer” Baric

Ralph Baric is a legend in coronavirology. He is a trailblazer of synthetic genomic manipulation techniques. Back in 2002, he published a breakthrough work, which marked a milestone in both the study of various mechanisms of natural viruses and in gain-of-function research. In their paper, the Baric group described creating a synthetic clone of a natural murine coronavirus:

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SARS-2003

Just a few weeks after the publication of the above work, the first SARS-CoV epidemic broke out. The Baric group sprang into action. By summer of 2003, they have submitted a paper on synthetically recreating SARS-CoV:

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SARS-2006

Baric was the first, but far from the last. Genetic engineering developed by leaps and bounds, creating newer and better tools. Other groups explored alternative synthetic virology techniques. For example, in 2006, Spanish researchers followed in Baric’s footsteps, also creating a synthetic SARS clone, but using an alternative approach (bacterial artificial chromosome):

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Strategy to assemble a SARS-CoV infectious cDNA clone as a BAC.
(A) Genetic structure of the SARS-CoV Urbani strain genome. Relevant restriction sites used for the assembly of the full-length cDNA clone are indicated. Numbers in parentheses indicate the genomic positions of the first nucleotide of the restriction endonuclease recognition sequence. Letters and numbers indicate the viral genes. L, leader sequence; UTR, untranslated region; An, poly(A) tail. (B) Construction of pBAC-SARS-CoV 5′-3′. After the selection of appropriate restriction sites, the intermediate plasmid pBAC-SARS-CoV 5′-3′ was constructed as the backbone for assembling the infectious cDNA clone. This plasmid includes the first 681 nt of the genome under the control of the CMV promoter, a multiple-cloning site containing the restriction sites selected for the final assembly of the infectious clone, and the last 975 nt of the genome, followed by a synthetic poly(A) tail (pA), the hepatitis delta virus ribozyme (Rz), and the bovine growth hormone termination and polyadenylation sequences (BGH). All these elements were precisely joined by overlapping PCR. The CMV promoter transcription start and the ribozyme cleavage site are shown. © Schematic diagram showing the five-step cloning strategy used for the assembly of the SARS-CoV full-length cDNA clone. The five overlapping cDNA fragments, named SARS 1 to SARS 5, were sequentially cloned into the plasmid pBAC-SARS-CoV 5′-3′ to generate the plasmid pBAC-SARS-CoVFL. Relevant restriction sites are indicated. The labels are as described for panel A.

Wuhan 2007

Let’s get back to 2007. That is when the Shi Zhengli group joined the synthetic virology race with a study of the spike protein of human and bat coronaviruses, trying to determine what exactly is responsible for the ability to skip from one species to another:

Chimera-2015

In light of the above experiments, it is not very clear what caused the uproar that followed probably the most famous gain-of-function virology paper. I am referring to the joint 2015 work of Shi Zhengli and Ralph Baric, in which they created a synthetic chimeric virus:

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(a) Schematic of the SHC014-CoV molecular clone, which was synthesized as six contiguous cDNAs (designated SHC014A, SHC014B, SHC014C, SHC014D, SHC014E and SHC014F) flanked by unique BglI sites that allowed for directed assembly of the full-length cDNA expressing open reading frames (for 1a, 1b, spike, 3, envelope, matrix, 6–8 and nucleocapsid). Underlined nucleotides represent the overhang sequences formed after restriction enzyme cleavage.
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Murine SARS-2007

One quick aside regarding the “murine virus MA15” from the above paper. That was not some kind of natural murine coronavirus, as one might think. It was a laboratory-modified human SARS-CoV, which back in 2007 the Baric group — possibly in competition with the Shi Zhengli group (remember their article from 2007) — turned into a real beast. To do this, they first iteratively “improved” it in mice, and when after several iterations it became maximally “effective”, they reproduced the observed mutations in a synthetic clone, and once again checked that it really does have increased virulence and lethality:

Baric-2008

Here is another example of the potential scientific rivalry between the Baric and Shi Zhengli groups. In 2008, the Baric group took the Bat-SCoV strain and replaced its RBD with an RBD from human SARS. That is, they essentially reproduced the work of Shi Zhengli’s group from 2007, except they didn’t limit themselves to pseudo-viruses, but created a real chimeric virus:

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(B) Schematic representation showing organization of the SARS-CoV and Bat-SCoV Spike proteins. The engineered Spike proteins are pictured below with the virus name to the left. Bat-SRBD includes all of the Bat-SCoV Spike sequence except that the Bat-SCoV RBD (Bat-SCoV amino acid 323–505) is replaced with the SARS-CoV RBD (amino acid 319–518) (GenBank accession no. FJ211860). Bat-SRBD-MA includes the MA15 Spike RBD change at SARS-CoV aa Y436H. Bat-SRBM includes the minimal 13 SARS-CoV residues critical for ACE2 contact, resulting in a chimeric RBD of Bat-SCoV amino acid 323I-429T and SARS-CoV amino acid 426R-518D. Bat-Hinge is Bat-SRBM sequence, with Bat-SCoV amino acid 392L-397E replaced with SARS-CoV amino acid 388V-393D. Bat-F includes nt 1–24057 of SARS-CoV (to Spike amino acid 855), with the remaining 3′ sequence from Bat-SCoV. To the right of the schematic representations, observation of transcript activity and approximate stock titers at passage 1 (P1) are indicated. ND indicates no infectious virus detected by plaque assay.

Baric-2016

The Baric group does seem to have its share of similar papers. For example, in 2016, they essentially repeated their collaboration with Shi Zhengli from 2015 to create a chimeric virus, only this time they inserted a spike protein segment into their mouse-adapted SARS not from RsSCH014, but from another strain Shi Zhengli found in Yunnan — its close relative Rs3367. Or, to be exact, from WIV1 — the laboratory clone of Rs3367 isolated at the Wuhan Institute of Virology in 2013:

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Baric-1990

Just so you appreciate how long Ralph Baric has been at this game — he was designing recombinant coronaviruses way before there were any DNA sequencing machines or other modern tools of genetic engineering. Here is his paper on the creation of “temperature mutants” from mouse coronavirus from 1990:

Wuhan-2017

The Shi Zhengli group has also not been idle since the famous 2015 paper. In 2017, they published a paper where they reported creating not one but 8 chimeric viruses — all made using transplanted RBDs from bat SARS-like viruses which they collected over a span of 5 years from the very cave around Kunming, Yunnan Province, where Shi Zhengli originally found Rs3367 and RsSCH014.

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Similarity plot based on the full-length genome sequence of civet SARS CoV SZ3.
Full-length genome sequences of all SARSr-CoV detected in bats from the cave investigated in this study were used as reference sequences. The analysis was performed with the Kimura model, a window size of 1500 base pairs and a step size of 150 base pairs.

Baric-2019

Ralph Baric also showed no signs of slowing down. At the end of October 2019, his group submitted for publication another paper on the importance of spike protein protease cleavage (remember the furin site?) to crossing the “barrier to zoonotic infection” by coronaviruses:

Gain-of-Function: Risky Business

Many people who first learn about the above research ask a very valid question: “But why?” Why do scientists create chimeric killer viruses? The politically correct answer is to develop preventive measures (drugs or vaccines) from possible natural chimeras and to understand the risks of their occurrence. Here, in fact, is what Baric, Shi Zhengli, and co-authors themselves wrote on this subject in their famous 2015 paper:

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Beware of Lab

Let’s now take a look at the lab leak hypothesis. But first, I will provide some historical context, including previous confirmed lab leaks, as many of those happened before. First and foremost, lab leaks of the first SARS-CoV: initially, in the summer of 2003 in Singapore, then in December 2003 in Taiwan, and in the spring of 2004 twice in Beijing.

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Inside America’s secretive biolabs

Vials of bioterror bacteria have gone missing. Lab mice infected with deadly viruses have escaped, and wild rodents…

www.usatoday.com

Possible Hallmarks of Lab Origin?

Let us now turn our attention back to the virus itself. Does it have any obvious signs of lab manipulation? First, a few words about what “obvious” means. Any mutation can arise naturally, and even if the amino acid insert that had created the furin site in CoV2 was not “PRRA” but “MADEINWVHANPRRA”, there would still be a non-zero chance that it arose by accident. But for us, and for any court, I think this would be enough to prove lab origin beyond a reasonable doubt.

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Schematic representation of SARS-CoV and Bat-SCoV variants.
(A) Schematic representation of SARS-CoV and Bat-SCoV (GenBank accession no. FJ211859) genomes and reverse genetics system. (Top) Arrowheads indicate nsp processing sites within the ORF1ab polyprotein (open arrowheads, papain-like proteinase mediated; filled arrowheads, nsp5 [3C-like proteinase] mediated). Immediately below are the fragments used in the reverse genetics system, labeled A through F. The fragments synthesized to generate Bat-SCoV exactly recapitulate the fragment junctions of SARS-CoV with the exception that the Bat-SCoV has 2 fragments, Bat-E1 and Bat-E2, which correspond to the SARS-E fragment.
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Codon Preferences

For these purposes, I decided to take a look at codon usage bias to check which strains look like CoV2 and RaTG13 the most. It is known that viruses tend to adapt their codon signature to the preferences of their hosts, so I expected to see RaTG13 exhibit a similar pattern to other bat viruses, and also hoped to see a difference from pangolin strains.

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The Nature Paper vs. the Lab-Made Hypothesis

But didn’t that Nature article refute the lab-made hypothesis? No, not really. There is no irrefutable evidence against it in the paper, just a loud “we don’t believe so” based on a shaky foundation. Judge for yourself — here are the authors’ key arguments in support of their conclusions:

On the 4% Genome Difference between RaTG13 and Cov2

Some critics of the lab-made hypothesis claim that the observed ~4% genetic difference between RaTG13 and CoV2 is too high to have possibly occurred in a lab if RaTG13 itself was used as a backbone. Observed mutation rates for RNA viruses vary widely — from 10⁻⁶ to 10⁻⁴ nucleotides per replication in vitro, and in humans CoV2 seems to mutate at a rate of 25 mutations per year. Thus, the logic goes, it would take years, if not decades, for two strains to diverge by 4%. While that is a valid point, there are several issues with that line of reasoning.

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Shi Zhengli-2020

As I was writing this post, a fresh paper co-authored by Shi Zhengli came out, in which the authors tested a peptide which they have been studying for some time before against CoV2. That peptide was meant to be a pan-coronavirus inhibitor, and its designed mode of action was to block the fusion of a spike protein with a cell membrane. The authors, of course, mention the new furin site of CoV2, and suggest that it may play an important role in the much more efficient penetration of CoV2 into the cell:

This is the End, Beautiful Friend

If you made it here by reading rather than scrolling, mad props to you. Hey, even if you scrolled, that’s cool too, and I apologize for the verbosity. I just didn’t anticipate that the rabbit hole would turn out to be a whole underground cave system. I hope that you found this deep dive into the world of virology interesting and enjoyed the exploration of the lab-made CoV2 hypothesis. In my opinion, the data I have presented, taken together, do not allow us to reject this possibility.

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How I Learned to Hate the GOF

I hope this post is not used to prematurely assign blame or propagate one-sided theories. What I do hope it highlights is the scale of dangerous gain-of-function research that has been and is going on in virology. The Covid-19 pandemic really exposed its huge risks in the face of few benefits: GOF research hasn’t protected us from this outbreak, hasn’t provided us with any effective treatments or vaccines in time to save hundreds of thousands of lives lost to CoV2, and if there is even a 0.1% chance GOF research caused the whole thing, that chance is too high.

WRITTEN BY

Yuri Deigin

Life mission: defeat aging. Here I write mostly in English, my Russian articles are on Habr. I am also on Facebook, LinkedIn, YouTube, etc.

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Life mission: defeat aging. Here I write mostly in English, my Russian articles are on Habr. I am also on Facebook, LinkedIn, YouTube, etc.

Published in Becoming Human: Artificial Intelligence Magazine

·Oct 2, 2019

Not just a copy, but truly still you

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When I first heard about the concept of mind uploading, I thought it meant creating a digital copy of one’s mind in a computer where it would then continue to live all on its own. Well, I thought, this is of little comfort to me, the original. Who cares how many of my clones are created, as if I — this particular instance of me — die, it would be the end of the only ‘me’ I have ever been a part of.

To counter that view, some say that a clone of you waking up and believing that it is you is the same as you waking up every morning — after all, you lose continuity with reality every time you go to sleep — but for me personally the above kind of mind uploading is still the least preferred. I would be willing to try it as a last resort in the case of, say, recovery after cryopreservation, but I would much rather upload my mind gradually. …

Read more in Becoming Human: Artificial Intelligence Magazine · 5 min read


Oct 2, 2019

What is a biomarker of aging and why do we need it?

We all probably know someone who “looks young for their age” and someone who “hasn’t aged well”:

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A biomarker of aging allows us to turn this subjective description into objective medical reality. It enables us to conclusively say: yes, you are 50 years old, but you have the health of a 35-year-old. But you, young man, should take better care of yourself — your biological age is 10 years higher than chronological age, and this is fraught with a 48% increase in the risk of death.

What does aging have to do with the probability of death? Everything. In humans, as in practically all mammals, aging is accompanied by an exponential increase in mortality…

Read more · 9 min read

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Mar 1, 2019

Exhumation not needed!

In the past few weeks, many new photographs of both Jeanne and Yvonne have emerged. While none of them offer any conclusive evidence for or against the identity switch, they do add new pieces to the puzzle. Moreover, a brilliant new insight from Philip Gibbs could mean that we can use an existing blood sample of Mme. Calment to conclusively establish whether it belongs to Jeanne or Yvonne. But more on that later.

First, I’d like to share something about Yvonne’s bow hat photo had been bothering me from the start. Take a look, does anything jump out at you?

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The hands. Look at the size of those hands. …

Read more · 11 min read

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Jan 6, 2019

(Note: To avoid work misattribution, please see the Authorship section at the end of this article)

In part 2 of my series of articles on Jeanne Calment, I wrote that she looks way too young on her ID card photo:

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The ID was issued sometime in the 1930s:

Read more · 9 min read

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Dec 25, 2018

(Note: To avoid work misattribution, please see the Authorship section at the end of this article)

If you haven’t read part 1 of the series on Jeanne Calment, it is best to start there.

Update: the ear analysis below is outdated. Please see this article for the current version.

In the comments to my Habr article on Jeanne Calment, user Rikkitik recommended that I look closely at her ears. After all, ear shape or even ear prints have long been used in forensic science for identification. …

Read more · 10 min read

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