https://t.co/TVXC4QOjt0
https://t.co/QnMiOrdNhx
https://t.co/ZK2vfwYEFj
in fact, adaptation in CaLu-3 actually reverses changes that happened in VERO E6. P681 and RRAR is fine-tuned to growth in CaLu-3 cell cultures. P681 guards the cardin-weintraub motif against cleavage in

cell lines.
https://t.co/vytn6YVRYQ
https://t.co/fjg6ZXc2KN
the FCS is perfectly stable in anything that isn't VERO E6 classic or 293T-ACE2. anything that had TMPRSS2 and grown in trypsin-free media stably maintains the FCS.
https://t.co/NUrJ8AndTx
in fact, the PRRARS, as opposed to other mutated cleavage sites--even the "perfect" H5CS--confers the greatest infectivity in CaLu-3 cells.
https://t.co/TVXC4QOjt0
in fact, even P681R or S686G changes were less fit in CaLu-3 compared to PRRA virus--the P681R virus show either no difference or is slightly less effective compared to the P681 virus, and the S686G virus
https://t.co/DNtIR17r4S
https://t.co/nDNNw9OaWd
show significantly less entry compared to PRRAR/S681 virus.https://t.co/9HCxNjyNAC similarily, both CaLu-3 and HELA-hACE2 show significantly reduced viral entry upon mutation of the FCS.
https://t.co/tAP9OQ9FYl
P681 is favored specifically becuse of the cardin-weintraub motif it leaves behind (pRRaRs(O-hexNAc)) compared to R681.
https://t.co/LqKsreg1tu
in deed, even the optimized RRRKR site fares LESS well compared to the unmodified PRRAR site for CaLu-3.
https://t.co/6XFCMUPUqe
once agaiP681 and P681 with RRARS in particular, optimizes CaLu-3 cell entry for the SARS-CoV-2 S.
https://t.co/9HX97A9T5S

https://t.co/H7UqiJ15v9
The final piece of the puzzle: it turned out that CaLu-3 cells DO glycosylate the S1-S2 in the presence of P681, which is necessary for all the previous mechanisms to function—in deed the live SARS-CoV-2 virus on CaLu-3
Cells leads to a significant fraction of S proteins that have not been cleaved, whereas almost no S remain uncleaved on pseudoparticles produced on HEK293T cells. https://t.co/UDOU3kDGlT
These uncleaved fractions indicate that intact cardin-weintraub motifs are formed during
CaLu-3 cell culture, and that these motifs are formed as the result of interference of furin cleavage in the cells by P681-mediated glycosylation of T678 and S685.
https://t.co/qbaazFZTIF

https://t.co/9O3WLoNiY0
In addition, G614 eliminated these motifs by allowing complete cleavage of the S protein, which is why it will not form during cell passage in CaLu-3. As O-linked glycosylation in The live virus is incomplete, it can not perform any of the proposed immunoevasive function
As claimed by Anderson et al, as it (with fraction <25% on CaLu-3) will not be able to shield most of the S trimers on virion (only <1/64 of the S trimers will be fully shielded given the fraction observed on CaLu-3) against Antibody or T cell recognition on the S1-S2 junction,
Whereas the intact Cardin-Weintraub motifs formed on each virion (~50 Heparan Sulfate binding sites formed for 100 S trimers per virion), alongside with the O-glycan epitopes ares sufficient to both confer an selective advantage (inhibition of cleavage in at least 20% of the S,
Binding to Heparan Sulfate proteoglycan coreceptors in culture) in the CaLu-3 cell line and confer an selective disadvantage in the presence of an innate immune system (SIGLEC binding by O-linked HexNAc glycans, PRR recognition of intact cardin-weintraub motif by macrophages) in
Live hosts—if P681 is so critical to shield the virus from the immune system, why it is destroyed in humans by P681R and P681H mutations? If P681 can not arise in passage, why it is so conserved in CaLu-3 cells? It is important to notice that while CaLu-3 cells are used
As a model for airway epithelial cells for passage experiments, viral stock preparation (after each passage) is still only performed in VERO E6 cells. This cell line had a much more pronounced preference of P681 over R681 compared to CaLu-3, especially if TMPRSS2 is expressed in
The cell line—therefore, if virus stocks are regularly prepared between passage experiments, per the PREEMPT proposal, the conjugate potential formed by VERO E6 and CaLu-3 cell lines will fixate P681 on the PRRA insert even faster than CaLu-3 alone, while the CaLu-3 passages
In between completely eliminates any VERO-derived mutations between passages.
In fact, the presence of mutations that both abolish S1-S2 glycosylation (destruction of the P681 residue) and disrupts the Cardin-Weintraub motif in all major human Variants-Of-Concern (B.1.1.7, B.1.617.2,A.23.1) in the form of an additional basic residue appended before the
XBBXBX motif, during the course of immune selection in live human hosts, runs directly against the idea that P681 and cell-specific O-linked glycosylation associated with it could “only be the result of immune selection in the presence of a functional immune system”—in stead it
is the exact opposite, specifically favored in the two PREEMPT-specified cell types (airway epithelial and VERO) and abhorred by immune selection in-vivo. https://t.co/elcdQBF9z3
https://t.co/tAP9OQ9FYl
O-linked glycosylation is only found in uncleaved fractions, which contains an intact XBBXBX Cardin-weintraub Heparan Sulfate binding motif that is favored above furin cleavage specifically in cell cultures. Uncleaved fraction is found both in VERO and CALU-3 cells, which
O-glycosylates the S1-S2 with fractions that are consistent with the uncleaved S in total S for these cell lines.

More from Daoyu

@franciscodeasis https://t.co/OuQaBRFPu7
Unfortunately the "This work includes the identification of viral sequences in bat samples, and has resulted in the isolation of three bat SARS-related coronaviruses that are now used as reagents to test therapeutics and vaccines." were BEFORE the


chimeric infectious clone grants were there.https://t.co/DAArwFkz6v is in 2017, Rs4231.
https://t.co/UgXygDjYbW is in 2016, RsSHC014 and RsWIV16.
https://t.co/krO69CsJ94 is in 2013, RsWIV1. notice that this is before the beginning of the project

starting in 2016. Also remember that they told about only 3 isolates/live viruses. RsSHC014 is a live infectious clone that is just as alive as those other "Isolates".

P.D. somehow is able to use funds that he have yet recieved yet, and send results and sequences from late 2019 back in time into 2015,2013 and 2016!

https://t.co/4wC7k1Lh54 Ref 3: Why ALL your pangolin samples were PCR negative? to avoid deep sequencing and accidentally reveal Paguma Larvata and Oryctolagus Cuniculus?
@franciscodeasis https://t.co/Sd9IslUCH5 a FCS need a FCS in the inoculum to exist. It can not arise de-novo as it will be destroyed instantly by the immune system.

https://t.co/UgXygDjYbW a fourth Sars-like CoV is live at the WIV. This fourth virus is an infectious clone, where engineering of the S1-S2 is used regularly as mean to generate a culturable virus in HAE cells. No VERO E6 here, and HeLa-hACE2 is the new VERO of the WIV

https://t.co/DtjyycKy1v
https://t.co/PG7LVnHfsy
Even with VERO E6, only half the time does passage lead to the loss of the FCS—smaller plaques need to be explicitly picked for that to be a certainty.

Marburg virus is a novel virus that escaped from the lab. https://t.co/OGQM6qV27l the only reason why it did not become a pandemic is due to it being too lethal to sustain asymptomatic transmission in humans.

The highest reported number of cases were in WuChang right on top of the old WIV headquarters, In contrast to the population density data of Wuhan—note that the place near the market had the highest population density in all of Wuhan, which make it the most optimal location for
@JackDempsey2_2 @pathogenetics @ggronvall

@pathogenetics @ggronvall And 100% of all the alleged market animal photos were taken in 2017.
https://t.co/Vh4dKy6rvI there is a reason why that animal paper refused to release the per-month data of animal sales in the wet markets—the sales were completely banned in 2018 when the consumption of

@pathogenetics @ggronvall Contraband animals were prohibited in an 2017 revision fo the CCP’s own “wildlife protection law”. This targets the main reason for consumption, which is to brag as a social status. No animals or their meat were ever sold in wet markets or online after that date. The only things

@pathogenetics @ggronvall Ever sold after the date were desiccated and sun-dried parts for use in jewelry and for medicine. They are always thoroughly dried out which would inactivate all enveloped viruses incl. all coronaviruses within them if they were present.

@pathogenetics @ggronvall https://t.co/2679qQiA0P
The claimed market origin is undermined by the presence of consistent positive SARS-CoV-2 detection in wastewater and in patient samples outside China in at least two different countries well into November 2019, which is before the first case in the wet
@AngloScot2 @WisdomRebel https://t.co/iHKwUoNLJ5

https://t.co/sxm17Eu9hV

https://t.co/FtjifSGItc
PRRA is highly purified in the CaLu-3 cell line when FBS is added to inhibit trypsin. Stu lied about that in his defense on the FCS identity. https://t.co/GzM7uA6vup the Gallaher article contain glaring

@WisdomRebel Mistakes and is completely impossible as HKU9 + RaTG13 only lead to a frameshift inactivated S and not a furin cleaved S.
https://t.co/gmDXK9OluN
The serological test the WIV claimed to be negative in Shi’s addendum is the exact same test they claimed positive in 2012 in the phD

@WisdomRebel thesis, and Stu once again lied about it. https://t.co/Uiy8U9BTYp significant evidence in the form of contaminated SRA datasets suggest that the DEFUSE grant have been funded in China, as the full-length HKU4 is not a part of the 2016-2019 grant, but is found before the start

@WisdomRebel Date of the 2020-2024 grant of June 2020. The WIV also contained several HKU3-like Coronaviruses, particularly in mice in an 2017 GEO experiment, which is consistent with the published HKU3 and batified mice experiment in the DEFUSE document (evidence from an already conducted

@WisdomRebel Experiment). Either DEFUSE or fast-tracked form of the 2020-2024 grant in 2019 would have lead to full-length Sarbecovirus clones being used, leading to SARS-CoV-2 in the lab—they used HKU4 in stead of HKU5 for the MERS spike experiment indicating that backbone sequence distance

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MASTER THREAD on Short Strangles.

Curated the best tweets from the best traders who are exceptional at managing strangles.

• Positional Strangles
• Intraday Strangles
• Position Sizing
• How to do Adjustments
• Plenty of Examples
• When to avoid
• Exit Criteria

How to sell Strangles in weekly expiry as explained by boss himself. @Mitesh_Engr

• When to sell
• How to do Adjustments
• Exit


Beautiful explanation on positional option selling by @Mitesh_Engr
Sir on how to sell low premium strangles yourself without paying anyone. This is a free mini course in


1st Live example of managing a strangle by Mitesh Sir. @Mitesh_Engr

• Sold Strangles 20% cap used
• Added 20% cap more when in profit
• Booked profitable leg and rolled up
• Kept rolling up profitable leg
• Booked loss in calls
• Sold only


2nd example by @Mitesh_Engr Sir on converting a directional trade into strangles. Option Sellers can use this for consistent profit.

• Identified a reversal and sold puts

• Puts decayed a lot

• When achieved 2% profit through puts then sold
1. Mini Thread on Conflicts of Interest involving the authors of the Nature Toilet Paper:
https://t.co/VUYbsKGncx
Kristian G. Andersen
Andrew Rambaut
Ian Lipkin
Edward C. Holmes
Robert F. Garry

2. Thanks to @newboxer007 for forwarding the link to the research by an Australian in Taiwan (not on

3. K.Andersen didn't mention "competing interests"
Only Garry listed Zalgen Labs, which we will look at later.
In acknowledgements, Michael Farzan, Wellcome Trust, NIH, ERC & ARC are mentioned.
Author affiliations listed as usual.
Note the 328 Citations!
https://t.co/nmOeohM89Q


4. Kristian Andersen (1)
Andersen worked with USAMRIID & Fort Detrick scientists on research, with Robert Garry, Jens Kuhn & Sina Bavari among


5. Kristian Andersen (2)
Works at Scripps Research Institute, which WAS in serious financial trouble, haemorrhaging 20 million $ a year.
But just when the first virus cases were emerging, they received great news.
They issued a press release dated November 27, 2019:

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