1/ Was the pandemic caused by SARS2 the result of vaccine research gone wrong? The following thread is the product of a DRASTIC* investigation: the possibility that SARS2 and RaTG13 might be two different vaccine strategies and that SARS2 leaked from a lab during its development.
2/ Which are typical features of a live attenuated vaccine (LAV)? LAV should infect a host with lower virulence and replication capability than the WT, trigger a pronounced adaptive immune response but be cleared quickly without latent infection and it must not revert to its WT.
3/ “Circuit breakers” are required in LAV and the more are integrated into the genome, the safer is the LAV. The resulting fitness attenuation must however retain immunogenicity and protection efficacy against WT challenges.
4/A central mechanism in mammals that controls both innate and adaptive immune responses is mediated by interferons (IFNs). LAV should therefore induce IFN, but rapid clearance after IFN induction would be desirable.
5/ SARS2 seems to have some signatures in its genome which indicate protection strategies, such as IFN-hypersensitivity when compared to the first SARS.
biorxiv.org/content/10.110…
ncbi.nlm.nih.gov/pmc/articles/P…
6/ Several viral proteins involved in IFN signalling in SARS2 seem to be affected by attenuation, such as NSP3 (less effective deubiquitinating domain due to 24 AA insertion upstream), ORF3b (premature stop codon)
7/ and truncation/changes in ORF6.
biorxiv.org/content/10.110…
8/ Bat immortalized kidney cell line RaK4324 expressing hACE could provide the environment for hACE2 adaptation and avoid at the same time IFN response.
9/ Moreover, the QTQTN motive proximal to the FCS is beneficial for virus entry in presence of Cathepsin, which is naturally produced by kidney cells.
s3-eu-west-1.amazonaws.com/itempdf7415535…
10/ Synonymous mutations were used in the past as strategy for attenuating viruses through codon deoptimization. By comparing RaTG13 with SARS2, an accumulation of synonymous mutations in spike around the FCS is clearly observed.
researchgate.net/publication/68…
jvi.asm.org/content/89/7/3… ImageImageImage
11/ Interestingly, the № of CpG dinucleotides of SARS2 is significantly lower than in SARS or MERS and may point as well towards attenuation.
12/ Only in certain regions (such as FCS) there is an accumulation of CpG that could direct an immune response towards S in an otherwise attenuated virus.
ncbi.nlm.nih.gov/pmc/articles/P…
link.springer.com/article/10.100…
13/ The use of recombination resistant strains is another strategy for LAV development and it can be achieved by manipulating the viral transcription regulatory networks (TRNs).
14/ In 2018 Baric published a paper where the typical TRS transcription leader and body sequences of SARS were substituted with a novel sequence (UGGUCGC) to reduce recombination in animal models as a live vaccine candidate.
nature.com/articles/s4200…
15/ This TRS leader sequence starts at nucleotide 1465 in SARS2 and could result in a novel viral RNA transcript that deletes part of the nsp2 protein in the ORF 1ab polyprotein.
heraclitusoncovid19.com/origin-of-covi…
16/ The same TRS is also found at nucleotide 1446 in RaTG13 but in no other CoVs. Baric refers in his paper to the cave where RaTG13 was collected and RaTG13 was fully sequenced in 2018 but its special TRS is not mentioned.
nature.com/articles/s4200…
17/ LAV should also not easily mutate and SARS2 seems to be quite resistant to mutations. This feature might have been achieved by selecting strains with RNA-dependent RNA polymerase (RdRp) with improved fidelity via experiments with mutagenic drugs such as ribavirin.
19/ Moreover, ORF10 is coded by a unique sequence in SARS2 and it is not essential. It is immunogenic and it might contribute in hijacking the host’s ubiquitination machinery but it is truncated and probably not expressed, representing another possible attenuation strategy.
21/ How to explain the presence of the FCS? This site (new for Sarbecovirus) could have been inserted to confer immunity not only to SARS-like CoVs but also to CoVs with FCS, such as MERS.
22/ The FCS in SARS2 has highly CpG-rich insertion (CGG-CGG) which is extremely rare as double instance in CoVs and deoptimizes the codon for replication
researchgate.net/publication/34… Image
23/ To avoid activation of the FCS, a shield of O-linked glycans was possibly planned, by inserting a leading proline in the sequence, but it failed to be synthetized, probably because of a N-linked glycan site just upstream of the S1-S2, blocking the required glycosyltransferase
25/ If the shield would have been present, not only the FCS would have been blocked but also the activity of Cathepsin and TMPRS2 would have been hampered.
26/ O-glycosylated S1-S2 junction would not be a superantigen and it won’t bind to NPR1, the “door” for the central nervous system.
pnas.org/content/early/…
biorxiv.org/content/10.110…
27/ Testing of stability and effect of FCS in the virus would have required cell lines not prone to its deletion, such as HEK293T, Human Airway Epithelia (HAE) cells or Organ-on-a-chip models.
pubmed.ncbi.nlm.nih.gov/30849247/
biorxiv.org/content/10.110…
28/ It is not possible to exclude that the original sequence coding for the FCS was designed to be inactive, but it mutated during human to human transmission.
ncbi.nlm.nih.gov/pmc/articles/P…
29/ Moreover, the FCS of CoV2 matches exactly to that of a Heparan Sulphate binding motif—XBBXBX.
sciencedirect.com/science/articl…
This particular feature confers to SARS2 the ability to efficiently bind to proteins.
30/ Not only ACE2 and TMPRSS2 expression, but also Heparan Sulphate binding could explain the striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures.
cell.com/cell/pdf/S0092…
nature.com/articles/s4159…
31/ In the hypothesis of SARS2 being developed as intranasal or self- spreading vaccine, this ability could reduce the viral load in the lung, where the FCS is more prone to be activated.
sciencedirect.com/science/articl…
32/ but it could also result in adverse reactions in some individuals, in particular those with an impaired antiviral immune response.
news-medical.net/news/20200616/…
33/ RaTG13 also presents codon deoptimization. Moreover, its RBM is not able to bind hACE2 and it is not found in any other CoV known. RaTG13 could be therefore the unfinished result of a different vaccine strategy, such as Virus-like particle (VLP).
34/ Development of several vaccine strategies against BtCoV was an active field of research at the WIV and DARPA before SARS2 outbreak.
35/ Recent ongoing research also involved experiments on self-spreading vaccines. The risk that an unfinished vaccine might have leaked during tests on animal models (e.g. lab worker infection, improper waste disposal) should therefore be not underestimated.
37/ *DRASTIC is a group of independent and not funded researchers working voluntarily together to investigate the origins of SARS2.

@CZilcho @ch_zimmer @interne41914499 @schnufi666
38/please unroll @threadreaderapp
We are not the only one taking in consideration RaTG13 as vaccine
"Prospect of Using RaTG13 Sarbecovirus As a Candidate Vaccine for COVID-19"
papers.ssrn.com/sol3/papers.cf…
Another sign of attenuation? " This new orf8 of 2019-nCoV does not contain known functional domain or motif. An aggregation motif VLVVL (amino acid 75–79) has been found in SARS-CoV orf8b
ncbi.nlm.nih.gov/pmc/articles/P…
...which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes [26], but this is absent in this novel orf8 of 2019-nCoV. Also confirmed in the last review from Shi:
nature.com/articles/s4157…
To develop a pan-corona vaccine was an active goal of EcoHealth Alliance before SARS2 outbreak, thanks @TheSeeker268 for the links
twitter.com/messages/11701…
twitter.com/messages/11701…
twitter.com/messages/11701…

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More from @Rossana38510044

22 Oct
propublica.org/article/here-a…
eu.usatoday.com/story/news/201…
If a SARS2 adapted to mice will be detected in nature we know where to look for its origin
"We previously developed a recombinant mouse adapted strain of SARS-CoV-2 (SARS-CoV-2 MA) capable of utilizing mACE2 for viral entry by remodeling the spike and receptor binding interface via reverse genetics"
nature.com/articles/s4158…
"Although SARS-CoV-2 MA mediated infection of wild-type mice, young adult mice did not display the major clinical manifestations or hallmarks of ALI"
Read 5 tweets
23 Sep
@__ice9 1. They had it since 2016 from Baric, Shi said in the webinar.
They tested other BtCoVs but still working on the data
"We performed in vivo experiments in transgenic (human ACE2 expressing) mice and civets in 2018 and 2019 in the Institute’s biosafety laboratory...
@__ice9 "the viruses we used were bat SARSr-CoV close to SARS-CoV. Operation of this work was undertaken strictly following the regulations on biosafety management of pathogenic microbes in laboratories in China."
sciencemag.org/sites/default/…
@__ice9 3. "The results suggested that bat SARSr-CoV can directly infect civets and can also infect mice with human ACE2 receptors. Yet it showed low pathogenicity in mice and no pathogenicity in civets. These data are being sorted and will be published soon."
Read 4 tweets
23 Sep
@Ayjchan @DecrolyE 1. Interesting. A paper from Baric, 2018 on the evaluation of recombinant resistant CoV for vaccine development, which cites (ref. 55) the cave where RaTG13 was discovered as harbouring...
nature.com/articles/s4200…
2. “zoonotic pools of CoVs that genetically resemble lethal human and animal CoVs, often with only a few percentage points of difference between the zoonotic and lethal human sequences”
3. But RaTG13 was the only SARS-like virus in that cave, quite far away to be lethal for humans. Mistake in the reference or another virus was in the same cave?
Read 7 tweets
29 Aug
1. Baric, 2019: "To address an unmet medical need for the treatment of current human CoV infections and to maximize pandemic preparedness, broad spectrum antiviral therapies are needed that are effective against current and future emerging CoV... pubmed.ncbi.nlm.nih.gov/31233808/
..."given the numerous examples of novel CoV emergence...Upon passage of MHV in the presence of (Remdesivir) RDV, resistance mutations arise in the RNA dependent RNApolymerase (RdRp) that confer resistance demonstrating that the RdRp is a target of RDV antiviral activity"
"These data further illuminate the breadth and antiviral activity of RDV against the CoV family and suggest RDV as a potential antiviral for current endemic and epidemic CoV as well as future emerging CoV."
Read 4 tweets
18 Jul
scimex.org/newsfeed/exper…

Holmes: "There is some unfounded speculation that this virus (RaTG13) was the origin of SARS-CoV-2. It was sampled from a different province of China (Yunnan) to where COVID-19 first appeared". And what about your pangolin CoVs? @edwardcholmes
"The level of genome sequence divergence between SARS-CoV-2 and RaTG13 is equivalent to an average of 50 years (and at least 20 years) of evolutionary change."
What about possible cell/ animal passaging in presence of drugs as Remdesivir? @edwardcholmes
"The genetic changes in the virus can be found in two other coronaviruses from bats and pangolins".Both not valid because the first disintegrated and it is not possible to verify, beside it was named with two different names and the second is contaminated with human sequences
Read 4 tweets
25 Jun
Biochemical evidence of furin specificity and potential for phospho-regulation at Spike protein S1/S2 cleavage site in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV
biorxiv.org/content/10.110…
The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous insertion was present in the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV.
We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage.
Read 5 tweets

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