Let Uncle Ralph educate you
Synthetic Viral Genomics:
Risks and Benefits for Science and Society

"Alternatively, “No See’m” sites can be used to insert foreign genes into viral, eukaryotic, or microbial genome or vector, simultaneously removing all evidence of the restriction sites that were used in the recombinant DNA manipulation"
By orientating the restriction sites as “No See’m”, the sites are removed during reassembly, leaving only the desired mutation in the final DNA product.
The dual properties of strand specificity & a variable end overhang that can be tailored to match any sequence allow for Esp3I sites to be engineered as “universal connectors” that can be joined with any other four nucleotide restriction site overhangs (e.g. EcoRI, PstX1, BamH1)
Seamless assembly (also called No See’m Sites (85)) cascades have been used to assemble full length cDNAs of the coronaviruses mouse hepatitis virus, transmissible gastroenteritis virus, infectious bronchitis virus and SARS-CoV (Refs: 85,86,87)
Type IIS restriction endonucleases recognize asymmetric binding sites & leave asymmetric ends
These enzymes can be used to create unique interconnecting junctions, which can be subsequently removed from final assembly product allowing seamless reconstruction of an exact sequence
With enzymes like Esp3I, interconnecting restriction site junctions can be located at ends of each cDNA & systematically removed during assembly of complete full-length cDNA product
Consequently, knowledgeable experts can theoretically reconstruct full length synthetic genomes for any of the high priority virus pathogens, although technical concerns may limit the robustness of these approaches.
Another approach might be to “humanize” zoonotic viruses by inserting mutations into virus attachment proteins or constructing chimeric proteins that regulate virus species specificity (viral attachment proteins bind receptors, mediating virus docking and entry into cells).
The pathogenicity of these chimeric coronaviruses is unknown
The pathogenicity of these chimeric coronaviruses is unknown
The pathogenicity of these chimeric coronaviruses is unknown
The pathogenicity of these chimeric coronaviruses is unknown
"Scapegoat option"
This powerful technique provides bioterrorists with a “scapegoat” option; leaving a sequence signature that misdirects efforts at tracking the true originators of the crime. Even better, the approach could be used to build mistrust &/or precipitate open warfare
Dual Use
Uncle Ralph summarises his findings (1)
Uncle Ralph Concludes (2)
Hence Ecohealth DARPA/DTRA spooks & virus thieves collaboration with Baric (UNC) Lipkin (Mailman) NIchols (Atlanta CDC) and USAMRIID (Bavari, Totura et al) & Jonathan Epstein's palpable concern about dual use references in the @USRightToKnow FOIA emails
A scary read for an experimental monkey!
Delving into the cold and calculating mind of a twisted genius?
Bring Uncle Ralph and his transgenic mice in for questioning!
unroll @threadreaderapp
You just read the Baric Paper, one of 6 in a series, the rest are available here in a 191 page document
Related Papers
1. Synthetic Genomics: Options for Governance (2008) https://t.co/A32jEfL1TQ
2. Sequence Screening - Robert Jones (2005)
3. Synthetic Biology as a Field of Dual-Use Bioethical Concern - Alexander Kelle
Related papers (2)
4. Sanghvi Y. A Roadmap to the Assembly of Synthetic DNA from Raw Materials.
5. Collett MS. Impact of Synthetic Genomics on the Threat of Bioterrorism with Viral Agents.
Related Papers (3)
6. Fleming DO. Risk Assessment of Synthetic Genomics: A Biosafety & Biosecurity Perspective.
7. Risk Governance of Synthetic Biology
8. US Competitiveness in Synthetic Biology
Related papers (4)
9. Ensuring security of synthetic biology
10. Synthetic biology: emerging research field in China
What rough beast? Synthetic biology, uncertainty,& the future of biosecurity (2016)
Back to Baric et Al
Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus (2003)
Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63 (2008)
For the Record (WIV)
In 2016, Shi and her team at the WIV, in conjunction with the New York-based EcoHealth Alliance, constructed a full-length clone of a bat coronavirus called SL-CoV WIV1. They assembled it in discrete segments.
They genetically engineered the virus using the pGEM®-T Easy Vector Systems to join the segments. This system, also available on the internet, gives researchers several options for how to remove GM inserts that can be seen as signatures of a lab-made virus.18
pGEM®-T Easy Vector Systems:
“Thus, several options exist to remove the desired insert DNA with a single restriction digestion.”
This shows that researchers at the WIV have the ability to genetically engineer viruses and remove the signatures of the genetic engineering.
WIV & EcoHealth Alliance published a paper in 2017 on how they genetically modified spike proteins of 8 bat coronaviruses, by cutting & pasting genetic material from other coronaviruses, so that the viruses infected the human ACE2 receptor
They used pGEM®-T Easy Vector Systems to join the segments to genetically engineer these viruses.
They showed how they can insert new spikes into viruses. The researchers state:
“Then any spike could be substituted into the genome of SARSr-CoV WIV1 through this strategy.”
This shows that researchers at WIV have the ability to genetically modify multiple coronaviruses to insert new spikes, and these new viruses cannot be detected as genetically engineered.
The research clearly shows that GOF researchers at WIV could assemble SARS-CoV-2 from bat coronaviruses, such as RaTG13 or similar & spike protein from Malayan Pangolins & insert multi-basic cleavage sites into precise regions of spike & leave no evidence of genetic engineering.
All the above about WIV research is taken verbatim from an excellent article by Andre Leu on the website of @OrganicConsumer
COVID 19: The Spike and the Furin Cleavage

More from Billy Bostickson 🏴👁&👁 🆓

@EricTopol @NBA @StephenKissler @yhgrad B.1.1.7 reveals clearly that SARS-CoV-2 is reverting to its original pre-outbreak condition, i.e. adapted to transgenic hACE2 mice (either Baric's BALB/c ones or others used at WIV labs during chimeric bat coronavirus experiments aimed at developing a pan betacoronavirus vaccine)

@NBA @StephenKissler @yhgrad 1. From Day 1, SARS-COV-2 was very well adapted to humans .....and transgenic hACE2 Mice

@NBA @StephenKissler @yhgrad 2. High Probability of serial passaging in Transgenic Mice expressing hACE2 in genesis of SARS-COV-2

@NBA @StephenKissler @yhgrad B.1.1.7 has an unusually large number of genetic changes, ... found to date in mouse-adapted SARS-CoV2 and is also seen in ferret infections.

@NBA @StephenKissler @yhgrad We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the ... Thus, this mouse-adapted strain and associated challenge model should be ... (B) SARS-CoV-2 genomic RNA loads in mouse lung homogenates at P0 to P6.
1/ Directed Evolution and Serial passage of RNA Viruses
A quick (I hope) thread on RNA viruses & some others, mainly for my own learning process, but also as evidence (as if it was needed) of what modern virology & microbiology is capable of doing to viruses in their laboratories

2/ In Vivo Continuous Directed Evolution
1 Recent advances in the field of in vivo continuous evolution
2 Advances in continuous culture of microbes enable continuous evolution
3 Viruses, prokaryotes, & eukaryotes mediate in vivo continuous evolution

3/ Continuous Directed Evolution of Biomolecules
By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

4/ CDE for Strain & Protein Engineering
Inducible hypermutator systems accelerate strain evolution without compromising genome integrity.
Site-specific in vivo mutagenesis strategies have facilitated continuous protein engineering.

5/ Targeted mutagenesis:
A sniper-like diversity generator in microbial engineering
Advances in the directed evolution of proteins
In vivo continuous directed evolution
1. The mysterious case of Eddie Holmes and the SARS-COV-2 genome sequence publication delay


Seems that Eddie was playing the "innocent card"

@TheSeeker268 @franciscodeasis @ydeigin @AntGDuarte @edwardcholmes @K_G_Andersen

2. Was the motive of https://t.co/nu7hNVhiwP’s rush release due to competition to get the first releaser credit for the sars-cov-2 genome?

3. @GISAID claims 'the access credentials of some of the persons listed as authors of Wuhan-hu-1 were among the access credentials used on January 10, 2020 to access the first genomes available on GISAID'

4. @GISAID formally wrote to the authors who acknowledged Virological,org’s post as the world's first release of the SARS-COV-2 genome, and penalised the authors of MN908947

5. but @GISAID didn't identify who exactly from Fudan University or University of Sydney ACCESSED and KNEW about IVDC's 'initial' submission of IVDC-01/05 on GISAID on 10th Jan, 2020, via login records of their GISAID account credentials.

More from Science

Hard agree. And if this is useful, let me share something that often gets omitted (not by @kakape).

Variants always emerge, & are not good or bad, but expected. The challenge is figuring out which variants are bad, and that can't be done with sequence alone.

You can't just look at a sequence and say, "Aha! A mutation in spike. This must be more transmissible or can evade antibody neutralization." Sure, we can use computational models to try and predict the functional consequence of a given mutation, but models are often wrong.

The virus acquires mutations randomly every time it replicates. Many mutations don't change the virus at all. Others may change it in a way that have no consequences for human transmission or disease. But you can't tell just looking at sequence alone.

In order to determine the functional impact of a mutation, you need to actually do experiments. You can look at some effects in cell culture, but to address questions relating to transmission or disease, you have to use animal models.

The reason people were concerned initially about B.1.1.7 is because of epidemiological evidence showing that it rapidly became dominant in one area. More rapidly that could be explained unless it had some kind of advantage that allowed it to outcompete other circulating variants.

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