Let Uncle Ralph educate you Synthetic Viral Genomics: Risk…

"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?
https://t.co/MrwxYK93cx
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
https://t.co/rjNZ8aDNDl

Related Papers
1. Synthetic Genomics: Options for Governance (2008) https://t.co/A32jEfL1TQ
2. Sequence Screening - Robert Jones (2005)
https://t.co/zOqMroYbVU
3. Synthetic Biology as a Field of Dual-Use Bioethical Concern - Alexander Kelle
https://t.co/Va1movPqvs

Related papers (2)
4. Sanghvi Y. A Roadmap to the Assembly of Synthetic DNA from Raw Materials.
https://t.co/4kWu4vzcXC
5. Collett MS. Impact of Synthetic Genomics on the Threat of Bioterrorism with Viral Agents.
https://t.co/OcaTmuCUBy

Related Papers (3)
6. Fleming DO. Risk Assessment of Synthetic Genomics: A Biosafety & Biosecurity Perspective.
https://t.co/JBcLVLr005
7. Risk Governance of Synthetic Biology
https://t.co/9BE9X5s0Fb
8. US Competitiveness in Synthetic Biology
https://t.co/OCZJdsXSjl

Related papers (4)
9. Ensuring security of synthetic biology
https://t.co/7kPE1OpbrS
10. Synthetic biology: emerging research field in China
https://t.co/T3BcaDvUsa
11.
What rough beast? Synthetic biology, uncertainty,& the future of biosecurity (2016)
https://t.co/XTlBXILEWh

Back to Baric et Al
Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus (2003)
https://t.co/k8PI1rbcm8
Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63 (2008)
https://t.co/bWhAv1wsC0

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
https://t.co/bDOMmhpJ1t