The COVID-19 coronavirus SARS-CoV-2 has a telltale “ fingerprint” that means it is highly more likely to have been made in a laboratory, new research has found.
Mathematical biologist Dr . Alex Washburne and colleagues Valentin Brutte plus Antonius VanDongen published their research in a preprint that will finds “ a high probability that SARS-CoV-2 may have originated as an infectious clone assembled in vitro ”.
They explain this evidence, which they term an “ endonuclease fingerprint”, is independent of the proof relating to the Furin Cleavage Site , which others have suggested is a ‘ smoking gun’ for the lab origin.
Dr . Washburne stresses that he is not alleging malign intent or even gain-of-function work in this paper. He writes : “ We find no evidence of SARS-CoV-2 being a bioweapon (on the contrary, this looks like an accident) or any gain of perform work. We find evidence suggesting SARS-CoV-2 may have been synthesised in the lab with known strategies, probably for normal pre-Covid research purposes. ”
Professor Francois Balloux has given the study their imprimatur on Twitter , writing: “ It is really an important piece of work. To me, this looks solid both conceptually and methodologically. I was given advance warning and could replicate the key findings. To the best of my knowledge, I confirm the reported designs are genuine. ”
To accompany the study Dr . Washburne has published a Substack plus a Twitter thread . The Twitter line is reproduced in full below – it can be a bit specialized in places, but the message it’s conveying should come through clearly enough.
The origin of SARS-CoV-2 is unknown. Some hypothesised two spillover events on the wet market, but methodological flaws make that work inconclusive . We need to understand the true origin of SARS-CoV-2 to prevent pandemics.
We examined whether SARS-CoV-2 was synthesised in a lab. We studied a common way of synthesising coronaviruses (CoVs) within the lab. This method was thought to not leave a finger-print. We found the fingerprint. That fingerprint is in the SARS-CoV-2 genome.
Here’s how you make a CoV in the lab. To make a 30, 000 base (30kb) RNA virus in the lab, you will need a 30kb DNA clone To put together a 30kb DNA identical copy, scientists glue together several smaller fragments.
A popular method for DNA set up is ‘ golden gate assembly ‘.
Fantastic gate assembly requires the particular DNA sequence have exclusive ‘ cutting’ sites (type IIS restriction sites). Reducing sites create three to four nucleotide (nt) ‘ sticky ends’. Sticky ends help you ‘ paste’ DNA segments collectively, ensuring faithful assembly of your 30kb DNA copy of a viral genome.
RNA viruses like CoVs are not under selection especially for this kind of cutting and pasting. So , wild viruses generally have cutting and pasting sites randomly scattered in their genome. Researchers building viruses inside a lab will often add or even remove cutting sites.
We collected examples of CoV infectious clones constructed with these type IIS cutting and pasting systems through 2000-2019. We found a clear pattern in how scientists tended to add or eliminate cutting and pasting sites.
Researchers tend to turn randomly-spaced restriction maps into regularly-spaced ones (A-B). Regular spacing comes from wanting fewer fragments (typically 5 to eight) while keeping the longest fragment measures low.
Digesting 70 CoVs with 200-plus restriction enzymes yields a ‘ wild kind distribution’, a null model for how long the greatest fragment may be as a function of the number of fragments [when the virus is wild]. The red container is the ideal range for reverse genetic systems used to make infectious clones.
CoVs engineered to become infectious clones will move from having restriction maps falling within the wild type distribution to being outliers under the wild-type distribution, falling within the lab-ideal range of come apart number & low longest-fragment-length.
Getting found this fingerprint, all of us examine specific cutting and pasting sites in the SARS-CoV-2 genome (BsaI/BsmBI).
BsaI and BsmBI are extremely popular enzymes for this type of in vitro assembly They likewise have many conserved sites in CoVs. Very useful for making chimeras.
The SARS-CoV-2 BsaI/BsmBI restriction map falls neatly within the ideal range for a reverse genetic program. It is an anomaly (bottom 1%) amongst wild type CoVs. It is a midpoint among engineered CoVs.
Digesting CoVs with only type IIS digestive enzymes that could be used for assembly, SARS-CoV-2 is an even greater outlier. It could in the bottom 1% max-fragment-length for all restriction enzymes It’s the single largest outlier (< 0. 07%) of 1, 491 type IIS digestions
We then tested the lab-assembly speculation. If SARS2 has a artificial origin via golden gate assembly, several other criteria should be met. For example: all sticky ends must be unique, non-palindromic and contain at least one A/T. SARS2 passed this check (60% chance of this).
The mutations isolating SARS-CoV-2 BsaI/BsmBI sites from the close relatives must almost all be silent mutations. Almost all 14 mutations in BsaI/BsmBI sites are silent. 84% of mutations in SARS2 and close relatives are silent, so 9% opportunity all 14 distinct mutations will be silent.
There’s a significantly higher concentration of silent mutations for each nucleotide within BsaI/BsmBI acknowledgement sequences than in the rest of the genome. P=0. 004 for BANAL52-SARS2 P=9e-8 for RaTG13-SARS2.
Such an idealised invert genetic system is unlikely to evolve by chance in the close relatives of SARS-CoV-2. There’s a 1% chance of unique RaTG13 mutants having as great or greater z-score and 0. 1% possibility for BANAL52.
Testing this from multiple angles, we could not really reject the hypothesis that SARS-CoV-2 has a synthetic origins. Each test also reduced the odds of SARS-CoV-2 aquiring a natural origin. The BsaI/BsmBI fingerprint of SARS-CoV-2 shows synthetic origin of SARS-CoV-2.
Please examine our manuscript for our careful language and limitations. These are important. For example , our answers are independent of the Furin Cleavage Site (FCS). While the RBD is docked in fragment five, we shine no gentle on the origin of the FCS.
Our study does not identify the laboratory. We hypothesise this limitation map would enable construction of chimeric viruses, similar to the recent questionable work done in Boston (but with a various method for within vitro assembly).
Our theory of the synthetic origin of SARS-CoV-2 can and should be examined. Further tests may deny our theory. We accepted these tests. Our program code is available on GitHub and point to future research that can reject our hypothesis or even refine our understanding of this problem.
Making chimeric viruses in vitro carries risks. We encourage transparency from researchers studying CoVs in Wuhan. We strongly encourage global coordination on biosafety.
We encourage open, civil and compassionate discourse on this important topic. This pre-print was not hurried. It was reviewed by many colleagues, truly world experts. We all thank them all immensely for feedback. This has been an amazing project. Yet, for apparent reasons, this is the saddest document I’ve ever written.