Lab Leak or Zoonotic Transfer? Leading Biologists Review COVID-19 Virus Origin Evidence

COVID Origin Analysis

Amid debate around the origins of SARS-CoV-2, leading global biologists have reviewed the scientific evidence to help clarify the origin of the virus that causes COVID-19 in humans.

  • Pre-print paper highlights links supporting zoonotic origin for the virus
  • Zero biological evidence exists for a laboratory leak
  • Focus on lab-leak distracting from work to prevent next pandemic

An international team of eminent biologists, led by Professor Edward Holmes from the University of Sydney and Professor Andrew Rambaut from the University of Edinburgh, has published a critical review paper on the origins of SARS-COV-2 as a pre-print on Zenodo.

The paper summarises and reviews the existing scientific evidence for the origin of the virus, which causes COVID-19 in humans, concluding that overwhelmingly its most likely origin is zoonotic – a transfer from an animal source to human infection. While the authors say that a laboratory accident “cannot be entirely dismissed” they emphasize that there currently exists zero evidence for such a laboratory origin.

Edward Holmes

Professor Edward Holmes. Credit: University of Sydney

Professor Holmes said: “Our careful and critical analysis of the currently available data provided no evidence for the idea that SARS-CoV-2 originated in a laboratory.”

The review paper says: “There is no evidence that any early cases had any connection to the Wuhan Institute of Virology (WIV), in contrast to the clear epidemiological links to animal markets in Wuhan, nor evidence that the WIV possessed or worked on a progenitor of SARS-CoV-2 prior to the pandemic.”

Rather, it argues that “there is substantial body of scientific evidence supporting a zoonotic origin for SARS-CoV-2.”

The 21 eminent scientists from universities and research institutes around the world warn that a focus on a highly improbable lab origin is distracting from the most urgent scientific tasks to “comprehensively investigate the zoonotic origin through collaborative and carefully coordinated studies.”

The authors warn that without a focus on this line of enquiry, the world will be “vulnerable to future pandemics” arising from new viruses.

Reference: “The Origins of SARS-CoV-2: A Critical Review” by Holmes, Edward C; Goldstein, Stephen A; Rasmussen, Angela L; Robertson, David L; Crits-Christoph, Alexander; Wertheim, Joel O; Anthony, Simon J; Barclay, Wendy S; Boni, Maciej F; Doherty, Peter C; Farrar, Jeremy; Geoghegan, Jemma L; Jiang, Xiaowei; Leibowitz, Julian L; Neil, Stuart J D; Skern, Tim; Weiss, Susan R; Worobey, Michael; Andersen, Kristian G; Garry, Robert F; Rambaut, Andrew, 7 July 2021, Zenodo.
DOI: 10.5281/zenodo.5075888

As well as the University of Sydney and University of Edinburgh the affiliations of the 21 authors include the University of Utah (US), University of Saskatchewan (Canada), University of Glasgow (UK), University of California Berkeley (US), University of California San Diego (US), University of California Davis (US), Imperial College London (UK), Pennsylvania State University (US), the University of Melbourne at the Doherty Institute (Australia), The Wellcome Trust (UK), University of Otago (New Zealand), Jiaotong-Liverpool University (China), Texas A&M University (US), King’s College London at Guy’s Hospital (UK), Medical University of Vienna (Austria), University of Pennsylvania (US), University of Arizona (US), Scripps Research Institute (US), Tulane University (US), Zalgen Labs (US).

The pre-print paper will be submitted to a leading journal for peer review and publication.

19 Comments on "Lab Leak or Zoonotic Transfer? Leading Biologists Review COVID-19 Virus Origin Evidence"

  1. Why am I not surprised?

  2. Great article!

  3. the 2006 SARS and other major outbreaks recently were all due to lab leaks. As someone who worked in the field many years, i think most people don’t realize how easily something like this can happen. Did the paper go over how there has been no evidence at all for a mediating host, or how the very odd furin cleavage sites came into being in this virus only but none of its closest relatives? And how China is extremely secretive and obstructive in the whole investigation. Think about it.

    • Carolyn L Zaremba | July 17, 2021 at 8:34 pm | Reply

      Your obvious prejudice against China is mistaken. China has NOT been “secretive and obstructive”. On the contrary, China told the world about the virus at the very beginning and revealed their isolation of the virus to the world — for free.

      • You communist loving rhetoric is laughable. You’ve contributed nothing to the comments other than show how blind you are

  4. I have read this new article. Four of the authors of, “The Origins of SARS-CoV-2: A Critical Review”, also wrote “Proximal Origin of SARS-COV-2”. One of their conclusions in this new 2021 article is,

    “This demonstrates beyond reasonable doubt that RaTG13 is not the progenitor of SARS-CoV-2, with or without laboratory manipulation or experimental mutagenesis.”

    This conclusion is spurious at best. They base their conclusion on their analysis of the 1AB gene. They disregard the rest of the genome. But don’t point out, that RaTG13 1AB gene is 96% identity similar to Covid-19 1AB gene, according to Zhou below. Slightly less than RmYN02, RpYN06 and PrC31’s, but pretty close. More importantly, RaTG13 S gene is 96% identity amino acid similar with Covid-19. RmYN02, RpYN06 and PrC31 S genes are only 76% and 63% similar to Covid-19, and their RBDs even less (60.91%). See Zhou below. Making them less likely Covid-19 immediate progenitor. The better science is represented by the articles,

    “A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein”, Hong Zhou, Xing Chen, and others

    “Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia”, by Supaporn Wacharapluesadee, Chee Wah Tan, and others

    “Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses”, by Hong Zhou, Jingkai Ji, and others

    Zhou stated,

    “Phylogenetic analysis of full-length genome sequences of representative sarbecoviruses revealed that SARS-CoV-2 (Covid-19) was most closely related to RaTG13, while RmYN02 and the Thailand strains formed a slightly more divergent clade.”

    That is a scientific fact. Full-length genome analysis points at RaTG13. For someone to state, “this demonstrates beyond reasonable doubt that RaTG13 is not the progenitor of SARS-CoV-2”, as these 21 authors do, is to exclude a RaTG13 related type bat, from 50 years ago, as the possible immediate progenitor of Covid-19. That would be bad science. Boni, Robertson, and Holmes own article, ‘Viral CpG Deficiency Provides No Evidence That Dogs ….’, https://academic.oup.com/mbe/article/37/9/2706/5870838oV-2, Figure 1, says Covid-19 and RaTG13 share about the same “genomic CpG deficiency (ICpG) versus viral genomic GC content”. The two coronaviruses are just that similar. RaTG13 related coronaviruses can’t be ruled out as possible backbone for Covid-19. The authors’ second conclusion,

    “the claim that the virus was already highly adapted to the human host, or somehow optimized for binding to human ACE2, is without validity”

    Again is a conclusion, that does not take into account the high genomic homology of 99.98%, of many of the first Covid-19 victims. Most first victims were all hit by the same highly toxic Covid-19 environment in part of the Hunan Seafood market. All animals had to be destroyed. No outpatient service for many of these first victims, straight to the ICU. Few mutations, small time period to last ancestor (tMRCA), Covid-19 was ready to go at the beginning of the outbreak.

    And the lack of identifiable immediate precursor coronaviruses, point at already highly adapted, not recently evolving from known precursor coronaviruses. Its divergence date says it had been in nature for at least 50 years already. Yes, already highly adapted. The binding affinity of 20% greater than related betacoronaviruses. More residues to bind human ACE2 in the Covid-19 RBD, leading to more van der Wals contacts, according to “Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2”, by Qihui Wang and others. Greater binding affinity. This says something about Covid-19’s “optimization”. Three O-glycans surrounding the furin cleavage site, to help Covid-19 run under the radar of human immune system detection. Not slightly “optimized”, but fully locked and loaded. The 21 scientists all full of logical arguments and plausible scenarios, but short on good science.

    Peter G.

  5. William Martin Readling | July 17, 2021 at 1:17 am | Reply

    Could it be that virologists fear that origin from a lab could break all their fun gain of function toys? Maybe lab origin would cause governments to close labs, meaning fewer virology jobs. This is sort of like asking insurance companies to decide if auto insurance should be mandatory, or physicians if private medical insurance should be mandatory.

    • Carolyn L Zaremba | July 17, 2021 at 8:35 pm | Reply

      Yours is a despicable and unscientific comment.

    • Is there a significance difference between intentional misinformation, and information given, that doesn’t quite reach the level of “best science”? And should this latter information be censored, even though it has been shown to be just as plausible and effective? And who determines what the “best science” is? Who fact checks, the fact checkers? Should those scientists who have the highest IQ’s, be held to the highest level of “scientific truth” and ethical standards in their daily work? And what is “scientific truth”, since science is ever evolving? Should the desire to sustain a livelihood in a given profession, justify ‘apparent’ half-truths and misinformation by those in that profession, to continue to make that profession a viable livelihood? Should a person who has common sense (Dr. Watson), who disagrees with persons of high IQ (Sherlock Holmes), be censored? Does having a high IQ, absolve those people from lying? Is having a high IQ, the gold standard for truth? Has it ever been? Sherlock Holmes’s Moriarty, and Kaczynski and Dahmer, were mastermind criminals, high IQ. Is there a way to meaningful discern the difference between “scientific information” given, intended to coverup the origins of a coronavirus, from just plain old half-truths intended to mislead for economic or other reasons? What would be the gold standard for such a determination, the high IQ’s of those who consistently present the half-truths, the level of nonsensibility of the half-truths, etc? Who would make that determination? In Christ Jesus.

  6. Great, we’ll written article but I’m still not convinced. On the one hand you have a wet market operating in the same way it has for about 2000 years, and just up the road there’s a germ research lab, however we are expected to believe the virus did not originate from there.
    I’m afraid in this case the old maxim stands, if it looks like a duck and quacks like a duck then it’s a duck. I rest my case!

  7. This is germ warfare. CC China has no problem with forced abortions. Do you think they have a problem with killing off the elderly? China has enormous problems with the size of their elderly population as well as feeding their people. The virus was a convenient win -win. Take out the elderly and sell the world ppe and a half assed cure. The WHO and Tedros Gerbreyssus have covered for China all along. Wake up, ccp China is the enemy of mankind.

  8. Why does it have to be an “either or” choice? Should we be considering a combination of the two? A inadvertent zoonotic transfer in the lab to the three workers who then went to the hospital, infecting people along the way in places like elevators, ER waiting rooms, etc, then those people spread it to the wet market near WIV and then it blossoms into the full scale nightmare that it has become.

  9. Adela Darling | July 21, 2021 at 6:12 pm | Reply

    Totally agree with you Chris.
    It’s not a distraction or improbable. I bet the odds of a lab leak are why higher then winning the lottery or actually dying of (a)Coronavirus.

  10. The question (zoonotic vs lab leak) is not simple; but simple risk vs gain/avoidance analysis suggests a strong leaning (big bubble) in the lab leaked/ gain of function/ vested interest/ big funds, quadrant. Or should that last list be in reverse order?!

  11. The House Foreign Affairs Committee Report Minority Staff report “The Origins of Covid-19: An Investigation of the Wuhan Institute of Virology” has been published at https://gop-foreignaffairs.house.gov/wp-content/uploads/2021/08/ORIGINS-OF-COVID-19-REPORT.pdf.

    First the report emphasizes the significance of Wuhan Metro Train Line # 2, in relation to the Wuhan Institute of Virology older location in the Wuchang district, and to local hospitals.

    P. 25 “When people get sick, they are likely to seek healthcare near their home or work. Each of the hospitals that saw a rise in traffic with patients complaining of COVID-19 symptoms are located within 6.5 miles of the WIV Headquarters and are connected by public transit lines…… six hospitals are clustered around the WIV Headquarters in Wuchang, Wuhan, and are connected to that facility via the Wuhan Metro Train (Line # 2)…..”

    Steven Quay recognized the significance of Metro Train Line # 2 in Covid-19 spread in his paper, “Where Did the 2019 Coronavirus Pandemic Begin and How Did it Spread?” https://zenodo.org/record/4119263, and https://zenodo.org/record/4642956

    The Foreign Affairs committee report then listed the following BSLs (biosafety labs) in Wuhan.

    P. 13 of the report states Wuhan National Biosafety Laboratory (WNBL) in Zhengdian Scientific Park houses twenty BSL-2, two BSL-3 and one BSL-4.
    P. 13 also states the Wuhan Institute of Virology older location in the Wuchang District of Wuhan, houses at least two BSL-2, and one BSL-3.
    P. 53 states Wuhan University houses a BSL-3, which was temporarily shut down in September 2019.
    The Wuhan CDC has one BSL-2 according to Filippa Lentzos 2020 article on Covid-19. A BSL not mentioned in the Foreign Affairs committee report.

    Which BSL lab, Covid-19 may have “leaked” from, you choose. They are all pretty close to Wuhan Metro Train Line # 2. Some probable causes the Minority Staff report posits for a coronavirus “leak” are listed below.

    p. 36 “According to an interview with Shi published by Science, all coronavirus experimentation, including infecting hACE2 mice and civets, was done at the BSL-2 and BSL-3 levels”. Not enough safety in such low safety laboratories for such experimentation implied.

    p. 58, at the BSL-4, “A defective hazardous waste treatment system and central air conditioning system would increase the likelihood of a lab employee (or several) becoming infected with SARS-CoV-2, as viral particles would be more likely to remain in the air for longer periods of time.” All of this “defectiveness” in a brand new 2018 building, built by the French, increased the likelihood of lab “leak”.

    On pages 33, 34 and 40 of the report, the most compelling and shocking “implied reason” for a laboratory leak, is that the Chinese scientists “are capable and have a history” of creating the coronavirus. The report spends twenty pages explaining this “capability and history”. And then in the sheerest sense of hypocrisy, the Minority Staff report reviews in detail American scientists “capableness and history” of creating and engineering coronavirus. The Minority Staff report cited the 2015 American scientist paper, “A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence”, the 2016 American paper, “SARS-like WIV1-CoV poised for human emergence”, and the 2005 American paper, “Development of mouse hepatitis virus and SARS-CoV infectious cDNA constructs” (textbook on engineering and cloning viruses using restriction sites), all unGodly knowledge, which laid the foundation for the possibility of a future “laboratory leak”. If not the intentional launch of a bioweapon. The only difference between the Chinese “capableness and history”, and the Americans “capableness and history”, seems to be Wuhan Metro Train Line # 2. Please correct me if I am wrong.

    Which of the 25 above Chinese laboratories Covid-19 may have “leaked” from, the choice is yours, implies the Minority Staff report. Most laboratories were on Wuhan Metro Train Line # 2. With one Metro stop (the Hankou station) near the Huanan Seafood Market. Another Metro stop (Jiedaokou station) near Dr. Steven Quay’s PLA Hospital, NO. 627 Wuluo Road, where he alledges first Covid-19 patients were identified. Another Metro Line # 2 stop near the old WIV Wuchang location. Yes, all of these laboratories are prime suspects, for laboratory “leak”. “Round up all the usual suspects” as captain Louis Renault (Claude Rains) says at the end of the movie “Casablanca”. But not the American laboratories, because they are not on Wuhan Metro Train Line # 2.

    Enough innuendos in this House Foreign Affairs Committee report (that took them only 8 or 9 months to produce) to insight a lynch mob. Thank Jesus and the God of our fathers, that our judicial system tries facts, and not allusions. Our judicial system places normal evidential weight on “who is capable and has a history of doing what”, and employs a higher standard of causal proof than this Minority Staff report evidences. At least this report concluded that some of those at the October 2019 Wuhan Military Games were probably infected with Covid-19. This is where the report should have begun its inquiry, and not ended. Who were those five American soldiers that got sick at the October 2019 Wuhan Military games, and were sent home early before the games ended? Were their 2019 lab specimen’s ever tested for Covid-19 specific antibodies? Answer this, and then a report can rise above the level of sleazy innuendo filled tabloid.

  12. In the article ‘The Proximal Origin of SARS-CoV-2’, the authors Edward Holmes, Andrew Rambaut and others state that Covid-19’s RBD (receptor binding domain) binds to human ACE2 in an “optimized manner (high affinity) “, but not an optimal manner. Therefore Covid-19 is “not the product of purposeful manipulation”.

    “While the analyses above suggest that SARS-CoV-2 may bind human ACE2 with high affinity (optimized), computational analyses predict that the interaction is not ideal and that the RBD sequence is different from those shown in SARS-CoV to be optimal for receptor binding….SARS-CoV-2 appears to be optimized for binding to the human receptor ACE2;”
    “The Origins of SARS-CoV-2: A Critical Review”, by Edward Holmes, Andrew Rambaut, Kristian Anderson, Robert Garry and others,

    Rambaut, Holmes, and Anderson base their conclusion regarding optimalness of Covid-19’s RBD/RBM (receptor binding motif), on the January 2020 Ralph Baric and Fang Li article, “Receptor recognition by novel coronavirus from Wuhan”. Rambaut, Holmes, and Anderson reference this latter article seven times in their own article.

    In January 2020, Baric and Fang Li listed optimal RBD to bind to human ACE2 as F442 F472 N479 D480 T487 using in vitro design. See figure 1B of the article, ‘Receptor Recognition…”. The January 2020 Baric and Fang Li research was actually based on 2011 Fang Li research, where he ‘designed’ optimal human RBDs at the University of Minnesota,

    “To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD “ . ‘Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus’, Fang Li and others, University of Minnesota, Department of Pharmacology, JBC Papers in Press, January 30, 2012, DOI 10.1074/jbc.M111.325803

    So the optimization of human RBDs (and civets and possibly pangolin RBD), in the context of optimal human ACE2 residues, is a Baric and Fang Li area of expertise. Rambaut, Holmes and Anderson are just the public face, of Baric an Fang Li optimization research.

    Covid-19 RBD/RBM not optimal, thus not created in a laboratory, according to these scientists.

    In August 2021, Rambaut, Holmes, Anderson, an Garry built on this Covid-19 not optimal theme, when they applied the words suboptimal , and ‘only optimal’, to the Covid-19 furin cleavage site, amino acids RRAR.

    ‘The SARS-CoV-2 furin cleavage site (containing the amino acid motif RRAR) does not match its canonical form (R-X-R/K-R), is suboptimal compared to those of HCoV-HKU1 and HCoV-OC43……There is no logical reason why an engineered virus would utilize such a poor (non-optimal) furin cleavage site, which would entail such an unusual and needlessly complex feat of genetic engineering.’
    “The Origins of SARS-CoV-2: A Critical Review”, by Edward Holmes, Andrew Rambaut, Kristian Anderson, Robert Garry and others,

    The canonical/preferred furin cleavage site is amino acid sequence RSRR, according to the authors, and research done by Gary Whittaker at Cornell University in 2013. But, in this ‘The Origin of SARS-CoV-2: Critical Review’ article above, it is the suboptimal cleavage site, RRAR-Covid-19, that is the bad, non-laboratory made, and impliedly the optimal, RSRR, that would be signs of ‘engineered’ laboratory made bioweapon.

    In 2014 Gary Whittaker at Cornell University stated that RSRR sites cleavages furin at a 100% rate per minute, according to Figure 4 of that article. .

    ‘We used fluorogenic peptides containing the canonical motif (RR-S-R-R-S) or with substitutions from positions P1′ through P7 (Figure 4, panel A). The canonical peptide was efficiently cleaved by furin (Figure 4), with average Vmax of 235 Relative Fluorescence Units (RFU) per minute…. Modifications of the P1 arginine
    in the canonical peptide, regardless of the residue tested, for example, glycine (G), methionine (M) or threonine (T), abrogate cleavage by furin. When P2 arginine is changed to histidine (H), there is complete inhibition (0% of canonical cleavage).’,
    ‘Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus’, Gary Whittaker and others, Cornell University, 2013, http://dx.doi.org/10.3201/eid1907.121094

    100% furin cleavage by the canonical RSRR site after a short period of time. Quick virus entry into human cells. Whittaker showed himself to an expert in optimizing and minimizing furin cleavage sites in 2014.

    Whittaker also used the PiTou algorithm to predict cleavage of a viral protein by human furin in many of his articles.

    “furin has the ability to cleave and activate viral proteins….PiTou utilizes a hidden Markov model, specifically targeting 20 amino acid residues surrounding furin cleavage sites and important for binding and solvent accessibility. The final score in PiTou is based on log-odds probability. ProP utilizes an artificial neural network to predict furin cleavage”,
    in the article ‘ Furin cleavage sites in the spike proteins of bat and rodent coronaviruses: Implications for virus evolution and zoonotic transfer from rodent species’, by Whittaker and others, June 2021,
    https://doi.org/10.1016/j.onehlt.2021.100282

    In 2021, Whittaker using the PiTou 2.0 furin prediction algorithm, stated that Covid-19 RRAR cleavage site had a PiTou score of 9.19. See Figure 1 in the article, “Functional evaluation of proteolytic activation for the SARS-CoV-2 variant B.1.1.7: role of the P681H mutation”, by Gary Whittaker and others, April 2021.

    See also Figure 5 in the article ‘Proteolytic Activation of SARS-CoV‑2 Spike at the S1/S2 Boundary Potential Role of Proteases beyond Furin’, by Gary Whittaker and others, https://dx.doi.org/10.1021/acsinfecdis.0c00701
    ACS Infect. Dis. 2021, 7, 264−272, where Covid-19 RRAR cleavage site shows a PiTou score of 9.19, about 66% on the canonical RSRR cleavage site.

    ‘We consider that SARS-CoV-2 could have originated via recombination with a currently unknown ancestor bat virus with a robust furin cleavage site (i.e., PiTou score: approximately 13−15), but one which cannot bind to a human receptor, and that in order to gain the ability to infect human cells, the furin cleavage site may have been downregulated.’

    An so we look for a bat with a robust furin cleavage site score, as the progenitor of Covid-19.

    In the 2014, Whittaker article, ‘Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis’, FCov-RM(I) with its canonical RSRR cleavage site showed a PiTou score of 14.4. BCov-Quebec with its RSRR a PiTou score of 14.1 See Table 1. Both considerably higher than Covid-19’s RRAR PiTou score of 9.19.

    So Covid-19 RRAR cleavage site doesn’t reflect optimization for being engineered, unless scientists wanted to create a ‘non-orthodox’ coronavirus, a ‘hobo’ coronavirus whose backbone couldn’t be traced, continually adding more African bats to its evolutionary history, two different types of Malaysian pangolans (from Guangxi-2017 and Guangdong-2019) to its evolutionary history, possibly mouse hepatitis genomic aspects in some of its features, a non-optimized S gene, a Mulligan’s stew type of coronavirus, to test the world’s preparedness, for a real zoological outbreak, or bioweapon attack, a Peachtree CDC, to Rising Sun CDC preparedness exercise, gone wrong.

    Another Gary Whittaker must read article, ‘ Furin cleavage sites in the spike proteins of bat and rodent coronaviruses: Implications for virus evolution and zoonotic transfer from rodent species’, June 2021, where presents the PiTou cleavage scores for bats and rodents.
    https://doi.org/10.1016/j.onehlt.2021.100282 in his Figure 1 and Figure 2.

    Rodent AcCov-JC34 (Shi Zhengli’s 2017 rodent from Yunnan province) has RRAR cleavage site, but a a PiTou score .15 One version of mouse hepatitus JVM has an RRAR cleavage site, but a PiTou of only 4.8.

    Another Whittaker 2021 article, “ SARS-CoV-2 spike and its adaptable furin cleavage site” is worth reading.

    ‘Coronavirus entry: how we arrived at SARS-CoV-2’, by Gary Whittaker, Susan Daniel, and Jean Millet, April 2021

    As a side note, in 2009, Whittaker ‘introduced’ RSRR cleavage site into the original SARS virus.

    “To examine the potential use of the SARS-CoV S1–S2 and S2_ positions as sites for proteolytic cleavage, we first introduced furin cleavage recognition sites at these locations by making the following mutations 664SLLRSTSQSI-SLLRRSRRSI671 (S1–S2) “
    “Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites”, Sandrine Belouzard, Victor Chu, and Gary Whittaker, 2009, Cornell University

    Inserting cleavage sites into coronaviruses is old hat for Whittaker.

    Gary should reexamine his cat coronavirus,

    FCoV-Sample 08 153990-1 TDLFEFVNHTQPRRARM

    Which he mentions in many of his articles, which is pretty close to Covid-19 PRRA S gene insert, and RRAR cleavage site. That P (proline) before the cleavage site, in conjunction with the cleavage site itself, is killing a lot of people. In Christ Jesus.

  13. the only true friend of Cardinal Raymond Burke | November 9, 2021 at 8:28 am | Reply

    Andrew Rambaut, Edward Holmes, K. Anderson and R. Garry authors of the 2020 article, “Proximal Origin of SARS-CoV-2”, are also co-authors of this 2021 article, “The Origins of SARS-CoV-2: A Critical Review”. They hypothesize Covid-19 originated in nature, not a laboratory. Walter Lipkin, one of the coauthors of ‘Proximal Origin’, name is not included in this 2021 ‘Critical Review’ article.

    This 2021 article lists at least three attributes of the Covid-19 RRAR furin cleavage site.

    (1) Its possible origin via copy choice recombination mechanism in cells coinfected with Covid-19 and HKU9 coronaviruse.
    (2) It is expressed in a -2 out of frame reading (two nucleotide shift towards the 5’ end) of the Spike gene
    (3) It is deleted after cell passage in some (not all) Vero E6 cells.

    In explaining the original presence of the RRAR cleavage site in the Covid-19 Spike gene, Rambaut, Holmes research group stated,

    “A near identical nucleotide sequence is found in the spike gene of the bat coronavirus HKU9-1, and both SARSCoV-2 and HKU9-1 contain short palindromic sequences immediately upstream of this sequence that are indicative of natural recombination break-points via template switching. Hence, simple evolutionary mechanisms can readily explain the evolution of an out-of-frame insertion of a furin cleavage site in SARS-CoV-2 (Fig. 2).”—–
    “The origins of SARS-CoV-2: A Critical Review”, by Edward Holmes, Andrew Rambaut, and many others, https://doi.org/10.1016/j.cell.2021.08.017

    Rambaut and Holmes twice reference William Gallaher’s article, “A Palindromic RNA sequence as a Common Breakpoint Contributor to Copy-Choice Recombination in SARS-COV-2”, in the quote above. It is Gallaher, a retired virologist who in 2020 first hypothesized that Covid-19 furin cleavage site was the result of a natural copy choice recombination mechanism in cells involved in viral replication.

    In February 20, 2020, Gallaher posted the following on the Internet, regarding the -2 out of frame reading for the RRAR furin cleavage site.

    “Then one looks at the actual RNA alignment. The “insert” is actually not in frame, but CTCCTCGGCGGG, or -2 out of frame. Again, who does that?” https://virological.org/t/naturally-occurring-indels-in-multiple-coronavirus- spikes/560/4

    Rambaut and Holmes 2021 “Critical Review” article, merely recapitulated what Gallaher had already observed in 2020. The -2 out of frame reading for the cleavage site, ie, shifted two nucleotides to the 5’ end, the complicatedness of creating such an out of frame site, implies that it was created in nature, and not a laboratory, for them.
    May 21, 2020, William Gallaher posted on the Internet,
    “Ten of the 12 nucleotides in the RRAR insert are identical to a sequence in the spike protein gene of Bat Coronavirus HKU9 isolated from a Rousettus fruit bat in Guangdong province in 2011.

    The “insert” is CT CCTCGGCGGG, the last ten identical to the sequence in HKU9.

    The sequence context has other similarities, i.e the “cagac” upstream and a “c” downstream of the “insert” as aligned here:” Tackling Rumors of a Suspicious Origin of nCoV2019

    Rambaut and Holmes focus on HKU9 coronavirus as the possible origin for Covid-19 RRAR cleavage site, must be read in the context of Gallaher’s 2020 proposals. The focus on the ‘CAGAC-CAGAT’ nucleotide sequence/’QTQT’ amino acid palindrome , prior to RRAR is key. Gallaher explained copy-choice recombination as it relates to these sequences as follows.

    “In single-stranded RNA viruses, recombination does not commonly take place by breakage and rejoining of two complete strands but rather by the polymerase slowing or stopping during transcription and jumping to another template strand – “copy choice” – during a mixed infection. This can take place in either direction of synthesis, even though the vast majority of replications are of the positive strand from the negative template….. The hypothesis being put forward here is that the RNA genome of members of the genus Betacoronavirus, including
    SARS-CoV-2, is organized into blocks of information bounded by short “breakpoint sequences”. A subset of these is identified here in multiple sites as CAGAC and CAGAT…..Alignment of SARS-CoV-2 in the furin insert region with a candidate source of the insert sequence, Bat-HKU9-1. Alignment begins at nt 23,601 of SARS-CoV, paired with nt 24,190 of Bat-HKU9-1, corresponding to a peptide region 29 amino acids prior to the beginning of the heptad repeat 1 region of SARS-CoV-2, 206 amino acids from the beginning of S2.”
    “A Palindromic RNA Sequence as a Common Breakpoint Contributor to Copy‑Choice Recombination in SARS‑COV‑2”, by William Gallaher, https ://doi.org/10.1007/s0070 5-020-04750 -z

    Gallaher hypothesized that a RRAR block of nucleotides near the heptad repeat 1 region of HKU9 bat coronavirus S gene, was copy choice inserted into Covid-19 as its RRAR furin cleavage site. That block of HKU9 nucleotides had the proper breakpoint sequence ‘CAGAC’ sequence’, prior to its RRAR sequence.

    But, not only are ‘cagac’ and ‘cagat’ breakpoint sequences for Gallaher’s recombination hypothesis, they bind SMAD proteins.

    “The CAGAC motif has been considered as the main binding element for Smad2/3/4 proteins, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. Positive and negative binding controls using the CAGAC and CAGAT sites”, in the article, ‘Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors’, by Pau Martin-Malpartida, Marta Batet, and others

    Positive and negative binding controls, sounds like battery terminals in a car. Yes, this CAGAC-CAGAT complex, better know as QTQT at the amino acid level, may be at the heart of the RRAR furin cleavage insertion.

    If CRISPR was used to insert the NSPRRA sequence into the Covid-19 S1|S2 spike gene location (instead of copy-choice), the CAS9 gene would encode a complementary sequence to that of the QTQT aa (CAGAC-CAGAT nt) motif, as the place where CAS9 molecule would possibly bind to a Covid-19 cDNA molecule, make a cut in that cDNA, let the cell’s Homology Directed Repair mechanism help insert the CAS9 frame containing the NSPRRA nucleotide sequence, and repair the cut Covid-19 cDNA . Non-Homologous End Joining (NHEJ)? The virologist would then convert the cDNA back to RNA.

    “To direct Cas9 to snip a specific region of DNA, scientists can simply change the sequence of the crRNA, which binds to a complementary sequence in the target DNA.” ‘What is CRISPR?’, by Aparna Vidyasagar , Nicoletta Lanese, October 2021, https://www.livescience.com/58790-crispr-explained.html

    “CRISPR “spacer” sequences are transcribed into short RNA sequences (“CRISPR RNAs” or “crRNAs”) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the CRISPR system – binds to the DNA and cuts it, shutting the targeted gene off. ” ‘Chapter 8 – Role of CRISPR/cas System in the Development of Bacteriophage Resistance’, by Agnieszka Szczepankowska,
    https://doi.org/10.1016/B978-0-12-394621-8.00011-X

    If only one QT in the Covid-19 Spike gene S1|S2 area, then perhaps copy choice as Gallaher and Rambaut proposed. But a QTQT double motif, is more like a docking station for a CrispR gene insertion operation.

    A recent Nemzer and Daoyo twitter conversation noted that only Covid-19 has the QTQT aa motif, to the exclusion of all other known sarbecovirus betacoronaviruses.

    “Note that SARS-CoV-2 is the only QTQTNS genome with two “CAGAC”s on the QTQTNS, indicating that it have been recoded—” Louis R Nemzer (@BiophysicsFL) / Twitter https://twitter.com › biophysicsfl and https://twitter.com/daoyu15/status/1450973210497806341?s=21

    This observation is supported by the fact that on the NCBI website, the Covid-19 nucleotides near the S1|S2 juncture in the spike gene shows, MN908947.3,

    23581 ttatcagact cagactaatt ctcctcggcg ggcacgtagt
    Q T Q T R R A R

    Deigin and Segreto show the nucleotide similarity of RaTG13 and pangolin QTQT amino acid sequences, in relation to Covid-19, in figure 2 of their article, ‘SARS-CoV-2′s claimed natural origin is undermined by issues with genome sequences of its relative strains’. RaTG13 and pangolin may have a CAAAC nt at that location, translated as QT aa.

    It is this QTQT aa motif, as well as the NSPRRA sequence that is being lost in some types of cell passage. Holmes and Rambaut wrote in ‘A Critical Review’,

    “The SARS-CoV-2 furin site is also lost under standard cell culture
    conditions, as is true of HCoV-OC43….. if low-fitness codons had been artificially inserted into the virus genome they would have been quickly selected against during SARS-CoV-2 evolution (but not during cell passage?)”, in the article “The origins of SARS-CoV-2: A critical review”, by Edward Holmes, Andrew Rambaut, and others, https://www.cell.com/cell/pdf/S0092-8674(21)00991-0.pdf

    This loss was first observed by some at the Guangdong CDC.

    “By sequencing the whole genome of SARS-CoV-2 from cell isolates and
    clinical samples, we identified two deletion variants that directly affect the furin cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). We could detect these two deletions in both cell-isolated strains and clinical samples….. By sequencing the whole genome of SARS-CoV-2 from cell isolates and clinical samples, we identified two deletion variants that directly affect the furin cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). We could detect these two deletions in both cell-isolated strains and clinical samples…. vivo” “Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2”, by Zhe Liu, Huanying Zheng, and many others, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China, and many others, posted online June 2020

    The consistent deletion of the Covid-19 cleavage site after ‘some’ types of Vero E6 cell passages, was reaffirmed by Yaara Finkel, Orel Mizrahi and others in their article, “The Coding Capacity of SARS-CoV-2”, September 2020. See their Extended Data Figure 4e, showing a Covid-19 TNSPRRAR deletion after cell passage. Not just the deletion of RRAR.

    On the other hand, the Mart Lamers research group attributed the deletion of the RRAR site (and QTQT aa) in some Vero E6 cells, to the presence of Trypsin, or complete cleavage of virus particles.

    “As TMPRSS2 expression prevented MBCS (multibasic cleavage site) mutations, we tested whether the addition of trypsin (0.7 mg/ml TPCK-Trypsin) would have a similar effect. Surprisingly, the addition of trypsin to VeroE6 cells, but not VeroE6-TMPRSS2 cells, led to deletion of the entire MBCS (multibasic cleavage site). This deletion may arise due to the complete cleavage (S1/S2 and S2’) of virus particles that are not bound to the cellular membranes, which would inactivate them….we show that the ectopic expression of the serine protease TMPRSS2 in VeroE6 cells prevented MBCS mutations. Virus propagation in Calu-3 cells, which naturally express serine proteases, also prevented cell culture adaptation.” —— “Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture
    Adaptation”, by Mart Lamers, and many others.

    But again not just RRAR sequence is being lost, but also the whole NSPRRA sequence. And in some instances the QTQT, the possible docking station for a Cas9 Crispr RNA insert delivery gene. The World World Health Organization addressed this issue.

    “when SARS-CoV-2 is propagated in Vero E6 cells, there is a risk that during the sequential passage of this virus for working stock generation, deletions may arise in critical virulence components of the virus, including the FCS (furin cleavage site)…… On the basis of this preliminary data, we encourage researchers producing stocks of SARS-CoV-2 to consider:
    — limitation of the number of passages in cell culture, using low
    MOI (multiplicity of infection), in an effort to maintain wild-type properties…….(the) WHO recommendations for cell culture production, including principles for good cell culture practice, need to be followed when propagating SARS-CoV-2 virus.” —–
    ‘A cautionary perspective regarding the isolation and serial
    propagation of SARS-CoV-2 in Vero cells’, by Simon Funnell, Ivana Knezevic, and many others, World Health Organization, Geneva, Switzerland, and others

    The World World Health Organization is more concerned about MOI (multiplicity of infection) during cell passage, than Covid-19 origin. The WHO cautions all virologists to limit the number of cell passages of Covid-19, or else!!! The fact remains that cleavage site loss during cell passage, is a critical attribute of Covid-19, and virologists should be rigorously pursue as to why. (heparin sulfate?, binding affinity?)

    Shouldn’t virologists cell passage Guangdong pangolin 2019 and RaTG13 Spike genes, to ascertain whether their QTQT amino acid sequences are lost during Vero E6 cell passage. To make sure there is no across the board innate low fitness in the S1|S2 areas of these spike genes, no artificialness. No other sarbecoviruses betacoronaviruses have this QTQT amino acid sequence, but Covid-19, pangolin 2019 Guangdong, and RaTG13. The three phylogenically closely related amigos in the pandemic origin.

    Has Rambaut, Holmes and Gallaher focus on the possible copy-choice “breakpoint sequences” CAGAC and CAGAT in Covid-19 and HKU9’s Spike genes, caused them to purposefully overlook the restriction enzymes EcoRI (gaattc) and BstEII (ggttacc) in that same Covid-19 Spike gene, near its RBM (receptor binding motif)? Which may point towards gene manipulation.

    “Interestingly, the SARS-CoV-2 S ORF contains two unique restriction sites EcoRI (gaattc) and BstEII (ggttacc) flanking the RBM sequence that are not present in other human CoVs (HKU1, OC43,MERS, SARS-CoV) and could facilitate such engineering. It is striking that the RaTG13 S ORF contains the identical EcoRI and BstEII restriction sites.” ———— “Decoding Covid-19 with the SARS-CoV-2 Genome”, Phoebe Ellis, Ferenc Somogyvári, and others, London Metropolitan University, UK, Department of Medical Microbiology and Immunobiology, University of Szeged, Hungary

    Li-Men Yan herself found BstEII restriction site at 1509 nt/503 aa (ggttacc) of Covid-19 Spike gene. EcoRI (gaatcc) at 1307 nt/435 aa of the same Spike gene. Figure 5a in her article, “Unusual Features of the SARS-CoV-2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route”.

    These restriction sites are located at the beginning and end of the Covid-19 RBM (receptor binding motif), as it is mapped in figure 2a in the article, ‘Furin: A Potential Therapeutic Target for COVID-19’, by Camrong Wu and others. I checked the NCBI database MN908947.3 entry for Covid-19, and Yan is correct. Restriction sites at these precise locations means that the Covid-19 RBM may have been laboratory swapped into the Covid-19 Spike gene.

    As Ralph Baric has stated these restriction enzymes allow you to synthesize, assemble cDNA (complementary DNA) together into a gene or genome.

    “The genome of the virus was synthesized as six contiguous cDNA fragments, as shown in Fig. 1A, and each subclone was flanked by class II restriction
    endonucleases that allow for directed assembly of a full length cDNA genome… Full-length RNAs were (afterwards) generated using in vitro transcription of ligated cDNAs”, in the article
    “A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant”, by Ralph Baric, Sudhakar Agnihothram, and others.

    What’s more plausible, synthesizing and assembling of the Covid-19 Spike gene using Crispr to insert the PRRA furin cleavage sequence, and the use of restriction enzymes in cell passage to swap in a different RBM. Or, the copy-choice recombination of a coinfected HKU9 bat coronavirus from Hong Kong, that is missing a key proline (P) from its insertion sequence, with a different lineage RaTG13 bat coronavirus from Yunnan 1500 miles away. To create a monster. You decide. In Christ Jesus.

  14. A note on Covid-19 cleavage site loss during some cell passages | November 12, 2021 at 5:22 am | Reply

    Some might recommend the use SELEX and aptamers diagnostics to determine each part of the Covid-19 spike gene selection intensity, in vitro or in vivo. Why Covid-19 loses its furin cleavage site during certain types of cell passage.

    “Aptamers are short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells.” —- ‘What is an Aptamer? – Aptamers and SELEX’, https://www.basepairbio.com/what-is-an-aptamer/

    “The basic scheme for in vitro selection experiments is outlined in Fig. 1. This process is referred to as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) when it is applied specifically for the selection against ligands, with the obtained molecules called aptamers [4]. Selection starts from construction of an initial oligonucleotide pool or library, which precedes the selection process. The first step may be a counter-selection against a matrix on which the target is immobilized to a non-target molecule. After removal of non-specifically bound aptamers, a pool of oligonucleotides is incubated with the target. Unbound sequences are removed; the target-bound oligonucleotides are collected, reverse transcribed into DNA and forwarded to PCR amplification. After several selection rounds, cloning and sequencing (Sanger or NGS) steps are performed followed by evaluation of target affinity of the enriched aptamers.”, on the Internet site, ‘The in vitro selection world’, Kenan Jijakli, Basel Khraiwesh, and many others, https://doi.org/10.1016/j.ymeth.2016.06.003

    More on aptamers use to help determine the Covid-19 furin cleavage site electrostatic, hydrogen bonding, thermo properties, affinity selection, etc attributes.

    “Aptamers recognize and bind to their targets through 3-dimensional shapes and various physiochemical interactions, similar to antibody binding mechanisms. These interactions include, but are not limited to, hydrophobic, electrostatic, hydrogen bonding, van der Waals forces, base stacking, and shape complementarity. These functional nucleic acid molecules or aptamers owing to their flexibility, small size, and reduced steric hindrance can recognize biomolecules with ease, offering vast potential in diagnostics…. one means to evaluate aptamer-target binding is by the measurement of binding constants …..The term affinity refers to the strength of interaction that may exist between an aptamer and its target and is often assessed by measuring the binding or association constant (Ka), which is inversely proportional to the dissociation constant (Kd)…… Fluorescence and ITC can measure Kd limited to nanomolar and spectrophotometry techniques can measure Kd in micromolar range. Recently, MicroScale Thermophoresis (MST), a novel low cost highly sensitive technique has been described by numerous aptamer—research groups that can estimate apparent dissociation constant in pico to nanomolar range”———— in the article, ‘Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity’, Priya Kalra, Abhijeet Dhiman, and others, Frontiers in Molecular Biosciences | http://www.frontiersin.org 1 May 2018 | Volume 5 | Article 41

    Yes, measure the affinity selection, Kd (dissociation), and other attributes of the Covid-19 furin cleavage site, and its neighbor amino acids, using aptamers, to help determine why it loses that site, under certain cell passages.

    Some scientists would possible use Circular Sequencing (CirSeq) or microfluidics to make such a determination.

    To measure site-specific negative selection, some would use high-resolution screening and sequencing of mutant libraries before and after selection, using Circular Sequencing (CirSeq). They recommend the use of microfluidics to observe individual virus particles and genomes, to quantify important biological heterogeneity and dynamics. In the article, ‘Mapping the Evolutionary Potential of RNA Viruses’, by Patrick Dolan, Zachary Whitfield and others, Cell Host & Microbe 23, April 11, 2018

    Some might use Next Generation Sequencing (NGS) to determine why Covid-19 loses its furin cleavage site during some Vero E6 cell passages.

    “In recently developed in vitro selection techniques, NGS is used after each selection round instead of being applied after the last selection round as in previous applications of sequencing to in vitro selection . NGS also enables comprehensive characterization of obtained aptamers, identification of their functional and rare motifs, and a comparison of functional motifs in each oligonucleotide population along with quantification of their abundance. NGS also allows for the study of the genetic evolutionary adaptation of oligonucleotide populations as the selection experiment proceeds” ————–in the article, ‘The In Vitro Selection World’, by Kenan Jijakli, Basel Khraiwesh, and many others, Laboratory of Algal, Systems, and Synthetic Biology, New York University Abu Dhabi, United Arab Emirates, http://dx.doi.org/10.1016/j.ymeth.2016.06.003

    Others might use direct coupling analysis (DCA), to analyze the Covid-19 loss of its furin cleavage site.

    “The application of direct coupling analysis (DCA) on an artificial library
    would give the possibility to generate data without the need of relying on natural evolution, paving the way for structure determination by artificial selection in vitro. By substituting natural with in vitro evolution, we explored a brand new application of DCA which overcomes the limitations that have so far hindered the generality and scalability of the method evolution”———— in the article. ‘Protein Structural Information and Evolutionary Landscape by In Vitro Evolution’, by Marco Fantini, Simonetta Lisi, and others, BioSNS Laboratory of Biology, Scuola Normale Superiore (SNS), Pisa, Italy, and others, Oxford University Press

    The scientific technology is there, to determine why Covid-19 loses its furin cleavage site under certain types of cell passage. For almost two years, American virologists and scientists have only used this technology to recommend therapeutics against Covid-19, not to determine Covid-19 origin. No faith in American scientists and virologists, put your faith in Jesus, put your trust in the Lord.

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