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 summarizes 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 a 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.

22 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.

  15. p. gutierrez - hopefully the full comment untruncated | April 26, 2022 at 4:22 am | Reply

    On February 26, 2022, George Gao of the Beijing China National CDC released for publication the preprint,
    1) ‘Surveillance of SARS-CoV-2 in the environment and animal samples of the Huanan Seafood Market’, by George Gao, William Liu, and many others, Chinese Academy of Sciences, National Institute for Viral Disease Control and Prevention, and others https://assets.researchsquare.com/files/rs-1370392/v1_covered.pdf?c=1645813311

    which contains the results of 33 environment samples collected January 1 or 12, 2020, from the Huanan Seafood Market (HSM) in Table 1 of the article, and results of samples collected on later dates. On the same day Michael Worobey and Christian Andersen study group released their preprint article below, that contained 33 positive environment samples from the HSM in their Table S2.

    (2) ‘The Huanan market was the epicenter of SARS-CoV-2 emergence’, by https://zenodo.org/record/6299116#.Yh_ErJhMFdh, by Michael Worobey, Joshua Levy, Pekar, and others

    Worobey and Anderson probably consulted with Dr. Gao, to get their list of environmental samples of the HSM.
    Dr. Gao’s Table 1 lists the Huanan Seafood Market positive environment samples for Covid-19, and he observed,

    “live viruses were isolated from samples F13 (wall) , F54 (ground), and B5 (ground), which were the only three samples with Ct values <30 (cycle threshhold) ….. samples F13 (wall) and F54 (ground) were from the stalls with confirmed patients ……..The genome sequences of two environmental samples, F13 (wall) and F54 (ground), were found to be highly identical to the reference strain HCoV/Wuhan/IVDC-HB-01 (WH01, sequence identity of 99.993%) and completely identical to the human stain Wuhan-Hu-1 (GenBank: NC_045512) …….Commercial products of swabs and virus preservation solution were used for the sampling (Disposable Virus Sampling Tube, V5-S-25, Shen Zhen Zi Jian Biotechnology Co., Ltd., Shenzhen, China). For environmental samples, sampling swabs were applied to smear the floors, walls or surfaces of objects and then preserved them in virus preservation solution.”

    Dr. Gao says F13 environmental sample (wall) is highly identical to WH01 (Wuhan person #1- NC_045512) isolate. WH01 isolate was collected December 26, 2019 (according to patient information table in the article, ‘Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding’, by Roujian Lu, Xiang Zhao, and others, Beijing National CDC, and others.

    Dr. Gao also indicated F13 (wall sample) had a Ct value of 23.85, and F54 sample (ground) a Ct value of 25.8 in his Table 1. Cycle threshold means the number of cycles required for the fluorescent signal to cross the threshold. Ct levels are inversely proportional to the amount of target nucleic acid in the sample, according to Igho Onakpoyo article ‘Sars-Cov-2 and Role of Fomite Transmission’. Wuhan person # 1 had a Ct value 23.967 according to the article ‘A new coronavirus associated with human respiratory disease in China’, Fan Wu, Su Zhao, and others, extended data figure 4. For a person aged 41 admitted December 26, 2019. F13 wall sample and Wuhan person #1 cycle threshold almost match and five days difference in the sampling.

    Wuhan person #1 who worked in seafood at HSM, bronchoalveolar lavage fluid (BALF) for late December 2019, “the viral load in the BALF sample was estimated by qPCR to be 3.95 × 108 copies per ml”. —- in the article, ‘A new coronavirus associated with human respiratory disease in China’, Fan Wu, Su Zhao, Bin Yu, and others, Shanghai Public Health Center and others. They named the isolate MN908947.

    In February 2022, Jonathan Pekar calculated tMRCA of lineage B Covid-19, as being mid-December 2019. (last time when all coronavirus alleles were still in their original source.)

    “We inferred the median tMRCA of lineage B to be 13 December (95% HPD: 29 November to 23 December) and the median tMRCA of lineage A to be 25 December (95% HPD: 17 December to 30 December) (Fig. 6A), under the unconstrained model…… our results indicate that lineage B was introduced into humans no earlier than November 2019, and lineage A cross-species transmission likely occurred within days to weeks of the first event.” —-in the article, ‘SARS-CoV-2 emergence very likely resulted from at least two zoonotic events’, by Jonathan Pekar, Andrew Magee, and other, University California San Diego,

    Time to most recent ancestor possibly mid-December 2019, according to Pekar. Wuhan person # 1 showed signs of virus on December 20, 2019 according to Roujan Lu study referenced above. Wuhan person # 1 had high viral titers December 26, 2019. Mid-December 2019 is a common time period for the two different studies. Others had signs of Covid-19 before then. How did that live F13 Covid-19 sample get on the wall in the HSM, possibly associated with Wuhan person #1 (according to Gao), is the purpose of this comment. According to the WHO, Covid-19 transmission occurs either by droplet transmission, airborne transmission, or direct contact.

    “Droplet transmission occurs when a person is in in close contact (within 1 m) with someone who has respiratory symptoms (e.g., coughing or sneezing) and is therefore at risk of having his/her mucosae (mouth and nose) or conjunctiva (eyes) exposed to potentially infective respiratory droplets. Transmission may also occur through fomites in the immediate environment around the infected person. Therefore, transmission of the COVID-19 virus can occur by direct contact with infected people and indirect contact with surfaces in the immediate environment or with objects used on the infected person (e.g., stethoscope or thermometer).

    Airborne transmission is different from droplet transmission as it refers to the presence of microbes within droplet nuclei, which are generally considered to be particles <5μm in diameter, can remain in the air for long periods of time and be transmitted to others over distances greater than 1 m.”———- in the article, ‘Modes of transmission of virus causing COVID-19: implications for IPC precaution recommendations’, by the World Health Organization, https://apps.who.int/iris/handle/10665/331601

    An Egyptian study group observed.

    “The aerosol particles can be divided into (1) ultrafine particles (less than 0.1micrometer-.000001 m), (2) fine particles (0.1-2 micrometers), and (3) coarse particles (greater than 2 micrometers). The size of PM in aerosols plays a pivotal role in determining their deposition site and mechanism at the respiratory system. The mechanisms of respiratory deposition are classified based on particles sizes into different mechanisms, namely diffusion, sedimentation, impaction, and interception. Particles with sizes smaller than 0.5 micrometers are deposited in the alveoli by diffusion mechanism…..Sedimentation is another deposition mechanism, which plays a significant role in the setting out particles of aerodynamic size between 1-5 micrometers in the smaller airways of bronchioles and alveoli where the airflow is low. Sedimentation is governed by gravitational forces, particle velocity and aerodynamic size…….Particles with sizes greater than 5 micrometers m are deposited at the bronchial regions by impaction. Impaction is highly dependent on aerodynamic diameter and mass. “ in the article, ‘Inhaled nano- and microparticles for drug delivery’, by Ibrahim El-Sherbiny, Nancy El-Baz, Magdi Yacoub

    Proper sizing the particles in the F13 wall sample is very important in determining how that Covid-19 got on that HSM wall, eg, sedimentation, impaction, etc..

    Physics behind how sample F13 may have gotten on the HSM wall —–

    Airborne transmission, also known as aerosols. F13 sample was collected by Gao from a vertical wall. Was there direct contact to contaminate the wall? Or perhaps, horizontal trajectory of droplets or aerosols being influenced by air flow, heat, rate of evaporation and other factors. A recent University of Missouri study concluded,

    “horizontal velocity of respiratory droplets depends strongly on human activity, age, and ambient environment. The trajectory of the exhaled respiratory droplets is affected by both the expired air flows profile and surrounding air flow patterns. Existing studies treated the exhaled air as a turbulent round jet , and the turbulent flow will enhance the heat and mass transfer between the droplet and the surrounding air. Therefore, respiratory droplets will likely evaporate faster than the simulated results in this study, and a larger fraction of respiratory droplets and viruses may remain airborne for a longer period of time…. airborne droplets can travel a horizontal distance of 2.64 m after 10 s, and 3.95 m after 30 s”,—–'Modeling the load of SARS-CoV-2 virus in human expelled particles during coughing and speaking’, by Yang Wang and others, University of Missouri

    Droplets and airborne transmissions should eventually drop to the ground (a horizontal surface) because of evaporation, unless air flow or direct contact caused the virions or particles to stick to the vertical HSM wall. An University College London study observed,

    “The adhesion mechanism of SARS-CoV-2 on environmental surfaces has yet to be adequately delineated, but it has been predicted that it is primarily driven by electrostatic attractions (e.g. pH, isoelectric point ( pI) and ionic strength), then hydrophobic effects, and minorly non-covalent bonds (e.g. van der Waals forces) which could all govern the binding of the S protein to solid surfaces.” — in the article, ‘Surface interactions and viability of Coronaviruses’, by Mehmet Onur Aydogdu, Esra Altun and others

    Was there some electrostatic attraction or hydrophobic effects between the Covid-19 source, and the part of the HSM wall where the F13 sample was collected? The section of Aydogdu’s above article, ‘SARS-CoV-2 adsorption mechanism on different inanimate surfaces’, is well worth reading. Did the density, inertia drag, and size of the particles play an intricate part in the Covid-19 collected from the HSM wall? In 1986, Paul Baron of the NIOSH observed,

    ‘The aerodynamic diameter (of a particle) is defined as the equivalent of the settling velocity diameter. For particles being measured 0.8 to 20 μm in diameter the particle settling velocity is sufficiently small that the viscous drag predominates…for larger particles the viscous and inertia drag are significant as indicated by the size of the particle ‘s Reynolds number in Table 1. …The Weber number is used to characterize breakup of droplets when they are in motion relative to air, is the ratio of the drag force to the surface tension force for a particle in motion.” —–in the article, ‘Calibration and Use of Aerodynamic Particle Sizer’, by Paul Baron, National Institute for Occupational Safety and Health, 1986

    The aerodynamic diameter of the (possible Covid-19) particles depends of the density of the particle in still air, according to Baron. Viscous, thick sticky consistency, between solid and liquid. Could F13 sample on the wall of HSM been a result of drag related and density of aerosoled or droplet particles? Baron continued,

    “When a droplet moves through the air at a high velocity, the drag force exerted by the air can be described by the equation ….”

    Viscous and inertia drag in conjunction with particle surface tension can create distorted particles, oblate spheroids, according to Baron.

    Recently, many studies have tested surfaces for Covid-19 but reported results in concentration of gene copies per μL, eg, Joshua Santarpia and Danielle Rivera and others in their article, ‘Aerosol and surface contamination…’. Molar concentration per unit volume. That has important medical and autopsy purposes.

    But determining and analyzing surface particles μm size (micrometer-.000001 m) and pleomorphism seems more practical regarding forensics, origin of the coronavirus, determining particles density, their Weber and Reynolds numbers, Womersley number associated with oscillatory flow capturing the importance of unsteady acceleration through the ratio of the oscillation frequency to viscous effects and perhaps trace those F13 sample particles possible source of origin.

    The Covid-19 virons themselves ——-

    Dr. Gao properly sized the Covid-19 virions themselves using transmission electron microscopy.

    “Negative-stained virus particles were generally spherical, pleomorphic and 60-140 nm (nanometers) in diameter.” (0.000000001 m).

    Verified by Dr. Na Zhu in February 2020. Pleomorphic means the occurrence of more than one distinct forms of a natural object, eg, oblong, star shaped, etc.

    “Electron micrographs of negative-stained 2019-nCoV particles were generally spherical with some pleomorphism (Fig. 3). Diameter varied from about 60 to 140 nm” (0.000000001 m) ——in the article, ‘A Novel Coronavirus from Patients with Pneumonia in China, 2019’, by Na Zhu, Dingyu Zhang, and others.

    A Zhejiang and Tsinghua Universities study in 2020 elaborated on the pleomorphism nature of Covid-19 virions.

    “SARS-CoV-2 virions (ID: ZJU_5) were collected on January 22, 2020 from a patient with severe symptoms and were propagated in Vero cells……Intact and unconcentrated virions were directly visualized from the supernatant by cryo-EM, showing ellipsoidal and spherical enveloped particles (Figure S1A), consistent with the observation of virions concentrated by ultracentrifugation through a sucrose cushion (Figure S1B). We modeled 2,294 virions as ellipsoids by meshing their lipid envelopes, measuring average diameters of 64.8 plus or minus 11.8 nm, 85.9 plus or minus 9.4 nm, and 96.6 plus or minus 11.8 nm (average – SD) for the short, medium, and long axis of the envelope, respectively…. Our observations of the structures and landscape of the intact SARS-CoV-2 are consistent with two other cryo-ET studies that were published at the same time (Turo_nova´ et al., 2020; Ke et al., 2020)”. —–in the article ‘Molecular Architecture of the SARS-CoV-2 Virus’, by Hangping Yao, Yutong Song, and many others, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou , Zhejiang Province, Tsinghua University, Beijing, and many others

    The study group visualized the Covid-19 virons using cryo-EM in their unconcentrated form/nature as ellipsoids and spherical. They then observed the Covid-19 virons as concentrated, using ‘ultracentrifugation through a sucrose cushion’, with the similar observational results. Their figure 4E shows the ellipsoidal Covid-19 virons as being oblong in nature. Certainly not the result of nebulization. Supplemental figure Figure S1 B shows spherical and oblong Covid-19 virons together. The study group also shows the inner packing of ribonucleoproteins in the Covid-19 virons. But they don’t show or size the possible surface particles that Covid-19 virions possibly existed in, or were transmitted in.

    A Sharchar-Berman study group observed a few years ago regarding ellipsoidal particles.

    “Whether natural or anthropogenic (e.g. biomass burning), the majority of inhalable airborne particles are intrinsically non-spherical (Kleinstreuer and Feng, 2013)…..For micron-sized airborne particles in the absence of electrostatic forces, transport dynamics are hence foremost governed by viscous drag, aerodynamic lift and gravity (in the negative y-direction;”, —–in the article ‘Transport of ellipsoid fibers in oscillatory shear flows: Implications for aerosol deposition in deep airways’, by Lihi Shachar-Bermana and others.

    Ellipsodal Covid-19 virons may be a product of nature, or other means. Particle dynamics study is necessary. In July 2021, Vincent Munster of the NIAID observed regarding the possible larger particles housing Covid-19 virons,

    “demonstration of true aerosol transmission of SARS-CoV-2 should only include particles less than 5 μm (micrometer=.000001 m), over longer distances and in the absence of any other potential transmission routes such as fomite or direct contact….Epidemiological data suggests that the principal mode of infection with SARS-CoV-2 is via airborne transmission”——-in the article, ‘Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS-CoV-2’, by Vincent Munster, Julia Port and others, NIAID Montana

    Munster did not address methods for determining surface particle sizes for Covid-19 environment samples. Only methods for collecting aerosol size particles. Those particles were measured using a Model 3321 aerodynamic particle sizer spectrometer from TSI.

    Methods for Collecting and Determining Surface Samples Particles Sizes —

    Similarly in 2020, the World Health Organization did not address collecting direct transmission environment surface samples, other than to say,

    “Environmental samples need to be taken using a swab with a synthetic tip and a plastic shaft (2,3,9-12). The swab specimen collection vials should contain 1-3ml of viral transport medium (e.g. protein stabilizer, antibiotics and buffer solution) including neutralizing buffer to counteract the effects of any residual disinfectant (e.g. Tween 80)………sequencing of environmental samples may be challenging and may need to be discussed with laboratories with coronavirus sequencing expertise.”— in the article, ‘Surface sampling of coronavirus disease (COVID-19): a practical “how to” protocol for health care and public health professionals’, (No. WHO/2019-nCoV/Environment_protocol/2020.1). https://apps.who.int/iris/bitstream/handle/10665/331058/WHO-2019-nCoV-Environment_protocol-2020.1-eng.pdf?sequence=1&isAllowed=y

    The WHO suggested using ‘swabs with synthetic tips’ to collect the environmental sample and ‘discuss with laboratories with coronavirus sequencing expertise’. If the WHO wants to standardize a method to collect and size Covid-19 surface particles, a Harvard University study group had a good technique and collected μm size (micrometer) particles as follows,

    “We used custom-designed Harvard Micro-Environmental Cascade Impactors (Demokritou et al., 2002) to collect simultaneous samples in three distinct size fractions: fine (less than or equal 2.5 μm-micrometer aerodynamic diameter), coarse (2.5–10 μm), and large (greater than or equal 10 μm) . The first impactor stage used a polyurethane foam (PUF) substrate to collect large particles. The second impactor stage used a smaller PUF substrate to collect particles in the coarse size range. The last stage collected fine particles on a 37-mm diameter glass-fiber filter We confirmed our hypothesis, finding positive samples in the fine size fraction and in a pattern indicating location-specific size distributions. A key finding is that the size distribution of particle-associated SAR-CoV-2 was related to the location where the particles were collected.”—— in the article, ‘Levels and particle size distribution of airborne SARS-CoV-2 at a healthcare facility in Kuwait’, by Rebecca A. Stern, Ali Al-Hemoud, and others

    If that Harvard Micro-Environmental Cascade Impactor can be used to collect particles from the HSM environment surface samples, then maybe it can be used to help size environment sample F13 (wall) and F54 (ground) particles. Would the key would be collecting samples on the polyurethane foam (PUF) substrate, native to the Impactor? What if no large (greater than or equal 10 μm – micrometer ) particles were found in the F13 wall sample? What if only nanometer sized Covid-19 virions are found, not housed in any particles whatsoever?

    A Singapore study group collected environment surface samples using Puritan EnviroMax Plus pre-moistened macrofoam sterile swabs, and seeming integrated the contents of those swabs into a NIOSH air sampler, to analyze particles greater than 4 μm in diameter.

    “Surface samples were collected with Puritan EnviroMax Plus pre-moistened macrofoam sterile swabs (25-88060) ……Particles collected with the NIOSH sampler are distributed into three size fractions. Particles greater than 4 μm in diameter are collected in a 15mL centrifuge tube, particles 1–4 μm in diameter are collected in a 1.5 mL centrifuge tube, and particles less than 1 μm (micrometer) in diameter are collected in a self-assembled filter cassette containing a 37-mm diameter, PTFE filter with 3 μm pores”, —-in the article ‘Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients’, by Po Ying Chia, Kristen Coleman, and others, National Centre for Infectious Diseases, Singapore, Singapore, Duke NUS Medical School, and others

    Seemingly, the Singapore group integrated Puritan surface collection swabs into NIOSH air sampler, to collect particles greater than 4 μm in diameter, utilizing a two-stage bioaerosol cyclone samplers provided by the National Institute for Occupational Safety and Health (NIOSH). Their methods section is fuzzy regarding this. Hopefully Gao, the WHO and Atlanta CDC can consider standardizing the Singapore’s or Rebecca Stern’s technique in collecting and sizing surface environmental particles, for documenting and tracing the origin of possible future outbrakes. Require environment sample photos to be published early. If the HSM was not aerosoled, droplet transmitted, nebulized, maybe the direct contact (large particle) source was present at the location.

    Lastly, digital drop PCR has been used to size Covid-19 aerosols into micrometre particles. Why not use digital drop PCR to size particles collected from surface environmental samples? In the article, ‘Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals’, by Yuan Liu, Zhi Ning, and others, used digital drop PCR to “size Covid-19 aerosols into submicrometre region (dp between 0.25 and 1.0 μm-micrometers) and the other in supermicrometre region (dp greater than 2.5 μm) aerodynamic particles into….. total of three size-segregated aerosol samples was collected using a miniature cascade impactor (Sioutas Impactor, SKC) that separated aerosols into five ranges (greater than 2.5 μm, 1.0–2.5 μm, 0.50–1.0 μm and 0.25–0.50 μm on 25-mm filter substrates, and 0–0.25 μm on 37-mm filters) at a flow rate of 9.0 l min−1”.

    A different impactor than the one used by the Harvard study group. Why not try using a similar technique regarding sizing environmental samples particle sizes? And standardize that method. Other surface collection of virus particles articles worth reading are,
    ‘Contact transmission of SARS-CoV-2 on fomite surfaces: surface survival and risk reduction’, Abhimanyu Tharayil, and others, Ghandi University, India and others, encyclopedia on virus surface factors, nothing on method of collection

    ‘Sustainability of Coronavirus on Different Surfaces’, Rajiv Suman, and others, very little on method used

    ‘ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load Specimens’, Tao Suoa, Xinjin Liub, and others used digital drop PCR to report copies/reaction

    ‘Stability of SARS-CoV-2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: A review’, by Hamada Aboubakr, Tamer Sharafeldin, and Sagar Goyal1, encyclopedia type of article environmental surfaces, very little on coronavirus surface collection methodology

    Nebulization:

    In 2020, the NIAID, Tulane University, Fort Dietrich study group used nebulizers to generate Covid-19 particles, and the Aerodynamic Particle sizer to size the particles generated.

    “The 3-jet (C3), 6-jet Collison (C6), or Aerogen Solo (AS) nebulizers were used for generation of viral aerosols ( MERS-CoV and SARS-CoV to SARS-CoV-2 )…… Aerosol particle size was determined using an Aerodynamic Particle Sizer solely or with the addition of a diluter (TSi, Shoreview, MN, USA). scanning electron microscopy was used to size virions” — ‘Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 in Aerosol Suspensions’, by Alyssa Fears and Robert Garry-Tulane, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Maryland, USA (M. Lackemeyer, J.K. Bohannon, R. Johnson) , US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA (A. Nalca, A. Totura, D. Dyer, B. Kearney), and others, Emerging Infectious Diseases • http://www.cdc.gov/eid • Vol. 26, No. 9, September 2020

    In 2020, the NIAID and Atlanta CDC generated Covid-19 aerosol particles using a Collison nebulizer.

    “Aerosols (<5 μm) containing SARS-CoV-2 (105.25 50% tissue-culture infectious dose [TCID50] per milliliter) or SARS-CoV-1 (106.75-7.00 TCID50 per milliliter) were generated with the use of a three-jet Collison nebulizer and fed into a Goldberg drum to create an aerosolized environment. The inoculum resulted in cycle-threshold values between 20 and 22, similar to those observed in samples obtained from the upper and lower respiratory tract in humans.”— in the article ‘Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1’, by Neeltje van Doremalen, Vincent Munster (National Institute of Allergy and Infectious Diseases-Hamilton, MT), Azaibi Tamin, Jennifer Harcourt, Natalie Thornburg, Susan Gerber,Centers for Disease Control and Prevention Atlanta, Georgia, N Engl J Med 382;16, April 16, 2020

    Vincent Munster of the NIAID used a standard spray bottle containing chemicals to help generate droplets and aerosols from hamsters inoculated with Covid-19.

    “particle size next validated the caging design using an aerodynamic particle sizer to analyze the aerodynamic size of particles (dynamic range from <0.5-20 μm) traversing from donor to sentinel cage. Droplets and aerosols were generated in the donor cage (20% (v/v) glycerol solution, sprayed with a standard spray bottle “, in the article, ‘Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS CoV-2’, by Julia Port, Claude Kwe Yinda, Victoria Avanzato, Jonathan Schulz, Myndi Holbrook, Neeltje van Doremalen, Carl Shaia, Robert Fischer, Vincent Munster, NIAID Montana

    Nebulization the hardway. As if they have nothing else to do at the NIAID in Montana other than inoculate hamsters with Covid-19. The article, ‘Utility of Three Nebulizers in Investigating the Infectivity of Airborne Viruses’, by Sadegh Niazi,a Lisa K. Philp,b Kirsten Spann, Graham Johnson, Queensland University of Technology — worth reading

    This comments supercedes the similar comment at https://scitechdaily.com/wuhan-lab-leak-theory-rare-genetic-sequence-doesnt-mean-the-covid-virus-was-engineered/ dated April 20, 2022. That similar comment at that website can be deleted, because of massive truncations in the comment, caused by less than and greater signs in its content, being interpreted as html delete content indicators, by the scitechdaily.com webserver platform. Those less than and greater signs have been removed from this copy of the comment, which should reflect the whole comment. Thank you scitechdaily.com website.

  16. p. gutierrez | May 12, 2022 at 7:02 am | Reply

    This is an update to my comment above, more critically thought out. p. gutierrez — May 12, 2022

    Dr. Gao’s Table 1 lists the Huanan Seafood Market positive environment samples for Covid-19, and he observed.
    “live viruses were isolated from samples F13 (wall) , F54 (ground), and B5 (ground), which were the only three samples with Ct values <30 (cycle threshhold) ….. samples F13 (wall) and F54 (ground) were from the stalls with confirmed patients ……..The genome sequences of two environmental samples, F13 (wall) and F54 (ground), were found to be highly identical to the reference strain HCoV/Wuhan/IVDC-HB-01 (WH01, sequence identity of 99.993%) and completely identical to the human stain Wuhan-Hu-1 (GenBank: NC_045512) ……. Notably, samples F13 and F54 were from the stalls with confirmed patients. All the results of successful virus isolation and the Ct values of the original samples revealed the existence of live SARS-CoV-2 with high titers in the environment of HSM……Commercial products of swabs and virus preservation solution were used for the sampling (Disposable Virus Sampling Tube, V5-S-25, Shen Zhen Zi Jian Biotechnology Co., Ltd., Shenzhen, China). For environmental samples, sampling swabs were applied to smear the floors, walls or surfaces of objects and then preserved them in virus preservation solution.”

    Gao says F13 environmental sample (wall) is highly identical to IVDC-HB-01 (WH01) , and completely identical to Wuhan-HU-1, NC_045512 isolate. IVDC-HB-01 (WH01) might have been EPI_ISL_402119 taken December 31, 2019, according to Table 5 in the article, ‘Nosocomial Outbreak of 2019 Novel Coronavirus Pneumonia in Wuhan, China’, by Xiaorong Wang, Qiong Zhou, and others. Table S1 in the article, ‘Supporting Information A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry’, by Mohamed El-Tholoth, and others says EPI_ISL_402119 was 49 years old.

    More importantly, Dr. Gao doesn’t explicitly state who NC_045512 is, the person ‘ completely identical’ to F13 sample. The NCBI entry for NC_045512.2 states,

    “The reference sequence is identical to MN908947. On Jan 17, 2020 this sequence version replaced NC_045512.1.”
    https://www.ncbi.nlm.nih.gov/nuccore/NC_045512

    So according to NCBI, NC_045512.2 reference sequence is identical to MN908947, which should ‘probably’ include the genomes identicalness.

    We know that MN908947 was a 41 year old who worked in seafood at HSM, admitted to the hospital December 26, 2019, “The patient studied was a 41-year-old man with no history of hepatitis, tuberculosis or diabetes. He was admitted to and hospitalized
    in the Central Hospital of Wuhan on 26 December 2019, 6 days after the onset of disease……. During admission, BALF (bronchoalveolar lavage fluid ) was collected and stored at −80 centigrade until further processing….the viral load in the BALF sample was estimated by qPCR to be 3.95 × 10 to the 8th power copies per ml…. This virus strain was designated as WH-Human 1 coronavirus (WHCV) (and has also been referred to as ‘2019-nCoV’) and its whole genome sequence (29,903 nt) has been assigned GenBank accession number MN908947”. —- in the article, ‘A new coronavirus associated with human respiratory disease in China’, by Fan Wu, Su Zhao, Bin Yu, and others, Shanghai Public Health Center and others.

    So NC-045512 identical to MN908947 isolate for 41 year old which may have been ‘completely identical’ (Gao) to F13 wall environment sample, and possibly identical to F54 ground environmental sample. Either F13 (wall) was on the wall of HSM stall December 26, 2019 when 41 year old was admitted to the hospital, or it was not. Same for F54 ground sample, being on the HSM ground. Gao stated the active samples had ‘high titers’. One example of ‘high titers’ is,

    “SARS-CoV-2 infectivity varies by case viral load, contact event type, and age. Those with
    high viral loads are the most infectious….predict the real-world likelihood of a SARS-CoV-2 infected individual infecting someone else……85.4% (197,677/231,497) of case-contact pairs with PCR-positive contacts, i.e., plausible onward transmission, had case viral loads of greater than or equal 10,000 RNA copies/ml (i.e. Ct less than or equal to 24.4)”, in the article, ‘ SARS-CoV-2 infectivity by viral load, S gene variants and demographic factors and the utility of lateral flow devices to prevent transmission’, by Lennard YW Lee, and others, University of Oxford

    I think it safe to say Wuhan-person-1 with viral load December 26, 2019 be 3.95 × 10 to the 8th power copies per ml had ‘high titers’. And as the ‘high titers’ of F13 and F54 started to decay (not reproducing as it does in humans) , many studies have calculated the half-life (time taken to achieve a 50% reduction in titre/viral load) for Covid-19, on fomite surfaces as being a matter of hours, to a matter of days. See Table 1 in the article below.

    “SARS-CoV-2 at a starting viral load and in a fluid matrix equivalent to that typically excreted by infected patients, remains viable for at least 28 days when dried onto non-porous surfaces at 20 °C and 50% relative humidity…… Recent data published on SARS-CoV-2 survivability on hospital PPE observed viable virus up to 21 days post inoculation on both plastic and N95 mask material when….”, in the article, ‘The effect of temperature on persistence of SARS‑CoV‑2 on common surfaces’, by Shane Riddell, Sarah Goldie, and others, Virol J (2020) 17:145 l

    Another article on the decay rate of Covid-19 fomite surfaces is ‘Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1’, by Neeltje van Doremalen, and others, National Institute of Allergy and Infectious Diseases, Hamilton, MT

    The expectation of F13 and F54 is a reduction in viral load based on heat and humidity and sunlight in the HSM environment, if the samples were physically present on December 26, 2019, to the time when the samples were actually taken by the China CDC January 1, 2020. Gao says F13 and F54 had ‘high titers’, only giving Ct values.

    Disregard the facts that Dr. Gao stated F13 (wall sample) had a Ct value of 23.85, and Wuhan-person-1 had a Ct value 23.967 from his December 26 sample, according to the article ‘A new coronavirus associated with human respiratory disease in China’, by Fan Wu, Su Zhao, and others, extended data figure 4. Forget these facts, and only say the words, ‘high viral titers’.

    If F13 (wall) was present December 26, 2019 in HSM, should it not reflect ‘middle or lower titers’ on January 1, 2020, due to half life decay. At least seven persons in ICU in Wuhan hospitals for Covid-19 in December 2019, according to the article, ‘A pneumonia outbreak associated with a new coronavirus of probable bat origin’, by Peng Zhou, Xing-Lou Yan, and many others, Wuhan Institute of Virology, and many others, Extended Tables # 1 and # 2. With the active HSM environment samples possibly playing a part. Only one patient non-HSM related. The World Health Organization 2020 study lists four clusters of early Covid-19, two directly involving HSM.

    “Cluster 2: there were 3 confirmed cases, all of whom were traders of the same stall in Huanan Market. Stall employee one, 40 years old, fell ill on 17 December 2019; stall employee two, 32 years old, fell ill on 19 December 2019; stall employee three, 57 years old, fell ill on 25 December 2019. It was a fixed stall in Huanan Market, dealing in frozen products such as pastry and soy products. Employee two was purchasing goods from the Baishazhou market and Huanan Market back and forth. Employee three was delivering goods in Huanan Market.”

    ‘Cluster 4: There were two confirmed cases, both of whom were employees of the same stall in Huanan Market, and both of them denied contact history of poultry and animals, as well as contact history of travel. Employee one, 56 years old, fell ill on 20 December 2019; employee two, 45 years old, fell ill on 26 December 2019. It was a fixed stall in the Huanan Market, dealing in aquatic products such as catfish’, in the article ‘WHO-convened Global Study of Origins of SARS-CoV-2: China Part’, Joint WHO-China Study
    14 January-10 February 2021 Joint Report – ANNEXES

    It is possible Wuhan-Hu-1 NC-045512/ MN908947 isolate can be found in one of these two WHO clusters of HSM workers. Perhaps Wuhan-Hu-1 was admitted to the hospital December 26, and one of his co-workers at HSM picked up a piece of contaminated half frozen dead fish, and threw it at the wall, on December 30 or 31, causing the ‘high titer’ effect on January 1, 2020 when Beijing CDC arrived. Or, an irate customer came back and threw the contaminated partially frozen stuff against the wall on December 30 or 31, 2019, ‘high titers’ on January 1, 2020 when Beijing CDC arrived. We don’t know. But if Wuhan-Hu-1 NC-045512/ MN908947 person transmitted to the F13 wall location by coughing, sneezing, of spitting on December 26, 2019, should we not expect ‘lower viral titers’ January 1, 2020? Does F13 wall sample and F54 ground sample because of ‘high titers’ represent a possible zoonotic transmission event for lineage B Covid-19?

    Jonathan Pekar would not rule out the possibility of different sources for lineages A and B Covid-19.

    “the pandemic most likely began with at least two separate zoonotic transmissions starting no earlier than November 2019, with a lineage A virus jumping into humans after the introduction of a lineage B virus.”—in the article, ‘SARS-CoV-2 emergence very likely resulted from at least two zoonotic events’, by Jonathan Pekar, Andrew Magee, Michael Worobey, and many others

    Sudhir Kumar sees one progenitor coronavirus for lineages A and B, but lineage B lacked lineage A variants as ancestors.

    “These facts support the inference that coronaviruses lacking ‘A’ variants were the
    ancestors of Wuhan-1 and other genomes sampled in December 2019 in China. Therefore, we conclude that Wuhan-1 was not the direct ancestor of all the early coronavirus infections globally.”—–in the article, ‘An Evolutionary Portrait of the Progenitor SARS-CoV-2 and Its Dominant Offshoots in COVID-19 Pandemic’, Sudhir Kumar , Qiqing Tao, and others

    The possible separate coronavirus source (Pekar) for lineage B in HSM, F13 wall and F54 ground, would not the size (in micrometers) of the particles containing the virons, rule in or out aerosol, droplet, or fomite original transmission, be used in conjunction with tMRCA to help ‘localize’ the original source in time? While Kumar would say one progenitor source coronavirus for lineages A and B is likely, lineage A coronaviruses were not ancestors to Wuhan-person-1 (lineage B). A BLAST run on NCBI (on three different dates) shows NC_045512.2 is 100% nucleotide identical to later date isolates,

    OK372407.1 collected July 20, 2020 Tennessee
    MZ722702.1 collected 12/21/2020 Los Angeles
    MW562722.1 collected 12/31/2020 Utah
    and identical to many other later date isolates. https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&QUERY=NC_045512.2&DATABASE=nr&MEGABLAST=on&BLAST_PROGRAMS=megaBlast&LINK_LOC=nuccore&PAGE_TYPE=BlastSearch

    Is this identicalness due a founder effect of a progenitor coronavirus, or does this identicalness mean that the source coronavirus for lineage B is still active and not restricted to HSM or its locale? Note this is a question, and not a conclusion.

    “a founder effect: a genetic bottleneck that occurs when, … a new type is established from a small isolated number of infections.”——- ‘Genetic Study Three Variants of SARS-Cov-2’ — sci-news.com

    Surprisingly Gao doesn’t reference, BetaCoV/Wuhan/IPBCAMS-WH-01/2019 EPI_ISL_40212 MT019529 sample taken December 23, 2019, earliest known human sample, when comparing environmental samples to human samples. Pekar and Worobey analyzed that sequence as being identical to Wuhan-person-1.

    “reanalysis of sequence data from the earliest sampled viruses found that three previously reported mutations in IPBCAMS-WH-01 were spurious, and the genome was, in fact, identical to the Hu-1 reference genome” .”—in the article, ‘SARS-CoV-2 emergence very likely resulted from at least two zoonotic events’, by Jonathan Pekar, Andrew Magee, Michael Worobey, and many others

    Other articles and helps that list early Covid-19 persons possibly related to the F13 environmental sample are,

    https://www.viprbrc.org/brc/viprStrainDetails.spg?ncbiAccession=MT019529&decorator=corona&context=1581947810403

    ‘Supporting Information – A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry’, by Mohamed el-tholoth and others, ——— associates assession numbers with age of persons epi_isl_402123 is 65 years taken on Dec 24, 2019 mt019529

    ‘Nosocomial Outbreak of 2019 Novel Coronavirus Pneumonia in Wuhan, China’,
    By Xiaorong Wang, Qiong Zhou, and many others ——– Table S5—associates NBCI accession numbers with their GISAID counterparts

    ‘WHO-convened Global Study of Origins of SARS-CoV-2:China Part
    Joint WHO-China Study 14 January-10 February 2021 Joint Report’——-on page 76, ‘Table 7. The overview of sequences from early patients (with onset date before 31 December 2019)’ and Table 5 on page 66

    ‘A Novel Coronavirus from Patients with Pneumonia in China, 2019’, by
    Na Zhu, Dingyu Zhang, and many others, Chinese Center for Disease Control and Prevention “Three adult patients presented with severe pneumonia and were admitted to a hospital in Wuhan on December 27, 2019. Patient 1 was a 49-yearold woman, Patient 2 was a 61-year-old man, and Patient 3 was a 32-year-old man.”
    ‘A pneumonia outbreak associated with a new coronavirus of probable bat origin’, by
    Peng Zhou, Xing-Lou Yan, and many others Wuhan Institute of Virology, and many others, Extended Tables # 1 and Table # 2 patient info and laboratory results.
    ‘Dissecting the early COVID-19 cases in Wuhan’ by
    By Michael Worobey, University of Arizona ‘WHO-convened global study of origins of SARS-CoV-2: China Part (2021)’

    Please integrate the reading of this comment, into my initial comment above. Thank you scitechdaily.com for posting my comments, which do not tell people what to think, but tries to piece together the most recent scientific findings, without taking them out of context.

  17. p. gutierrez | May 20, 2022 at 3:50 am | Reply

    This a correction or update to my May 12 comment above. p. gutierrez 5/20/2022

    When Dr. Gao stated, ‘the Ct values of the original samples revealed the existence of live SARS-CoV-2 with high titers in the environment of HSM’, he was probably referring to Covid-19 infectivity, and not viral load.

    A Los Alamos and San Diego Univ study shows viral load is measured in RNA copies per ml. Whereas ‘viral titers’ is measured in plaque forming units per milliliter, and usually peaks four days after infection, according to Figures 1-3, in the article, ‘Modeling Within-Host Dynamics of SARS-CoV-2 Infection: A Case Study in Ferrets’, by
    Naveen Vaidya, Angelica Bloomquist, and Alan Perelson. This Los Alamos and San Diego study group mathematically calculated a ‘viral load’ of 10 to the 8th power RNA copies per milliliter, after inoculation of ferrets with F13 wall sample coronavirus by Chinese study group in February 2020. And calculated ‘viral titers’ of 10 the 4th power plaque forming units per milliliter, four days after inoculation.

    ‘The infected cell death rate, d, and virus clearance rate, c, estimated by our model were 5.20 per day and 6.10 per day, respectively. These estimates indicate that infected cells turned over in about 5 h on average, and the free virus gets cleared from the body in about 4 h on average. Using Model 2, we estimated k (the rate of cell transfer from the eclipse phase to the productively infected phase) to be 7.88 per day, which implies that the infected cells started producing virus about 3 h after they were infected…..during the post-peak
    phase, the infectious virus decayed with a per capita decay rate of 1.95 per day (Table 4)
    and a half-life of 8.54 hours…….We compared the viral dynamics generated due to infection by the two types of viruses, F13-E and CTan-H. As discussed above, these virus types represent viruses in the environment (seafood market ofWuhan) and viruses within an infected person, respectively. Successful establishment of infection by both viruses (R0 greater than in both cases) implies that environmental transmission may also be an important route in addition to direct transmission.’

    F13 wall sample can infect humans environmentally, according to Los Alamos, but it takes four or 5 days within a person to peak to ‘high titers/infectivity’ regarding human cells. And so as one analyzes the ‘high titres’ of the F13 wall environmental sample, its decay rate, cell death rate, its apparently ‘infectivity freshness’ on January 1, 2020, when the sample was collected by Beijing, all of this has to be kept in mind.

    This Los Alamos and San Diego University 2020 study, merely analyzed the data produced in the study by Harbin Veterinary Institute, ‘Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS–coronavirus 2’, by Jianzhong Shi, Zhiyuan Wen, and others, that inoculated ferrets with the F13 wall environmental sample provided by the Beijing CDC. Their Figure 1 G shows a ‘viral titer’ of about 10 to the fourth power (10,000) plaque forming units per milliter ‘viral titer’ on day two after inoculating ferrets with F13 sample provided by Beijing CDC. ‘High viral titers’. What about possible inoculated pangolins December 31, 2019?

    Also recommended reading, ‘The basic reproductive number and particle‑to‑plaque ratio: comparison of these two parameters of viral infectivity’, by Winston McCormick and Leonard Mermel

    Also recommended, ‘Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS-CoV-2’, by Emelissa Mendoza, Kathy Manguiat, and others

    Below is a correction to my May 12 comment above. It stated ‘EPI-ISL_40212’. It should have read.

    “Surprisingly Gao doesn’t reference, BetaCoV/Wuhan/IPBCAMS-WH-01/2019 EPI_ISL_402123 (GISAID) MT019529 (NBI) age 65 years old, sample taken December 23, 2019, earliest known human sample,”

    In the March 2020, Cornell University article, ‘Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop’, by Javier Jaimes, Gary Whittaker, and others, Figure 4 shows their interest in Huanan Seafood Market F13 environmental sample, all three F13 isolates taken by Beijing.

    BetaCoV/Wuhan/ I V D C – H B – envF13– 2 0 /2020|EPI_ISL_408514, BetaCoV/Wuhan/IVDC-HB-envF13/2020 |EPI_ISL_408511, and BetaCoV/Wuhan/IVDC-HBe n v F 1 3 – 2 1 / 2 0 2 0 | E P I _ I S L _ 4 0 8 5 1 5.

    Gary Whittaker has written at least ten articles on Covid-19 furin cleavage site, and is very interested in F13 wall sample, just like Los Alamos study group.

    Also the article, ‘Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study’, by Nanshan Chen, Min Zhou, Xuan Dong, and others, a good write-up of who they consider to be patient 1, 61 years old, and who they consider to be patient 2, 69 years old

    The following is an article that tracked the early Covid-19 patients in Huanan Seafood Market, worth reading.
    https://www.jenniferzengblog.com/home/2020/2/26/tracking-the-source-of-novel-coronavirus-gene-sequencing-when-the-alarm-goes-off

    Dr. Gao’s Figure 3A genetic analysis of WH01, much different from NC-045512 is what is being swept under the rug.

    ‘A plaque-forming unit (PFU) is a measure used in virology to describe the number of virus particles capable of forming plaques per unit volume.’

    My comment at https://thebulletin.org/2021/08/covid-19-lab-leak-theory-gain-of-function-is-a-hot-topic-but-a-bad-explanation/

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