Wuhan Lab-Leak Theory: “Rare” Genetic Sequence Doesn’t Mean the COVID Virus Was Engineered

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The theory that the COVID-19 pandemic was triggered by the Sars-CoV-2 virus being leaked from the Wuhan Institute of Virology in China was recently given new life following an explosive article in the Wall Street Journal (WSJ) in which the authors claimed “the most compelling reason to favor the lab leak hypothesis is firmly based in science.” But does the science really support the claim that the virus was engineered in a laboratory?

Understanding the origin of a viral outbreak can provide scientists with important information about viral lineages and allow steps to be put in place to avoid similar outbreaks in the future. As such, the origin of Sars-CoV-2 has been debated from the beginning of the pandemic and remains an active topic of discussion among scientists.

It has long been known that viruses similar to the original Sars-CoV that causes Sars are found in bats. These viruses are well studied in China, where the 2002 Sars outbreak originated. But related viruses have been found globally.

Unsurprisingly, coronaviruses are again involved in a pandemic, the third such event in the 21st century – first Sars, then Mers, now COVID-19. While a natural origin seems likely – and many have long warned about the danger of wildlife circulating viruses – scientists shouldn’t jump to conclusions.

An important way scientists can determine the origin of a virus is by looking at its genome. In the WSJ article, the authors, Prof Richard Muller, an astrophysicist, and Dr Steven Quay, physician and chief executive of Atossa Therapeutics, claim Sars-CoV-2 has “genetic fingerprints” of a lab-origin virus. They say that the presence of a particular genetic sequence (CGG-CGG) is a sign that the virus originated in a lab.

To understand the claims being made, we must first understand the genetic code. When a virus infects a cell, it hijacks the cellular machinery, providing instructions (genome) to produce more copies of itself. This genome comprises a long series of molecules called nucleotides, each of which is represented by the letters A, C, G or U.

A group of three nucleotides (known as a codon) provides the instruction for a cell to make an amino acid, the most basic molecular building block of living things. Most amino acids are encoded by several different codons. CGG is one of six possible codons that instruct the cell to add the amino acid arginine.

The authors of the WSJ article argue that Sars-CoV-2 originated in a lab based on the presence of a “CGG-CGG” sequence. They claim this is a “readily available and convenient” codon pair that scientists prefer to use to produce the amino acid arginine. But to anyone with an understanding of the techniques required for genetic modification, this double-CGG is usually no more difficult or easy to produce than any other pair of codons that encode arginines.

No reason CGG-CGG had to be made in lab

The authors claim that the CGG codon appears less frequently than the other five possible codons in betacoronaviruses (the family of coronaviruses to which Sars-CoV-2 belongs). If we look at related coronaviruses, the CGG codon encodes about 5% of all arginines in Sars-CoV compared with about 3% of all arginines in Sars-CoV-2. Though CGG is less common than other codons, the authors’ argument fails to provide a reason that the double-CGG sequence could not exist naturally.

The authors argue that recombination (when viruses that infect the same host share genetic material) was the most likely way in which Sars-CoV-2 was able to obtain the double-CGG sequence. They note that the double-CGG codon pair is not found in other members of this “class” of coronavirus, so natural recombination could not possibly generate a double-CGG. However, viruses do not just depend on preassembled segments of genetic material to evolve and expand their host range.

The authors also claim that mutation (random copying errors) is unlikely to generate the double-CGG sequence. But viruses evolve at a rapid rate, so much so that the accumulation of mutations is a common inconvenience of virological studies. Recombination is one way in which viruses evolve, but the authors’ dismissal of mutation as a source of viral change is an inaccurate description of reality.

The final claim that the first sequenced Sars-CoV-2 virus was ideally suited to the human host neglects evidence of viral circulation in local animal populations, animal-to-animal transmission, and the rapid evolution that is driving the increasing transmissibility of the newer variants. If the virus was ideally adapted to humans, why is so much further evolution evident?

Disappointingly, many other media articles appear to have accepted and repeated the claims from the WSJ piece. The origin of Sars-CoV-2 may remain unresolved, but there is no evidence presented in the WSJ piece that scientifically supports the concept of a lab leak of a genetically engineered virus.

Written by

  • Keith Grehan – Postdoctoral Researcher, Molecular Biology, University of Leeds
  • Natalie Kingston – Research Fellow, Virology, University of Leeds

Originally published on The Conversation.The Conversation

12 Comments on "Wuhan Lab-Leak Theory: “Rare” Genetic Sequence Doesn’t Mean the COVID Virus Was Engineered"

  1. Gordon Jenkins | July 7, 2021 at 7:09 pm | Reply

    Perhaps Spin Doctors really are worth the extra expense.

    • Gordon Jenkins | July 7, 2021 at 7:27 pm | Reply

      Let’s not forget the two Wuhan Biologists, who were working at Canada’s Level 4 Lab in Winnipeg, Manitoba, sending samples directly to the Wuhan Lab and “disappeared” with the Canadian Government refusing accountability and disclosure to the Canadian Parliament. The two Michaels waiting in China for release. Political Hot Potato in desperate need of China-Positive Spin to make it all go away.

  2. Putting the WSJ article aside, the codon argument is just one arm of the “lab-leak” theory. Even if the Sars-CoV-2 virus RNA is completely natural and non-engineered, it still might have leaked from a lab in which it was stored after being cultivated. Many will demand proof of such “slanderous ideas” but those that have paid attention understand that any evidence has likely been destroyed already by the CCP.

  3. p. gutierrez | July 8, 2021 at 9:29 pm | Reply

    You use the term “finger prints” of lab origin. When I read Dr. Quays article ‘Bayesian Analysis of SARS-CoV-2 Origin’ version 3 March 29, 2021 ( https://zenodo.org/record/4642956), the real genomic ‘fingerprints’ are the restriction sites that he found in the RaTG13 genome. Pages 132 and 133 state, “Examination of RaTG13 identified two Esp3I cleavage (restriction) sites in the Spike Protein gene …..The 5′ restriction site in RaTG13 begins at aa residue 455L, identified by Andersen et al, Nature, 2020, as the start of the ‘receptor-binding domain (RBM) ACE2 contact residues’…..The 3’ restriction site in RaTG13 is at residue 980L. (the end of heptad repeat 1 domain)….. This is a frequency of these site at their exact location being here from a natural process of approximately one in a billion.”
    For a restriction site (a laboratory ‘fingerprint’) to be found at the beginning of the RBM (receptor binding motif), is pretty amazing. The Covid-19 RRAR cleavage site is within the bounds of these two ESPI restriction sites, supposing RaTG13 is the backbone coronavirus for Covid-19. Restriction digestion may have been used to cut DNA at those sites for removal, and fragments of other DNA may have been pieced together like building blocks via ligation. Dr. Quay (on page 131) sites Ralph Baric’s 2005 article, “Development of Mouse Hepatitis Virus…..”, as to how this may have been done.—————-
    In her report # 1, Dr. Li-Men Yan found EcoRI and BstEII restriction sites (‘genomic fingerprints’) in Wuhan person 1 Covid-19 genome. “Two restriction sites are present at either end of the RBM of SARS-CoV-2, providing convenience for replacing the RBM within the spike gene. A. Nucleotide sequence of the RBM of SARS-CoV-2 (Wuhan-Hu-1). An EcoRI site is found at the 5′-end of the RBM and a BstEII site at the 3′-end.” Quote from Yan’s article, “Unusual Features of the SARS-CoV-2 Genome Suggesting Sophisticated Laboratory Modification….”. Coincidence, both Quay and Yan found restriction sites (‘genomic fingerprints’) at the start of the RBM, in different genomes. If true, more possible evidence of S gene laboratory tampering. Many good scientists say that the Covid-19 RBM is pangolin origin. It would have been easy to insert the (downstream) cleavage site, at the same time as a possible RBM swap.—————-
    In their February 2020 paper, “Functional assessment of cell entry and receptor
    usage for SARS-CoV-2 and other lineage B betacoronaviruses”, Vincent Munster and Michael Letko, of the NIAID in Montana, stated, “We replaced the RBD (receptor binding domains) of full-length clade 2 and 3 spike with the consensus clade 1 RBD and tested pseudotypes on cells expressing ACE2…… For SARS-CoV spike, silent mutations were introduced around codons 308 and 519 to form KpnI and XhoI (restriction) digest sites (to facilitate RBD replacements)……Spike (gene) RBDs were appended with regions of the target spike (gene) backbone to facilitate In-Fusion cloning and synthesized as double stranded DNA fragments….. RBD inserts were resuspended in water and In-Fusion cloned into gel-purified, digested spike backbone vectors (Takara).” State of the art S gene engineering, clone RBDs into S gene backbone vectors from other bats, cut and paste. If you want to reverse engineer how the Covid-19 RRAR cleavage site may have originated, consult the experts. And, test the five Wuhan Hunan Seafood environmental samples genomes available in GISAID, for restriction sites (‘genomic fingerprints’).

  4. Matt Ledbetter | July 10, 2021 at 8:19 am | Reply

    It also might be worth mentioning that Science Tech Daily is headquartered in Zhang Jingan, China.

  5. Theories, theories everywhere and not a DOI to link.

    I can’t wait to see which field YouTube will offer a double secret PhD in next, and how many will die from the advice of borderline schizophrenics who knew nothing on the topic a month ago.

  6. Using a virus as a weapon of war is within the tolerance of the People’s Liberation Army.

  7. There are several flaws in this discussion of Quay’s work. It is worth noting that Quay is a world leading expert in genetics with 100s of papers and 1000s of citations, whereas the authors of this discussion are humble post docs. The closing argument here, that the virus is still rapidly mutating to increase infectivity is false, as Quay has made clear. SARS CoV 2 was 99.6% adapted to infect human ACE2 from the first cases of the pandemic, which as Quay points out is incredibly unlikely for a pathogen emerging from nature. Quay also points out that ONLY 1% of all mutations of SARS-COV2 since the start of the pandemic are relevant to the infectivity of the virus (99% of mutations occurring in parts of the virus unrelated to infectivity). Quay is obviously aware of the arguments presented here re random mutations, but has assigned probabilities and liklehoods to these, and assessed them to be extremely unlikely.

  8. Ching Ching Ching | July 28, 2021 at 8:45 pm | Reply

    Well , why not exterminate some bats 🦇 if it’s been a f**kin problem for so long ?

  9. This posting is regarding the best 2020/2021 articles on the structure of the Covid-19 furin cleavage site.

    Some have found the Covid-19 furin cleavage site to exist as a disordered loop in the Covid-19 spike gene.

    “the loop containing the cleavage site (residues 676–689) is disordered, in both cleaved and uncleaved forms, the observation of the intermediate form, and much lower thermal stability of the cleaved protein”, ———in the article
    ‘SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin cleavage effects’, by Antoni Wrobel, Donald Benton, and others, Structural Biology of Disease Processes Laboratory, and others.

    “The comparative analysis of the intrinsic disorder predisposition of spike protein from SARS2, SARS, and Bat CoV revealed that the furin-like cleavage site of SARS spike is incorporated in the longer disordered region 676TQTNSPRRARSVAS691 , which is not present in spike proteins from SARS and Bat CoV. The presence of disorder in a region containing a polybasic (furin-like) cleavage site is an extremely important point, as an intrinsic disorder at the cleavage site is crucial for efficient protease action—–‘Insights on the Structural Variations of the Furin-Like Cleavage Site Found Among the December 2019–July 2020 SARS-CoV-2 Spike Glycoprotein: A Computational Study Linking Viral Evolution and Infection’, by Marni Cueno, Miu Ueno, and others, Department of Microbiology, Nihon University, Tokyo, Japan, and others

    Many have found that part of the QTQTNSPRRAR ‘longer’ disordered region of the cleavage site is being lost/deleted in some cell passage experiments.

    “The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates….. Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS (furin cleavage site), and is less accessible to host proteases, such as TMPRSS2.”——-in the article, ‘QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis’, by Michelle Vu, Kumari Lokugamage, and others, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, and others.

    One of the best linear photo depictions for the Covid-19 furin cleavage site and RBD (receptor binding domain), are figures 1a and 1b in the article, ‘Enhanced Binding of SARS-CoV‑2 Spike Protein to Receptor by Distal Polybasic Cleavage Sites’, by Baofu Qiao and Monica Olvera de la Cruz, Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois https://dx.doi.org/10.1021/acsnano.0c04798

    The photo depictions of the cleavage site and RBD are worth examining, and the article is worth reading in leisure time.

    Qiao and Cruz stated that the Covid-19 RBD (receptor binding domain) is about 10 nanometers (nm) from the furin cleavage site, in Covid-19’s S gene, ‘distal’.

    “all-atom explicit solvent molecular dynamics (MD) simulations reveal that the spike protein polybasic cleavage sites, which are distributed
    approximately 10 nm away from the RBD,”

    Most scientists agree on this fact. Wei Lei of Nantong University observed,

    “As shown in Fig. 1, the inserted FCS (furin cleavage site) is spatially located at a random coil loop region with no structural proximity to other part of the
    structure of the S protein.” — see reference below

    Qiao and Cruz observed that the three arginines (R amino acids) in the Covid-19 furin cleavage site at 682, 683, and 685, are positively charged,

    “the substitution of positively charged arginine with negatively charged glutamic acid increases protein hydration ……Negatively charged glutamic acids (Glu) were included to neutralize the positively charged arginine residues at the polybasic cleavage sites”

    But the Covid-19 spike trimer is overall negatively charged.

    “the SARS-CoV-2 spike protein trimer and ACE2 are both highly negatively charged with a net charge of −21e and −28e, respectively”

    The Erasmus Medical Center Rotterdam study group says the alanine (A amino acid) in the Covid-19 furin cleavage site is non-polar.

    “The SARS-CoV-2 S1/S2 cleavage site contains three basic arginines inter-
    rupted by a non-polar alanine (RRAR) and is therefore referred to as a multibasic cleavage site (MBCS).” —–in the article, ‘Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation’, by Mart Lamers, Anna Mykytyn, and others, Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands, and others.

    The Virginia Commonwealth University study group observed that the RBD is connected via peptides, to the downstream residues in the Spike gene.

    “The RBD (receptor binding domain) is tethered to the SD1/SD2 domain by two peptide strings: one from the NTD (approximately residues 327-335) and the other connected to the SD1 domains (approximately residues 527-535). These peptide “strings” are stabilized or “tied together” by beta strand structures near the S1/S2 interface.”——— in the article, “Static all-atom energetic mappings of the SARS-Cov-2 spike protein and dynamic stability analysis of “Up” versus “Down” protomer states”, by Michael Peters and others, Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, and others.

    And, as one moves closer to the furin cleavage site at amino acid 682, there is a 630 loop of amino acids in the Covid-19 spike gene structure, between the RBD (receptor binding domain), and the furin cleavage site.

    “A structural element, designated the ‘630 loop’, in CTD2 is largely disordered in the S trimer of the Wuhan- Hu-1 strain but is ordered in the presence of the D614G mutation and plays an important role in stabilizing the S trimer”, ——— in the article, ‘Mechanisms of SARS- CoV-2 entry into cells’, by Cody Jackson, and others.

    Trp633 and Tyr636 of are residues in this 630 loop in the Covid-19 spike gene, according to figure 4d in the article, ‘Structural impact on SARS-CoV-2 spike protein by D614G substitution’ by Jun Zhang, Yongfei Cai, and others

    As one moves downstream past this 630 loop, one approaches the amino acids in the ‘surburbs’ of the Covid-19 furin cleavage site.

    “The spike protein residues N657, N658, E661, Y660, T678, N679, S680, R682,
    R683, R685, S689, and Q690 strongly interact with the Furin protease (Figure 2B).” —–Naveen Vandakari, ‘Structure of Furin Protease Binding to SARS-CoV‑2 Spike Glycoprotein and Implications for Potential Targets and Virulence’

    The best ‘structural chain’ mapping of the Covid-19 furin cleavage site residues, are Figures 9C and 10A in the article, ‘Comprehensive Structural and Molecular Comparison of Spike Proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, and Their Interactions with ACE2’ by Ma’mon Hatmal, Walhan Alshaer, and many others, Department of Medical Laboratory Sciences, Faculty of Applied Health Sciences, The Hashemite University, Zarqa, Jordan, Department of Immunology, School of Medical Sciences, Universiti Sains Malaysia, and others.

    This article is worth reading, the furin cleavage site mapping well worth examining.

    In 2020, the Virginia Commonwealth University study group reaffirmed that each Covid-19 spike (S) gene consists of three chains, or protomers.

    “The SARS-CoV-2 Spike protein structures considered here consist of three chains or protomers (A, B, and C chains) of which one chain is given in the so-called “Up” state of its RBD and the remaining two chains are in their “Down” state………”—–in the article, “Static all-atom energetic mappings of the SARS-Cov-2 spike protein and dynamic stability analysis of “Up” versus “Down” protomer states”, by Michael Peters and others, Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, and others.

    The article is worth reading.

    The Covid-19 Spike gene’s ‘A chain’ includes 1-1120, the ‘B chain’ includes 1121-2240, and the ‘C chain’ includes 2241-3360, according to the article, “Dynamic Asymmetry Exposes 2019-nCoV Prefusion Spike”, by Susmita Roy and others, J. Phys. Chem. Lett. 2020, 11, 7021−7027 supplementary materials https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427122/

    Article is worth reading, but their interchain and intrachain analysis contained in their supplementary materials, includes little if any information regarding the Covid-19 furin cleavage site.

    The best photo showing the three chains of the Covid-19 S gene is figure 1d of the article, ‘Structure of Furin Protease Binding to SARS-CoV‑2 Spike
    Glycoprotein and Implications for Potential Targets and Virulence’, by
    Naveen Vankadari, J. Phys. Chem. Lett. 2020, 11, 6655−6663

    Wei Li of Nantong University, examines the Covid-19 furin cleavage amino acids PRRAR (681-684) as they are seen across the three chains A, B. and C, whether the residues are exposed or hidden, in Table 4 in the article ‘Delving deep into the structural aspects of a furin cleavage site inserted into the spike protein of SARS-CoV-2: A structural biophysical perspective’. Table 1 of the article examines the furin cleavage amino acids side chain bindings.

    Also, regarding Covid-19 Spike gene intrachain chain interactions, the Virginia Commonwealth University study group intimates there may be partial atomic and van der Wals interactions among the PRRAR residues, without explicitly saying so.

    “we observe conspicuous, strong partial atomic charge and van der Waals interactions between the R group of GLN564 in the SD1 domain with backbone atoms of the three sequential residues ALA520-PRO521-ALA522 in the RBD domain that were also seen in the cross chain glue point interactions. These specific atom-atom interactions are shown in more detail in Figs 7 and 8. Note the PRO521 interactions are dominated by van der Waals interactions in both instances (see S7 and S8 Tables in S1 File).”, in the article ‘Static all-atom energetic mappings of the SARS-Cov-2 spike protein and dynamic stability analysis of “Up” versus “Down” protomer states’, by Michael Peters, and others Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America, College of Biological Sciences, University of Minnesota, Minneapolis, 3 NASA Ames Research Center, Moffett Field, California

    This article is worth reading, but could have been better written.

    In 2020, Wei Li (of Nantong University) examined the PRRAR (681-685) furin cleavage structure and determined,

    “With a close inspection of all tables in the supplementary file supplementary.
    pdf,
    1. no salt bridge or hydrogen bond was structurally identified for the
    three basic residues at FCS.
    2. no hydrogen bond was structurally identified for Arg682 or Pro681.
    3. one hydrogen bond was structurally identified for Ala684 with
    Arg685.
    4. two hydrogen bonds were structurally identified for Arg683 with
    Thr604 and Asn679.
    5. four hydrogen bonds were structurally identified for Arg685 with
    Val687, Lys310 and Ala684.”—–in the article, ‘Delving deep into the structural aspects of a furin cleavage site inserted into the spike protein of SARS-CoV-2: A structural biophysical perspective’, by Wei Li, Institute of Special Environmental Medicine, Nantong University, Nantong City, Jiangsu Province, People’s Republic of China, Biophysical Chemistry 264 (2020)

    This article is well worth reading. As of January 2022, this remains one of the best analysis of the structure of the Covid-19 furin cleavage site.

    Another structural feature of Covid-19 is that there are multiple copies of the Spike gene in the membrane of a Covid-19 viron.

    “S protein is assembled as a homotrimer and is inserted in multiple copies into the membrane of the virion giving it its crown-like appearance……..SARS- CoV-2, like MERS- CoV, belongs to the first category: its S protein is cleaved by proprotein convertases such as furin in the virus-producer cells. Therefore, the S protein on the mature virion consists of two non-covalently associated subunits: the S1 subunit binds ACE2 and the S2 subunit anchors (fuses) the S protein to the membrane…… This perplexing observation
    suggests that the acquisition of a furin-cleavage site by SARS-CoV-2 may have been a recent event….., SARS-CoV-2 rather acquired a different mutation, D614G, to stabilize the S protein and slow S1 shedding.”——–in the article, ‘Mechanisms of SARS- CoV-2 entry into cells’, by Cody Jackson, Michael Farzan, and others, Nature Reviews | Molecular Cell Biology, volume 23 | January 2022

    The article ‘The sequence at Spike S1/S2 site enables cleavage by furin and phospho‑regulation in SARS‑CoV2 but not in SARS‑CoV1 or MERS‑CoV’, by Mihkel Örd, and others shows the rate of cleavage by furin, for RRAR, “the novel coronavirus SARS-CoV2 site was cleaved very efficiently (Fig. 1d–f).”

    see my Internet BtKy72 Covid-19 postings.

  10. p. gutierrez - comment April 2022 | April 20, 2022 at 5:08 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 Gao, to get their list of environmental samples from the HSM.

    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.”

    Gao says F13 environmental sample (wall) is highly identical to WH01 (Wuhan person #1- NC_045512) isolate, which 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

    Reviewing the bronchoalveolar lavage fluid (BALF) for Wuhan person #1 who worked at HSM, “The viral load in the BALF sample was estimated by qPCR to be 3.95 × 108 copies per ml .”—- ‘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.

    “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, and others

    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, and had high viral titers December 26, 2019. Mid-December 2019 is a common time period for the two different studies. Others had signs 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

    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 article 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. Could how the Covid-19 got on the HSM wall have been partially due to particle drag and particle density? 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 its importance.

    But determining and analyzing surface particles μm size (micrometer-.000001 m) seems more practical to help determine particles density, their Weber and Reynolds numbers, and perhaps trace those particles possible source of origin.

    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.

    “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.

    But Dr. Gao did not size the possible surface particles, that Covid-19 virions possibly existed in, or were transmitted in. Nor elaborate on the pleomorphisms observed. In July 2021, Vincent Munster of the NIAID observed regarding the larger particles containing Covid-19 virons,

    “demonstration of true aerosol transmission of SARS-CoV-2 should only include particles 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 >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 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 analyzing surface environmental samples.

    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) and the other in supermicrometre region (dp > 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 (>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. Why not try using the same regarding sizing environmental samples particle sizes? Other surface collection of virus particles articles worth reading are,

    ‘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 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

    As if they have nothing else to do at the NIAID in Montana 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

    Comments at https://scitechdaily.com/lab-leak-or-zoonotic-transfer-leading-biologists-review-covid-19-virus-origin-evidence/ worth reading

  11. p. gutierrez ---truncated part of above comment | April 20, 2022 at 10:37 am | Reply

    (the above comment was truncated in processing. The important truncated part is below.)

    ………existed in, or were transmitted in. Nor elaborate on the pleomorphisms observed. In July 2021, Vincent Munster of the NIAID observed regarding the larger particles containing Covid-19 virons,

    “demonstration of true aerosol transmission of SARS-CoV-2 should only include particles 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 >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 <1 μm 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,……………………………….

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