James Lyons-Weiler, PhD 2-2-2020

UPDATE – 2/9/2020 – IPAK HAS CONDUCTED FURTHER, IN-DEPTH STUDIES OF THE GENOMIC AN PROTEIN SEQUENCES OF THE 2019-nCoV CORONAVIRUSES AND THEIR RELATIVES AND HAVE COMPELLING RESULTS OF A KEY SIGNATURE USEFUL FOR IDENTIFYING A PARTICULARLY PATHOGENIC CORONAVIRUSES LINEAGE. GIVEN THAT WE HAVE FOUND THIS SIGNATURE, A FUNCTIONAL MOTIF FINGERPRINT, PRESENT IN THE HK-3 CoV FROM 2005, WE BELIEVE THIS EXONERATES RECOMBINATION IN THE LAB AS A SOURCE OF THE VIRUS. THIS DOES NOT EXONERATE ACCIDENTAL RELEASE, HOWEVER. WE ARE WORKING TO PUBLISH OUR FINDINGS.

IN THE INTEREST OF TRANSPARITY, WE ARE KEEPING THE ARTICLE BELOW AS ORIGINALLY PUBLISHED FOR POSTERITY AND PROVENANCE. – JLW

Dr. Marc Wathelet commented that he was puzzled about my report of a spike protein gene homologous to part of the pShuttle-SN vector, given that spike glycoproteins are found in bat coronavirus. He urged me to analyze the homology (sequence similarity) of the SARS-like spike protein element I reported with other spike proteins, saying that any scientist working on coronviruses would be surprised if there were not a spike protein.

I replied in comment that I, too, would expect protein sequence level homology due to shared conserved domains, but assured him that I would undertake further genome sequence-level (nucleotide) analysis as the location of the novel sequence relative to the other spike proteins is certainly of interest.

A few recent publications (sent to me by followers/readers) contained further bat coronavirus accession numbers, and SARS accession numbers, so I procured the spike protein coding sequence (CDS) of these from NCBI’s nucleotide database and aligned them using Blast, with the sequence from the first 2019-nCoV protein as the anchor. (Oddly, that Genbank entry does not label the S protein CDS as a spike glycoprotein, instead annotating it only as a “structural protein”).

The resulting massive alignment confirms a major unique inserted element in 2019-nCoV not found in other bat coronaviruses, nor in SARS in the homologous genomic position:

This is why full genome phylogenetic trees cannot tell the full story of recombinant viral evolution.

Blasting the novel sequence region against all non-viral sequences (to pick up vector technology) again results in pShuttle-SN (no surprise) but now this time is also picked up a recombinant coronavirus clone Bat-SRBD spike glycoprotein gene from UNC, USA. (Genbank entry) and other synthetic constructs.

As I published earlier, before anyone points fingers at the Chinese, note that recombinant viruses have been in play in laboratories all across the world in many nations.

The overlap occur at the 3′ end of the novel region (search restricted from 21600-22350 bp in the query 2019-nCoV sequence originally blasted against the other coronavirus CDS. It could arguably merely be that I selected too large a region; I chose the region visually to include the fully potentially inserted sequence including any homologous vector elements at the 5′ or 3′ end.

It is worth pointing out that due to the length of overlap, the sequence strength is considered moderately strong: highly significant E-value, high %identity, but short sequence length. These findings cannot be considered strong validation for obvious reasons: produced by the same analyst, using (part) of the same data. Spike proteins determine receptor binding for entry into cells, and 2019-nCoV appears to, like some bat species SARS coronavirus, target ACE2 receptors [1].

For those tracking closely, I confirmed that the novel inserted sequence in the large alignment above is the same as the novel sequence I reported a few days ago. The sequence of interest is here.

[1] Hou et al., 2010. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry Arch Virol 155:1563-1569

https://www.msi.umn.edu/~lifang/otherflpapers/bat-ace2-archivesofvirology-2010.pdf

These results do show, however that the novel sequence is not likely present in other coronaviruses.

Thus, it still seems prudent that this inserted sequence in 2019-nCoV become the focus on urgent research, and that laboratory sources be included in the search for the origins of 2019-nCoV and potential targets for treatments and expected pathophysiology in patients infected with 2019-nCoV.

I am grateful to Dr. Wathelet for this inquiries and requests for additional clarification.

RECOMMDED RELATED:

CLEAR EVIDENCE it is NOT a Bioweapon. So What’s All This About ACE2 and nCoV-2019 (COVID-2019)?

2019-nCov Vaccine Recommended Readings

Why over the next two weeks the world will learn how bad the 2019 nCoV Coronavirus Pandemic will be

80 thoughts on “Moderately Strong Confirmation of a Laboratory Origin of 2019-nCoV

  1. Dr. Lyons-Weiler: Thank you once again for the important work you do, and for your clear explanation of your findings about this novel viral entity that even a non-scientist can grasp.

  2. It’s getting incredibly difficult to get any accurate information on what’s going on in China. Eve news outlet has their own spin. I ran across this on Yahoo:

    Cocktail of flu, HIV drugs appears to help fight coronavirus: Thai doctors
    https://www.yahoo.com/news/cocktail-flu-hiv-drugs-appears-124753996.html

    BANGKOK (Reuters) – Thai doctors have seen success in treating severe cases of the new coronavirus with combination of medications for flu and HIV, with initial results showing vast improvement 48 hours after applying the treatment, they said on Sunday.

    The doctors from Rajavithi Hospital in Bangkok said a new approach in coronavirus treatment had improved the condition of several patients under their care, including one 70-year-old Chinese woman from Wuhan who tested positive for the coronavirus for 10 days.

    The drug treatment includes a mixture of anti-HIV drugs lopinavir and ritonavir, in combination with flu drug oseltamivir in large doses.

    Based on what I have been reading about Coronavirus, I don’t know what to make of this.

    1. codetalker: It is indeed difficult to trust any information about this. I have read that there have been no fatalities among those below age 55, that the mean age of fatalities is 75, and that many exhibit serious lung pathologies. This, in one of the most polluted cities in China. Something worrisome is going on here, but the WHO hyperbole makes it sound like the rest of the viral scares: SARS, H1N1, Zika, and so forth. Tremendous economic hit for China, coming at the beginning of the Lunar New Year, when so many travel.

      1. “I have read that there have been no fatalities among those below age 55, that the mean age of fatalities is 75, and that many exhibit serious lung pathologies.”

        I read that too. Also, there are some who are testing positive for the virus but have few to no symptoms. What symptoms they are exhibiting is more like the Flu. I think if the individual suffers from a chronic illness like lung problems because of the pollution they may be more likely to have a sever case which leads to their death. You’re right abut Swine Flu, SARS, H1N1, Zika and other scares. At the end of the day after various companies are given huge amounts of money to develop vaccines-the MSM just seems to lose interest in reporting because what they hyped didn’t come to pass.

      2. codetalker: Right you are. What is fascinating to me is Dr. Lyons-Weiler’s investigation of the origins of this novel form. The WHO and others are using this for political and economic reasons. We may, in the future, be required to be vaccinated to get on a commercial flight. That is where this is headed. Greater control of the public for greater profit for the private sector, while sweeping the damage under the rug. So very important that we applaud and support honorable scientists who shine a bright light on these nefarious activities by seeking truth. They will not succeed, any more than any of the dictatorial regimes of the Twentieth Century succeeded for very long, except the Kim dynasty in North Korea. The populist movements arising around the world have been partially in response to Obama’s GHSA, which seeks to forcibly vaccinate everyone. The result has been a rising awareness of the risk of vaccine injury.

  3. Gary, the mandatory vaccine plan has been in motion for decades if you read the reporting of Dr. Tenpenny. Healthy People 2020 was initiated over 35 years ago:

    Healthy People 2020 and the Decade of Vaccines
    https://vaxxter.com/healthy-people-2020-and-the-decade-of-vaccines/

    It’s easy to blame every administration that comes into office saying they’ll take a look at the vaccine issue, just as trump did and Obama did but the reality is for any administration it’s a low low priority issue. Healthy People began in 1979:

    -Surgeon General Julius B Richmond chronicled the state of health in America, then defined five quantitative goals for public health. The document came to be referred to as Healthy People. With the help of Assistant Surgeon General Michael McGinnis, the formal publication was released in 1979. In 1980, a companion piece written by the Institutes of Medicine – Promoting Health/Preventing Disease: Objectives for the Nation – set forth 226 specific, measurable health objectives. This was the action plan for the Healthy People goals. These two documents, considered to be “landmarks” in the history of public health, became Healthy People 1990.

    The pharmaceutical industry is the largest lobbying group in Washington and has been for years outspending even the oil companies. For decades very few elected officials have been willing to take on the vaccine issue from the sides of parents because like Doctors, they are fearful of losing their livelihoods. It’s very easy to place blame on Obama, trump has washed his hands of the issue which has completely blindsided Robert Kennedy Jr who was to head a task force on vaccine.

    The only way to take back control of medical autonomy for everyone is what is happening now- grassroots organizations such as ICAN, The Highwire and other groups across the country who are now making headway to the point that the WHO has acknowledge in their vaccine summit in Geneva in December that the front line of Doctors is very wobbly because they cannot answer questions parents are asking so parents are refusing to vaccinate. The WHO has also acknowledge that people questioning vaccines are not spreading misinformation-it’s information that Doctors cannot refute and censoring such as on facebook & twitter is not the approach to take.

    Don’t look to any administration to help. Don’t look for any politician to help. They like that nice cushy job with lifetime benefits after they leave office. After all it was the Bush administration that had the provision that a vaccine maker can’t be sued put in a bill late in a session that passed because there was not debate or discussion before the vote.

      1. James, Im merely a layperson who has found his way here…I understand you arent an epidemioligist but do you have any views on the potential for this to spread globally?

      2. Dan,
        Thank you for your comment and question. The threat of which you ask really needs to asked in terms of
        the threat associated with mortality from 2019-nCoV.

        Given the scenarios, it all hinges on which scenario (hypotheses) bears out:
        (1) Natural recombination – looks like this is not likely at all. Killer
        recombined coronaviruses are, however, to be expected, and reduction of risk
        of zoonotic transfer from animals to humans must be minimized (food choice, fully
        cooked food, gloves that prevent cuts during food prep, sanitary food prep).
        Elizabeth Tillier’s molecular phylogenetic work that shows that bats are primates
        must be kept in mind: recombination among coronaviruses between humans and other primates
        would be most likely possible. But in this case, the natural origin hypothesis seems dim
        to me.
        (2) Lab-origin – seems most likely.
        (2a) Lab-origin, vaccine type.
        (2a1) Lab-origin, vaccine type, escaped.
        If this is an experimental vaccine type, escaped from a lab, it is by definition contagious
        (as would any coronavirus be) and the lab may have been working to modify the transmissibility.
        Such modifications could have resulted in a super-transmissible virus, which also happens to
        have more pathogenic capacity. The R0 does appear elevated. Such a virus would infect anyone, everyone,
        but would not be overly lethal to the entire population. A lab worker could have picked it up.
        (2a2) Lab-origin, vaccine type, administered, tested on humans in China.
        If this scenario is true, then the rest of the world need not worry so much. Vaccines of
        this type (SARS, etc) have notoriously bad reputations because the animal studies resulted
        in high mortality in older animals upon secondary challenge. Since the rest of the world
        was not vaccinated w/an experimental coronovirus w/an extra SARS-like spike protein, we would
        not be sensitized and the older population (50+) would not be at increased risk.
        (3) Lab-origin, bioweapon, escaped or released.
        If this is a bioweapon, it’s poorly designed. A decent weaponized coronvirus (which I
        recommend against) would be tailored to target 18-25 yos to take out troops. It would not
        have such a long latency period; it would be design to quick quickly, have much higher
        transmissibility, low latency period, and it would tend to give those infected little chance
        to recover yet seem to require intensive urgent care releasing massive amounts of infected
        body fluids. This is not likely a bioweapon.

        Overall, if we all act as though we, and everyone we meet, is infected, and do not shake hands,
        hug or kiss, use the “Ebola elbow bump” to greet (in short, keep your fluids to yourself), don’t
        share food or utensils, carry and use tissues, and throw them in the bin (as HMG suggests), wipe
        down all common public surfaces w/bleach wipes (door knobs, door handles, keypads, elevator buttons,
        bathroom fixtures, light switches) throughout the day, we can all work together to
        bring R0 down to below 1.0. And we should do these things anyway during flu season.

        While 2019-nCoV will spread worldwide, I don’t think we’ll see a widespread pandemic of deadly disease.

  4. Dear Dr. Lyons-Weiler:

    I, together with my family, appreciate what you did on this. Your contribution is extremely valuable. I think. all infected, especially those in China, deserve the truth.

    Best,

    Samuel

  5. Are the Pangolin virus sections overlapping the region you thought was from pShuttle and a better explanation for the insert than pShuttle or are they in distinct regions of the insert and both present? Can you do an analysis which shows the alignment of the pangolin virus part and the pShuttle part on the same diagram?

  6. Dear Dr. Lyons-Weiler,

    Thank you for the interesting and informative reads.
    I was wondering, if it truly is an escaped vaccine, what does that imply for reinfection amongst the general population?
    Let’s say a normally healthy person gets infected, but eventually gets healthy again after some period. Are there chances for them to pick up the virus again, causing a reinfection leading to the severe immunopathologic lung issues mentioned in the other article? What if that person gets reinfected by a mutated strain instead?

    Jeff

  7. Dr. Lyons-Weiler:

    Thank you for your explanations and citations.

    Given 2019-nCoV would make a poor bioweapon, but shows signs of being a vaccine, what contagion would such a vaccine target?

    Might 2019-nCoV be an as-yet unperfected vaccine for a bioweapon? If possible, what characteristcis might such a bioweapon have?

  8. Hi I’m not a scientist or a Dr, I have a little trouble understanding about the proteins and how you can tell that they may have a lab origin? thank you, ps sorry if my question is too vague.

  9. Many thanks Dr Lyons-Weiler, for your detailed response. I note that we still don’t have any deaths due to infection from anyone outside of Wuhan. I think we will likely have a much clearer idea if it is potentially 2a or 2a2 within a few weeks. thanks again.

  10. Dr. Weiler,

    I appears to me you discount the possibility that this is a Biological weapon ( “# 3”) too easily.

    Why do I say this?

    a) It seems to infect Males more than females. ( Males are more likely to serve in combat in the PLA). If they are slightly older, then you might just take out the leadership ( officer corps), a favored tactic since 1776.

    Even if many of the younger healthy males survive, they will be weaker and sicker for a very long period. While this can infect your troops if you were to invade, in a war with country like China, you should not expect to invade without huge exchanges of Nuclear weapons, Biological warfare, and Chemical agents that would cover any PLA retreat, or break up an attacking forces advance.

    That is why this “asymmetrical warfare” ( aka Dirty sneaky tricks to Isolate China) can not be ruled out. They are simply to powerful to take on headfirst.

    b) The death rate appears to be between 11% and 15% when given the best treatment known today. ( A mix of Anti flu and and HIV drugs, all of which are Anti-virals). ON the battlefield these drugs will not be so available. We do NOT really know the exact death rate, but we should be more concerned about the death rate for those who receive no ( or poor) treatment.

    In one of these studies it was reported that 10% had heart damage….

    https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30183-5/fulltext

    https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30211-7/fulltext

    These drugs may even come into short supply if this virus is not contained in the near future, let alone in a conflict. Supply chain disruptions are already happening.

    c) A long incubation period, during which the infected is contagious is exactly what is needed to spread the weapon to the maximum number of the opposing side.

    It is not actually necessary that the opposing troops die. In fact that they are sick, makes them a burden on the opposing side. It was well known in WWII that wounding a soldier on an opposing side tied up multiple soldiers in the effort to evacuate and treat. Let alone the resources needed to treat an injury or sickness. Almost all patients of 2019 N-Cov have a fever, many with breathing and coughing issues…Just how effective will that young man be on the battlefield?

    In the mean time China is being isolated by friend and foe alike. That may be the real goal. However that may backfire in the long run. This could spread very easily ( and is spreading currently outside China) and there is always retaliation to consider.

    I Know you are a scientist, but please try and think like a strategoi.

    A simple Lewi.

    1. One reason why I discount the bioweapon origin includes that it would be reckless to make such an accusation given that
      we have a humanitarian situation first. That’s not a scientific reason, but even scientists are human.

      However, we simply do not have a real handle on fundamental and important statistics and characteristics of 2019-NCoV.
      We don’t know the complete number of cases in China, some experts assess it now (2/5/2020) as high as 200,000
      We don’t know how many of the >20,000 cases that are symptomatic and the possibly >180,000 that are asymptomatic might crash after 10-
      14 days of illness into critical condition, nor how many will die. We know the most sick will get a diagnosis given that universal
      screening is not feasible.
      For these reasons, we also have little knowledge of a secure R0 statistic. The suspected asymptomatic prodromal period of 5-7 days leaves us
      with estimates of R0 ranging as far as 2-8. Because we do not have a viable per-case death rate estimate (# dead / # infected) and a viable
      R0, it is difficult to say this is a bioweapon per se. It may have been a laboratory experiment TOWARDS a bioweapon gone wrong.

      I am not ruling out a bioweapon, my assessment is that it is very unlikely.

      Even if it is one, I would like to encourage a humanitarian response for all countries with high case rates and critically ill, and
      our collective goal should be to reduce R0 < 1 via self social isolation techniques NOW, instead of waiting for the evidence in the form of hundreds of thousands of cases outside of China. Don't share food, don't shake hands, or high-five. Use the Ebola fist bump (bump elbows). Don't put your hands above your shoulders. Wash your hands frequently. All public facilities and companies should disinfect common shared surfaces hourly: door knobs, light switches, menus, condiment containers, etc. Let' not stir international strife or racism simply because this outbreak originated from China. The Chinese started the concern that it originated from eating bats, but if it has a laboratory origin, such an outbreak could have started in the US, Germany, Hong Kong, Japan, Australia - all places with labs that have created recombined viruses of one sort or the other with the SARS CoV Spike protein toward vaccine development.

      1. Absolutely agree we should not resort to “justified Racism”. By simply “closing” a border you might be simply delaying entry of that pathogen…but people have a habit of fleeing quarantine, and eventually ( and actually currently) many Countries are dealing with “clusters” of peoples infected with 2019 N-COV. So the “cat is out of the bag” to some extent that remains to be seen.

        I would propose it would be best that all travelers from ANY country that has reported cases of 2019 N-COV be in quarantine for at least two weeks.

        I suspect that many countries will simply fail to report publicly, especially if the cases are isolated. The “word on the street” in South Africa is currently covering up at least one case. I don’t like to report rumors, but that is how the word on Chernobyl got started.

        We really do not know, and may never know exactly where this virus came from. The Chinese Government ( CCP/ PLA) would have reasons to conceal what they know. If it escaped from their laboratory, then that is obvious they would want to cover that up.

        If,on the other hand, the virus was intentionally spread, they might also wish to keep that secret. Blaming this on “bat soup” etc., avoids public pressure to respond. This will give China the option to respond in the way they wish and when they wish.

        It is early to know with 100% certainty death rates, transmission rates, or even cure rates. But the early studies do give us some kind of idea of the seriousness of the problem. I see you take this very serious as well, as you propose we conduct ourselves like we are in a Ebola Outbreak…this is very wise advice. Maybe we should bow like Asians ( and some of my Near Asiatic Ancestors) handshakes are bad western habit IMHO. My global travels have proven to myself that most humans are not very hygienic and cannot avoid touching their noses and eyes, even I have difficulty with that.

        It is good you keep your “human perspective”, but we should not let our emotions (totally) rule our rational side. I think the older and wiser we get, the more we realize humans are not as rationale as we think we are! If we keep this in mind, we can overcome our irrational fears.

        All the best,

        A simple Lewi

      2. Hi, Dr. Lyons-Weiler: , another scenario need be considered:
        scenarios 2. 2b2
        Lab origin. The gene modified testing animals were stolen by lab technicians and sold to the nearby “seafood & wildlife market”.
        My reasoning:
        1. It is not rare in China that lab technicians steal, eat or even sell lab testing animals. Some have been arrested for illegally profiting from it.

        https://mp.weixin.qq.com/s/EFmWDzzkd5hAqPsH0aG8Rg

        2. Wu Han biologist already did some modification on the virus from bat, which could make it transmit able to human.

        https://mp.weixin.qq.com/s/1Hoe4emhtmL1c5vdxC5aHA

        3. The research facility (lab) is close to the seafood and wildlife market where the SARI started。
        I maybe wrong. I hope I am wrong.
        Shiyu

  11. I think there have been many who died under age of 50 and the actual situation in Wuhan is far worse than you can tell from the official report/statistics. I am actually very concerned with the slow reaction of cdc in U.S. which in many ways resemble how government officials thought in Wuhan two weeks ago.

  12. 1) The difference between the Wuhan coronavirus from other coronaviruses is intriguing, but certainly not alarming. When you examine the genome sequence of any species with close relatives, you will find mostly similar sequences but some divergent regions as well. That is why they call it a NEW coronavirus.
    2) Your claim about the similarity between the INS1378 sequence and the pSHUTTLE-SN vector is deliberately misleading. pSHUTTLE-SN is pSHUTTLE plus a Spike gene fragment from SARS-CoV and therefore, the similarity is between the SPIKE gene fragments from two different coronaviruses, what is the big deal? I bet there are billions of vectors containing viral gene fragments that scientists have constructed and these cloned sequences will show similar or even identical sequences to many of the viruses that are infecting people, so you are going to tell people that all these viruses are man-made?

    1. The data we have analyzed show that part of a viral genome that otherwise would be classified as a bat coronavirus contains
      a SARS spike protein. We now have a match that INS1378 is over 96% similar to a SARS spike protein.

      The rest of the scientific community as ruled out natural recombination as a potential source.
      As an expert in molecular evolutionary genetics, I simply cannot rule out a laboratory origin of this virus, especially given
      that we know with 100% certainty that much research has been going on in labs around the world putting SARS spike protein into
      other viruses to study immunogenicity for a vaccine.

      Rest assured we are only reporting what we see in the data. No one has offered other plausible, evidence-based explanations
      for the presence of a SARS-CoV Spike protein in a bat coronavirus. It is certainly possible that recombination occured in a human being.
      The high match to a known SARS spike protein is remarkable. Its specific genomic location is compelling.

      My assessment:
      Natural recombination in the wild or in humans – 5-10%
      Laboratory origin – 90-95%
      -Vaccine-related >99%
      -Bioweapon <1%

      I am obviously willing to admit that my assessment is incorrect. We need disclosure
      of all laboratory CoV sequences from all laboratories that have been conducting any
      kinds of CoV recombination research TO RULE OUT (i.e., to falsify) a laboratory origin.

      Thank you for your comment.

  13. you guys fell into the pitfalls of bioinformatics research.
    1, Due to data updating issue, the default BLAST search against nr database would not return the hit of RaTG13 genome, which is a naturl Bat CoV (Bat SARS-like coronavirus RsSHC014, complete genome,GenBank: KC881005.1) , there was no such long insertion as mention in this assay.
    2, Due to the display issue of NCBI BLAST MSA VIEWER, if you choose the default hit number settings, the matched alignment with natural sequence in the region socalled “pShuttle-SN vector insertion” would not be shown in the MSA, even they actually exists. You could find lot of natural virus sequnces with high homology to the pShuttle-SN vector sequence, e.g. MK211376.1,MK211375.1, AY862402.1.

    Anyway, we should learn how to use BLAST first.

    1. *correction:
      2, Due to the display issue of NCBI BLAST MSA VIEWER, if you choose the default hit number settings, the matched alignment with natural sequence in the region socalled “pShuttle-SN vector insertion” would not be shown in the MSA, even they actually exists. You could find lot of natural virus sequnces with high homology to the pShuttle-SN vector sequence (AY862402.1), e.g. MK211376.1,MK211375.1

      AND, we could identify highly homologous sequences of “insertion sequence” in couple of natural virus sequences, e.g. MK211376.1,MK211375.1, AY864806.1, AY278554.2 and et.al.. you could use the ENTREZ QUERY AS DATABASE SEARCH SET: “entrez query: txid10239[Organism:exp] not wuhan”.

  14. correction: you fell into the pitfalls of bioinformatics research, and should learn how to use BLAST first.

    1, Due to data updating issue, the default BLAST search against nr database would not return the hit of RaTG13 genome, which is a naturl Bat CoV (GenBank: MN996532.1) , there was no such long insertion as mention in this assay.

    2, Due to the display issue of NCBI BLAST MSA VIEWER, if you choose the default hit number settings, the matched alignment with natural sequence in the region socalled “pShuttle-SN vector insertion” would not be shown in the MSA, even they actually exists. You could find lot of natural virus sequnces with high homology to the pShuttle-SN vector sequence (AY862402.1), e.g. MK211376.1,MK211375.1

    1. Jason, thank you but that is incorrect. I never searched against the default nr database. I specifically excluded viral sequences using a query with what is now called the “middle fragment” and found SARS spike proteins and pShuttle-SN. You don’t have to worry about my bioinformatics skills. I teach classes on how to use NCBI’s bioinformatics tools and was scientific director of the Bioinformatics Analysis Core at the University of Pittsburgh. Look up who Webb Miller was; he along with Dr. Nei were my post-doctoral advisors at Penn State University during my AP Sloan Computational Molecular Biology Post-doctoral years, and I’ve designed alignment algorithms myself.

      1. Thanks for your reply, could you answer questions briefly and directly?
        1, a naturl Bat CoV (GenBank: MN996532.1) genome contain the socalled “insertion sequence”
        2, we could identify highly homologous sequences of “insertion sequence” in couple of natural virus sequences, e.g. MK211376.1,MK211375.1, AY864806.1, AY278554.2 and et.al.. you could use the ENTREZ QUERY AS DATABASE SEARCH SET: “entrez query: txid10239[Organism:exp] not wuhan”.

      2. Jason, Curious that was collected in 2013 and was not a hit in my previous coronavirus – specific blasting. The fact that it’s military lab submission from a sample from 2013 does not help. We need a NEW sample from the wild to rule contamination, mistake sample labels, and misleading data. I’m doing an in-depth analysis of Spike proteins in comparison to pShuttle and the middle fragment . “Highly homologous” is not sufficient: all analyses must include the genomic location, including the original Wuhan sequence that had a gap compared to other coronaviruses. The original analysis shows, requires consideration and explanation. Why would the first paper on it find a sequence with zero match to any database? Why would my analysis exclude Viridae point to SARS spike glycoproteins and to pShuttle-SN? I’m doing an in-depth analysis of Spike proteins in comparison to pShuttle and the middle fragment.

      3. you just ignore many natural sequences containing the socalled insersion sequences by mean, many of them are not submitted by military lab. The socalled insertion sequence is just a sars virus sequence.
        What’s your purpose?

      4. That’s how it appears now – but the original paper reported a sequence they could not align, and a second publication
        mentioned it as a mysterious “middle fragment” and then concluded. I hope NCBI will look into the provenance of these specific sequences.
        If they werre updated, they should have been versioned appropriately.

  15. Have you realized that pShuttle-SN contains the cDNA for SARS spike protein and is NOT an empty cloning vector?
    Your blast just hits the cDNA for SARS spike but nothing else.
    No any indication of laboratory origin!

    1. Yes I know pShuttle-SN is not an empty vector. It contains a SARS-like spike protein encoding sequence. Search EXCLUDING viruses. The full story will require more data since pShuttle-SN has absolutely been used in the lab to transfer the Spike protein into other viruses.

  16. Have you performed the alignment with GenBank: MG772933.1 (a naturl Bat CoV)?
    I did not find any significant gap.

    Also, have you realized that pShuttle-SN contains the cDNA for SARS spike protein and is not an empty vector?
    You blast just hits the cDNA for SARS spike protein.

      1. If align the Wuhan 2019 nCov and SARS pShuttle, there are three 10nt, 11nt and 11nt sequences aligned to the vector backbone. How often could this happen if 2019 nCov is a natural originated?

  17. Dear Dr. Lyons-Weiler,

    Is it possible that the insertion is coming from an intermediate host between bats and human? I am in no way an expert in virus biology, but I assume that the manipulation of viral genetic information under laboratory setting can leave behind other evidence. Is there any other evidence other than the unusual insertion?

    Sincerely,

    Jack

    1. We have no data that supports that, as far as we know, but we cannot absolutely rule that out. However, recombination in nature, in the lab, or in humans are all possibilities, the very most likely place where humans would come into contact in my view in the lab. We also, however, cannot yet rule out that an infected animal escaped a lab or that person working in a lab was infected. ALL LABS should sequence ALL SARS and bat SARS-like coronaviruses and deposit them ASAP to help w/the molecular epidemiology.

      1. Almost all papers I’ve read starting with the Lancet hypothesize there was an intermediate species between bats and humans. To me, the fact that genomic analysis can’t identify the there species jump should be at least a little disconcerting. Furthermore, how likely is a three species jump and what precedent is there for such a bizarre sequence?

  18. Can you please publish your alignment of the spike protein from the 2019 nCoV to the pShuttle vector? It seems to me the S protein from the nCoV is very different from the one in the vector, which is because what’s in the vector is a truncated version of SARS spike protein. This will disprove your conspiracy therory.

    1. James,
      Your use of the term “conspiracy theory” places your discussion outside of the realm of science.

      Let’s stay focused on hypotheses, please.

      And see https://jameslyonsweiler.com/2020/02/06/molecular-epidemiology-of-spike-protein-sequences-in-2019-ncov-origin-still-uncertain-and-transparency-needed/

      I would question the original paper reporting the sequence they could not match to anything, and the study that called it the “middle fragment”.

      My cursory analysis pointed to pShuttle-SN as “similar”, as is evidenced by the tree w/Spike proteins. Far more information is now available and the direct
      link to the old pShuttle-SN sequences seems less supported. However, the oddities in the database updating that you knew about no doubt played a factor.

      1. This comment is BS. You should know given your background that the cloning vector contains the cloned genetic fragment. Failing to do so is inexcusable or deliberately misleading.

      2. Incorrect. Not all cloning vectors contain a SARS Spike protein element. I think you are not placing sufficient emphasis on the TYPE of cloned genetic fragment. People want to to know WHY recombination research in viruses with such dangerous proteins is ongoing – especially gain of function research.

  19. I’m not sure why you said that the gene was “ Oddly, that Genbank entry does not label the S protein CDS as a spike glycoprotein, instead annotating it only as a “structural protein“. The ORF(21563-25384) is clearly annotated as “Spike protein”! Please confirm.

    1. James you are course correct and I did not annotate WHICH sequence call it only a “structural protein”. I apologize and will look into this.
      All of the Spike proteins are properly labeled, but one and I will search to see where I saw it. I apologize for not providing the accession
      to which I referencing.

  20. Well argued, Informative and convincing.

    The PRC state-run media and institutes have locked the 2019-nCoV origin with a specific natural source such as bat in Yunnan Province of China, heavily denied any possibility re virus-escape from lab.

    I would view it as a knowingly leak of lab-made virus/bio-weapon and by non-state actors. The PRC can have poorly gene-edited babies, certainly they can have poorly designed / managed bio-weapons.

  21. Dear Dr. Lyons-Weiler, You have made a big mistake by ignoring the fact that the pShuttle-SN vector actually contains a cloned gene fragment (1378bp). This truncated gene is actually the S1 subunit of spike protein from SARS-CoV, the domain responsible for interaction with ACE2. In addition, this 1378bp region is present in almost all coronavirus, at least in the B sub-family. Don’t ever trust too much of your meta-analysis. You also need to analyze individually the specific domains. The receptor-bind domain of coronavirus is one of the most variable region of the genome. Your blast search failed to pick up this region was mainly due to the poor similarity.
    You should have analyzed the pShttule-SN before you made the claim. Your claim has been misleading and has already caused big confusion and panic to general public.

    1. Thank you for your feedback, Yinghong. I DID analyze pShuttle-SN, if you re-read the analysis and found that it is clustered with the Spike proteins (See figure). The mere existance of cloning vector that contains a SARS spike protein is a cause of concern given that so many have warned against gain-of-function research in these very viruses. I’m certain that the human toll is sufficient cause for panic. The fact that many are attributing the increased virulence to the ACE2 receptor binding potential due to differences in the 2019-nCoV spike protein is cause for concern. Remember that two peer-reviewed studies reported a fragment that they could not align, and I found the match to the Spike protein. It’s not merely the match – it’s also the clinical presentation that matches that of animals who had previously been exposed to recombinant SARS spike protein-based vaccines. Wouldn’t it be wonderful if all of the attention on the exact nature of differences in the 2019-nCoV Spike protein compared to the rest of the B sub-family of Coronaviruses gave a hint at a treatment such as an antiviral?

  22. Dear professor,
    I knew little about virus proteins. But I blasted the biggest ORF in the 1378bp (from ATGGAAAGTGAGTTCAG to end),and I found this ORF possesed high homologous with SARS protein and especially, there are quite a number of silent mutations at the third base, just like an artifical design for some purpose.
    This probelm may be too big to hide.

    1. Silent mutations at third positions should outnumber the first and second position nucleotide position changes.

      A statistical test of your hypothesis would be to compute the pairwise distance for 1st, 2nd and 3rd positions only
      for the pair of coronaviruses in question, and of for coronaviruses that have the same overall distance and see if there
      is an equivalent distribution of changes in all three positions. The sequence of interest would stick out if there was
      falsification of data as you imply, from an excess of the 3rd position changes.

  23. It is easy to explain the low score alignment region:
    S-protein of the virus determines the host species. The first 300 amino acid of S-protein of the Wuhan virus is aligned to other coronaviruses as well as SARS’. However, the nucleic acid sequence of the fragment is not aligned to any of the S-protein, except for Wuhan virus.The only reasonable explanation is the amino acid sequence was manually reverse translated into nucleic acid sequence

    1. Interesting hypothesis, however there appears to be a truncated NTD motif. And other changes that may be unique to the pathogenicity –
      traceable back to 2005.

      I think the hypothesis of lab origin is falsified, working on the write-up now.

    2. Thanks Stanley. Extremely valuable insight … would be good to see your claim examined by virology specialists… I think you caught the good doctor a little flat footed.

  24. Dear Dr. Lyons-Weiler, 80 percent I think you are right. Except science analyze also many other reasons available…

      1. As they should be. Unforunately, their analysis does not add new information and does not rule out laboratory escape.

        I have ruled out labroatory origin w/out their input based on extensive phylogenetic analysis.

      2. Are you planning to publish your findings somewhere? Love to see you put put that into action.

      3. Dr. Lyons-Weiler;

        You state:
        “does not rule out laboratory escape. … I have ruled out labroatory origin”

        Logically, I would expect something that “escaped” from a laboratory, was therefore previously in a laboratory, and thus had the laboratory as its origin.

        The paper (“Rebuttal to the claim of formation via laboratory
        recombination”) makes the statement “The pShuttle-SN should
        not be called as a vector but a plasmid generated from Adeno-X(TM) to study SARS-CoV.”

        That sounds like a laboratory engineered change, yes? Albeit for the purposes of study, but a laboratory origin nonetheless.

        But I gather there is some nuance I’m missing. Could you kindly explain how something that escaped *from the laboratory* is not considered to have *orginated in the laboratory*, please?

      4. 1. Bring a bat into the lab. Isolate the virus. Grow it in a test tube. Infect yourself along the way. Bring it home. =Lab escape.

        2. Isolate a virus. Add a gene. =Lab modified.

        Lab origin does require lab modified

      5. Thank you;
        “2. Isolate a virus. Add a gene. =Lab modified.”

        Does not “a plasmid generated from Adeno-X(TM) to study SARS-CoV” qualify as lab modified?

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