Thread by @WuhanChupacabra

archive.md/2sAST
No matter how you slice the data the issue is simply that the low total abundance species effect is easily countered against by comparison of correlation within the positive samples themselves

The most raccoon dog DNA are actually found in negative stalls and samples—and it failed to correlate at all within W6-29-33 (negative correlation) or within the positive samples, or within any samples.
archive.md/csYBM

Homo Sapiens is in every sample and was the only reliably and consistently positively correlated species within W6-29-33.
archive.md/JSQvc

It is all toilets and sampler boot prints.
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The stall with most positive samples out of all sampling was W4-26-28, the stall closest to the toilets.
archive.md/gvHfw archive.md/RirQ7

In fact in both Jan01 and in Jan12 the stall with most positive samples were the stall closest to the toilets—W4-28(2/2), outside surface of W4-26-28(5/6) for Jan01, and W6-29-33 in Jan12 was the closest sampled stall at that day to the toilets.

archive.md/CTP3i archive.md/ETjzS archive.md/BWZJL
The rules: must be contacted by a sampler. 🥼👖👢=positive.
archive.md/NeybM archive.md/2PM9Y archive.md/RirQ7
Must not be frequently handled by a vendor. 🥩🥬🍄🛁📻☎️📦🧺=negative.

Both sampler-linked contamination and differential stability of SARS-CoV-2 RNA on surfaces causes the results.

sciencedirect.com/science/articl…
SARs-CoV-2 RNA stability: Fresh PPE> New bangladesh banknotes > Old bangladesh banknotes.
nature.com/articles/s4159…
medrxiv.org/content/10.110…
Wastewater from toilets>high touch surfaces in train stations.
imperial.ac.uk/news/213133/im…
High touch=low stability.

archive.md/rSaO9 archive.md/ef2JW This is such an obvious problem, if the issue with toilets was not properly addressed the "wildlife stalls" myth just become toilet-associated contamination mediated by samplers, they bleached the toilet area before sampling it.

archive.md/LJzSO archive.md/4cCHG archive.md/YGDiK Q61 is caused by cross-contamination.

Q70 was also caused by cross-contamination.

Q37 is caused by contaminated sampler suits touching the lip of the sample tube during sampling of an awkward position (thus contaminating the medium only at the second withdrawal for NGS) and then confirmation bias on snakes caused specific, poorly controlled sampling that

onlinelibrary.wiley.com/doi/10.1002/jm…
Contaminated the stall with artifacts in a later date, even though the location of Q37 was no longer positive. As reads align over the ORF1ab qPCR primers, negative PCR indicate contamination of sample happened after PCR and before NGS.

Jan01 sampling there were also negative.

PCR-/NGS+ mean contaminated sample—the convention is to verify RNA-seq results with qPCR after determine which sequence to look at and design primer on. NGS especially the Illumina platform (43bp libraries) are highly prone to process-linked and cross-contamination due to their

Complicated library preparation process. Cross-contamination is a form of systematic bias,
archive.md/LJzSO
And it occurred Alongside one less systematic process-linked contamination instance as well in Jan12 sampling.

The sample positivity in the market is more predicted by object type than epidemiology—none of the “everyday objects” Even those used by human cases in human case stalls were positive, even stalls with positive samples and no wildlife DNA in these samples.
archive.md/NeybM

Sampler👖👢🥼=positive. archive.md/CTP3i
Vendor🔪🥩🧺📦🥛📱🪜🛁📥=negative.
archive.md/ETjzS
archive.md/BWZJL
archive.md/NeybM
archive.md/KLkHS

SARS-CoV-2 RNA is actually highly unstable on high-touch surfaces, such as vendor items, knives and chopping blocks, cashiers, phones, monitors, ladders, fishbowls, boxes and baskets. It is most stable within sewage and on fresh sampler PPE.

We do know that if something is frequently handled (vs not handled and clean), nature.com/articles/s4159…
The stability of SARS-CoV-2 RNA is reduced from over a month to less than 3 days.
ncbi.nlm.nih.gov/pmc/articles/P…

hutchisonintl.com/product-page/c…
Unfortunately, whatever “non-inactivating virus preservation solution” of Gao et al didn’t contain an RNAse inhibitor. (See the list of components).

Once inside the liquid medium, RNAses is free to diffuse and if present, destroys all cell-and virion-free RNA and SARS-CoV-2 virion RNA within minutes and hours during sample transportation and storage.
archive.md/NeybM

medrxiv.org/content/10.110…
imperial.ac.uk/news/213133/im…
This applies to objects and conditions for public spaces that are outside the market as well. Toilet contamination lead to positives, direct handling lead to negative results.
archive.md/BWZJL archive.md/2PM9Y

Of course, the real problem here is that raccoon dogs are just not susceptible at all to natural infections by any strain of SARS-CoV-2.
archive.md/yyX0Z archive.md/4rVph archive.md/iw1Pz archive.md/DChUL

No evidence at all that any non-human animal shed the SARS-CoV-2 in that “stall”.
archive.md/T2u0S

archive.is/QWngz
Also, A20 was the least consistent sample out of all Gao et al “samples” in the “data”. archive.md/ANS4Q

In fact, the other major challenge for archive.md/q4ZDV archive.md/QkFbk fur farmed species in general is that fur farming is practiced only in Northern China, where no SC2r-CoV is found in bats—and their low pricing have meant that any kind of smuggling of wild-

Caught specimens unprofitable, restricting any such supply to within the municipal borders of Wuhan—again all negative.

archive.md/HIJ9o archive.md/2sAST
It arrived on two legs. Not four legs.
sciencedirect.com/science/articl…
vis.csh.ac.at/sars-ani/
archive.md/e3615 archive.md/vWjZl
archive.md/LJzSO

They in fact, did trace to the suppliers to the HSM. Results are all negative.
academic.oup.com/ve/article/8/1…
Raccoon dogs that were tested in China were also negative, the same result as their European bretheren. pubmed.ncbi.nlm.nih.gov/35298912/

archive.md/n9o0f archive.md/vL6U4 archive.md/7doR8 archive.md/JSQvc

archive.md/bzeP1 archive.md/Ttn6P

Bad ACE2, failure for correlation,
archive.md/n9o0f archive.md/vL6U4 archive.md/0A24q
archive.md/2sAST archive.md/bzeP1 archive.md/Ttn5P
No species other than human
archive.md/JSQvc
archive.md/csYBM
archive.md/T2u0S

archive.md/8JhAb are responsible for shedding the SARS-CoV-2 in the market.

Query “which species shed the virus in the stall W6-29-33” give the same result as querying “which species shed the virus in the positive samples”.
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archive.md/JSQvc
–Both normalizes against bad spatial distribution and unrelated confounding causes.

archive.md/LJzSO
Them not banning fur farming clearly indicate that they weren’t thinking them as “public health hazards”.
As long as there is even a single non-food use of a species, it is simply relabeled as “livestock” and returned to the markets.

https://twitter.com/McWLuke/status/1346437062732509185?s=20

ncbi.nlm.nih.gov/pmc/articles/P……Investigation using serological testing of SARS-CoV-2 in foxes, jackals or wild boars, as well as animals in the zoo in Croatia, found no evidence of either SARS-CoV-2 RNA or neutralizing antibodies to SARS-CoV-2.

sciencedirect.com/science/articl…
They tried to test their raccoon dog and fox farms—all of which are found in northern China because fur animals need a winter to have their pelts to mature—and zero infections were found. They are all kept where bats with the right CoVs were completely

Absent and where transmission from bats to any of these farmed canids are not possible.
academic.oup.com/ve/article/8/1…

Freuling is fraudulent. journals.plos.org/plosone/articl…
pubmed.ncbi.nlm.nih.gov/36423168/
ncbi.nlm.nih.gov/pmc/articles/P…
Despite active surveillance in Europe, no reports of raccoon dog infections were seen and no replication of Freuling et al were ever confirmed.

mdpi-res.com/d_attachment/v…… woah.org/app/uploads/20…
fao.org/animal-health/…
woah.org/app/uploads/20…
vis.csh.ac.at/sars-ani/

https://twitter.com/AlexBerenson/status/1638546828839747584

eur-lex.europa.eu/legal-content/…
Despite monitoring programs.
woah.org/app/uploads/20…
who.int/europe/publica…
who.int/europe/publica…
who.int/europe/publica…
who.int/europe/publica…
who.int/europe/publica…
who.int/europe/publica…
who.int/europe/publica…

journals.asm.org/doi/full/10.11…
no natural infections of raccoon dogs detected here either.

journals.plos.org/plosone/articl…
ncbi.nlm.nih.gov/pmc/articles/P…

https://twitter.com/RealChrisCRISPR/status/1637344975242379265?s=20

the only law that was passed in China in response to the pandemic mentioned et markets in only one article: “It is forbidden to introduce used experimental animals into the market”. “禁止将使用后的实验动物流入市场”.
npc.gov.cn/npc/c30834/202…
chinalawtranslate.com/biosecurity-la…

Minimal fines: CNY 200000 (US $29850+)—20 times the maximal fines for selling equivalent wild animals seen in the Xiao Xiao list) for selling or otherwise allowing used experimental animals into the marketplace. They clearly were concerned on 🐁🐒>🦝🦔

Despite surveillance, no raccoon dogs were ever found naturally infected anywhere in the world. As for the other species? Not even experimental evidence of infection is found.
archive.md/4rVph archive.md/yyX0Z archive.md/DChUL

Nor were old-world deer or muntjacs either.
pubmed.ncbi.nlm.nih.gov/35456800/ biorxiv.org/content/10.110…
ncbi.nlm.nih.gov/pmc/articles/P…
onlinelibrary.wiley.com/doi/full/10.11…
ncbi.nlm.nih.gov/pmc/articles/P…
https://t.co/flshtGbl2r
biorxiv.org/content/10.110…
No fox infections before Omicron too.

ncbi.nlm.nih.gov/pmc/articles/P…

There is a systematic form of bias with cross-contamination—same stall are processed on same rack too. archive.md/LJzSO

Why only the stall that is closest to the toilets was positive? The same reason why within that stall only Homo Sapiens reads show consistent and reliable positive correlation with SARS-CoV-2 reads.

All everyday objects (Cashiers, Fishbowls, Chopping blocks and knives, waster cups, phones, monitors, boxes and baskets) in the market as well as all sites where contact with sampler suits, pants and boots are unlikely (surface of food, water in basins) were negative.

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Did they practice aseptic techniques during sampling? Not really. Samplers and cleanup workers go side by side on same days. Most weren’t even researchers—volunteers that have poor knowledge or no knowledge at all on keeping the scene uncontaminated. The samplers brought in

The main source of contamination, not vendors or animals. archive.md/ETjzS
Outer layers of sampler gloves, sampler boot covers left discarded on the site, also happens to be some of the most contaminated samples in the market.

In Jan 01, the most positives were found in stall W4-26-28, the stall closest to the toilets—outside of W4-26-28, 2/2 W4-28 and 3/4 W4-26 (5/9 for all W4-26-28 samples). In Jan12, W6-29-33 is the stall closest to the toilets that were sampled at that time.

(2/2 positive for W4-28, 5/6 positive for outside surface of W4-26-28, 5/9 for W4-26/28) This is characteristic of sampler-linked contamination with the toilets as the main contamination source. archive.md/gvHfw

SARS-CoV-2 RNA is most stable in sewage and things associated with the toilets, and least stable on objects receiving frequent handling.
sciencedirect.com/science/articl…
nature.com/articles/s4159…
archive.md/2PM9Y archive.md/RirQ7

archive.md/ETjzS archive.md/BWZJL
Must be contacted and contaminated by a sampler, must not be frequently and directly handled by a vendor.

Regardless of which slice you use, there is just no evidence at all of positive correlation between raccoon dogs and SARS-CoV-2. archive.md/A892b archive.md/7doR8 archive.md/Ttn5P archive.md/0A24q

Contradicting what C-C claims; the “positive sewage and waster samples” have a host composition that is characteristic of the toilets—and not of the wildlife stalls.

archive.md/NeybM toilets and infected samplers. archive.md/ETjzS archive.md/BWZJL archive.md/KLkHS
Contamination and cross-contamination. archive.md/YGDiK All sewage well samples including those with reads say toilets not wildlife stalls.

archive.md/Sokty archive.md/FskYn
when most if not all your samplers and cleanup workers were private contractors (Jiangwei......) or volunteers you enlist on an emergency, without any serious training on scene preservation or on aseptic techniques

(doubtful that even the WCDC itself received any training on scene preservation or the techniques necessary to prevent unconscious contamination or cross-contamination between objects or from themselves to the objects during sampling, as this is a strictly forensic technique that

only specialist forensic investigators in the police department in China receive mandatory training on), then you are most likely getting far more that were caused by your own contamination compared archive.md/gvHfw to any contamination that was originally there.

These are volunteers, not professional forensic investigators—they are neither trained for evidence preservation or scene preservation, nor are they even experienced in aseptic techniques before other than some simple instructions. These breaks down rapidly once the work begun,

Leading to contamination happening everywhere and becoming the primary and likely only source of positive samples within the market. Then there are decontamination efforts happening inbetween sampling, including the bleaching of the entire toilet area prior to any samples that

May propose an alternative hypothesis than “illegal wildlife trade” were being taken. Also, the list of samples in Gao et al notably did not contain a sample from the toilets themselves. archive.md/rSaO9

Details on object type, touch status and research articles regarding skin nucleases and stability of SARS-CoV-2 RNA in relation to human touch. archive.md/2PM9YThis extend to other surfaces outside the market as well. archive.md/RirQ7
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