Thread by @stevenemassey

Zhengli Shi of the WIV constructed a dangerous Gain of Function MERS chimera that contaminated pre-pandemic rice sequence datasets from Wuhan

Potential violation of the Biological Weapons Convention

NIAID + USAID provided funding Parallels with SARS2 🧵 #DRASTIC

@RepBradWenstrup @DrJBhattacharya @joniernst 1/ In this thread, I'll show that a dangerous Gain of Function chimera, involving insertion of MERS spike into a novel merbecovirus backbone, can be positively attributed to Zhengli Shi of the Wuhan Institute of Virology, and that NIAID + USAID funds contributed to its creation

2/ A preprint describing the analysis has been 'on hold' for almost 3 weeks at arxiv, in another case of suppression of 'alert' reports.

In the meantime, a copy can be found here:

zenodo.org/records/179232…

3/ Abbreviations used:

GOF Gain of Function
IC infectious clone
CoV coronavirus
WIV Wuhan Institute of Virology
MERS Middle Eastern Respiratory Syndrome
FCS furin cleavage site
CMV Cytomegalovirus
HDV Hepatitis D virus
BGH Bovine growth hormone

4/ Previously, @humblesci Daoyu @AlmanaLepiz2252 @ydeigin @BiophysicsFL @quay_dr and myself published a paper that described a novel merbecovirus IC contaminating pre-pandemic rice sequencing datasets (NCBI Bioproject PRJNA602160) from Wuhan

fortunejournals.com/articles/disco…

5/ The novel merbecovirus was termed 'HKU4r-HZAU-2020'

'HKU4r' indicates it was related to bat CoV HKU4 (a MERS-related merbecovirus)
'HZAU' refers to Huazhong Agricultural University, Wuhan, who sequenced it
'2020' is the year it was uploaded to the NCBI

6/ The HKU4r-HZAU-2020 genome was flanked by plasmid sequences, indicating it was an IC 👇

The plasmid sequences allow the virus genome to be expressed and 'live' virus recovered when the IC is transfected into a mammalian cell line

7/ ICs allow 'live' viruses known only from their sequences to be generated

This applies to viruses identified in metagenomic datasets, ancient and extinct viruses (such as 2018 'Spanish' flu), and synthetic, genetically enhanced viruses

8/ The construction of ICs can be considered 'Dual Use Research of Concern' (DURC) if they may potentially be used for protective purposes, or for malign, offensive purposes

This especially applies if the ICs have enhanced pathogenicity using dangerous GOF

9/ Of particular concern was evidence of an additional MERS-chimera in the datasets, with the MERS-CoV spike inserted into the HKU4r-HZAU-2020 backbone

The chimera is referred to here as 'HKU4r-HZAU-2020-MERS(S)'

10/ The HKU4r-HZAU-2020-MERS(S) chimera represents dangerous GOF, because MERS spike receptor binding domain (RBD) has superior affinity for the human receptor than that of HKU4r-HZAU-2020 likely increasing human infectivity. Below is a figure from our paper

11/ MERS-CoV spike also possesses a FCS (PRSVR) at the S1/S2 boundary, which HKU4r-HZAU-2020 lacks

This is also expected to increase infectivity in humans compared to HKU4-CoV, as Zhengli Shi and Peter Daszak demonstrated in Yang et al (2015)

journals.asm.org/doi/10.1128/jv…

12/ Not only that, as described in the same study, MERS-CoV spike has an additional cleavage site for human endosomal cysteine protease (hECP), in comparison to HKU4 (and HKU4r-HZAU-2020), that also increases infectivity in humans compared to HKU4-CoV

13/ The presence of each of these 3 features mean that insertion of the MERS-CoV spike into the HKU4r-HZAU-2020 backbone would have a high probability of increasing human infectivity of the chimera, and so represents dangerous GOF

14/ Given the inherent unpredictability of chimera creation, there is always a possibility that such a chimera might increase mortality and/or transmissability compared to MERS-CoV itself

MERS-CoV has a 22-36 % mortality rate

15/ Our work contributed to the updated German Intel assessment of a lab leak origin of COVID19 from 80-95 %

https://x.com/stevenemassey/status/1899928979232166103?s=20

16/ I subsequently traced the provenance of the HKU4r-HZAU-2020 IC and HKU4r-HZAU-2020-MERS(S) chimera, as our paper did not identify the source lab

17/ Pre-pandemic there were 2 approaches to constructing CoV ICs
developed by Baric, and Luis Enjuanes / Volker Thiel (Grok)

Zhengli Shi interacted with both Baric and Enjuanes

18/ Baric used in vitro ligation, while Enjuanes used a Bacterial Artificial Chromosome (BAC) plasmid approach

According to Baric's Congressional testimony, Shi did not use his approach, apparently preferring the BAC approach instead

19/ Pre-pandemic no group had published CoV ICs in Wuhan other than Zhengli Shi (Grok)

I examined all Shi's CoV IC work. In 2016 she published a novel CoV IC plasmid called 'pBAC-CMV', with Peter Daszak of EcoHealth Alliance

journals.asm.org/doi/10.1128/jv…

20/ pBAC-CMV was constructed using the BAC method pioneered by Luis Enjuanes of Centro Nacional de Biotecnología, Spain, a colleague of Zhengli Shi

This involved adding the CMV promoter, and HDV ribozyme / BGH terminator to a pBeloBAC11 plasmid backbone

21/ The work creating pBAC-CMV was funded by NIAID grant R01AI110964 'Understanding the risk of bat coronavirus emergence' of which Daszak was PI and Shi co-PI

documentcloud.org/documents/2105…

22/ Using the description of pBAC-CMV in Shi's 2016 paper 👇 it can be shown that the use of pBelo-BAC11 as a backbone, restriction sites (with 1 difference), CMV, HDV and BGH sequences, and PCR primer sites perfectly match those in the HKU4r-HZAU-2020 IC

23/ In my preprint I give exact details and provide an annotated sequence of the HKU4r-HZAU-2020 IC illustrating its corresponding pBAC-CMV features

Here is a figure that shows the key features of the HKU4r-HZAU-2020 IC

24/ Consequently, the plasmid flanking the HKU4r-HZAU-2020 genome can be identified as pBAC-CMV, whose Ultimate Origin is Zhengli Shi's lab

25/ pBAC-CMV was not widely shared, and only one publication without Shi as co-author mentions the use of pBAC-CMV (to express a MERS-CoV replicon), supplied by Shi

These observations imply that the Proximal Origin of the HKU4r-HZAU-2020 IC is Shi's lab

26/ Enjuanes has so far has avoided scrutiny

It is an open question what technical input Enjuanes contributed to the creation of pBAC-CMV, and the HKU4r-HZAU-2020-MERS(S) chimera discussed below

He has a strong interest in MER-CoV proteolytic sites
x.com/i/grok/share/s…

27/ Given a lab leak scenario, Baric alluded to the possibility of European tech input in his testimony to Congress, but failed to elaborate

However, his reference to 'baculoviruses' is misleading, Shi used BACs not baculoviruses

28/ After identifying the plasmid flanking HKU4r-HZAU-2020 as pBAC-CMV, I then considered the HKU4r-HZAU-2020 genome itself

HKU4r-HZAU-2020's RdRp gene has a 100 % match over 399 bp to HKU4-related isolate 152762 (Accession MN312732)

29/ Isolate 152762 was collected by Shi from lesser bamboo bat Tylonycteris pachypus, from Southern China

Its RdRp sequence was published in Latinne et al (2020)

nature.com/articles/s4146…

30/ Its collection and sequencing was funded by NIAID grant R01AI110964 and USAID PREDICT GHN-A-OO-09-00010-00, as reported in Latinne et al

31/ HKU4r-HZAU-2020 has a 25bp poly(A) tail

This motif is characteristic of Shi's constructs:

A 25 bp poly(A) tail was added by Shi to the 3’ end of a MERS-CoV replicon inserted into pBAC-CMV, and to a HKU5-CoV IC

32/ The RdRp sequence match confirms that the HKU4r-HZAU-2020 IC was constructed by Zhengli Shi

This confirms that the HKU4r-HZAU-2020-MERS(S) chimera was constructed by Zhengli Shi

33/ By mapping the reads from Bioproject PRJNA602160 to pBeloBAC11, it can be shown that the pBeloBAC11 backbone of the HKU4r-HZAU-2020 IC is complete and without breaks

This means the HKU4r-HZAU-2020 IC is circular and functional

34/ Circular BAC plasmids are transformable and better at transfection, compared to linearized plasmids

This presents a biosafety hazard, and a possible means by which a lab worker could become infected via exposure to the IC DNA

35/ Baric in his Congressional testimony, claims that his method avoids that possibility, presumably partly because the ligated product is not clonable into bacteria, and also because it was ligated in BSL3

36/ The MERS-CoV spike sequences found in Bioproject PRJNA602160 are fused to HKU4r-HZAU-2020 sequences, indicating a chimera

The absence of 5 and 6 cutter restriction sites at the junctions indicate that No See'm technology, pioneered by Baric, was used

37/ The MERS-CoV spike sequence has a SNV, C21695T

Significantly, the SNV is located in the only BsaI site in MERS-CoV spike

The SNV ablates the BsaI site, which would be necessary if BsaI were used for insertion of the MERS-CoV spike into the HKU4r-HZAU-2020 backbone

38/ This is only 1 of 2 SNVs in the MERS-CoV spike sequence, and confirms the use of BsaI in No See'm manipulation of the MERS-CoV spike sequence during construction of the chimera

39/ Baric's justification for the No See'm approach is that it avoids the need to modify a wild type virus sequence

However, this argument seems incorrect, as the approach often involves removal of inconvenient sites, as in the example provided here

What is the true purpose of No See'm ?

40/ Baric, in a working paper on biological weapons, notes that the No See'm approach can be used to insert foreign genes into a virus, leaving no evidence of the manipulation

jcvi.org/sites/default/…

41/ The insertion of a foreign gene sequence could still be detected using phylogenetic approaches. However, No See'm would allow the insertion of smaller sequences motifs, such as proteolytic cleavage sites, without detection

42/ The HKU4r-HZAU-2020-MERS(S) chimera was likely a complete IC

This is because Shi ligated entire ICs in one go, not in a stepwise fashion

The presence of an inserted spike gene therefore implies the presence of the complete IC

43/ As co-author of Yang et al (2015), Zhengli Shi was well aware of the FCS and hECP cleavage sites in MERS-CoV spike, and their effects increasing human infectivity compared to HKU4-CoV

44/ From the Year 5 report of grant R01AI110964, Shi would also have been aware of the enhanced affinity of MER-CoV RBD for the human receptor DPP4 compared to HKU4r-CoVs, due to her chimera experiments where she added HKU4r-CoV RBDs to a MERS-CoV backbone

45/ Given these 3 factors, Shi would therefore have been aware of the risk of inserting MERS-CoV spike into a HKU4-related backbone, and that such a chimera would have represented dangerous GOF, as the new spike would have enhanced the human infectivitiy of HKU4r-HZAU-2020

46/ I strongly suspect that 1 of the 4 HKU4r-CoVs that had their RBDs inserted into the MERS-CoV backbone in the Yeatr 5 report was HKU4r-HZAU-2020

Peter Daszak, EcoHealth Alliance and NIAID should release the RBD sequences, and the flanking spike sequences

47/ A scientific reason for Shi's insertion of MERS-CoV spike into a novel merbecovirus backbone is elusive

I discuss the difficulties in understanding a scientific purpose for the construction of the chimera in my preprint

48/ If anyone who can think of a scientific rationale for construction of the chimera please let me know

Given no protective benefit to its construction, it appears to violate Article I of the Biological Weapons Convention (BWC)
ihl-databases.icrc.org/en/ihl-treatie…

49/ Sadly, the USA may have violated Article III of the BWC by contributing to construction of the chimera via NIAID / USAID

I do not think this was the will of the US taxpayer or their Representatives, and so there should be an inquest into how this was allowed to happen

50/ Presumably, the WIV Institutional Biosafety Committee (IBC) approved construction and use of the HKU4r-HZAU-2020-MERS(S) chimera

This may help to explain the unwillingness of the WIV to undergo inspection in the wake of the COVID19 pandemic

51/ Alternatively, its construction may have gone ahead without the knowledge of the WIV IBC

They results reported here suggest Zhengli Shi may have conducted additional undeclared dangerous GOF experiments on novel bat coronaviruses from S. China prior to the pandemic

52/ The analysis allows the following conclusions to be made:

i) The WIV was making undisclosed infectious clones of novel beta-coranaviruses from Southern China immediately prior to the pandemic

53/ ii) The WIV was conducting dangerous GOF on novel and undisclosed beta-coronaviruses via manipulation of the spike gene using No See’m technology

54/ iii) The WIV made a chimera inserting a spike sequence that had enhanced binding to its human receptor, and which included a FCS at the S1/S2 boundary, lacking in the original spike

The spike donor MERS-CoV has 22-36 % mortality

55/ iv) The WIV leaked complete transfectable infectious clones and chimeras into other experiments

v) WIV and EcoHealth Alliance virus hunting expeditions sampled the novel CoV used to create the undisclosed dangerous GOF chimera

56/ vi) The WIV used grants from NIAID and USAID to fund work that led to the creation of a dangerous GOF chimera that incorporated key pathogenicity factors from MERS-CoV

57/ vii) The WIV Institutional Biosafety Committee apparently approved dangerous and reckless GOF work on novel bat coronaviruses, which may explain the WIV’s reluctance to undergo an audit

58/ viii) Coronavirus infectious clone technological innovations by Western scientists were adopted by WIV researchers in their pursuit of dangerous GOF

59/ ix) A scientific rationale for creation of the HKU4r-HZAU-2020-MER(S) chimera is elusive, and so it may violate the Biological Weapons Convention

60/ Creation of the HKU4r-HZAU-2020-MERS(S) chimera has several parallels with lab leak theories for the origin of SARS2, as follows:

a) use of a novel undeclared betacoronavirus from S. China as a backbone, sampled via PREDICT

61/ b) a FCS was acquired at the S1/S2 boundary, absent in all members of the backbone lineage

c) enhanced binding of spike RBD for the human receptor was acquired

62/ d) a BsaI site was used for virus genome construction

e) Zhengli Shi and Peter Daszak were instrumental in creation of the chimera, with US funding

63/ f) a leak of a transfectable dangerous GOF infectious clone occurred, confirming lax biosafety

g) an environment where dangerous GOF coronavirus chimeras could be constructed with or without approval of the WIV's Institutional Biosafety Committee

64/ If SARS2 is an (escaped) IC then Shi's pBAC-CMV is its likely expression vector

Consequently, the pBAC-CMV sequence may inform considerations of the SARS2 genome and its restriction sites
biorxiv.org/content/10.110…

65/ If you would like to make the preprint describing this work censor-proof, using the method of @Kevin_McKernan, please zap sats to the lightning address 👇

Alternatively, send to bitcoin address:

bc1qypgcyeyx54vgz59qlwdp39gxvj4jkzpd3tvkgf

@Kevin_McKernan 66/ These will be used to setup a Bitcoin node, and for the transactions on bitcoin and a file storage blockchain

Kevin's method harnesses the censor resistant properties of bitcoin to preserve sensitive scientific reports 👇

#DeSci
anandamide.substack.com/p/immortalizin…

@Kevin_McKernan 67/ h/t @humblesci @AlmanaLepiz2252 @ydeigin @BiophysicsFL @quay_dr @BillyBostickson @R_H_Ebright DRASTIC, USRTK, and all the dogged Origins investigators

"When I grow up, I gonna be a DETECTIVE"

@Kevin_McKernan @humblesci @AlmanaLepiz2252 @ydeigin @BiophysicsFL @quay_dr @BillyBostickson @R_H_Ebright @threadreaderapp unroll