'Conincidentally' we showed pUC57 was also used to clone a genome fragment from an unpublished pangolin-related SARS2r-CoV from China 🧵

h/t @humblesci @Daoyu15 @ydeigin @quay_dr

mdpi.com/2673-8007/2/4/…

x.com/Engineer2The/s… 2/ This striking conincidence indicates that the novel pangolin-related coronavirus we identified from contaminated datasets (GX_ZX45r-CoV) was likely part of an infectious clone (IC), given that one of the reads was ligated to pUC57

From our paper:

I have published a paper on my analysis of the RaTG13 dataset

RaTG13 is the closest phylogenomic relative of SARS2

Transcriptome and GO enrichment analysis indicates the likely source of the dataset is a bat mating plug, rather than a fecal swab 🧵

mdpi.com/2036-7481/15/3… A mating plug provenance resolves the long standing conundrum over the low amounts of bacteria in the RaTG13 sample, noted by @MonaRahalkar and others, which is inconsistent with a fecal swab as reported by the WIV, who collected the sample

A leak of polio from a research facility is indicated by a poliovirus genome sequence generated from a sample collected in 2014 in China by the Wuhan Institute of Virology

The sequence is 99 % identical to that of a polio strain from 1954 🧵
academic.oup.com/ve/article/10/… 2/ Chesnais et al sequenced 3 poliovirus genomes recovered from 60 year old historic samples generated by Albert Sabin at the Pasteur Institute, Paris

Sabin is the father of the oral polio vaccine, created from attenuated polio strains

Why are virologists freely allowed to anonymize lethal synthetic viruses, but developers are put in jail for writing code that anonymize bitcoin transactions ?

Genetically enhanced infectious clones present a much higher risk than anonymous monetary transactions 🧵 @R_H_Ebright @SenGaryPeters @COVIDSelect @BiosafetyNow @CharlesRixey @HSGAC_GOP @RepBradWenstrup @RepRaulRuizMD Virologists often synthesize infectious clones (ICs), which are used to produce live infectious viruses

To make an IC of a coronavirus, due to its large size constituent fragments need to be synthesized, and then ligated together to form a complete genome

Unravelling the RaTG13 Dataset's Origin

The RaTG13 dataset has overexpressed transcripts which indicate it was mis-sampled from a bat mating plug

This provides a reasonable explanation for anomalies in the dataset, described as a 'fecal swab' 🧵 2/ RaTG13 is (still) phylogenomically the closest coronavirus backbone to SARS2

There has been a longstanding controversy regarding the origin of the RaTG13 dataset

RaTG13 is described as being generated from a Rhinolophus affinis (Intermediate Horseshoe bat) fecal swab

Re-watching this video, it becomes clear why virologists have lobbied so hard to detach the names of novel pathogens / strains from the location where they arise

"the disease is the same name as the lab" 😆

https://twitter.com/stclairashley/status/1803952107902456202

Detaching regional / place names from pathogens is a form of anonymization eg delta, omega etc

Why would virologists want to use names which are arbitrary and non-informative ? geographic information is an important part of epidemiology and helps understand origin and spread

Some further insights into the SARS2 spike sequences found in Pseudomonas aeruginosa datasets, recorded as being sampled in 2019 🧵 2/ Complete SARS2 spike gene sequences were found in contigs generated from Pseudomonas aeruginosa cultures sampled in 2019, by @iximeno

The spike sequence displayed codon optimization and lacked the furin cleavage site

Differential gene expression analysis of the controversial RaTG13 dataset reveals strong similarity to the RaTG15 dataset, also described as generated from a Rhinolophus affinis 'rectal swab' from the Mojiang Mine

This indicates they have a common, undefined source 🧵 2/ The source of the RaTG13 dataset has been a key puzzle of the C19 Origin debate

RaTG13 was sequenced by the Wuhan Institute of Virology prepandemic in 2017/2018 and remains the closest related CoV backbone to SARS2

The Zhang group of Fudan University have identified and validated two A-B intermediate SARS2 genomes from the early pandemic

This provides a key to understanding the origin of COVID19 🧵 2/ In their new paper, the Zhang group sequence 343 new SARS2 genomes from the early pandemic (sampled up to Oct 2020). The genomes were obtained from COVID19 patients in the Shanghai Public Health Center
academic.oup.com/ve/advance-art…

Was Baric aware of the work on the human α-ENaC furin cleavage site (FCS) at the University of North Carolina (UNC) ? 🧵

In a striking coincidence, the human α-ENaC FCS is exactly the same as that of SARS2, as first noted by Anand et al in 2020
elifesciences.org/articles/58603 2/ Furin cleavage of human α-ENaC has been studied by M.Jackson Stutts, who is based at the UNC School of Medicine

Ralph Baric is also based at UNC School of Medicine, and also has an interest in lung disease. Was he aware of this work ?

rupress.org/jgp/article/15…

What was the restriction enzyme BsmBI, itemized in the DEFUSE budget, to be used for? 🧵

3 possibilities:

i) To make an spike ectodomain expression vector
ii) To insert recombinant S genes into VRP vectors
iii) To make infectious clones (ICs) 2/ I will consider each of these possibilities as follows

A potential explanation for the alanine (A684) in the SARS2 FCS ?

Alanine mutational scanning is used to systematically mutate residues in functional sites, to determine if they ablate function, so indicating their importance 🧵

en.wikipedia.org/wiki/Alanine_s… 2/ In the recent FOIA-ed DEFUSE documents released by @emilyakopp and @USRightToKnow there is a section that proposes to 'ablate' 'human-specific cleavage sites' introduced into SARSr-CoV spike proteins

Bombshell !

From an early draft of DEFUSE:

"The BSL-2 nature of work on SARSr-CoVs makes our system highly cost-effective relative to other bat-virus systems"

https://twitter.com/emilyakopp/status/1736848388350459913

Baric was aware of the lax safety standards at the WIV, in the following draft note:

" In china, might be growin these virus under bsl2. US reseachers will likely freak out"

But put his name to the proposal anyway

This statement about DEFUSE by Eddie Holmes is misleading. DEFUSE describes the identification of potential FCSs in the spike gene of SARSr-CoVs. FCSs are typically found at the S1/S2 boundary...... 🧵 2/ To quote DEFUSE: 'We will analyze all SARSr-CoVs for appropriately conserved proteolytic cleavage sites in S2 AND (my capitals) for the presence of potential furin cleavage sites'

When were the only two known pre-pandemic SARS2-related live isolates (attributed to pangolins) isolated ? And when were they sequenced ?

And why does one of them have SARS2 reads in its sequence file ? 🧵 2/ There has been a lot of interest in the provenance of the RaTG13 sequence, as it is still the closest related backbone to SARS2

ZC45 and ZXC21 were the only other known SARS2-related genome sequences generated in China pre-pandemic

Or were they .......?

A couple of snippets from the surprisingly sparse recent ODNI report on the origin of C19 consisted of the claim that the WIV worked on pangolin samples in 2019, and that there was PLA involvement in coronavirus research there 🧵

2/ Do they have some forensic genomic data or inside sources ?

Unfortunately maybe not - it seems that the pangolin claim may have derived from the recent 2023 paper by ZLS and Yigang Tong on infectivity characteristics of live pangolin isolate MpCoV-GX

journals.asm.org/doi/full/10.11…

A new animal coronavirus, possibly from bamboo rat, is present in abundance in sample Q61 from the Huanan Seafood Market

This find, however, stands in stark contrast to the single SARS2 read present in the same sample, which has a high proportion of raccoon dog sequences 🧵 2/ Sample Q61 was generated by Liu et al in their survey of the Huanan Seafood Market

It has generated discussion for having a high level of raccoon dog reads, which has been postulated by some workers as an intermediate host for SARS2
nature.com/articles/s4158…

New coronaviruses in raccoon dog sample Q61 !!

On Blast searching individual reads from Q61 that matched the human OC43 and HKU1 genomes, the closest matches are to bamboo rat and rodent CoV sequences 🧵

h/t @babarlelephant @humblesci 2/ Previously I had found reads that mapped to human OC43/HKU1 coronaviruses, interpreting this to mean that human coronaviruses were present in the raccoon dog sample Q61

https://twitter.com/stevenemassey/status/1647286339367481346?s=20

What is the true trace level of SARS2 in the raccoon dog sample Q61 from the Huanan Seafood Market ?

Here, in a refined analysis, I show that human common cold coronaviruses are present in quantities greater than SARS2 in Q61 🧵 2/ Q61 is a key sample described by Liu et al in their survey of the Huanan Seafood Market, as it contained large quantities of raccoon dog sequences, leading some to claim that it was evidence that raccoon dogs were the source of the SARS2 pandemic
nature.com/articles/s4158…

I've identified numerous vertebrate RNA viruses in the Q61 raccoon dog sample from Liu et al's survey of the Huanan Seafood Market

Their % genome coverages are substantially higher than that of SARS2, and many are associated with animals identified previously in the dataset 🧵 2/ The Q61 (Env_0576) NGS dataset was mapped against all virus genomes from NCBI, using our coronascan mapping procedure

This revealed an impressive array of RNA viruses (Accessions in purple)

(note, the entries are ranked according to % coverage)

In their survey of the Huanan Seafood Market, Liu et al conclude that there is no evidence for a zoonotic transmission of SARS2 at the market

Out of curiosity, I did a deep dive into sample Q61 (Env_0576), which they report as having a high proportion of raccoon dog reads 🧵 2/ I ran a mitoscan analysis, a procedure developed with @humblesci , that was first described by Csabai and Solymosi in their Antarctic soil preprint that identified early SARS2 sequences associated with a variety of potential mammalian cell lines 👇
researchsquare.com/article/rs-133…