Thread by @petelincoln48

The Road to SARS-Cov-2

1999 paper from the Dutch group of Peter Rottier from Utrecht University with a revealing title Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier.

Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture.

Shi Zhengli coauthored a paper Peter Rottier in 2005, (but her current address was listed at Shanghai Institute). That article itself is quite curious — in it the authors investigated what exactly allows viruses to expand their species tropism:

Only a relatively few mutations in its spike protein allow the murine coronavirus to switch from a murine-restricted tropism to an extended host range by being passaged in vitro.

One such virus that we studied had acquired two putative heparan sulfate-binding sites while preserving another site in the furin-cleavage motif.

It is interesting that the furin site in that virus (SRRAHR | SV) is similar to the site in CoV2 (SPRRAR | SV), although in CoV2 it is cut more efficiently due to dual arginines (this is what makes it a polybasic site it has multiple basic amino acids in the RxxR sequence)

2002 — BEFORE the outbreak of the first SARS-CoV. Ralph Baric published a breakthrough work, which marked a milestone in both the study of various mechanisms of natural viruses and in gain-of-function research.

In their paper, the Baric group described creating a synthetic clone of a natural murine coronavirus:

In essense, the authors have “translated” the RNA virus into the language of DNA (using reverse transcriptase), which enabled them to manipulate its genome with the help of existing genetic engineering tools.

Having created 7 such cDNA provirus segments, the authors then stitched them together “seamlessly” after which they transcribed their construct back into RNA, which was then translated into virus particles in other cells.

2002- , a year before SARS a patent was obtained by the ‘Godfather’ of coronavirus, Ralph Baric. Ralph Baric is a professor of microbiology and immunology at the Gillings School of Global Public Health at the University of North Carolina at Chapel Hill .

While the patent for the vaccine process used for pigs, it can also cover human vaccines made from the same processes

2002- No See’em’ technology developed that allows reverse engineering on viruses to be used without leaving any traces (Yount et al. 2002).

2003-Just a few weeks after the publication of the above work, the first SARS-CoV epidemic broke out. The Baric group sprang into action. By summer of 2003, they had submitted a paper on synthetically recreating SARS-CoV:

Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses that contained the expected marker mutations inserted into the component clones

Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor…

Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

2004- Shi Zheng Li joined an international team of researchers to collect samples from bat colonies in caves near Nanning, the capital of Guangxi.

Shi Tou Cave on the outskirts of Kunming, the capital of Yunnan, where they conducted intense sampling during different seasons over five consecutive years.

The efforts paid off. The pathogen hunters discovered hundreds of bat-borne coronaviruses with incredible genetic diversity. “The majority of them are harmless,” Shi says.

But dozens belong to the same group as SARS. They can infect human lung cells in a petri dish and cause SARS-like diseases in mice

2006- an American team had inserted RRSRR into the spike protein of the first SARS-CoV back :
To facilitate cell–cell fusion activity, we mutated the wild-type SARS-CoV glycoprotein to construct a prototypic furin recognition site (RRSRR) at either position.

2007. Shi Zhengli group joined the synthetic virology race with a study of the spike protein of human and bat coronaviruses, trying to determine what exactly is responsible for the ability to skip from one species to another:

A series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone.

That is, the authors inserted different segments from the human SARS-CoV spike protein into the spike protein of the bat virus. At the same time, they tried to replace shorter fragments, including just the RBM

2007 the Wuhan Institute of Virology and Shi Zhengli published a study in the Journal of Virology in which they combined a SARS-like virus from bats with human immunodeficiency virus (HIV) and created a chimera virus capable of infecting human cells

2010, Shi Zhengli’s team published a paper to examine the sensitivity of different types of bat ACE2 to human SARS-CoV spike protein (S protein) using live SARS virus and HIV (AIDS) pseudovirus.

In the experiment, they also changed several key amino acids of bat ACE2 to test its binding to the S protein and constructed the HIV pseudovirus HIV / BJ01-S with the SARS virus BJ01-S protein.

2012- Shi’s team had been called in to investigate the virus profile of a mine shaft in Yunnan’s mountainous Mojiang County—famous for its fermented Pu’er tea—where six miners suffered from pneumonialike diseases and two died.

Although Shi said in July 2020 the fungus turned out to be the pathogen that had sickened the miners, she says it would have been only a matter of time before they caught the coronaviruses if the mine had not been promptly shut.

Two months later she said she suspected they had an unknown virus

2013- Analyzing samples obtained in 2011-2012 In Shitou Cave—a coronavirus strain was found that came from horseshoe bats with a genomic sequence nearly 97 percent identical to the one found in civets in Guangdong

The finding concluded a decade-long search for the natural reservoir of the SARS coronavirus.

Shi Zhengli and Peter Daszak announced their discovery of RsSHC014 in their 2013 paper, “Isolation and Characterization of a Bat SARS-Like Coronavirus that Uses the ACE2 Receptor,”

and stated that it was found during “a 12-month longitudinal survey (April 2011–September 2012) of SL-CoVs in a colony of Rhinolophus sinicus at a single location in Kunming, Yunnan Province, China.”

2013, Chinese scientists demonstrated the capability to insert a furin polybasic cleavage site, similar to that of COVID-19, into a protein, an article which cited US patent “Insertion of Furin Protease Cleavage Sites in Membrane Proteins and Uses Thereof.”

The holder of this patent is Dennis T Brown out of the UNC , the same UNC that Ralph Baric is based

One of the authors of that 2013 article, Chinese scientist Shibo Jiang, has a joint appointment at the Lindsley F. Kimball Research Institute in New York and Fudan University in Shanghai and is a long-time collaborator of Zheng-Li Shi and Ralph Baric in coronavirus research

2014 Shi Zhengli received a $665,000 grant from NIH for a study titled The Ecology of Bat Coronaviruses and the Risk of Future Coronavirus Emergence as well as $559,500 more from USAID (a DOD partner/CIA front) for a study titled Emerging Pandemic Threats PREDICT_2

2014-The closest coronavirus relative to SARS-CoV-2 with a furin site is the HKU5 strain, isolated by the Shi Zhengli team in 2014 in Guangzhou from bats of the genus Pipistrellus . But it is a very distant relative — their spike proteins share only 36% similarity

It was found that all Spike with a SARS-CoV-2 Spike sequence homology greater than 40% did not have a furin cleavage site (Figure 1, Table 1), including Bat-CoV RaTG13 and SARS-CoV (with sequence identity as 96.2% and 78.6%, respectively).

2015 near Shi Tou Cave Shi’s team collected blood samples from more than 200 residents in four of those villages. It found that six people, or nearly 3 percent, carried antibodies against SARS-like coronaviruses from bats

Only one had traveled outside of Yunnan prior to the sampling, and all said they had seen bats flying in their village.

2015-Baric and Shi Zheng Li coauthor paper in Journal of Virology-“Two Mutations Were Critical for Bat-to-Human Transmission of Middle East Respiratory Syndrome Coronavirus”

They compared the virus surface spikes of MERS-CoV and a related bat coronavirus, HKU4. Although HKU4 spike cannot mediate viral entry into human cells, two mutations enabled it to do so by allowing it to be activated by human proteases.

These mutations are present in MERS-CoV spike, explaining why MERS-CoV infects human cells

2015- November 9 - Ralph Baric and Shi Zhengli published a study (The Ecology of Bat Coronaviruses and the Risk of Future Coronavirus Emergence ) in which they created a synthetic chimeric virus:

Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone.

The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2) and replicate efficiently in primary human airway cells

Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis.

On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo

In other words they took the spike-like protein from RsSHC014, which Shi Zhengli isolated from Yunnan bats in 2011, and inserted it into a murine-adapted variant of SARS-CoV for subsequent in vivo experiments.

They also created a recombinant clone of the same RsSHC014 strain and found that the SHC014-MA15 chimera was more virulent than SHC014 itself, even in human cells and that the ability of a spike protein to be cleaved by proteases (including furin) can have an impact on virulence.

Simon Wain-Hobson, a virologist at the Pasteur Institute in France, expressed this concern deeply, telling Nature: “If the (new) virus escapes, no one can Predict its path.”

2017, Shi and her colleague Cui Jie discovered that the SARS coronavirus likely originated in a population of cave-dwelling horseshoe bats in Township, Yunnan based on Serological testing of nearby residents.

2017-no known bat/human coronaviruses contain the exact sequence of -PRRAR/SVA- that is present in the SARS-CoV-2 Spike protein,

a similar -RRAR/AR- sequence has been observed at the S1/S2 junction of the Spike protein in a rodent coronavirus, AcCoV-JC34, which was published by Dr. Shi Zhengli in 2017

It is evident that the legitimacy of -RRAR- as a functional furin-cleavage site has been known to the WIV experts since 2017.
The furin-cleavage site in the SARS-CoV-2 Spike protein may not have come from nature and could be the result of genetic manipulation.

2018-the Beta- coronavirus RaTG13 was fully sequenced in 2018 (Zhou et al. 2020c) but only published after the identification of SARS-CoV-2 (Zhou et al. 2020b) and more unpublished sequences existed in a WIV database that was restricted after the beginning of the pandemic

2018-ZC45 and ZXC21 are bat coronaviruses that were discovered (between July 2015 and February 2017), isolated, and characterized by military research laboratories in the Third Military Medical University and the Research Institute for Medicine of Nanjing Command

The data and associated work were published in 2018. Some suggest this may be the backbone/template for the creation of SARS-CoV-2,

S2 of SARS-CoV-2 shares a high sequence identity (95%) with S2 of ZC45/ZXC21. In stark contrast, between SARS-CoV-2 and ZC45/ZXC21, the S1 protein, which dictates which host the virus can infect, is much less conserved with the amino acid sequence identity being only 69%.

2018-2019-Shi Zhengli and her collaborators are also funded by the U.S. military. Peter Daszak /EcoHealth Alliance currently receives more money from the Department of Defense’s DTRA for Scientific Research Combatting Weapons of Mass Destruction than any other military contractor

In 2018 “The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity,” that was published with military scientists and DTRA funding.

A virus that has co-evolved with the bat’s antiviral system is completely out of its element in the human....That’s why it is so deadly — the human immune system is overwhelmed by the inflammatory response

The bat immune system responds very differently from ours to viral infection. Instead of attacking and killing an infected cell, which leads to a cascade of inflammatory responses, the bat immune system can starve the virus by turning down cellular metabolism

The military is using its experiments on bat immunity to “develop drugs that dampen down inflammation and arrest the virus by depriving it of what it needs to grow rather than trying to kill it outright. But you can also make viruses more deadly by “passaging” them through bats

Shi’s other DTRA funded paper, “Filovirus-reactive antibodies in humans and bats in Northeast India imply zoonotic spillover,” was published in 2019.

The study alarmed the public by finding “the presence of filovirus (e.g. ebolavirus, marburgvirus and dianlovirus) reactive antibodies in both human (e.g. bat hunters) and bat populations in Northeast India, a region with no historical record of Ebola virus disease.”

In addition to military funding through DTRA, Shi’s paper was co-authored by two U.S. military scientists, Christopher C. Broder and Eric D. Laing of the Uniformed Services University of the Health Sciences, Department of Microbiology and Immunology.

2019-External access to batvirus.whiov.ac.cn which is administered by Shi Zheng Li ended on Sept 12, one month before Event 201. Batvirus.whiov.ac.cn had been online for a few years. Sequenced virus data includes viruses found and sequenced as part of funding from US govt

Part of batvirus.whiov.ac.cn data was confidential and password protected. That private data includes:

(1) Sampling data of viruses not yet sequenced and (2) sequences of viruses that have not yet been the object of publication. (3) sampling data from sites that the WIV prefers not to make public.

Partial funding of samples collection and sequencing via the NIH does not secure ownership of resulting data. Key aspects of the WIV research were partially funded by the NIH/NIAID via EcoHealth and USAID /PREDICT 2

2019 Shi Zhengli authors a review article in Nature Reviews Microbiology 181–192(2019) reporting on of 89 new “novel bat coronavirus” strains that use the same receptor as the coronavirus known as Middle East Respiratory Syndrome (MERS).

That study was jointly funded by the Chinese government’s Ministry of Science and Technology, USAID and the U.S. National Institute of Health

January 23, 2020, Shi Zhengli team published an article on bioRxiv preprinted version of the platform titled, “A pneumonia outbreak associated with a new coronavirus of probable bat origin.” The paper was subsequently published in the journal Nature on February 3.

The article stated that they found that the sequence of the novel Coronavirus was 96.2% identical to that of the Coronavirus numbered RaTG13 derived from Yunnan horseshoe bats.

We then found that a short region of RNA-dependent RNA polymerase (RdRp) from a bat coronavirus (BatCoV RaTG13) — which was previously detected in Rhinolophus affinis from Yunnan province — showed high sequence identity to 2019-CoV2

We carried out full-length sequencing on this RNA sample (GISAID accession number EPI_ISL_402131)

But then in November of 2020 an addendum was issued

Between 2012 and 2015, our group sampled bats once or twice a year in this cave and collected a total of 1,322 samples. From these samples, we detected 9 that were designated betacoronaviruses on the basis of partial RdRp sequences.

All of the nine betacoronaviruses are SARSr-CoVs, one of which (sample ID4991; renamed RaTG13 in our Article to reflect the bat species, the location and the sampling year) was described in a 2016 publication.

The partial RdRp sequence (370 bp) of ID4991 was deposited in GenBank in 2016 under accession number KP876546. All of the identified bat SARSr-CoVs are distantly related to SARS-CoV based on partial RdRp sequences.

In 2018, as the next-generation sequencing technology and capability in our laboratory had improved, we performed further sequencing of these bat viruses and obtained almost the full-length genome sequence (without the 5′ and 3′ ends) of RaTG13

In 2020, we compared the sequence of SARS-CoV-2 with our unpublished bat coronavirus sequences and found that it shared a 96.2% identity with RaTG13.

A little bit different than what they stated in January where it sounded like they only did full sequencing in 2020

2022-A publication showed the RBD from RaTG13, a SARS-CoV-2-like virus extracted from the horseshoe bat species R. affinis, detectably bound human, mouse and rat ACE2 orthologs .....

but did not interact with four of five ACE2 orthologs isolated from various horseshoe bat species, including one of two ACE2 variants isolated from R. affinis itself

This finding fuels suspicion that the reported sequence of RaTG13 could have been fabricated to shift the attention away from ZC45/ZXC21