American scientists planned to work with the Wuhan Institute of Virology to engineer novel coronaviruses with the features of SARS-CoV-2 the year before the virus emerged from that city, according to documents obtained by US Right to Know.

While rare in nature, these features were central to the esoteric research interests of the scientists working with the Wuhan lab, those documents show.

Scientists divided over the so-called “lab leak” and natural origin hypotheses have for years pored over the arcane language of a US-China research proposal called “DEFUSE” describing coronavirus engineering experiments.

The DEFUSE grant proposal was led by EcoHealth Alliance President Peter Daszak.

Now, drafts and notes uncovered through the Freedom of Information Act reveal fresh details about the intended research.

Specifically, the scientists sought to insert furin cleavage sites at the S1/S2 junction of the spike protein; to assemble synthetic viruses in six segments; to identify coronaviruses up to 25 per cent different from SARS; and to select for receptor binding domains adept at infecting human receptors.

The genome of SARS-CoV-2, the virus that causes Covid-19, matches the viruses described in the research proposal:

SARS-CoV-2 has a furin cleavage site positioned in the spike protein at the S1/S2 junction. The furin cleavage site supercharged the virus into the worst pandemic pathogen in a century. Virologists have yet to identify one in any other related coronavirus.

SARS-CoV-2 can be divided into six contiguous genomic pieces by the restriction enzymes Bsal and BsmBI. These restriction enzymes occur in nature but can also be used in the lab to splice viruses. A trio of scientists estimated in a 2022 analysis that the likelihood of seeing the pattern found in SARS-CoV-2 in nature would be remote. Orders for one of these restriction enzymes, BsmBI, can be found in the documents.

SARS-CoV-2 emerged highly infectious without evolving much in humans. The virus “came out of the box ready to infect.” The receptor binding domain appeared “finely tuned” for the human ACE2 receptor, yet had little genetic variation when first spilling over into humans, presenting a difficult “paradox” to virologists who sought to prove it emerged naturally. The documents confirm the scientists working with the Wuhan lab sought to select receptor-binding domains that bind well to human ACE2 in their research.

The genome of SARS-CoV-2 falls within the range of a 25 per cent genetic difference from SARS.

The documents reveal for the first time that a virologist working with the Wuhan lab planned to engineer new spike proteins – in contrast with the collaboration’s public work to insert whole spike proteins into viral backbones.

Language in the proposal indicates this work may have involved unpublished viruses, generating unpublished engineered spike proteins.

This American virologist, University of North Carolina Prof Ralph Baric, was set to engineer 20 or more “chimeric” SARS-related viral spike proteins per year of the proposal, and two to five full-length engineered SARS-related viruses.

Documents previously reported by US Right to Know show that some of the experimentation could secretly occur in Wuhan at a lower biosafety level than specified in the grant, apparently to save costs.

The documents challenge an argument made by the National Institutes of Health and some virologists against the relevance of the research proposal to the origins of the pandemic.

They have argued that this US-China scientific collaboration only planned to engineer viruses starting with viral backbones already in the public literature and that these viral backbones are too dissimilar to have played a role in the pandemic.

The new documents however reveal that the scientists planned to use new reverse genetics systems and test viruses in vivo — in other words, to engineer live viruses with novel backbones.

The documents describe the SARS-related viruses to be studied in the grant as posing “a clear-and-present danger of a new SARS-like pandemic.”

The documents do not prove a precise step-by-step instruction manual for how SARS-CoV-2 was generated in the lab. The genomes of some of the SARS-related viruses the scientists planned to work with remain unknown.

But they do describe experiments that could have generated the virus’ rare properties. They detail the scientists’ interest in working with viruses precisely like SARS-CoV-2.

The grant proposal was submitted to the Defence Advanced Research Projects Agency, which rejected the project. Whether the research was funded through other means remains unknown. Baric had engineered unknown spike proteins by the time the proposal was submitted.

Nonetheless, the documents suggest that some of the data central to the worst pandemic in a century may be found not only in China but also in the US.

“When the Wuhan Institute of Virology published their first paper on the pandemic virus, they made no mention of the unique furin cleavage site despite having recently authored DEFUSE, declaring that they were on the lookout for these concerning features in novel SARS-like viruses,” Broad Institute molecular biologist Alina Chan said.

“We need to get all of the exchanges between the Wuhan Institute of Virology and its US collaborators in 2018 and especially 2019 – the year of the pandemic.”

While the language of the formal DEFUSE proposal called for the insertion of “human specific proteolytic cleavage sites” in a portion of the spike protein called the “S2′,” earlier drafts of DEFUSE were more explicit.

Proteolytic cleavage sites is a more general term than furin cleavage sites. It refers to a combination of amino acids that allow enzymes to cleave the spike protein, which helps viruses like SARS-CoV-2 enter human cells. However proteolytic cleavage sites can be activated by a variety of enzymes, not just furin.

This language in the final, formal proposal has been a sticking point in the debates over the DEFUSE documents.

Scripps Institute virologist Kristian Andersen – an advocate for the natural origin theory – has argued that DEFUSE holds little relevance to the origins of Covid-19 because the grant calls for the introduction of proteolytic cleavage sites at the S2′ position of the spike protein.

However, the earlier drafts show the scientists’ particular interest in furin cleavage sites. The documents suggest the scientists were not yet certain about the relative importance of the other cleavage sites.

Earlier drafts also precisely specify the positions of the intended insertions, including ones that correspond to the S1/S2 junction, not just at S2′.

“Tissue culture adaptations sometimes introduce a furin cleavage site which can direct entry processes, usually by cleaving S at positions 757 and 900 in S2′ of other CoV, but not SARS,” the grant reads.

The position 900 is the S2′ site and position 757 is the S1/S2 site. A comment on the proposal clarifies that this language cites Baric’s “Figure C.”

Figure C shows a furin cleavage site at the S1/S2 boundary, precisely where the furin cleavage site in SARS-CoV-2 is found.

The documents suggest the research group had identified a way to isolate SARS-related coronaviruses with cleavage sites or to insert them, some scientists say.

• The Defender report / By Emily Kopp, an investigative reporter with US Right to Know