Scientists from Wuhan and the US were planning to create new coronaviruses that did not exist in nature by combining the genetic codes of other viruses, proposals show.
Documents of a grant application submitted to the US Defense Advanced Research Projects Agency (Darpa), leaked last month, reveal that the international team of scientists planned to mix genetic data of closely related strains and grow completely new viruses.
A genetics expert working with the World Health Organisation (WHO), who uncovered the plan after studying the proposals in detail, said that if Sars-CoV-2 had been produced in this way, it would explain why a close match has never been found in nature.
So far the closest naturally occurring virus to Sars-CoV-2 is a strain called Banal-52, which was reported from Laos last month and shares 96.8 per cent of the genome. Yet scientists expect a direct ancestor to be around a 99.98 per cent match – and none has been found so far.
The Darpa proposals, leaked to the pandemic origins analysis group Drastic, show the team had planned to take sequences from naturally occurring coronaviruses and use them to create a brand new sequence that was an average of all the strains.
The grant application, submitted in 2018, states: "We will compile sequence/RNAseq data from a panel of closely related strains and compare full length genomes, scanning for unique SNPs representing sequencing errors.
"Consensus candidate genomes will be synthesised commercially using established techniques and genome-length RNA and electroporation to recover recombinant viruses."
Explaining the proposal, a WHO collaborator, who has asked not to be named for fear of reprisals, said: "This means that they would take various sequences from similar coronaviruses and create a new sequence that is essentially the average of them. It would be a new virus sequence, not a 100 per cent match to anything.
"They would then synthesise the viral genome from the computer sequence, thus creating a virus genome that did not exist in nature but looks natural as it is the average of natural viruses.
"Then they put that RNA in a cell and recover the virus from it. This creates a virus that has never existed in nature, with a new 'backbone' that didn't exist in nature but is very, very similar as it's the average of natural backbones."
The source said it was noteworthy that the cut-off for generating such an average sequence was viruses that only had 5 per cent genetic divergence from each other.
Last year, scientists at the Wuhan Institute of Virology said they had found a strain named RaTG13 in bat droppings in a cave in Yunnan province in 2013 which was a 96.1 per cent match to Sars-CoV-2. It means RaTG13 could have been included in a set of viral genomes to help create an average sequence.
Although the grant proposal was rejected in 2018, the Wuhan database of viral strains was taken offline prior to the Covid outbreak some 18 months later, meaning it is impossible to check which viruses the team was working on or had created. Wuhan scientists have consistently denied creating Sars-CoV-2 in a lab.
The WHO source added: "If Sars-CoV-2 comes from an artificial consensus sequence composed of genomes with more than 95 per cent similarity to each other… I would predict that we will never find a really good match in nature and just a bunch of close matches across parts of the sequence, which so far is what we are seeing.
"The problem is that those opposed to a lab leak scenario will always just say that we need to sample more, and absence of evidence isn't evidence of absence. Scientists overall are afraid of discussing the issue of the origins due to the political situation. This leaves a small and vocal minority of biased scientists free to spread misinformation."
The proposal was submitted by the British zoologist Peter Daszak on behalf of a consortium which included Daszak EcoHealth Alliance, the Wuhan Institute of Virology, the University of North Carolina and Duke NUS in Singapore.
The plans are in addition to other proposals made in the Darpa documents, including inserting a section into existing viruses to make them more infectious to humans and inoculating wild bats with aerosolised engineered spike proteins from viruses.
Experts said if the ultimate aim of the proposal was to create a pan-coronavirus vaccine, constructing an "ideal" average virus would have been a good starting point.
Daszak, currently a member of the WHO team investigating the pandemic's origins, was also behind a letter published in The Lancet which dismissed suggestions that Covid did not have a natural origin as a conspiracy theory.
The WHO source said he had struggled to raise the issue of a lab leak with other scientists and had been warned not to go on the record with his concerns.
The proposal team has been approached for comment but had not responded at the time of publication.
Why scientists think Covid-19 came from nature
Earlier this year, a team of scientists published a pre-print study finding there was scant evidence to suggest that the virus had either been deliberately engineered, or had escaped from a culture researchers may have been growing.
"All of the evidence points toward a natural, zoonotic origin," Otago University virologist and review co-author Dr Jemma Geoghegan told the Herald at the time.
She and the other authors noted how the virus' documented epidemiological history – which began at the Wuhan Huanan Seafood Wholesale Market – was comparable to other coronavirus outbreaks linked to animal markets.
The researchers also said there was still no evidence that any of the early cases had any connection to the Wuhan Institute of Virology, nor was there any evidence the institute had or worked on a progenitor of Sars-CoV-2 virus before the pandemic.
"The suspicion that Sars-CoV-2 might have a laboratory origin stems from the coincidence that it was first detected in a city that houses a major virological laboratory that studies coronaviruses," they said.
Going deeper, the researchers addressed speculation about the supposedly unusual molecular make-up of Sars-CoV-2.
In one of the earliest major studies into the virus, scientists analysed its genetic template for spike proteins, which it used to grab and penetrate the outer walls of human and animal cells.
More specifically, they focused on its receptor-binding domain (RBD) - a kind of grappling hook that grips on to host cells - and what's called the furin cleavage site, a molecular can opener that allows the virus to crack open and enter host cells.
This earliest research suggested the RBD portion of the spike proteins had evolved to effectively target a molecular feature on the outside of human cells called ACE2 - a receptor involved in regulating blood pressure.
The Sars-CoV-2 spike protein was so effective at binding the human cells, in fact, that the scientists concluded it was the result of natural selection, and not the product of genetic engineering.
Yet many theorists have surmised that the furin cleavage site was so unusual it must have been artificially inserted by scientists.
The review authors did note the cleavage site was absent from the closest known relatives of Sars-CoV-2, but they added that wasn't surprising, given the lineage leading to the virus had been poorly sampled.
Further, they added, furin cleavage sites were common in other coronavirus spike proteins, including the last coronavirus to spark a global incident - MERS-CoV – and also feline alphacoronaviruses, most strains of mouse hepatitis virus, and endemic human betacoronaviruses.
A near identical nucleotide sequence to Sars-CoV-2's had also been found in the spike gene of the bat coronavirus HKU9-172 – and there were indications the evolution of both viruses had involved "recombination", or the natural exchange of genetic material.
"Hence, simple evolutionary mechanisms can readily explain the evolution of an out-of-frame insertion of a furin cleavage site in Sars-CoV-2," the researchers said.
They added there was "no logical reason" why an engineered virus would use such a poor furin cleavage site as Sars-CoV-2's.
That would have entailed an "unusual and needlessly complex" feat of genetic engineering, for which there were few precedents.
"Further, there is no evidence of prior research at the WIV involving the artificial insertion of complete furin cleavage sites into coronaviruses."
Although they said the possibility of a lab accident still couldn't be entirely dismissed, they concluded the explanation remained "highly unlikely, relative to the numerous and repeated human-animal contacts that occur routinely in the wildlife trade".
"Failure to comprehensively investigate the zoonotic origin through collaborative and carefully coordinated studies would leave the world vulnerable to future pandemics arising from the same human activities that have repeatedly put us on a collision course with novel viruses."
- Additional reporting: NZ Herald