Posts Tagged ‘National Institutes of Health’

The Lab Leak Hypothesis Revisited

Sunday, May 16th, 2021

If you’ve been following the blog since 2020, you know that we’ve looked at

the lab
leak
hypothesis
several
times.

Now Nicholas Wade, a science writer who’s worked on the staff of Nature, Science and the New York Times has taken a long look at the possibility the Wuhan Coronavirus did indeed leak from the Wuhan Institute of Virology.

Early on, several actors did their best to push the possibility of the lab leak hypothesis off the table:

From early on, public and media perceptions were shaped in favor of the natural emergence scenario by strong statements from two scientific groups. These statements were not at first examined as critically as they should have been.

“We stand together to strongly condemn conspiracy theories suggesting that COVID-19 does not have a natural origin,” a group of virologists and others wrote in the Lancet on February 19, 2020, when it was really far too soon for anyone to be sure what had happened. Scientists “overwhelmingly conclude that this coronavirus originated in wildlife,” they said, with a stirring rallying call for readers to stand with Chinese colleagues on the frontline of fighting the disease.

Contrary to the letter writers’ assertion, the idea that the virus might have escaped from a lab invoked accident, not conspiracy. It surely needed to be explored, not rejected out of hand. A defining mark of good scientists is that they go to great pains to distinguish between what they know and what they don’t know. By this criterion, the signatories of the Lancet letter were behaving as poor scientists: they were assuring the public of facts they could not know for sure were true.

It later turned out that the Lancet letter had been organized and drafted by Peter Daszak, president of the EcoHealth Alliance of New York. Dr. Daszak’s organization funded coronavirus research at the Wuhan Institute of Virology. If the SARS2 virus had indeed escaped from research he funded, Dr. Daszak would be potentially culpable. This acute conflict of interest was not declared to the Lancet’s readers. To the contrary, the letter concluded, “We declare no competing interests.”

Virologists like Dr. Daszak had much at stake in the assigning of blame for the pandemic. For 20 years, mostly beneath the public’s attention, they had been playing a dangerous game. In their laboratories they routinely created viruses more dangerous than those that exist in nature. They argued they could do so safely, and that by getting ahead of nature they could predict and prevent natural “spillovers,” the cross-over of viruses from an animal host to people. If SARS2 had indeed escaped from such a laboratory experiment, a savage blowback could be expected, and the storm of public indignation would affect virologists everywhere, not just in China. “It would shatter the scientific edifice top to bottom,” an MIT Technology Review editor, Antonio Regalado, said in March 2020.

Next came another attempt to declare that the Wuhan coronavirus couldn’t have been the result of a lab due to certain characteristics.

A second statement which had enormous influence in shaping public attitudes was a letter (in other words an opinion piece, not a scientific article) published on 17 March 2020 in the journal Nature Medicine. Its authors were a group of virologists led by Kristian G. Andersen of the Scripps Research Institute. “Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus,” the five virologists declared in the second paragraph of their letter.

Unfortunately this was another case of poor science, in the sense defined above. True, some older methods of cutting and pasting viral genomes retain tell-tale signs of manipulation. But newer methods, called “no-see-um” or “seamless” approaches, leave no defining marks. Nor do other methods for manipulating viruses such as serial passage, the repeated transfer of viruses from one culture of cells to another. If a virus has been manipulated, whether with a seamless method or by serial passage, there is no way of knowing that this is the case. Dr. Andersen and his colleagues were assuring their readers of something they could not know.

The discussion part their letter begins, “It is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus”. But wait, didn’t the lead say the virus had clearly not been manipulated? The authors’ degree of certainty seemed to slip several notches when it came to laying out their reasoning.

The reason for the slippage is clear once the technical language has been penetrated. The two reasons the authors give for supposing manipulation to be improbable are decidedly inconclusive.

First, they say that the spike protein of SARS2 binds very well to its target, the human ACE2 receptor, but does so in a different way from that which physical calculations suggest would be the best fit. Therefore the virus must have arisen by natural selection, not manipulation.

If this argument seems hard to grasp, it’s because it’s so strained. The authors’ basic assumption, not spelt out, is that anyone trying to make a bat virus bind to human cells could do so in only one way. First they would calculate the strongest possible fit between the human ACE2 receptor and the spike protein with which the virus latches onto it. They would then design the spike protein accordingly (by selecting the right string of amino acid units that compose it). But since the SARS2 spike protein is not of this calculated best design, the Andersen paper says, therefore it can’t have been manipulated.

But this ignores the way that virologists do in fact get spike proteins to bind to chosen targets, which is not by calculation but by splicing in spike protein genes from other viruses or by serial passage. With serial passage, each time the virus’s progeny are transferred to new cell cultures or animals, the more successful are selected until one emerges that makes a really tight bind to human cells. Natural selection has done all the heavy lifting. The Andersen paper’s speculation about designing a viral spike protein through calculation has no bearing on whether or not the virus was manipulated by one of the other two methods.

The authors’ second argument against manipulation is even more contrived. Although most living things use DNA as their hereditary material, a number of viruses use RNA, DNA’s close chemical cousin. But RNA is difficult to manipulate, so researchers working on coronaviruses, which are RNA-based, will first convert the RNA genome to DNA. They manipulate the DNA version, whether by adding or altering genes, and then arrange for the manipulated DNA genome to be converted back into infectious RNA.

Only a certain number of these DNA backbones have been described in the scientific literature. Anyone manipulating the SARS2 virus “would probably” have used one of these known backbones, the Andersen group writes, and since SARS2 is not derived from any of them, therefore it was not manipulated. But the argument is conspicuously inconclusive. DNA backbones are quite easy to make, so it’s obviously possible that SARS2 was manipulated using an unpublished DNA backbone.

He then links to another piece that demolishes these assertions in more pungent detail.

Wade continues:

The Daszak and Andersen letters were really political, not scientific statements, yet were amazingly effective. Articles in the mainstream press repeatedly stated that a consensus of experts had ruled lab escape out of the question or extremely unlikely. Their authors relied for the most part on the Daszak and Andersen letters, failing to understand the yawning gaps in their arguments. Mainstream newspapers all have science journalists on their staff, as do the major networks, and these specialist reporters are supposed to be able to question scientists and check their assertions. But the Daszak and Andersen assertions went largely unchallenged.

Section in which Wade notes that no supporting evidence of intermediate virus host transmission to support the natural origin theory snipped.

Why would anyone want to create a novel virus capable of causing a pandemic? Ever since virologists gained the tools for manipulating a virus’s genes, they have argued they could get ahead of a potential pandemic by exploring how close a given animal virus might be to making the jump to humans. And that justified lab experiments in enhancing the ability of dangerous animal viruses to infect people, virologists asserted.

With this rationale, they have recreated the 1918 flu virus, shown how the almost extinct polio virus can be synthesized from its published DNA sequence, and introduced a smallpox gene into a related virus.

These enhancements of viral capabilities are known blandly as gain-of-function experiments. With coronaviruses, there was particular interest in the spike proteins, which jut out all around the spherical surface of the virus and pretty much determine which species of animal it will target. In 2000 Dutch researchers, for instance, earned the gratitude of rodents everywhere by genetically engineering the spike protein of a mouse coronavirus so that it would attack only cats.

Virologists started studying bat coronaviruses in earnest after these turned out to be the source of both the SARS1 and MERS epidemics. In particular, researchers wanted to understand what changes needed to occur in a bat virus’s spike proteins before it could infect people.

Researchers at the Wuhan Institute of Virology, led by China’s leading expert on bat viruses, Dr. Shi Zheng-li or “Bat Lady”, mounted frequent expeditions to the bat-infested caves of Yunnan in southern China and collected around a hundred different bat coronaviruses.

Dr. Shi then teamed up with Ralph S. Baric, an eminent coronavirus researcher at the University of North Carolina. Their work focused on enhancing the ability of bat viruses to attack humans so as to “examine the emergence potential (that is, the potential to infect humans) of circulating bat CoVs [coronaviruses].” In pursuit of this aim, in November 2015 they created a novel virus by taking the backbone of the SARS1 virus and replacing its spike protein with one from a bat virus (known as SHC014-CoV). This manufactured virus was able to infect the cells of the human airway, at least when tested against a lab culture of such cells.

The SHC014-CoV/SARS1 virus is known as a chimera because its genome contains genetic material from two strains of virus. If the SARS2 virus were to have been cooked up in Dr. Shi’s lab, then its direct prototype would have been the SHC014-CoV/SARS1 chimera, the potential danger of which concerned many observers and prompted intense discussion.

Snip.

Dr. Baric had developed, and taught Dr. Shi, a general method for engineering bat coronaviruses to attack other species. The specific targets were human cells grown in cultures and humanized mice. These laboratory mice, a cheap and ethical stand-in for human subjects, are genetically engineered to carry the human version of a protein called ACE2 that studs the surface of cells that line the airways.

Dr. Shi returned to her lab at the Wuhan Institute of Virology and resumed the work she had started on genetically engineering coronaviruses to attack human cells.

How can we be so sure?

Because, by a strange twist in the story, her work was funded by the National Institute of Allergy and Infectious Diseases (NIAID), a part of the U.S. National Institutes of Health (NIH). And grant proposals that funded her work, which are a matter of public record, specify exactly what she planned to do with the money.

The grants were assigned to the prime contractor, Dr. Daszak of the EcoHealth Alliance, who subcontracted them to Dr. Shi. Here are extracts from the grants for fiscal years 2018 and 2019. “CoV” stands for coronavirus and “S protein” refers to the virus’s spike protein.

“Test predictions of CoV inter-species transmission. Predictive models of host range (i.e. emergence potential) will be tested experimentally using reverse genetics, pseudovirus and receptor binding assays, and virus infection experiments across a range of cell cultures from different species and humanized mice.”

“We will use S protein sequence data, infectious clone technology, in vitro and in vivo infection experiments and analysis of receptor binding to test the hypothesis that % divergence thresholds in S protein sequences predict spillover potential.”

What this means, in non-technical language, is that Dr. Shi set out to create novel coronaviruses with the highest possible infectivity for human cells. Her plan was to take genes that coded for spike proteins possessing a variety of measured affinities for human cells, ranging from high to low. She would insert these spike genes one by one into the backbone of a number of viral genomes (“reverse genetics” and “infectious clone technology”), creating a series of chimeric viruses. These chimeric viruses would then be tested for their ability to attack human cell cultures (“in vitro”) and humanized mice (“in vivo”). And this information would help predict the likelihood of “spillover,” the jump of a coronavirus from bats to people.

The methodical approach was designed to find the best combination of coronavirus backbone and spike protein for infecting human cells. The approach could have generated SARS2-like viruses, and indeed may have created the SARS2 virus itself with the right combination of virus backbone and spike protein.

It cannot yet be stated that Dr. Shi did or did not generate SARS2 in her lab because her records have been sealed, but it seems she was certainly on the right track to have done so. “It is clear that the Wuhan Institute of Virology was systematically constructing novel chimeric coronaviruses and was assessing their ability to infect human cells and human-ACE2-expressing mice,” says Richard H. Ebright, a molecular biologist at Rutgers University and leading expert on biosafety.

Wade also discusses the history or viral lab leaks:

Dr. Daszak was possibly unaware of, or perhaps he knew all too well, the long history of viruses escaping from even the best run laboratories. The smallpox virus escaped three times from labs in England in the 1960’s and 1970’s, causing 80 cases and 3 deaths. Dangerous viruses have leaked out of labs almost every year since. Coming to more recent times, the SARS1 virus has proved a true escape artist, leaking from laboratories in Singapore, Taiwan, and no less than four times from the Chinese National Institute of Virology in Beijing.

One reason for SARS1 being so hard to handle is that there were no vaccines available to protect laboratory workers. As Dr. Daszak mentioned in his December 19 interview quoted above, the Wuhan researchers too had been unable to develop vaccines against the coronaviruses they had designed to infect human cells. They would have been as defenseless against the SARS2 virus, if it were generated in their lab, as their Beijing colleagues were against SARS1.

A second reason for the severe danger of novel coronaviruses has to do with the required levels of lab safety. There are four degrees of safety, designated BSL1 to BSL4, with BSL4 being the most restrictive and designed for deadly pathogens like the Ebola virus.

The Wuhan Institute of Virology had a new BSL4 lab, but its state of readiness considerably alarmed the State Department inspectors who visited it from the Beijing embassy in 2018. “The new lab has a serious shortage of appropriately trained technicians and investigators needed to safely operate this high-containment laboratory,” the inspectors wrote in a cable of 19 January 2018.

The safety level required for research may also have been a factor.

The real problem, however, was not the unsafe state of the Wuhan BSL4 lab but the fact that virologists worldwide don’t like working in BSL4 conditions. You have to wear a space suit, do operations in closed cabinets and accept that everything will take twice as long. So the rules assigning each kind of virus to a given safety level were laxer than some might think was prudent.

Before 2020, the rules followed by virologists in China and elsewhere required that experiments with the SARS1 and MERS viruses be conducted in BSL3 conditions. But all other bat coronaviruses could be studied in BSL2, the next level down. BSL2 requires taking fairly minimal safety precautions, such as wearing lab coats and gloves, not sucking up liquids in a pipette, and putting up biohazard warning signs. Yet a gain-of-function experiment conducted in BSL2 might produce an agent more infectious than either SARS1 or MERS. And if it did, then lab workers would stand a high chance of infection, especially if unvaccinated.

Much of Dr. Shi’s work on gain-of-function in coronaviruses was performed at the BSL2 safety level, as is stated in her publications and other documents. She has said in an interview with Science magazine that “The coronavirus research in our laboratory is conducted in BSL-2 or BSL-3 laboratories.”

He also finds the natural origin hypothesis lacking in supporting evidence:

Beta-coronaviruses, the family of bat viruses to which SARS2 belongs, infect the horseshoe bat Rhinolophus affinis, which ranges across southern China. The bats’ range is 50 kilometers, so it’s unlikely that any made it to Wuhan. In any case, the first cases of the Covid-19 pandemic probably occurred in September, when temperatures in Hubei province are already cold enough to send bats into hibernation.

What if the bat viruses infected some intermediate host first? You would need a longstanding population of bats in frequent proximity with an intermediate host, which in turn must often cross paths with people. All these exchanges of virus must take place somewhere outside Wuhan, a busy metropolis which so far as is known is not a natural habitat of Rhinolophus bat colonies. The infected person (or animal) carrying this highly transmissible virus must have traveled to Wuhan without infecting anyone else. No one in his or her family got sick. If the person jumped on a train to Wuhan, no fellow passengers fell ill.

It’s a stretch, in other words, to get the pandemic to break out naturally outside Wuhan and then, without leaving any trace, to make its first appearance there.

For the lab escape scenario, a Wuhan origin for the virus is a no-brainer. Wuhan is home to China’s leading center of coronavirus research where, as noted above, researchers were genetically engineering bat coronaviruses to attack human cells. They were doing so under the minimal safety conditions of a BSL2 lab. If a virus with the unexpected infectiousness of SARS2 had been generated there, its escape would be no surprise.

So too is the evidence from the spike proteins:

The initial location of the pandemic is a small part of a larger problem, that of its natural history. Viruses don’t just make one time jumps from one species to another. The coronavirus spike protein, adapted to attack bat cells, needs repeated jumps to another species, most of which fail, before it gains a lucky mutation. Mutation — a change in one of its RNA units — causes a different amino acid unit to be incorporated into its spike protein and makes the spike protein better able to attack the cells of some other species.

Through several more such mutation-driven adjustments, the virus adapts to its new host, say some animal with which bats are in frequent contact. The whole process then resumes as the virus moves from this intermediate host to people.

In the case of SARS1, researchers have documented the successive changes in its spike protein as the virus evolved step by step into a dangerous pathogen. After it had gotten from bats into civets, there were six further changes in its spike protein before it became a mild pathogen in people. After a further 14 changes, the virus was much better adapted to humans, and with a further 4 the epidemic took off.

But when you look for the fingerprints of a similar transition in SARS2, a strange surprise awaits. The virus has changed hardly at all, at least until recently. From its very first appearance, it was well adapted to human cells. Researchers led by Alina Chan of the Broad Institute compared SARS2 with late stage SARS1, which by then was well adapted to human cells, and found that the two viruses were similarly well adapted. “By the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV,” they wrote.

Even those who think lab origin unlikely agree that SARS2 genomes are remarkably uniform. Dr. Baric writes that “early strains identified in Wuhan, China, showed limited genetic diversity, which suggests that the virus may have been introduced from a single source.”

A single source would of course be compatible with lab escape, less so with the massive variation and selection which is evolution’s hallmark way of doing business.

The uniform structure of SARS2 genomes gives no hint of any passage through an intermediate animal host, and no such host has been identified in nature.

Proponents of natural emergence suggest that SARS2 incubated in a yet-to-be found human population before gaining its special properties. Or that it jumped to a host animal outside China.

All these conjectures are possible, but strained. Proponents of lab leak have a simpler explanation. SARS2 was adapted to human cells from the start because it was grown in humanized mice or in lab cultures of human cells, just as described in Dr. Daszak’s grant proposal. Its genome shows little diversity because the hallmark of lab cultures is uniformity.

Proponents of laboratory escape joke that of course the SARS2 virus infected an intermediary host species before spreading to people, and that they have identified it — a humanized mouse from the Wuhan Institute of Virology.

Then there’s the furin cleavage question (much technical description snipped):

Viruses have all kinds of clever tricks, so why does the furin cleavage site stand out? Because of all known SARS-related beta-coronaviruses, only SARS2 possesses a furin cleavage site. All the other viruses have their S2 unit cleaved at a different site and by a different mechanism.

How then did SARS2 acquire its furin cleavage site? Either the site evolved naturally, or it was inserted by researchers at the S1/S2 junction in a gain-of-function experiment.

Snip.

It’s hard to explain how the SARS2 virus picked up its furin cleavage site naturally, whether by mutation or recombination.

That leaves a gain-of-function experiment. For those who think SARS2 may have escaped from a lab, explaining the furin cleavage site is no problem at all. “Since 1992 the virology community has known that the one sure way to make a virus deadlier is to give it a furin cleavage site at the S1/S2 junction in the laboratory,” writes Dr. Steven Quay, a biotech entrepreneur interested in the origins of SARS2. “At least eleven gain-of-function experiments, adding a furin site to make a virus more infective, are published in the open literature, including [by] Dr. Zhengli Shi, head of coronavirus research at the Wuhan Institute of Virology.”

Then there’s the question of identical DNA sequences:

The functional reason why SARS2 has a furin cleavage site, and its cousin viruses don’t, can be seen by lining up (in a computer) the string of nearly 30,000 nucleotides in its genome with those of its cousin coronaviruses, of which the closest so far known is one called RaTG13. Compared with RaTG13, SARS2 has a 12-nucleotide insert right at the S1/S2 junction. The insert is the sequence T-CCT-CGG-CGG-GC. The CCT codes for proline, the two CGG’s for two arginines, and the GC is the beginning of a GCA codon that codes for alanine.

There are several curious features about this insert but the oddest is that of the two side-by-side CGG codons. Only 5% of SARS2’s arginine codons are CGG, and the double codon CGG-CGG has not been found in any other beta-coronavirus. So how did SARS2 acquire a pair of arginine codons that are favored by human cells but not by coronaviruses?

Proponents of natural emergence have an up-hill task to explain all the features of SARS2’s furin cleavage site. They have to postulate a recombination event at a site on the virus’s genome where recombinations are rare, and the insertion of a 12-nucleotide sequence with a double arginine codon unknown in the beta-coronavirus repertoire, at the only site in the genome that would significantly expand the virus’s infectivity.

For the lab escape scenario, the double CGG codon is no surprise. The human-preferred codon is routinely used in labs. So anyone who wanted to insert a furin cleavage site into the virus’s genome would synthesize the PRRA-making sequence in the lab and would be likely to use CGG codons to do so.

“When I first saw the furin cleavage site in the viral sequence, with its arginine codons, I said to my wife it was the smoking gun for the origin of the virus,” said David Baltimore, an eminent virologist and former president of CalTech. “These features make a powerful challenge to the idea of a natural origin for SARS2,” he said.

His conclusion:

the available evidence leans more strongly in one direction than the other. Readers will form their own opinion. But it seems to me that proponents of lab escape can explain all the available facts about SARS2 considerably more easily than can those who favor natural emergence.

It’s documented that researchers at the Wuhan Institute of Virology were doing gain-of-function experiments designed to make coronaviruses infect human cells and humanized mice. This is exactly the kind of experiment from which a SARS2-like virus could have emerged. The researchers were not vaccinated against the viruses under study, and they were working in the minimal safety conditions of a BSL2 laboratory. So escape of a virus would not be at all surprising. In all of China, the pandemic broke out on the doorstep of the Wuhan institute. The virus was already well adapted to humans, as expected for a virus grown in humanized mice. It possessed an unusual enhancement, a furin cleavage site, which is not possessed by any other known SARS-related beta-coronavirus, and this site included a double arginine codon also unknown among beta-coronaviruses. What more evidence could you want, aside from the presently unobtainable lab records documenting SARS2’s creation?

Proponents of natural emergence have a rather harder story to tell. The plausibility of their case rests on a single surmise, the expected parallel between the emergence of SARS2 and that of SARS1 and MERS. But none of the evidence expected in support of such a parallel history has yet emerged. No one has found the bat population that was the source of SARS2, if indeed it ever infected bats. No intermediate host has presented itself, despite an intensive search by Chinese authorities that included the testing of 80,000 animals. There is no evidence of the virus making multiple independent jumps from its intermediate host to people, as both the SARS1 and MERS viruses did. There is no evidence from hospital surveillance records of the epidemic gathering strength in the population as the virus evolved. There is no explanation of why a natural epidemic should break out in Wuhan and nowhere else. There is no good explanation of how the virus acquired its furin cleavage site, which no other SARS-related beta-coronavirus possesses, nor why the site is composed of human-preferred codons. The natural emergence theory battles a bristling array of implausibilities.

Toward the end, he lists those who are to blame for the outbreak, a subject that came up in congressional hearings, including western virologists who obtained grants for the Wuhan Institute of Virology to conduct gain-of-function research. Guess who’s name came up?

The considerable evidence in favor of the lab leak hypothesis is why numerous medical researchers have signed an open letter in science asking that the lab leak hypothesis be seriously explored.

There’s plenty of evidence for the lab leak hypothesis, and only the word of China, its paid lackeys, and its enablers against it…

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