“Understanding the Risk of Bat Coronavirus Emergence” - a Merogenomics’ NIH grant review
Disclaimer: it was pointed out that our earlier definition of logarithmic growth was not correctly defined, and this has been changed accordingly. Special thank you to Dr. Robert Goodman!
Research of pandemic potential
Two National Institute of Health (NIH) grants that funded the work in Wuhan were finally made publicly available. They were not provided voluntarily though. This information, for some reason was never released by the NIH at the start of pandemic. Why? Who knows? The way they became available was through the Freedom of Information Act, used by a seemingly random news source, The Intercept, who was first to break the story on these NIH grants, and the type of research performed in Wuhan. It is the Intercept that finally provided access to these two NIH grants, not any public agency. And they had to sue the NIH to get access to their Freedom Of Information Act request for these “public” documents and they are staggering!
We looked at them in quite some detail and wanted to share this deep analysis from probably some of the most significant documents relating to what type of work was done in Wuhan prior to the pandemic. Ironically, that information comes from the US, not China - but we still get some very good details of what work was done in Wuhan. What emerges is that potentially, enormously risky experiments did take place, and they were funded by the US.
Prior to the release of these NIH grants documents, the type of work performed on coronavirus had already been suspected based on the available evidence (for example, actual published manuscripts). These NIH grants tie it together that funding was granted towards the genetic engineering of coronaviruses, specifically selected for their ability to infect humans. If this sounds like shocking news to you, this much had already been deduced from publicly available information. These grants confirm that what was guessed at before, appears to have been exactly what happened. And who paid for it? It looks like the US tax payers. And who asked for the money? It looks like Dr. Peter Daszak as his name is on every page in the document as the Program Director/Principal Investigator. We get to him below.
The most important finding is that these grants supported the very type of scientific research that had an embedded risk of producing exactly the type of pandemic the world is currently battling.
The grants clearly demonstrate that the type of work that was performed had the potential to generate novel types of SARS-like coronaviruses, and that these viruses were specifically engineered to infect humans. The way this was achieved was by discovering coronaviruses in nature, predominantly of bat origin. These new coronaviruses were then investigated for their genetic material and ability to infect different cells, including human cells. Viruses with the ability to interact with human cells were focused on. The ability to infect human cells is driven by the spike protein of the coronaviruses. Taking advantage of many new insights into the natural variation of coronaviruses, the researchers started creating different constructs of coronaviruses, sometimes by purposefully mutating them. Ostensibly this was done in order to understand the biology behind possible future threats to humans that could emerge naturally like the original SARS-CoV did. But it was the final set of experiments that were the most dangerous for the possibility of creating a pandemic - these viruses were used to infect mice that were genetically engineered with human receptors for the virus. Even without any engineering of the coronavirus, infecting genetically modified mice that possessed human receptors to try to identify the most infectious strain towards humans is where the pandemic risk was introduced. From the still limited information available, these experiments appear as extremely dangerous. Were there any safety precautions to minimize the threat to exposed humans or were any biological engineering steps taken to ensure these tested viruses posed no threat? We cannot say, but we do not see any being listed in these grants.
Below are the quotes from one of the grants - spanning funding for six years - to explain in detail how the above conclusion was made.
What exactly are these NIH grants documents?
These grants are a series of documents from the National Institutes of Health that had been first approved and awarded to the EcoHealth Alliance (a NY based research organization directed by Dr. Peter Daszak, the principal investigator listed on the grant). One grant, the older of the two, that is the focus of this article, titled "Understanding the Risk of Bat Coronavirus Emergence", was for new research starting June 2014, and subsequently renewed for further funding in 2015, 2016, 2017 and 2018 (with the project originally ending in 2019, oops, right prior to the pandemic start, but extended to 2025 with the subsequent rude pandemic interruption). Thus, the entire document is 528 pages long, but is made up of 6 grants with lots of the same information being reiterated over and over.
If you need the breakdown of this grant, here it is:
|Budget period||Document pages||Funds awarded|
|2014-2015||p.1-152||$666,442 (what a number, probably what jinxed it!)|
For a cool total of $3,748,715 from just one granting agency for research including the purposeful creation of new coronaviruses that are infectious to humans.
The stated purpose of the funded research was very broad and large in scope though, in essence to study the risk of the emergence of future coronaviruses that could spillover to humans. This included: monitoring populations for potential infections with coronaviruses from bats and other wildlife; including specimens from live animal markets; predictive modelling of how bat coronaviruses could evolve and spread; and finally, the most contentious component that is currently creating controversy, attempting to learn how coronaviruses could potentially infect humans by performing engineering experiments on isolated viruses to see how they could potentially infect humans. This is the part we will focus on in our quotes below. Throughout the grant years you can see how the research progressed and became more sophisticated with constantly growing capabilities for generating novel coronavirus constructs.
It is quite a remarkable story so let’s do our deep dive, especially since Merogenomics did not really find a good breakdown of the information contained in these grants anywhere. Note: anything inside the quotes that is encompassed by square [brackets] are our additions or comments for clarity.
Grant selected quotes and interpretation
p.92: “We request support for in vitro infection experiments using pseudoviruses carrying the spike proteins (wild type or mutants) or live viruses in cell lines of different origins, binding affinity assays between the spike proteins (wild type or mutants) and different cellular receptor molecules, and humanized mouse experiments.”
Why is this important: it reveals that the spike protein was being manipulated through mutagenesis, then inserted into viruses, tested for their ability to bind specific receptors, and tested on humanized mice. This is really important to understand, because in essence we are talking about engineering a virus that is then being infected into mice where these mice themselves are engineered to mimic some human biology, in this case, as we will discuss below, they are engineered to have human ACE2 receptors so as to bind virus spike proteins. If such mice are infected with such an engineered virus, this allows the virus to naturally evolve inside mice.
But, even more importantly, if such a virus is then isolated from the mice and used to infected another set of such humanized mice in multiple such rounds of virus isolation and mice infection - this is referred to as serial passaging - then the virus can go through its own natural evolution to look indistinguishable from viruses found in nature, because in essence it was manipulated by nature inside live mice. It could be basically impossible to tell that such a virus was originally a synthetic construct. However, to be clear, serial passaging does not appear to have been performed. Infected mice were euthanized within three weeks post infection.
These aims are reiterated on p.124 with the addition that experiments with humanized mice are to be done especially “if viruses are identified of significant human infection potential”. On p.125 we also learn that these pseudoviruses engineered to host the coronavirus spike proteins were actually HIV. Later on, as you will see, the authors created their own coronavirus to test new constructs.
p. 114: “With bat-CoVs [bat coronaviruses] that we've isolated or sequenced, and using live virus or pseudovirus infection in cells of different origin or expressing different receptor molecules, we will assess potential for each isolated virus and those with receptor binding site sequence, to spill over. We will do this by sequencing the spike (or other receptor binding/fusion) protein genes from all our bat-CoVs, creating mutants to identify how significantly each would need to evolve to use ACE2, CD26/DPP4 (MERS-CoV receptor) or other potential CoV receptors. We will then use receptor-mutant pseudovirus binding assays, in vitro studies in bat, primate, human and other species' cell lines, and with humanized mice where particularly interesting viruses are identified phylogenetically, or isolated. These tests will provide public health-relevant data, and also iteratively improve our predictive model to better target bat species and CoVs during our field studies to obtain bat-CoV strains of the greatest interest for understanding the mechanisms of cross-species transmission.”
Why is this important: it reveals that data was being generated to understand how coronaviruses from bats, already known to be a potential threat to human health safety could be even more dangerous in terms of their ability to infect humans, so that this information could be then used to screen such occurrences in nature. In fact, this is reiterated and highlighted with following statement: “The results will provide information whether bat-CoVs could use known bat and human ACE2, DPP4 or other known CoV receptors to enter cells, and allow us to determine critical receptor binding sites, viral host range, and to better predict the capacity of our CoVs to infect people” (p.125, underlined by authors). This suggests that while aims of the study might have been noble ones, they were also potentially very dangerous because it involved manipulation of the virus components, with a special focus on the spike protein. It was especially dangerous because the outcomes of this manipulation were tested against human cells, and once again, humanized mice, meaning those mice with certain cellular components mimicking humans. In other words, testing viruses for a direct capacity to infect humans, and hence very dangerous research in the unlikely event of a lab leak.
p. 124: “We will sequence the spike (or other receptor binding/fusion) protein genes from all bat-CoVs we identify, creating mutants of these to identify how significantly each would need to evolve to use ACE2 or CD26/DPP4 (receptor for MERS).”
Why is this important: it specifically confirms how much focus and attention was made on understanding the spike protein and how to make it more infectious by enhancing its ability to bind the ACE2 receptor – the exact receptor that SARS-CoV-2 uses to infect our cells. Once again, it points to the actual level of danger of conducting such experiments in the event that such engineered constructs escaped the lab.
p.126: “To evaluate pathogenicity of bat-CoVs we will perform in vivo infection experiments in humanized mice modified to carry human ACE2 or DPP4 gene in the Wuhan Institute of Virology BSL-3 animal facility. […] Mouse body temperature will be monitored with implanted microchips (LifeChip Bio-thermo, Destron Fearing), and mice will be weighed and observed for clinical signs of illness daily. Dead or moribund mice will be euthanized, organs harvested and sectioned. Live animals will be euthanized at three weeks post-inoculation and organs harvested. We will test for neutralizing antibodies against bat-CoVs on days 10, 15 and 21 pi [post infection]. We will collect nasal washes, oral swabs, and rectal swabs, and urine every two days and quantify virus using qRT-PCR. We will conduct routine histology, immunohistochemistry, qRT-PCR, and virus isolation on tissues. This work will provide information about viral pathogenicity, tissue tropism, transmission route, and infection symptom.”
Why is this important: it clearly spells out how many potential exposures there were to these infectious viruses, engineered to be more infectious to humans, that were actually taking place - specifically in the Wuhan Institute of Virology - including infected animal dissection and analysis that we all have become familiar with since the start of pandemic, and all of which carry the risks of laboratory staff infection. On p.132 we also learn that the mice were also studied to determine “whether these mice are capable of shedding infectious virus.” There is also no confusion that this dangerous aspect of working with humanized mice was indeed performed in Wuhan.
We learn more details on the humanized mice on p.145 in the support letter from Dr. Ralph Baric who apparently had engineered the mice and who provided the following description: “We have developed transgenic mouse models in the C57BL/6 mice, expressing hACE2 [h=human] in ciliated cells from the FOXJ1 promoter [promoters are genetic regions that regulate to what extent a nearby gene is used]. Unlike other epithelial cell promoters […], hACE2 expression from FOXJ1 should be specific to the airway epithelium. […] Inoculation of these mice with wild type SARS-CoV resulted in lethal respiratory tract infections characterized by high virus titers […], hemorrhage, severe pneumonia and acute respiratory distress syndrome between days 2-7 post infection.”
In other words, these mice were engineered to be highly susceptible to infection by coronaviruses that could bind to human ACE2 receptors.
However, from the description of these studies, it looks like the authors generated pseudoviruses using HIV as a vehicle for delivering mutated spike proteins, or by using isolated bat coronaviruses to infect humanized mice. At this stage it does not appear that the researchers went as far as actually mutating the spike protein of the isolated bat coronaviruses that were used to infect mice. These will become apparent in later years of work. But the way the experiments are described at this early stage, they do not look like gain-of-function work, per se. The hazard at this stage would have to come from either the HIV pseudovirus with mutated spike protein, or the novel bat coronaviruses used to infect humanized mice. But we will get to much more complex constructs later on.
p.126: “Pl [principal investigator] Daszak will oversee all aspects of the project management. He is an experienced manager, with over 15 years of federally-funded research experience. Prof. Shi, based at the Wuhan Institute of Virology, will oversee all laboratory testing and analyses. Prof. Shuyi Zhang will manage field sampling work.”
Why is this important: it clearly spells principal players involved including Dr. Peter Daszak as the main manager. However, Dr. Daszak was also the principal scientist promoting the natural origin of the virus, while denouncing any possibility of a lab leak theory, clearly a conflict of interest considering that it was under his helm that the manipulation of coronaviruses took place in Wuhan, and therefore clearly opening the possibility that the pandemic could be a result of a lab leak.
The fact that Dr. Daszak has gone to such length to mobilize the scientific community at the start of pandemic to denounce the possibility of the virus being either engineered or a possible lab leak, and to pronounce that SARS-CoV-2 could only be of natural origin is deeply disingenuous in light of the actual research he was in charge of, and in clear contradiction to what we have now learned as in fact a possibility that should have been considered from the start. This does not paint a good picture of Dr. Daszak as a beacon of truth, legitimacy and openness considering how long and how difficult it was to bring the contents of this grant to public light. How such an individual was subsequently charged with managing the national narrative of the pandemic origin is beyond belief - pointing to the grave investigative incompetence of the American intelligence and journalistic communities.
p.150: “Viral isolates will remain at the Wuhan Institute of Virology initially. Isolates, reagents and any other products, should they be developed, will be made available to other NIH-funded researchers via applicable Wuhan Institute of Virology and EcoHealth Alliance Material Transfer Agreements and/or licensing agreements.”
Why is this important: it states that any engineered viruses that were generated through mutagenesis of spike protein were kept in the Wuhan Institute of Virology. We do not know if they were shared with other facilities.
p.161: “The following experiments will be undertaken in Year 2:
1. Animal infection experiment with SARS-like CoV
Option 1. Virus infection through ACE2 humanized mouse. Human ACE2 promotor (9-10 kb) and ACE2 will be inserted into an expressing vector and sent to a commercial company to generate transgenic mice. The stably expressed human ACE2 mice will be used for virus infection.
Option 2. Virus infection through SARS-CoV susceptible animals such as ferrets.”
Why is this important: it states that the dangerous experiments of infecting humanized mice are to be continued, with additional, new generation of such genetically modified humanized mice for use in China. Once again, such experiments allow the virus to potentially mutate to forms that more likely could infect humans if any infected mouse happens to help select a specific more advantageous virus variant. Such experiments are dangerous due to the possible risk of an accidental infection with a virus that could be evolved to be more dangerous to humans.
p.166: “Table 1 Bat Samples collected for CoV surveillance in 2014”
Why is this important: this table lists where all the samples were previously collected, including the site in Mojiang, Yunnan, now infamous for a previous infection of miners with a mysterious and deadly respiratory disease, a topic which we previously covered. That included 156 samples collected in May 2014, 96 samples in July-Sept 2014, and 171 samples in Oct 2014. Mojiang was in fact extensively visited site by the authors in those early days.
p.171: “We amplified the full-length S gene of the novel SL-CoV [SARS-like coronavirus] detected in a Rhinolophus sinicus colony in Yunnan Province. In addition to our previously reported Rs3367 and RsSHC014, we now have 24 new full-length S gene sequences from 22 samples. Phylogenetic analysis showed that these SL-CoV are diverse, and identified two strains of novel SL-CoV more closely related to SARS-CoV than Rs3367 [author emphasis]. Our new strains named Rs4841 and Rs4874 share the highest homology to SARS-CoV than any other known SL-CoV, including those we published previously in Nature.”
Why is this important: this shows how many novel coronaviruses were being identified with numerous spike proteins being genetically sequenced. As the spike protein is the main candidate protein for viral infection, and such diversity provides authors with much ammunition to attempt to manipulate the spike protein to generate new constructs.
It is also important because the authors proudly identify the new bat coronavirus Rs4874. Later on, as the SARS-CoV-2 pandemic started in China, we learned from the very first publication of the genome sequence of SARS-CoV-2, that the strain Rs4874 is one of the most closely related viruses to SARS-CoV-2, and especially with one of the highest sequence similarities in the spike protein. This demonstrates how early the authors were already in possession of very close relative to SARS-CoV-2 virus that emerged in the same city where these dangerous experiments were being conducted.
Furthermore, the Wuhan Institute of Virology research group spearheaded by Dr. Zhengli Shi had also published information that these bat coronaviruses Rs4841 and Rs4874 can bind to human ACE2 receptors! Thus, the picture emerges that the authors were in a possession of SARS-CoV-2 closely related viruses that could bind ACE2 human receptors and they have infected mice - with human ACE2 receptors - with such viruses where they could mutate further. By the way, the Chinese research group who published that first SARS-CoV-2 genome sequence was promptly shut down by the Chinese authorities after that likely unauthorized event.
p.174: “Our results show that SL-CoV-WIV16 [their own lab grown Rs4841 coronavirus] can grow in human alveolar basal epithelial (A549), […] and human lung carcinoma (NCI-H292)”.
Why is this important: this demonstrates that one of the coronaviruses was shown to infect and grow inside human lung cells. There is no denial of the danger potential of such virus at this point.
p.189: “No funds are provided and no funds can be used to support gain-of-function research covered under the October 17, 2014 White House Announcement (NIH Guide Notice NOT-OD-15-011). Per the letter dated July 7, 2016 to Mr. Aleksei Chmura at EcoHealth Alliance, should any of the MERS-like or SARS-like chimeras generated under this grant show evidence of enhanced virus growth greater than 1 log over the parental backbone strain you must stop all experiments with these viruses and provide the NIAID Program Officer and Grants Management Specialist, and Wuhan Institute of Virology Institutional Biosafety Committee with the relevant data and information related to these unanticipated outcomes.”
Why is this important: first, this clearly indicates that NIH was aware of the fact that gain-of-function research was indeed taking place on the account of their awareness that MERS-like or SARS-like chimeras were being generated. Chimeras refers to combination of genetic material from different sources. Gain-of-function research refers to creating novel pathogenic constructs that can be more infectious as a consequence of their synthetic generation. It also clearly states how NIH was defining gain-of-function research: synthetic constructs that as a consequence of their creation experience increased growth (enhanced ability to replicate). In this case it is defined as greater than 1 log, where 1 log means a factor of 10, thus in other words, increased growth of at least 10-fold compared to the starting infectious agent. This is the first such mention in the document, the third year into the commenced work, and indicates that in the third year of this work, creation of novel, more infectious constructs could not take place. However, in the prior two years of research, no such restriction was in place. However, of the 6 years funded by NIH, only funding of third year appears to impose such a restriction.
p.195: “The following experiments will be undertaken in Year 2: [we think this is their typo and should have said 3. We all make them!]
- Humanized mice with human ACE2 receptors will be infected with WIV1 and the two rescued chimeric SARS-like coronaviruses to determine the tissue tropism and pathogenicity of bat SL-CoV
- Isolation of novel bat coronaviruses. Live virus or pseudovirus will be used to infect cells of different origin or expressing different receptor molecules. Spillover potential for each isolated virus will be assessed.
- An infectious clone of full-length MERS-CoV will be constructed using reverse genetic method. Using the S sequence of different MERS-related viruses identified from Chinese bats, the chimeric viruses with S gene [gene that codes for the spike protein] of bat MERS-related coronaviruses and backbone of the infectious clone of MERS-CoV will be constructed to study the receptor usage and infectivity of bat MERS-related coronavirus.
- Surveillance of infection in human populations by SARS-like CoVs. This work will be performed at locations in Yunnan, Guangxi, and Guangdong provinces, in previously identified areas with human populations of high risk of exposure to bats. PCR and ELISA will be used, respectively, for detection of viral replicase gene and antibodies against the viral nucleocapsid protein.”
Why is this important: first, work with humanized mice continues, now also clearly stating that they are to be infected with chimeric SARS-like coronaviruses. Recall that chimeric means including genetic information from different sources. The search for more coronaviruses is to be continued, continuously building up the potential option for further generation of chimeric coronaviruses. Now the authors also plan to create a MERS-CoV which happens to be the deadliest coronavirus ever isolated, as well as additional synthetic combinations with novel spike proteins. Finally, surveillance of bat coronaviruses that spill-over into human population is to continue in China’s three most southern landlocked provinces, known to be the territory with bat populations in close proximity to humans, but far from centrally located Wuhan. Authors previously published info on WIV1 and had shown that it is 99.9% identical to Rs3367 and was shown to use human ACE2 for cell entry.
p.206: “In Year 2, we continued surveillance for novel SARS-like CoVs from bats in Yunnan and Guangdong provinces and obtained full genome sequence for 11 CoV isolates. Full genome analysis of these CoV isolates was completed, including phylogenetic and recombination analyses. Importantly, recombination analysis of the full-length SL-CoV genome sequences from a single bat population revealed that frequent recombination events among different SL-CoV strains occur. Several SL-CoVs that are more genetically similar to SARS-CoV (2003) than any previously discovered were also identified from bat populations in Yunnan province.”
Why is this important: finally the research team graduated to genomics, obtaining full genome sequences of whole coronaviruses instead of just the spike protein. Even more closely related SARS-CoV bat coronaviruses are isolated (from Yunnan province, where the mysterious Mojiang infection took place). Finally, the authors comment that bat coronaviruses have high prevalence of genetic recombination (meaning different coronaviruses can mix their genetic material). This demonstrates how much potential coronaviruses have to rapidly and dramatically mutate themselves in a natural environment (inside a living host if co-infected by more than one type of coronavirus). In fact, on p.209 we find out that “direct progenitor of SARS-CoV was generated from recombination of WIV16 with Rf4092”!
p.206: “Genomic phylogenetic analysis showed that the SL-CoVs detected in this colony [a specific single bat colony in Yunnan, but which one is not identified] are more closely related to SARS-CoVs from other geographic regions, especially three isolates, WIV16, Rs4874 and Rs4231”.
Why is this important: this information identifies the most closely related coronaviruses to SARS-CoV to that date which helps to find out how closely related these viruses are to SARS-CoV-2 because all these viruses were actually plotted for genetic relatedness by the Chinese research group that published that first SARS-CoV-2 genome sequence. This statement was followed by definitely the best figure in the grant (although we took a better resolution version from one of their published manuscripts), a plot that analyzes the similarity of genetic sequences between different coronaviruses in relation to how related are they to SARS-CoV (strain SZ3 which was identified previously in civets).
We discussed this type of plot in detail before when comparing most related coronaviruses to SARS-CoV-2 (which includes a comparison of SARS-CoV-2 with the above shown WIV1 as well as SARS-CoV strain BJ01 which is closely related to SARS-CoV strain SZ3 that all the above bat coronaviruses are compared against, so check it out if you want to see how similar these viruses are to current SARS-CoV-2). In essence, the lines indicate how close is the genetic sequence of the different identified bat coronaviruses to SARS-CoV. The closer the genetic relation, the closer the line is to value of 100% identity. The further away the genetic relation, the smaller the value, and you can see this as the valleys in the above graph. By the way, that big dip in sequence similarity you see happens to cover the genome region that codes for the spike protein. This shows you that across the genomes of these identified bat coronaviruses, the spike protein can be highly variable and different from one another. The two genomes that exhibit the least dip in that region are Rs4874 and Rs4231. Since SARS-CoV infects humans via binding to ACE2 (same as SARS-CoV-2) you can appreciate from this plot why a coronavirus like Rs4874 can also bind to human ACE2. This shows you how sophisticated the scientific analysis had become and how much new knowledge was being generated by the authors.
p.210: “Additional Year 2 items for Specific Aim 3:
- The infectious clone of WIV1 was successfully constructed using reverse genetic methods;
- Two chimeric bat SARS-like coronavirus strains were constructed by replacing the S gene in the backbone of WIV1;
- Permission to import mice with human ACE2 to China was obtained, so as to conduct the experimental infections proposed in our R01 [type of NIH grant] specific aims.”
Why is this important: this information shows the sophistication level of being able to construct completely new coronaviruses. In this case, the coronavirus labelled WIV1 is stated to have been constructed using reverse genetic methods which means that a virus isolated from nature was reconstructed in a biological system (usually bacterial system) to allow its generation at will. Once the genetic sequence of the naturally isolated virus is decoded and known, it is engineered into a vector (such as bacterial plasmid, a circular DNA) that would allow easy manipulation of the genetic code, easy replication of that genetic code in a bacterial cell host, and easy long-term storage of such biological constructs (freezing long-term for example). The easy manipulation is confirmed by the second bullet point, which states that S gene was being manipulated by swapping one S gene from one coronavirus for another S gene from a different coronavirus. This confirms that the authors have mastered the ability to completely synthesize the SARS-like coronaviruses in any shape and format desired.
p.233 “The results of the 3rd year of our R01 work are detailed below. They include: […]
- The finding of serological evidence of spillover of bat SARS-like CoVs in 6 people in Yunnan. […]
- Receptor binding domain sequences from 37 new bat SL-CoVs that shows S proteins are more diverse than previously thought. […]
- Use of our reverse genetics system to identify 3 more novel SL-CoVs with potential to directly infect people.”
Why is this important: the first point actually points to the support of the authors theory that the spill-over risk of bat coronaviruses to the human population is significant, allowing identification of positive individuals even in a relatively small sample of random inhabitants in southern China (where the bat species with potentially dangerous coronaviruses are found). Second point helps to illustrate the importance of the research that was being undertaken by this group. It also points to an ever-increasing repertoire of coronavirus knowledge, including the emerging appreciation of how diverse and complicated these viruses can be. Third point speaks for itself, generation of novel coronaviruses that could infect people.
p.236: “Observations by research staff in live animal markets in Guangzhou found wildlife to be plentiful, although no bats were seen for sale during the observation period. In contrast, wildlife was not found in live animal markets at the sites we visited in either Yunnan or Guangxi. This is a change from previous research visits to the same or similar communities, when bats, rodents and wild boar could be found. Locals in Yunnan and Guangxi attribute the change to conservation law enforcement. The success of conservation enforcement may have moved hunting and trapping underground and made the capture of local wildlife less economically feasible than other income generating activities.”
Why is this important: this helps to showcase that the animal market could be a less likely explanation of how the pandemic started, than as originally suggested.
p.251: “In Year 3 we established an effective and economic reverse genetics system for bat SL-CoV which can be applied to efficiently rescue SL-CoVs that are difficult to culture. This can be used to explore the functions of newly identified SL-CoV genes, as well as to assess pathogenesis of novel bat SL-CoVs.”
Why is this important: this spells out once again that a system of developing and manipulating any coronavirus was established. A mechanism that allows designing novel viruses has been created, allowing the study of viruses that otherwise would not be available for such work due to the difficulty of keeping these viruses alive and propagating in cultures.
p.251: “we conducted full-length genome sequencing of 11 novel SL-CoVs detected in a single bat habitat in Yunnan province, which included strains highly similar to human/civet SARS-CoV in the most variable genes (N-terminal domain and RBD [receptor binding domain] in the S gene, […]). Based on recombination analysis, we hypothesized that the direct progenitor of the pandemic SARS-CoV may originated from this location after sequential recombination events at multiple genomic positions.”
Why is this important: further evidence of the growing understanding of coronaviruses’ genetics by the research group – it had advanced enough to commence establishing hypotheses of prior coronaviruses origins. This is important because the authors also are studying virulence of these strains, and understanding how genetic components might be swapped between viruses, in relation to virus infectious capacity, which starts to point to specific genetic components of importance for their value towards human infection.
p.251: “Among the 11 newly identified SL-CoVs, three different strains namely Rs4874, Rs7327 and Rs4231 contained no deletions in the RBD region but their RBD sequences varied from each other. […] Rs7327's S protein varies from that of WIV1 and WIV16 at three aa [amino acids] residues in the receptor-binding motif, including one contact residue (aa 484) with human ACE2.”
Why is this important: further information on the 11 new virus genomes decoded, and the discovery of the importance of residue 484 for binding human receptor. This same residue will later turn out to be very important in some of the variants of concern during the pandemic (alpha, beta and gamma variants, or the UK, South African and Brazilian variants if you need the older designations). It really shows you how advanced the researchers’ knowledge was ahead of time prior to the pandemic.
p.251: “Using the reverse genetic system we previously developed, we constructed two chimeric viruses with the WIV1 backbone replaced with the S gene of Rs7327 and Rs4231, respectively. Vero E6 cells [cells that are interferon-deficient so they do not secrete anti-viral interferon when infected by viruses] were respectively infected with Rs4874, WIV1-Rs4231S and WIV1- Rs7327S, and efficient virus replication was detected by immunofluorescence assay in all infections. To assess the usage of human ACE2 by the three novel SL-CoVs, we conducted virus infectivity studies using Hela cells with or without the expression of human ACE2. All viruses replicated efficiently in the human ACE2-expressing cells.”
Why is this important: this is most direct language thus far showing that synthetic versions of viruses were being generated that could infect human cells. Whether this constitutes gain-of-function (increased infectivity) should be a mute point, as it clearly demonstrates that new, synthetic viruses with the potential to infect humans were being made in the lab. Immunofluorescence is a technique allowing virus detection and quantity measurement through binding of the virus with fluorescent antibodies.
p.253: “Mice with human ACE2 have been imported to China and have been bred for one generation in Wuhan Institute of Virology. […] The animal infection experiments are planned to be conducted in following years to study the pathogenicity of diverse SL-CoVs and MERS-related CoV that we identified in Chinese bats.”
Why is this important: this confirms that the technology of genetically humanized mice was imported to Wuhan, allowing for further development of viruses with a potential to infect humans. In addition to SARS-related coronaviruses, the authors also indicated that MERS-related coronaviruses were also identified and pursued for further genetic understanding. MERS was the deadliest coronavirus to ever infect humans. However, MERS does not use ACE2 receptor to infect human cells.
p.268: “If any experiments proposed in this award result in a virus with enhanced growth by more than 1 log compared to wild type strains, you must notify your NIAID Program Officer and Grants Management Specialist immediately. Further research involving the resulting virus(es) may require review by the Department of Health and Human Services in accordance with the Framework for Guiding Funding Decisions about Proposed Research Involving Enhanced Potential Pandemic Pathogens.”
Why is this important: this shows that gain-of-function appears to be no longer prohibited but rather carefully monitored by the granting agency.
p.272: “The wildlife reservoirs of SARS-CoV were identified by our group as bat species, and since then hundreds of novel bat-CoVs have been discovered (including >260 by our group).”
Why is this important: indication of the sheer number of coronavirus genomes to play with by the research group while generating new synthetic constructs.
p.298: “Using the reverse genetic methods we previously developed, infectious clones with the WIV1 backbone and the spike protein of SHC014, WIV16 and Rs4231, respectively, were constructed and recombinant viruses were successfully rescued. In Year 4, we performed preliminary in vivo infection of SARSr-CoVs on transgenic mice that express hACE2. Mice were infected with 105 pfu [plaque forming units, a measure of viral quantity] of full-length recombinant virus of WIV1 (rWIV1 [recombinant indicates generated using controlled system, as opposed to isolated from nature]) and the three chimeric viruses with different spikes. Pathogenesis of the 4 SARSr-CoVs was then determined in a 2-week course. Mice challenged with rWIV1-SHC014S have experienced about 20% body weight loss by the 6th day post infection, while rWIV1 and rWIV-4231 S produced less body weight loss. In the mice infected with rWIV1-WIV16S, no body weight loss was observed (Fig. 35a). 2 and 4 days post infection, the viral load in lung tissues of mice challenged with rWIV1-SHC014S, rWIV1-WIV16S and rWIV1-Rs4231 S reached more than 106 genome copies/g and were significantly higher than that in rWIV1-infected mice (Fig. 35b). These results demonstrate varying pathogenicity of SARSr-CoVs with different spike proteins in humanized mice.”
Why is this important: this is probably as close as we might ever come to a smoking gun with regards to any possibility of linking the research and development of synthetic coronavirus constructs and the potential emergence of the pandemic, especially if these experiments were performed in Wuhan. This section indicates that different coronaviruses’ segments were fused together (becoming “chimeric”) and studied for increased pathogenicity in humanized mice. This section clearly spells out that such mice were studied for viral loads, which are complicated studies, with a virus of clear potential to infect humans - strongly pointing to the high level of risk undertaken with such studies.
What is not clear is where these studies took place, as similar research with chimeric SHC014 strain was previously published by the research team of Dr. Ralph Baric at the University of North Carolina who is one of the participating parties in this grant awarded research. At the time the work of Baric was criticized for the unnecessary danger of creating novel infectious viruses. If this work was delivered in Wuhan, as this grant indicates they were set up for such experiments, this could help link the origin of the pandemic with a novel coronavirus with numerous novel features and highly adapted for human infection to a lab leak theory. Both locations are listed as performing experiments using mice. On p.514 we learn “At the University of North Carolina (US Government select agent certified laboratory), some virus growth studies will be conducted in primary human airways, comparing wildtype SARS-CoV, WIV1 and various SARSr-CoV WIV1 chimeric virus growth kinetics. Wildtype SARS-CoV strain research will not be conducted at the Wuhan Institute of Virology.” Indeed, the work described here does not revolve around the original SARS, only novel coronavirus constructs. Recall that the Rs4231 coronavirus had one of the most closely related spike proteins to the original SARS, while the more damaging SHC014 strain is the one isolated earlier. Figure 35 is reproduced below. DPI refers to days post infection.
p.331: “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.”
Why is this important: this points once again to the advanced knowledge of the research group that allowed deep enough understanding of the spike protein to start predicting the threat to human populations based on spike protein differences between different coronaviruses.
p.483: “We will characterize the propensity of novel SARSr-CoVs to infect people in vitro using primary human airway epithelial cells and in vivo using the transgenic hACE2 mouse model. We will use mAb [monoclonal antibodies, or antibodies of one type as opposed to a mixture of different antibodies] and vaccine treatments to test our hypothesis that SARSr-CoVs with 10-25% divergence in S protein sequences from SARS-CoV are likely able to infect human cells, and to evade mAb therapeutics and vaccines.”
Why is this important: this is definitely getting interesting with this further evidence pointing to the degree of how much the coronaviruses were investigated for their potential to infect humans. This is the first mention of studying the viruses using human epithelial cells (cells lining your respiratory tract) besides using humanized mice, and testing the degree to which such viruses not only could be infectious to humans, but also the degree to which such viruses would also resist treatment. Once again, research with an incredible pandemic risk to human populations in an event of lab leak. As the authors stated themselves on p.486 “these strains likely use hACE2 but could escape existing vaccines and immunotherapeutics and represent significant public health threats.” If such natural viruses were deemed to pose significant public health threats, so would the laboratory viruses further engineered to be even more pathogenic.
p. 485: “In collaboration with Ralph Baric (UNC), we used the SARS-CoV reverse genetics system to generate a chimeric virus with a mouse-adapted SARS-CoV backbone expressing SHC014 S protein with 10% sequence divergence from SARS-CoV S. This chimera replicated in primary human airway epithelium, using the human ACE2 receptor to enter into cells.”
Why is this important: this sheds more information on the SHC014 strain spike which was determined to infect human cells with its importance revealed immediately below. This particular quote does appear to refer to the work published by Baric who did use SARS-CoV (MA15) strain that was specifically developed through serial passaging to infect and kill mice and reproduce SARS-like disease post infection (mice otherwise were not affected by SARS). However, SARS-CoV (MA15) is not the same backbone for chimeric experiments as the WIV1 strain described above and below, which was identified and developed in Wuhan.
p.486: “Infection of rWIV1-SHC014S caused mild SARS-like clinical signs in the transgenic hACE2 mouse model that weren't reduced by immune-therapeutic monoclonals that attenuate SARS-CoV pathogenecity. Vaccination against SARS-CoV did not reduce severity of clinical signs in mice subsequently infected with rSARS SHC014S. We found 2/4 broad human mAbs against SARS-CoV RBD cross-neutralized WIV1, but none could efficiently neutralize SHC014 which is less similar to SARS-CoV in the RBD [receptor binding domain, or the area of spike protein needed to bind to human ACE2 receptors].”
Why is this important: this is a further description of the synthetic viral construct with potential to infect humans, producing clinical symptoms in animal models similar to SARS and resistant to neutralization (preventing viral infection) by antibodies.
p.491: “Of the expected 100-200 novel SARSr-CoV strains, we will down-select to prioritize for further characterization based on S genes that are: i) different from SHC014, WIV1, SARS-CoV with diversity ranges of 10-25%; ii) have virus S RBD that could use human/bat receptors; iii) have recombinant chimeric spikes indicative of gene flow between clade I and II strains; iv) have bat ACE2 receptors that might select for spike RBDs that can use human receptors for entry (15/18 conserved residues in human/bat ACE2 molecules that bind SARSs-CoV S RBD domains are likely more efficient receptors than 3/18 conserved sites). Using structural models based on the SARS S glycoprotein, the extent and location of antigenic variation will be annotated onto the structure, further resolving the locations of highly evolving and conserved structural motifs/epitopes that function in protective immunity.”
Why is this important: this describes further research expectations with the search for more dangerous coronaviruses with a potential to infect humans and introduces additional sophisticated tools allowing for modeling of the spike protein. The constant growth of knowledge about coronavirus’ spike protein in relation to human infection and immune protection is evident.
p.494: “Infectious clones with the S gene of novel SARSr-CoVs and the SARSr-CoV WIV1 genome backbone using the reverse genetic system developed in our previous R01 [previously approved grants]. The correct infectious BAC [bacterial artificial chromosome, a biological system allowing storage and manipulation of large segments of genetic code] clones will be screened by BAC DNA digestion with appropriate restriction enzyme or PCR amplification. The chimeric viruses will be rescued in Vero cells and then verified by sequence analyses. Our research group is well versed in coronavirus reverse genetics.”
Why is this important: this is bragging rights by the authors of their capabilities of creating coronaviruses of desired design from scratch. Ironically, prior to these grant applications, a scientist who defected from China were making claims that SARS-CoV-2 virus was of synthetic design but at the time such claims were outright dismissed. Some of the condemning evidence they put forth was the tell-tale hallmarks of restriction enzyme cut sites in SARS-CoV-2, although that in itself does not amount to much. Restriction enzymes are molecular scissors that can cut DNA at a specific site through recognition of specific DNA code. Use of restriction enzymes is standard for mixing different genetic fragments from different sources, such as synthesizing chimeric viral constructs. In light of current statements in the NIH grants, those prior accusations are just that much more interesting to read now.
p.495: “hACE2 transgenic mice will be injected with SARS-CoV mAbs, and infected with chimeric bat SARSr-CoVs. Clinical signs and morbidity will be assessed and tissue pathology examined and compared with mice without treatment of mAbs to determine the therapeutic effect on SARSr-CoV infection, and protection of SARSr-CoV by wildtype SARS-S based vaccines assessed as described. We will sequence full length genomes of high risk strains that are antigenically distinct and escape SARS cross neutralization, synthetically reconstruct a small subset (1-2) and evaluate the ability of nucleoside analogues to inhibit growth in HAE cultures and/or in vivo.”
Why is this important: this is the last information we have for this grant prior to the pandemic interrupting the work, but it clearly indicates further work in selecting viruses with a potential to infect humans while at the same time being as resistant to potential treatment as possible, and once again it points to the ease of ability to take a coronavirus isolated from nature and create a synthetic version. SARS cross neutralization refers to the use of antibodies known to inhibit the infection capacity of the SARS virus while also being able to inhibit other coronaviruses. Hence, escape from such antibodies indicates coronaviruses that are mutated in such way that they can be more infectious to humans. Again, just dangerous work.
p.514: “The proposal will use a SARSr-CoV molecule clone designated WIV1 during the course of these studies, which is NOT a select agent. This strain has not been shown to cause human disease or be transmissible between humans. All recombinant DNA work will use the bat SARSr-CoV WIV1 molecular clone.”
Why is this important: this appears to be only information with regards to safety precautions being taken, however, this is extremely weak argument. From content, “select agent” appears to be defined as an agent that is known to be dangerous to humans, such as previous SARS. However, arguing that WIV1 coronavirus is safe because it does not infect humans becomes irrelevant when such virus has a spike protein replaced with one that is designed to allow the virus to infect humans. Authors are arguing for the safety of the virus, while ignoring the simple fact that this otherwise “safe” virus is reconstructed to be dangerous to humans. The spike protein after all is how these coronaviruses are infecting humans, and therefore the spike protein is the primary influencing factor determining how infectious a virus is to humans. In addition, the claim that WIV1 has not been shown to be transmissible in humans is also somewhat duplicitous because the authors themselves showed that WIV1 strain of coronavirus could infect humanized mice resulting in lung high viral loads, clearly pointing to a potential danger to humans (figure 35 above), not to mention that it was these same authors that demonstrated that WIV1 strain was the first bat coronavirus identified to not only bind to ACE2 but could bind to human ACE2. Thus, at the very least, this is indicative of a potential to infect humans.
p.516: “Importantly, we are not proposing to genetically manipulate SARS-CoV over the course of this proposal. However, we are proposing to genetically manipulate the full-length bat SARSr-CoV WIV1 strain molecular clone during the course of the proposal, which is not a select agent, has not been shown to cause human infections, and has not been shown to be transmissible between humans.”
Why is this important: this simply points further to the fact that the SARSr-CoV WIV1 coronavirus used as a template for developing further constructs can no longer be dismissed as a safe agent, if the new constructs will have newly mutated spike protein that is specifically selected to be more infectious to human cells, and more resistant to therapeutic protection.
To summarize, the NIH grants reveal that the work performed in Wuhan along with other institutes was geared towards generating coronaviruses more infectious to humans to learn about potential future threats which could then be subsequently monitored. This type of information makes a lab in Wuhan as the starting place of the current pandemic more plausible in light of the type of dangerous research with human pandemic potential that was being conducted in that city.
This also begs the question as to why did it take so long to have this information publicly available, with more than a year and a half since the pandemic’s start? After all, these were documents of publicly funded grants granted by NIH, an institute that has a carefully crafted image of great transparency, one of the reasons why the institute affords such a great public trust. With the information in these documents of the type of research being conducted, one could speculate that such an obvious assumption that the pandemic had to be of natural origin would not be as palpable if this information was immediately available. What is worse, if you chose to make the assumption that it is not a coincidence that the pandemic started in Wuhan where coronaviruses were being engineered to be more infectious to humans, then it would appear that accidentally, research in China funded by the US government started a global pandemic. You can see that such assumptions would implicate the world’s two greatest superpowers in unintentionally producing the pandemic through conducting insanely dangerous research (which was banned in the US for these very reasons – hence welcomed in China which appears to have a much higher risk tolerance in what type of scientific experiments can be performed).
If the type of dangerous experiments undertaken in Wuhan and the subsequent pandemic emerging from there are indeed mere coincidences, it is still inappropriate that information was attempted to be withheld or covered up.
We applaud the fact that such information is coming to light. The journalistic work of The Intercept had an immediate effect leading to members of Congress introducing legislation to freeze federal funding for gain-of-function research in China. How about anywhere? When it comes to global crises, it is quite irrelevant where the issue originated. What becomes much more important is how to prevent future crises, and for that we need as much transparency as possible so we know how information should be properly analyzed. Transparency from authorities tasked with governing complex systems is also a great method in winning public trust and support. Whatever conclusions might be made based on these NIH reports in terms of the pandemic’s origin is only important for one purpose: how do we help the global community, every citizen of our planet, be safer and live a life with the greatest dignity that we as a humanity can create for all of us? Definitely we need to consider stricter measures for what kind of scientific research is being conducted. No research should ever be considered that threatens humanity as a whole, such as work on viruses with global pandemic potential. If we were to allow ourselves to assume that this pandemic was an outcome of dangerous research being conducted, the alleged benefits of such dangerous research as gain-of-function or creating novel viruses with infectious potential clearly has been outstripped by the losses incurred due to the outbreak of a global pandemic. Perhaps this will also help us transcend to other aspects of life that afford us the safety of humanity as a whole, be they concerns with regard to pollution of the planet or equitable use of our global resources.
In these confusing and sometimes confrontational times, it is good to hope that humanity instead is still constantly improving.
This article has been produced by Merogenomics Inc. and edited by Jason Chouinard, B.Sc. Reproduction and reuse of any portion of this content requires Merogenomics Inc. permission and source acknowledgment. It is your responsibility to obtain additional permissions from the third party owners that might be cited by Merogenomics Inc. Merogenomics Inc. disclaims any responsibility for any use you make of content owned by third parties without their permission.
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