A rational analysis of the new lethal brain virus experiments reveals unseen inconsistencies as well as substantial covert biorisk implications (PART I)

The identified inconsistencies and vulnerabilities also contradict each of the expected benefits of GoF(type) work

Welcome to my substack! This will be my first “real” (scientific) post. I have been digging into issues related to the novel brain virus. To the best of my knowledge, these have not been identified elsewhere. What I have discovered will be shared in 3 parts.

An overall summary is as follows:

• When potential risks of gain of function experiments were first recognized, there was a clear definition of GoF experiments. Essentially, they encompassed everything that made a (potential) pathogen more nasty. In recent years, Dr Fauci has vehemently denied the original conception of GoF, pretending that they are defined differently. (But all of this is another story….)

• This note critically appraises the purported benefits that ostensibly justify GoF work.

• This is exemplified via the lab development of a novel SARS-CoV-2-related pangolin coronavirus that can result in 100% mortality in human ACE2-transgenic mice, potentially caused by late-stage brain infection.

• The main reason for the substantially enhanced lethality of the virus is likely attributable to basic lab experiments.

• As this novel brain virus was not intentionally engineered by the Scientists at the Beijing University of Chemical Technology, some may argue that their experiments are not GoF (even though the virus gained the ability to be 100% lethal).

• Regardless of what does and does not count as GoF, as is highlighted below, even within the framework of the published brain-virus experiments, there are no aspects whatsoever that align with the expected benefits of GoF work.

• The overarching analysis of these experiments provided below reveals inherent inconsistencies, challenges, and insights that provide a rational proof as to why such lab experiments, in fact, contradict the anticipated benefits of GoF(type) work, instead creating substantial biosafety/biosecurity risks and vulnerabilities related to all the claimed benefits.

In today’s post (Part 1), I will begin with the motivation for my analysis, the main findings obtained by the Chinese Researchers on the new brain virus, how these deadly viruses emerged, and the substantial pathogenicity differences between the variants in question. An independent analysis of this reveals some issues and challenges. Part 2 will then dig deeper into the potential of dangerous mutations obtained via cell culture experiments and will investigate if there are other explanations for why the pangolin CoVs became so virulent. Part 3 will then tie all of this together, including the issues and challenges that I discovered, what all of this means for biosafety/biosecurity and related to expected benefits of such GoF-type research. I don’t want to discourage readers of the first two parts, but the most important findings will be presented in Part 3.

1.1. Motivation

The news about the novel brain virus, posted January 04, 2024, on the Preprint Server, has been shocking. There, Chinese Scientists reported that a specific novel coronavirus strain, after it was studied in the lab ended up being 100% lethal in ‘humanized’ mice, raising substantial concerns for humans should such a virus get out of the lab. The progenitor SARS-CoV-2-related pangolin coronavirus, however, was previously reported incapable of causing severe disease.

Many are now wondering: What made those viruses so lethal? Why is such type of work not forbidden?

John Campbell, in two YouTube videos, may have touched on a nerve when discussing this preprint. Within a very short time, his first video garnered over 700k views and 60 likes. But it was quickly taken down by YouTube. When he reposted essentially the same video that too, gained enormous attention but was also taken down by YouTube.

What can he possibly have said that was so offensive, seeing that he was merely discussing what is in the open domain? After all, the article in question had been published on ResearchGate.

Of course, Campbell highlighted the risks of such experiments, which he described as a potential existential threat to humanity. Since the novel variant was able to kill all the humanized mice, he believes that a similar fate would befall humans should they be infected with this virus. The only potential application, he concluded, is as a bioweapon.

Now, the authors of the preprint never claimed they had set out to construct a killer virus. By contrast, they maintain that their work is critical to assess the potential spillover risk of these types of CoVs into humans.

Practically, it does appear that their viral variants are the result of some gain-of-function experiments. Indeed, they were the first to report the “surprising” high pathogenicity of the virus -- which had not been seen in its precursors. It would seem obvious that this depicts GoF experiments - as the novel variants are radically different, killing all the tested mice (whereas its precursors were reportedly attenuated).

However, in recent years, Dr. Fauci and colleagues have been adamant in promoting the notion that GoF experiments are something different altogether, creating much confusion and fruitless debate.

It seems that all the discussions as to what does and does not comprise a GoF experiment are major distractions from what is practically happening. Many high-ranked officials maintain the necessity of laboratory experiments - GoF work, if you wish - for specific expected benefits which are usually portrayed as so critically important that they would outweigh any potential risks of a lab escape/release into the open environment.

For years, it has been argued that GoF experiments are critically essential to prepare for the next pandemic. The claims have been that, specifically, such work should help to

(1) Predict spillover potentials of new pathogens,

(2) Help prepare so-called countermeasures (e.g. vaccines).

(3) Additionally, in recent years, we have observed yet another reason that apparently seemed to have justified GoF experiments: it’s to purportedly help prove the origin of SARS-CoV-2 (i.e., ostensibly from nature).

In this note, I will show major flaws with these assertions based on the example of the new brain coronavirus (CoV), based on the preprint by Song and collaborators. A key focus will be trying to comprehend what exactly made those viruses so lethal. As it turns out there is some uncertainty in the published literature that made things even more challenging.

1.2. What are the main findings of the new study by Song et al.?

Again, the preprint study by Song et al. demonstrates the ability of a new virus to replicate in lung and brain tissues in humanized mice. The SARS-CoV-2-related pangolin variant “GX_P2V(short_3UTR)” was derived from a previously passaged virus that had mutated in cell culture and then successively cloned. All of the four test mice, engineered to express human ACE2 receptors, when injected with variants of the new virus, were killed within eight days. The authors suggest that the cause of death may be severe neurological infection (brain infection).

How are those deadly viruses obtained?

That is, of course, the key question. According to Song and colleagues, the deadly brain virus(es) materialized as follows:

• Before the pandemic, two SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins were already known. One of them, the pangolin CoV GX_P2V, later drew special attention regarding its potential role as a precursor of SARS-CoV-2. It purportedly was previously cultured. Specifically, “The genome of coronavirus GX_P2V (GenBank accession number MT072864) was determined by using RNAs from the first passage of the GX_P2V sample.”

• This first-passage variant was not regarded an isolate of this virus. Thus,  Lu et al. reported the isolation of the original GX_P2V sample via serial passaging (more on this in Part II). Consequently, the eighth passage of GX_P2V was “partially purified from [Vero] cell culture supernatants by ultra-centrifugation.”

• The first important finding by  Lu et al. is that this serially passaged virus had rapidly adapted to Vero 6 cells.

• Lu et al. report that, compared to the single cell-passaged sample, the multiply passaged GX_P2V isolate had two mutations: one nonsynonymous mutation in the final amino acid codon of the nucleoprotein gene and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3′-terminus untranslated region (3′-UTR). 

• The GX_P2V variant, after this 8^th passage, was termed GX_P2V(short_3UTR).

GX_P2V(short_3UTR) is the variant that was the basis of the experiments by Song et al. in humanized mice, where it turned out to be so deadly.

However, it is important to note that the main focus of their preprint is not on GX_P2V(short_3UTR). In fact, Song and colleagues further modified GX_P2V(short_3UTR) before they did their experiments on the mice. Specifically,

• Song et al. cloned GX_P2V(short_3UTR) through two successive plaque assays.

• Doing so resulted in eight viral clones which then were chosen for next-generation sequencing.

• These clones, when compared with the genome of the original mutant analyzed by  Lu et al., all shared four identical single-ntd mutations.

• One of these clones, Clone 7, named GX_P2V C7, was randomly selected for further analysis - to assess its pathogenicity in hACE2 mice.

1.3. Drastic Differences in the Pathogenicity between these Viral Variants

The individual variants have shockingly different pathogenicities, as detailed by the published analyses by  Zheng-Li Shi et al., Lu et al., and Song et al.

Pathogenicity of GX_P2V, analyzed by Zheng-Li Shi et al.

It is important to note that it was previously clearly established that the original GX_P2V virus is not a human pathogen. This observation was stressed by Song et al., pointing to Ref. [5] in their preprint. The latter refers to the work by Zheng-Li Shi and collaborators, who had found that this variant, while able to infect mice expressing human ACE2, did not cause severe disease. (Ostensibly, the pangolin virus MpCoV-GX, said to be isolated from a smuggled Malayan pangolin, is the same as GX_P2V, which, too, was described as originating from an anti-smuggling operations in 2017).

Pathogenicity of GX_P2V(short_3UTR), analyzed by  Lu et al.

Lu and colleagues, based on the non-pathogenic nature of GX_P2V investigated the serially passaged variant (that is, GX_P2V(short_3UTR)) for its potential to serve as a live attenuated vaccine against SARS-CoV-2. This idea was based on the following findings:

• The observed highly attenuated character of GX_P2V(short_3UTR):  Lu et al. report that this variant could infect all five of the tested in vitro and in vivo infection models. However, it did so in a highly attenuated manner. For example, while it caused cell lysis in non-human primate cells, there was only mild cell damage, whereas no cytopathic effects or plagues were observed in Calu3 cells. As for animal models, GX_P2V(short_3UTR) infected golden hamsters and BALB/c mice but was highly attenuated.

• Golden hamsters infected intranasally had a short duration of productive infection in pulmonary, not extrapulmonary, tissues. This infection was reported to induce “neutralizing antibodies” against both GX_P2V and SARS-CoV-2. (More could be said about this, but but will not be discussed here).

• Of note, it has been previously suggested that the wild-type GX_P2V virus is an attenuated SARS-CoV-2 vaccine strain, even though the virus was initially identified in dead pangolins.

Pathogenicity of the cloned GX_P2V(short_3UTR) (that is, GX_P2V C7), analyzed by Song et al.

Recall that the focus of the preprint by Song et al. was on GX_P2V C7. As stated, the most disturbing finding by Song et al. is that this variant is 100% lethal in humanized mice. The virus was able to replicate in the lung and brain tissue of the genetically engineered humanized mice, killing all of them. Specifically, the study found the following strong indicators that the killing of the mice was not the result of poor experimentation itself:

• Detection of viral RNAs: This was done by quantifying GX_P2V N gene copies. While there may be some questions about this, the authors reported that they found significant amounts of viral RNA in the brain, lung, turbinate, eye, and trachea of the GX_P2V C7 infected mice; high viral RNA loads on days 3 and 6 post-infection (dpi) in lungs; on 3 dpi high viral RNA, and by 6 dpi, exceptionally high viral RNA loads in all four infected mice in brain samples; substantial viral RNA loads was also detected in the turbinate.

• Detection of infectious viral titers: This was measured by plaque-forming assays. Accordingly, the authors report significant tissue differences, with the highest titers detected in the lung, brain, and turbinate. Of note here is the brain where the study identified severe brain infection: (the highest infectious titers were detected on day 6, which was even significantly higher than that on day 3).

• Pathological alterations: infected mice showed significant neurological alterations, including shrunken neurons visible in the cerebral cortex and focal lymphocytic infiltration around the blood vessels. By contrast, neither mock-infected mice nor those infected with the inactivated GX_P2V C7 strain showed any clinical symptoms at all.

• Immunohistochemistry (staining for the viral N protein) also identified viral antigens in the lung and brain of the infected mice, in contrast to the controls. (Again, this may raise some questions given that this was based on the detection of an anti-SARS-CoV-2 N-specific Ab, but I don’t have enough info on this).

No clarity as to which variants demonstratively did not cause disease

To assess the troubling fact of GX_P2V C7’s extreme lethality, Song et al. stress its difference to GX_P2V. This is interesting, as their report deviates from what has been described by Lu et al.

That is, Song and colleagues specifically contrast the high pathogenicity of the cloned GX_P2V(short_3UTR) variant with GX_P2V. However, there is no clarity in the published literature.

• Firstly, Song and colleagues emphasize that both GX_P2 and another SARS-CoV-2-related pangolin CoV they studied can infect  “both golden hamsters and hACE2 mice.” Yet, they stress that in contrast to this other CoV, GX_P2 “does not appear to cause obvious disease in these animals.” One of the references is their own previous work (Lu et al.) -- which, however, analyzed GX_P2V(short_3UTR) and not GX_P2V (see Figure 1 below).

• Since they referred to Ref. [6] in their work (which is Lu et al.), which they described as their own findings (l 104 in the preprint), they may have more information than what is published.

• It may be that neither GX_P2V nor GX_P2V(short_3UTR) are (highly) pathogenic in the tested animals (notably, these include hACE2 mice). This remains uncertain, however, as seen from the final analysis of Song et al: “The high pathogenicity mechanism of GX_P2V C7 in hACE2 mice, in the absence of the wild-type GX_P2V control, requires further investigation.” Note that they keep referring to GX_P2V as control rather than GX_P2V(short_3UTR).

Fig. 1: Inconsistent literature depiction of characteristics of (variants of) the new brain virus.

Pathogenicity of the uncloned GX_P2V(short_3UTR), analyzed by Song et al.

Song et al. seem to want to highlight that the disparate pathogenicity, in hACE2 mice, may to a large part be related to the genetic differences between GX_P2V C7 and GX_P2V.

This may suggest that much of the “surprising” pathogenicity of this variant was only identified in their new preprint and exclusive to this particular variant (i.e, GX_P2V C7) that was studied therein.

However, this is not the case. Perhaps somewhat hidden in the preprint by Song et al. is their following finding:

“[A]ccording to additional infection experiments, the uncloned GX_P2V(short_3UTR) also resulted in 100% mortality in hACE2 mice.”

Somewhat surprisingly, this means that despite the focus of this study being on  GX_P2V C7 - which is the “cloned” version of the passaged GX_P2V(short_3UTR) mutant - the extremely high mortality rate concerns the “uncloned” mutant as well.

The most important features of the viral variants in question are summarized in Fig. 2 below.

Fig 2: Summary of the viral variants implied in the genesis of the novel brain virus.

This has immediate major biorisk implications:

1. The risks that those viral variants themselves pose to humanity (e.g. lab escape).

2. The challenge of identifying/declaring key characteristics of the variants. From what is published, it is unclear which were the first deadly variants.

3. The radical difference in pathogenicity of the individual variants, with this same disparate pattern applying to several in vitro and in vivo models. In other words, there was no slow adaptation of these viruses, i.e. with them gradually increasing their pathogenic potential. Rather, these viruses changed rapidly from not being able to cause (severe) disease to being 100% lethal, raising the prospect that the underlying mechanisms could either be accidentally or deliberately repeated.

4. The fact that it was considered important - and legitimate - to perform such experiments. Thus, it would likely be possible to camouflage dangerous research via seemingly less harmful viruses: whilst research with such viruses would not be regarded as dangerous, simple  lab experiments could quickly and effectively turn those viruses into clandestine bioweapons.

Parts 2 and 3 will dig deeper into these and additional issues of the experiments that led to the novel “killer viruses.”