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A pangolin coronavirus decimates “humanized” mice: what do you need to know?
Published: January 29, 2024 10:44am EST
Author
Anne Goffard
Doctor, University Professor – Hospital Practitioner, University of Lille
Disclosure statement
Anne Goffard is a virologist at the University of Lille and a doctor at Lille University Hospital. She received funding from I-Site ULNE, ANR, CNRS and Lille University Hospital. She is deputy mayor of Lille in charge of Universities, Research, Students and Pandemic Risk Management.
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A publication put online at the beginning of January 2024 on the BioRχiv site (pronounced, in Anglo-Saxon, “bioarchive”) caused a lot of ink to flow. It must be said that the story she tells is particularly intriguing.
Titled “Lethal Infection of Human ACE2-Transgenic Mice Caused by SARS-CoV-2-related Pangolin coronavirus GX_P2V(short_3UTR)” , it involves mice, pangolins, as well as a virus that is the cousin of an old acquaintance, the SARS-CoV-2 coronavirus.
In this scientific article not yet peer-reviewed (this is an important point to emphasize), researchers from the Beijing University of Chemical Technology report the results of their experiments. The latter consisted of infecting “humanized” mice with a virus derived from a coronavirus initially isolated in pangolins.
Result: a week later, 100% of the rodents were dead, the virus having invaded not only their lungs, but also their brains. How to interpret these results ? Should we be worried? Decryption.
Backtracking
To fully understand the context in which these experiments were carried out, we need to go back a few years. In 2020, precisely.
At that time, Chinese research teams were publishing pangolin coronavirus genome sequences. These came from viruses isolated from samples taken between 2017 and 2019, from animals seized during anti-smuggling operations. Before this discovery, no pangolin coronaviruses were known. However, the fact of having found coronaviruses in pangolins is not surprising, since these viruses infect mammals.
Two distinct strains were then isolated by the Chinese teams, after infection of cells in culture: the pCoV-GD01 strain (from a sample taken in 2019) and the GX-P2V strain (sample dated 2017).
Following the discovery of the two pangolin coronaviruses, other work was carried out to characterize them: complete sequencing of their genome, infection of different cell lines, infections of humanized animals. It was thus discovered that the genomes of these two viruses have significant homologies with that of SARS-CoV-2.
Remember that if the pangolin once appeared on the list of suspects that may have served as an intermediary between the natural reservoir of SARS-CoV-2 (still unknown, even if bats are suspected) and humans, it was later exonerated . Indeed, viruses with much closer sequences have been identified in certain bats (in particular the RaTG13 coronavirus, isolated from the horseshoe bat Rhinolophus affinis , which is found in particular in south-west China, more than 1,500 km from Wuhan, where the first SARS-CoV-2 infections probably occurred).
The conclusions of the studies carried out on the pCoV-GD01 and GX-P2V strains were published between 2020 and 2023, after peer review. In other words, scientists, experts in the field, but who did not participate in this work, analyzed these results and the way in which they were obtained, and judged them to be trustworthy.
It must be emphasized again that the new study has not yet undergone this evaluation process, which is the usual route for any “serious” scientific publication.
A new study that still needs to be verified
The work published in January 2024 is currently what we call, in scientific publishing jargon, a “preprint”: it has not yet been peer-reviewed. This is not abnormal, because BioRχiv, the site where it was deposited, is precisely intended to host such publications. They can, after having been uploaded, be the subject of comments.
This way of proceeding allows information to be circulated more quickly to specialists, who can comment on it, and sometimes also be used to establish the precedence of a discovery.
However, information contained in preprints (regardless of the site hosting it) should not be considered completely reliable until it has passed peer review and been published in a peer-reviewed journal. reading.
What information is contained in this new study?
Mutated pangolin viruses, sure, but not intentionally
During previous work, published in 2020 and 2023, the pangolin viruses that had been isolated were cultured on cells in the laboratory.
Over the course of these successive cultures, their genome underwent mutations, some of which were found to confer an advantage to the viruses that possessed them. These mutants therefore prospered, passing on their mutations to their descendants.
This natural phenomenon classically occurs when viruses are cultivated in vitro , whatever they may be: it has already been observed in the case of HIV or the hepatitis C virus, for example. Several selection mutants have been obtained in this way, but one of them is of particular interest to us.
Called GX-P2V (3'UTR), it has the particularity of having a genome amputated by 104 nucleotides, the “building blocks” of the RNA of which the genome of coronaviruses is made. This missing piece is normally located in the non-coding region located at one of its ends (3'UTR).
Another particularity of this mutant: the authors discovered that when they used it to infect mice, all of the sick animals died in 7 to 8 days. In dead animals, a significant amount of viral RNA was found in the lungs and brain. According to the authors, it was probably the brain damage that killed the rodents.
However, the infected mice were not just any mice, but so-called “humanized” mice. In other words, mice genetically modified to produce, on the surface of their cells, the human ACE2 receptor. The same one that allows the SARS-CoV-2 coronavirus at the origin of the 2020 pandemic to recognize human cells and infect them…
When the researchers further analyzed the GX-P2V(3'UTR) genome, they found the presence of mutations in the ORF1ab, S, and N genes. ORF1ab is a part of the genome conserved in coronaviruses, which helps produce proteins essential to the life cycle of these viruses. The S gene codes for the Spike protein (the “key” which allows the coronavirus to enter the cells it infects, by interacting with a receptor located on their surface). Finally, the N gene is used to produce the nucleocapsid protein, which is associated with and protects the genetic material of the virus. This latter protein is usually very immunogenic (capable of inducing an immune response).
Let us remember again that this study is currently being reviewed by peers, and that the comments of the members of the reading committee are not yet known.
If we confine ourselves to the information contained in the preprint, the Chinese team seems to have characterized a pangolin coronavirus capable of infecting humanized cells in mice, with the consequences of severe lung and especially brain lesions. A capacity that is not common among coronaviruses.
However, it should be noted that this study has several limitations.
The first, and not the least, is that these results are in contradiction with the results of another study, previously published by a Chinese team (published in a peer-reviewed journal, therefore having passed the evaluation by pairs). This work demonstrated that the GX-P2V mutant was responsible for mild infections in humanized mice .
We can certainly assume that this contradiction could be linked to increased virulence of the GX-P2V(3'UTR) mutant, different from its "parent", GX-P2V. However, it is currently difficult to be certain, because the infected mice in the new study appear different from those used in other studies. Produced by a Chinese company (Beijing SpePharm Biotechnology Company), we do not know their particularities well. Perhaps they would also have reacted differently to GX-P2V infections... This is one of the comments that the reviewers could make to the authors: why not use the mice usually used in animal research?
It might also be interesting to know how these humanized mice would react to SARS-CoV-2 infection (as severely? More or less severely?). No comparison is presented in the study.
Furthermore, this work concerns a selection mutant, not a natural virus. Selection mutants arise when culturing viruses in vitro, in the absence of pressure from the host immune system. It is therefore unclear how this virus would behave if confronted with the natural pressure of the human immune system.
Additionally, the infections were carried out on humanized mice expressing the human ACE2 receptor, which is not a natural model either. Naturally, in fact, mice express an ACE2 receptor which is not recognized by SARS-CoV-2, they are therefore not infected by this virus. By humanizing mice, we force their organism to express the human ACE2 receptor (hACE2): the rodents therefore become susceptible to SARS-CoV-2. However, the distribution of hACE2 on their tissues or the quantity of receptors expressed are probably overestimated compared to what is observed in humans.
Finally, animal models have long been used to try to understand diseases, to test vaccines, drugs, etc. But even if the disease developed by the animal resembles that observed in humans, the model can never be directly transposed. During viral infections, for example, viral proteins interact with cellular structures, which are species specific. The results obtained in an animal model must therefore always be considered with caution.
The questions posed by this study
Beyond the purely "biological" questions which persist concerning the specificities and the potential dangerousness of this "shortened" GX-P2V virus, this work is an opportunity to return to a debate which continues to agitate the scientific community: that of “gain of function” experiments, which involve manipulating viruses to intentionally make them more virulent.
It should be noted that in the present case, this study aimed to characterize the properties of a mutant virus obtained incidentally in the laboratory: this was not produced following intentional manipulation.
Gain-of-function experiments consist of evolving the virus studied artificially in order to give it new properties. This may, for example, be the ability to infect a new host species that it did not previously infect. The idea is then to analyze the modifications which allowed it to acquire these new capabilities.
To achieve this, we can either introduce new genes into the genome of the virus, by modifying it using genetic editing tools (such as CRISPR technology), or by cultivating it under certain conditions (with antiviral drugs, to make emerge from resistant mutant viruses, for example), or by simply cultivating it on cells, for several generations, and selecting the viruses presenting the characteristic that we want to study (increased virulence, for example; we “accelerate” and we are, in a way, directing natural evolution).
In 2014, an American team had already sparked controversy by constructing a chimeric flu virus that was part avian flu, part Spanish flu . The scientific community then mobilized to ban these experiments.
Controversy still exists among scientists about this type of experimentation. Some consider that they are essential to advance knowledge, better understand the dangers posed by the evolution of certain viruses, and enable the development of vaccines or drugs. Others claim that this type of work should be banned, because it represents too great a risk for humanity: we are never safe from a handling error which could lead to the dissemination of a virus thus modified .
A scenario which, let us remember, cannot be completely ruled out in the case of the emergence of SARS-CoV-2, even if no solid evidence has so far been able to support the thesis of a laboratory accident. Since its emergence in 2019, the question of biosecurity of virology laboratories and infrastructures intended to study the most dangerous viruses, particularly in China, remains relevant. The World Health Organization is responsible for this, however its experts continue to encounter difficulties in gathering all the information necessary to elucidate the origins of SARS-CoV-2, information that we still do not have.
To conclude, let us emphasize that in France, as well as in Europe and the United States, gain-of-function experiments on the SARS-CoV-2 coronavirus are prohibited. However, the work presented in the publication discussed here would likely not have fallen under this ban, as the mutated virus was not intentionally mutated.
Peer evaluation of these results, as well as possible replications of this work, will make it possible to determine whether there is really a need to monitor the GX-P2V(3'UTR) variant more closely.
One thing is certain: the question of the circulation of viruses between species and the risks of viral emergence remains more than ever raised, particularly in our time, when globalized exchanges are combined with profound environmental changes. Remember that more than two thirds of epidemic outbreaks originate from the passage of a pathogen from animals to humans ...
http://theconversation.com/un-coron...s-souris-humanisees-que-faut-il-savoir-222065
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Lethal Infection of Human ACE2-Transgenic Mice Caused by SARS-CoV-2-related Pangolin Coronavirus GX_P2V(short_3UTR)
https://www.biorxiv.org/content/10.1101/2024.01.03.574008v1
