Lessons not learned

"On reading," by Simon Wain-Hobson, is a weekly discussion of scientific papers and news articles around gain of function research in virology.

Persimmon halves

Since January 2024, Dr. Wain-Hobson has written weekly essays for Biosafety Now discussing risky research in virology. You can read his entire series here.

On reading numerous papers on dog, cat and bird flu viruses.

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There is reason to be concerned about the biosafety levels at which work on a number of animal flu viruses is being performed. Let’s start with a 2024 paper from a Japanese group on the H3N8 canine influenza flu viruses (CIV). In this study, to investigate the potential of the H3N8 CIV in human infections, we investigated its adaptivity to human respiratory cell cultures. By the 16^th passage (cycle of growth) on the human A549 cell line commonly used by the influenza crowd, they recovered a virus that showed much more efficient growth at approximately 10,000-fold higher titers than the wild-type virus. Here, wild type is jargon for the initial virus.

They did the experiment three times and succeeded each time by the 16^th passage. Genome analysis from all three viruses showed a medley of 5-9 mutations compared to the wild type with just three common to all viruses. Despite a huge amount of mutagenesis work, none of the common mutations explained well how these viruses grew indicating that there are multiple ways for a virus to adapt to a new environment. We’ve known this for years which is recapitulated nicely in the title of Paul Simon’s song, Fifty ways to lose your lover.

Their conclusion? These findings suggest that the H3N8 CIV could possess the potential to mutate into a virus that may infect human respiratory cells. Emphasis added here and below. Tremendous precision.

It turns out this study is a simple variant of a previous 2022 paper of theirs on a feline H7N2 flu virus. Again, they performed 16 passages of the virus on human A549 cells in lab culture. The three parallel experiments yielded viruses with different sets of mutations with no one mutation common to all three passaged viruses.

We conclude that the H7N2 feline IAV [influenza A virus] has the potential to become a human pathogen by adapting to human respiratory cells... Our findings may contribute to the prediction and assessment of the risk of animal viruses for the emergence of a pandemic virus. In no way does this work help the prediction of viral emergence. None of the three feline viruses had the same constellation of mutations – Paul Simon again. Not only are they not thinking up front, they are misrepresenting their own findings.

The feline H7N2 flu virus work was performed in a BSL2 lab which is standard fare for an animal flu virus. Presumably the same lab was used for the earlier mentioned dog H3N8 flu work, although it was not mentioned in the paper. For the genesis of several novel human adapted respiratory viruses, On reading considers this to be insufficient. It is the result of the experiment that matters for biosafety, not the starting point. BSL3 is more appropriate.

In a recent essay (Potential animal pandemic pathogens), we discussed the properties of the more prominent canine flu virus, H3N2, that is now transmissible between ferrets by the airborne route, something in common with human flu viruses. For the moment, it is not yet a problem for humans. Another Chinese group has just published a paper on this virus. By the 18^th sequential infection of mice they recovered a virus that was lethal for all mice which resulted from just two mutations. No doubt there was bias and a premium on selecting viruses from diseases animals throughout the sequential mouse infections (Observer bias).

Interestingly, when they injected the lethal mouse virus back into dogs, they found it was more pathogenic - exhibited significantclinical symptoms from 3 dpi, [days post infection] including reduced food intake, depression, tachypnea, mucous discharge, and coughing, compared to the original H3N2 virus. Was this a dangerous GOF experiment?

A line from the contemporary French philosopher Michel Onfray helps answer this question. When you're a toad, the most beautiful thing on earth is a female toad. From the dog perspective, this result is dangerous GOF research.

The authors conclude that our findings underscore the importance of ongoing surveillance for the PA (S184N) mutation in H3N2 flu viruses. They buy into the flawed notion that lab experiments can predict how a virus can evolve nature, that a dog virus serially injected into mice can recapitulate evolution in nature. Even when their own work on dog and cat viruses shows there are many ways to adapt to growth on human cells.

To cap of the discussion, neither of the two mutations identified in this mouse study of dog H3N2 influenza showed up in the huge Chinese study that featured in a recent essay (Potential animal pandemic pathogens). It’s possible the mouse model was not a good backdrop or else Fifty ways to lose you mind is at work again.

Finally, a recent paper from China looked at bird flu H4N1 adaptation to mammals in the lab culture and in mice. They find that their H4N1 isolate grew reasonably well and picked up a couple of mutations during extended growth on human cells. It’s a minor paper which struggles to get to the point fast. H4N1 isn’t yet a threat for Sapiens although a search for antibodies to bird flu H4 and H11 proteins in Lebanon suggested that backyard poultry growers may have been previously infected with H4 and H11 avian influenza viruses.

Once again, the important point is that the work with human cell line adapted flu virus was performed in a biosafety level 2 lab when a BSL3 lab would have been appropriate.

The discussion section of this paper repeats a formula that is trundeld out regularly. They mention that their own work shows that H4N1 bird flu virus is indicative of its low pathogenic profile. They cite a 2012 paper showing that with the addition of a furin cleavage site indicating that the H4 subtype AIV [Avain Influenza Virus] has the potential to mutate into a highly pathogenic phenotype.

Please note the following: Given the increasing prevalence in mammalian hosts globally and the notable annual rise in isolation frequency coupled with the capability for rapid mutation of H4N1 AIV observed in this study, H4 subtype AIVs may be evolving into a highly pathogenic form. This necessitates vigilance and further surveillance.

Obviously if the human population increases, so does the chicken population especially with the trend away from eating red meat. And with it H4N1 bird flu. Everything shows it is a low pathogenicity virus that can spillover to humans but nothing like as dangerous as H5N1 or H7N9 bird flu viruses. For the moment.

Yet despite this reassuring news, as H4N1 is a mutation machine it may be evolving into a highly pathogenic form. May is more than vague and the only evidence in its favor is a gain of function experiment published in 2012.

Then the punch line: This necessitates vigilance and further surveillance which is a euphemism for saying keep funding work on my virus, aka lobbying. And that’s it. And this lesson from authors who should have done the work in a BSL-3 lab.

This sort of drum beating has been going on for years. We’ll get back to this in another essay.

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We have mentioned several times virology’s Chernobyl moment, possibly the result of a poorly attenuated flu vaccine. Now, what do you make of this from a 2014 paper on the bird flu H9N2 virus? However, the 82.1% seropositive rate to a Hong Kong H9N2 virus, belonging to the Ck/Beijing/94 lineage, provided evidence of a broad and recent chicken vaccination program in Taiwan. To date, the Ck/Beijing/94-like H9N2 viruses are highly prevalent mainly in mainland China but have not been reported in Taiwan, nor were they detected in our surveillance. This further supports the possibility that, while apparently prohibited, vaccination with an H5N2 virus (potentially a bi- or trivalent vaccine including H6N1 and/or H9N2 viruses) has occurred and that this insufficiently inactivated or inadequately attenuated live-virus vaccine is the source of the H5N2 viruses found in chickens in Taiwan.

In the case of H5N2 in Taiwan, the reemergence of a North American vaccine strain virus in Asia after approximately 10 years is a strong indication that a vaccine may be the source of this virus. The currently emerging H5N2 variants in Taiwan are suggestive of possible reintroductions from a vaccine source, and some are developing signs of evolving into a highly pathogenic strain.

It sounds as though they know more than they mention.

People are using a poorly attenuated virus things to control bird flu but are in fact spreading the virus. The problem is that flu viruses invariably mix if the chicken gets a double flu virus infection. This can make viruses that undo the attenuation. This is not new.

The above examples show a very poor knowledge of virus evolution from an experimental standpoint. The work hasn’t a hope of providing useful information to public health officials. Nothing compared to the situation for Andes virus. It’s easy lab work which is why it is done.

One of the consequences with high profile papers like the Fouchier and Kawaoka publications in top scientific journals is that they attract copycat research and more papers. Scientists see this as topical and fundable research area and pile in. It happens in all areas of science. All. The GOF controversy surrounding the H5N1 bird flu viruses and the nearly three year long moratorium prevented much copycat research.

However, post COVID the term ‘pandemic preparedness’ has galvanized researchers everywhere which is understandable. What we are seeing is GOF 2.0 reemerge under the guise of pandemic preparedness. Anything is game. Once again, the potential for any virus to spillover to humans is being studied proactively.

Quantitation is crucial in science. Yet all the authors of these studies can conclude is this or that virus could, may, might, potentially, perhaps, one day jump host due to at least fifty different genetic permutations or set off a pandemic. They couldn’t put a probability on their thoughts if they tried. In a future essay we’ll see the extraordinary limitations of these experimental approaches that do not recapitulate viral evolution for a moment. They are science fiction compared to what happens in nature.

And as readers have found out, much of this work generating novel human respiratory viruses is being done in biosafety level 2 labs. We can’t even say Houston, we have a problem. It’s more United Nations, we have a problem.

Conclusion

Funders, you’re being conned. Use your money for better, more difficult projects, perhaps riskier in terms of success not danger, that might have a chance of advancing the field. Journals, you’re being duped. Don’t publish work that has no public health relevance. Stop this madness.

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Aside

We’ll take a break here and regroup in September. Enjoy your holidays.

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Simon Wain-Hobson is an emeritus professor at the Institute Pasteur, Paris, from which he retired in 2021. He and his colleagues were the first to sequence the genome of HIV, and Wain-Hobson has published more than 230 papers on virology and cancer.