A study published in Nature Communications uses genome editing in chickens to control avian influenza transmission.
Prof Timm Harder, Laboratory Head of the Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany and OIE Reference Expert for Highly pathogenic avian influenza and low pathogenic avian influenza (poultry), said:
“Infections with highly pathogenic avian influenza virus (HPAIV) of the lineage gs/GD have now taken on the character of a panzootic disease in wild birds and poultry. Protective measures can currently be implemented almost exclusively in poultry. Due to the increased incursion risks, management and biosecurity measures alone are no longer considered sufficient to protect poultry populations. Vaccination against HPAI is therefore promoted as a complementary pillar of prevention, but remains associated with a high surveillance effort. Therefore, further alternatives in the prevention of this disease are highly welcome. The use of resistant poultry species is one of such alternative measures; a highly interesting example of the generation of chickens that are no longer able to replicate avian influenza viruses due to alterations of a host cellular protein is presented by Idoko-Akoh et al.
Efficiency of gene editing
“The initial changes (two-amino-acid substitution) in ANP32A prevented infections when a low viral dose was used; however, at higher doses, resistance (not: immunity!) was breached because the bulk of inoculated viruses already contained viral variants that had mutations to bypass the altered ANP32A protein. Subsequently, some of these mutations turned out to be identical to those that can also confer increased mammalian/human adaptation. This is a critical point that may indicate that viruses exist that can evolve further resistance bypassing mutations. However, the usual ‘challenge’ dose of ten to the power of six infectious viral units used in vaccine trials was not achieved here; 1000- or 100-fold lower doses were used. Further Crispr/Cas-assisted adaptations of other chicken ANP32A-related proteins were required to generate extended resistance. This resistance aims to be generic, that is, to protect the chicken from any avian influenza viruses. It remains to be seen how these chickens will fare against the much more aggressive, highly pathogenic avian influenza viruses such as H5N1; these were not tested here.
Transfer of protection to embryos/chicks
“Breeding lines have been generated that are homozygous for the resistance mutations and thus there would be Mendelian heritability of the resistance.
Implications for the chicken
“The authors make no conclusive statement on this, and only long-term trials would be able to show such effects.
Application and legal/ethical hurdles
“According to EU law, organisms whose genomes have been modified using the CRISPR/Cas method are to be considered genetically modified organisms (GMOs). Their use would therefore require a genetic engineering approval and, under current law, they could only be kept in a genetic engineering facility. Free-range husbandry would then be equivalent to a release project. Without appropriate legal adjustments, mass use would certainly be inconceivable.
Effect on global avian influenza burden
“This is an important point: it would only marginally affect the current threatening situation of a panzootic with HPAIV H5N1, because the driver of this panzootic are water-living wild birds interacting with domesticated waterfowl populations. However, in countries with lower economic power, chickens play a major role in enriching the human diet; here, many direct chicken-human contacts also occur, for example, at live poultry markets. If resistant chickens were used here, the risk of human exposure to avian influenza would certainly be reduced considerably. This would be particularly significant if AI viruses became established, which would have a much higher propensity for zoonotic transmission than those currently circulating.”
Dr Colin Butter, Associate Professor and Programme Leader in Bioveterinary Science, University of Lincoln, said:
“The report is the first demonstrating resistance to avian influenza infection by editing of the bird’s genome rather than the previously reported approach of engineering in a bespoke “decoy RNA” system.
“Whereas the editing of a single target gene in birds did reduce shedding and transmission of the challenge virus, this was overcome by the natural selection of mutant viruses. This assessment was made by measuring viral shedding from the throat, possibly a little strange given that avian influenza is usually transmitted by the faecal oral route.
“Deletion of the target gene, or multiple genes of the same family, appeared to have a more robust effect in tissue culture. As the authors note, deleting these from the animal would likely affect the bird’s growth and health, thereby preventing testing in birds.
“The principal of editing host genomes for resistance to specific pathogens is exciting and this approach has also been deployed in other host species, including resistance of pigs to a number of viruses. Effective resistance to highly pathogenic avian influenza, such as is circulating presently in the UK, could be of very significant benefit. However, further work would be required to test protection conferred by these genetic alterations against these much more aggressive circulating field viruses.”
Prof James Wood, Head of Department of Veterinary Medicine, University of Cambridge, said:
“This study has been conducted by a highly respected collaborative group of international scientists and provides an important proof of principle for the genetic control of avian influenza – and its transmission between chickens. The study shows significant, but not absolute resistance of chickens to an avian flu virus of low virulence after they had one gene mutation introduced into breeding lines. On introduction of 3 gene mutation into chicken cells, there was complete resistance to both low and high virulence viruses, including a representative of the viruses causing such devastating disease in wild bird species and farmed poultry in the UK and the rest of Europe over the last few years. This is a breakthrough and provides important proof of principle of the utility of utilising gene editing in introducing genetic resistance to disease caused by influenza in farmed animals; it builds on previous studies and the findings are important outputs from a long term carefully planned research programme.
“The use of gene edited, disease resistant chickens in farming would be an exciting advance, and the pyramidal structure of the global poultry industry could allow rapid introduction of these influenza resistant chickens across many countries and continents. The research demonstrated that there were no obvious deleterious effects on the health and welfare of the chickens produced, but these findings would need first to be demonstrated on a larger scale before introduction into commercial farming.
“There are some other important considerations and caveats with this work, which the researchers openly considered. They demonstrated that chickens with a single gene edit that were not totally resistant to avian flu could in fact select for viruses that had some relative advantages in human cell systems. Even if the disease resistant benefits of birds with the single gene edit are clear, their ability to select for viruses that could be more dangerous in humans is something to be avoided. This is where it is important that the scientists also demonstrated that chicken cells that had 3 genetic mutations introduced did not support any virus growth at all. Clearly, it is important that the same benefits need to be demonstrated in birds, rather than just in cells that carrying 3 genetic changes. And the same checks on the health of birds carrying these 3 genetic changes would need to be carefully considered as well. It would further be highly valuable to determine if these changes were advantageous in turkeys and ducks, other important food species – and it will be important to demonstrate protection in animals against the more virulent viruses circulating as well.
“Avian influenza in farmed poultry has caused massive economic and human health impacts over the last 20 years or more. These findings could provide a resilient solution that is not specific to different strains of virus, as is the case for vaccines which are all still strain specific. However, there are still many detailed considerations, some of them regulatory in some parts of the world that will need to be addressed before this work translates into clear applied benefit. The costs of this disease globally do however make the potential benefits coming from this research highly attractive to pursue.”
‘Creating resistance to avian influenza infection through genome editing of the ANP32 gene family’ by Alewo Idoko-Akoh et al. was published in Nature Communications at 16:00 UK time on Tuesday 10th October.
Prof James Wood: “I have published and work with one of the authors of the study and have also received research funding for work on AI from BBSRC and Defra. I am employed by Cambridge University as Alborada Professor of Equine and Farm Animal Science.”
Dr Colin Butter: “I work on avian influenza infection but have not published with the authors nor have any conflicting interests.”
For all other experts, no reply to our request for DOIs was received.