J. Virology: Genetic Resilience or Resistance in Poultry Against Avian Influenza Virus: Mirage or Reality?

2005 cover of CSIRO’s Livestock Research Magazine

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Like the creation of a truly universal flu vaccine, the quest for an avian flu-resistant chicken (and other poultry species) is one of the holy grails of influenza virology. 

Nearly 13 years ago, in CSIRO: The Quest For Flu Resistant Poultry, I featured a 2005 Livestock Research Magazine cover (above), where Cambridge University virologist Laurence Tiley had stated, “The tools to make poultry resistant to flu infection already exist”.

His bigger concern was whether they could persuade the public of the benefits of Genetically Modified (GM) poultry, which remains a touchy subject. 

By 2012, additional work had been done on the creation of transgenic chickens; which would hopefully not only be resistant to H5N1, but could pass that resistance onto their chicks (see Silencing the bird flu gene: scientists prep live hen trials in The Conversation).

But just as predictions of a commercially available universal flu vaccine being just 5 years away have thus far proved optimistic, the road to flu-resistant poultry has hit repeated roadblocks.  

Progress continues to be made, however, on both fronts (see 2023's NIH: Clinical Trial of Universal Hexavalent Flu Vaccine Candidate Begins), and today we have a lengthy - and at times highly technical - review of recent strides in developing flu resilient and/or resistant chickens.

While we appear to be getting closer to those goals, there are still a lot of obstacles to overcome.  And assuming resistant poultry can eventually be produced, there is still the matter of acceptance by the public. 

Those looking for a deep-dive will want to follow the link and read the review in its entirety (warning: pack a lunch).  I'll have a brief postscript after the break. 

Genetic resilience or resistance in poultry against avian influenza virus: mirage or reality?

Authors: Paula R. Chen, Stephen N. White, Lianna R. Walker , Darrell R. Kapczynski , David L. Suarez  david.suarez@usda.govAuthors Info & Affiliations

https://doi.org/10.1128/jvi.00820-25

 ABSTRACT

The unprecedented global spread of the highly pathogenic avian influenza (HPAI) virus in wild birds, poultry, and mammalian species has challenged our control efforts. Alternative approaches to limit avian influenza viruses (AIV) include the development of resilient or resistant chickens. Genetically resilient birds may become infected but can overcome disease, whereas resistant birds prevent virus attachment or entry and do not become infected. 

The most intensively studied host gene is myxovirus-resistance (Mx), which is expressed via the interferon pathway. Both sensitive and resistant chicken Mx genotypes have been described, but this only provides limited resilience. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been demonstrated as a host cofactor for AIV replication via interaction with the polymerase. Small nucleotide changes within this gene have demonstrated some promise for the establishment of disease resilience. Certain MHC-defined genetic chicken lines have demonstrated increased resilience with higher innate immune responses, but HPAI-infected birds still have high morbidity and mortality. 

Alternatively, gene-edited or -transgenic chickens have had some success in increasing resilience. This strategy allows flexibility to include foreign genes, modification of existing genes, or combined approaches to block critical steps in the viral life cycle. Some candidate genes include solute carrier 35A1 (SLC35A1), retinoic acid-inducible gene I (RIG-I), and toll-like receptors 3 and 7 (TLR3/7), but animal testing needs to be conducted. 

Furthermore, existing hurdles for technology transfer to commercial application from either naturally occurring resistance genes or foreign genes remain high and will require acceptance by both the poultry industry and consumers.

        (SNIP)    

CONCLUSIONS

Many groups using multiple different approaches have tried to identify genetically resistant poultry with no success. Several genes have been identified that may increase genetic resilience, but because of the high virulence and genetic variability of HPAI viruses, no genes on their own are likely to provide enduring resilience to disease.

However, other genes involved in AIV infection in chickens are likely to be identified and validated. An even greater obstacle is to introduce these genetic changes into commercial lines, where genetic trade-offs for disease resistance will need to be weighed against many other production traits.

Future work will clarify these tradeoffs in the form of the potential correlated responses to selection for naturally occurring, gene-edited, or transgenic alleles. We note that gene editing and/or transgenic approaches can be used alone, in combination with naturally occurring variants and breeding, or as the first discovery step to implement through similar natural genetic variants and selective breeding. If successful, any of these approaches would provide additional tools to combat AIV and be immensely valuable to the poultry industry.

       (Continue . . .)

 

Finding ways to make livestock innately flu resistant - without the need for repeated vaccinations or a steady diet of antivirals - is an incredibly important goal, and worthy of pursuit. 

We've seen how quickly the overuse of antivirals can lead to resistance (see Nature's China's chicken farmers under fire for antiviral abuse), and vaccines must be continually updated if they are to be effective (see J. Virus Erad.: Ineffective Control Of LPAI H9N2 By Inactivated Poultry Vaccines - China).

While I don't expect a `genetic solution' will become available anytime soon, we do need to be taking the `long view' on avian flu control, since pharmacological solutions are both finite and incredibly fleeting. 

Cambodia Reports 4th H5N1 Infection In Siem Reap Province In Last 7 Days

 

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A week ago, we learned of a 41 year-old woman in critical condition with H5N1 from a small village in Siem Reap Province in Northern Cambodia. There were reportedly sick and dying chickens in her village. 

Two days ago, Cambodia Reported 2 More Human H5N1 Cases; both next door neighbors of the index case. 

This morning the Cambodian MOH is reporting a 4th case - also in Siem Reap Province - but this time about 3 km distant from the previous 3 cases.  Once again, this patient reportedly had contact with sick or dead chickens. 

Since the MOH lists this as the 11th case of 2025, there appears to be at least one case we are missing.  Earlier this month details on a case from last May were belatedly released, and so it is possible that information on this case is still in the pipeline. 

I've posted the screenshot from the Cambodia MOH Facebook page, followed by a translation.  I'll have more after the break. 

Kingdom of Cambodia

Nation Religion King

Ministry of Health

Press Release

Bird flu case in 36-year-old woman

The Ministry of Health of the Kingdom of Cambodia would like to inform the public: There is another case of bird flu in a 36-year-old woman who was confirmed positive for the H5N1 avian influenza virus by the Pasteur Institute of Cambodia on June 30, 2025. The patient lives in Daun Keo village, Daun Keo commune, Puok district, Siem Reap province and has symptoms of fever, cough, shortness of breath and difficulty breathing.

This is the 11th case for 2025 in the Kingdom of Cambodia and the 4th case in Siem Reap province (living in a different village, approximately three kilometers away from the previous 3 cases of bird flu). The patient is currently under intensive medical care. Investigations revealed that the patient had a sick and dead chicken at home, which the patient had handled and touched directly, and then buried it.

The emergency response teams of the national and sub-national ministries of health have been collaborating with the provincial agriculture departments and local authorities at all levels to actively investigate the outbreak of bird flu and respond according to technical methods and protocols, find sources of transmission in both animals and humans, and search for suspected cases and contacts to prevent further transmission in the community. They have also distributed Tamiflu to close contacts and conducted health education campaigns among residents in the affected villages.

The Ministry of Health would like to remind all citizens to always pay attention to and be vigilant about bird flu because H5N1 bird flu continues to threaten the health of our citizens. We would also like to inform you that if you have a fever, cough, sputum discharge, or difficulty breathing and have a history of contact with sick or dead chickens or ducks within 14 days before the start of the symptoms, do not go to gatherings or crowded places and seek consultation and treatment at the nearest health center or hospital immediately. Avoid delaying this, which puts you at high risk of eventual death.

How it is transmitted: H5N1 bird flu is a type of flu that is usually spread from sick birds to other birds, but it can sometimes be spread from birds to humans through close contact with sick or dead birds. Bird flu in humans is a serious illness that requires prompt hospital treatment. Although it is not easily transmitted from person to person, if it mutates, it can be contagious, just like seasonal flu.

How to prevent:

• Do not touch or eat sick or dead chickens and ducks. Wear gloves and a mask or cover your nose with a scarf before handling chickens for food. Then blanch them in boiling water before plucking their feathers.
• Adhere to hygiene practices, wash hands frequently before handling food, especially after touching animals, cleaning poultry feathers, or other objects that may be sources of contamination.
• Cook food thoroughly before eating, especially meat, poultry, and eggs. Do not eat raw or undercooked eggs, and keep raw and cooked foods separate. Clean cooking utensils properly.
• If there are many sick or dead chickens at home or in the village and they have symptoms of fever, cough, sputum discharge, or difficulty breathing, please urgently seek consultation and treatment at the nearest health center or hospital to avoid delay, which puts you at high risk of eventual death.

Therefore, the public is requested to be aware and take care of their health in the above preventive measures. The Ministry of Health will continue to provide information regarding public health issues on the Ministry of Health's official social media channels, as well as the official Facebook page of the Department of Communicable Disease Control and the website www.cdcmoh.gov.kh.

For more information, please contact the Ministry of Health's emergency hotline number 115 toll-free.

Tuesday, 6th day of the Asah month, year of the Monkey, B.E. 2569, Phnom Penh, July 1, 2025

This cluster - the first extended one we've seen in quite some time - is reminiscent of the type of bird flu activity we commonly saw between 2004-2016, particularly in places like Indonesia, Egypt, and Cambodia; large die offs of poultry, followed by community clusters of illness. 

Which may be due - at least, in part -  to the fact that these recent Cambodian cases appear to be due to a new reassortment of an older clade of the H5N1 virus (2.3.2.1c), recently renamed 2.3.2.1e.

While summertime outbreaks of avian flu are a bit unusual, the closer one gets to the equator, the more likely influenza is to circulate year-round.  Siem Reap Province is only about 13 degrees N. Latitude. 

Given the frequent contacts reported with sick or dead poultry, there is no evidence to suggest human-to-human transmission of the virus. 

Nevertheless, every human infection is another opportunity for the virus to better adapt to human physiology.  So we watch these cases - and clusters - with considerable interest. 

Stay tuned. 

 

ECDC: Updated Reporting Protocol for Zoonotic Influenza Virus

 

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A year ago, in the wake of the discovery of 3 (now > 6 dozen) human infections with H5N1 in the United States, the ECDC issued guidance for member nations on Enhanced Influenza Surveillance to Detect Avian Influenza Virus Infections in the EU/EEA During the Inter-Seasonal Period.

In that summary, the ECDC pointed out:

Sentinel surveillance systems are important for the monitoring of respiratory viruses in the EU/EEA, but these systems are not designed and are not sufficiently sensitive to identify a newly emerging virus such as avian influenza in the general population early enough for the purpose of implementing control measures in a timely way.

A conclusion not unlike what we saw in the 2023's UKHSA Technical Briefing #3, which found that it might take weeks - and hundreds of cases - before community spread of a novel flu could be confirmed using standard surveillance (see UK Novel Flu Surveillance: Quantifying TTD).

Last October, the ECDC released two additional guidance documents:

• ECDC Guidance For Testing & Identification Of Zoonotic Influenza Infections In Humans In The EU/EAA

• Surveillance & Targeted Testing for the Early Detection of Zoonotic Influenza in Humans During the Winter Period in the EU/EEA

As with similar guidance we've seen from the U.S. CDC, these are non-binding recommendations, and not all member states have the same capacity for testing and managing of cases. 

The ECDC uses EpiPulse - an online portal for European public health authorities to collect, analyze, and share infectious disease data - integrating several previously independent surveillance platforms; (The European Surveillance System (TESSy), the five Epidemic Intelligence Information System (EPIS) platforms and the Threat Tracking Tool (TTT). 

Over the weekend the ECDC published an updated, 28-page protocol for reporting zoonotic influenza infections to the TESSy database. I've reproduced the introduction below:

Introduction

An event of a human case infected with an influenza virus deriving from an animal source should be reported within 24 hours to the Early Warning and Response System (EWRS) which will cover the International Health Regulations (IHR) notification for EU/EEA countries. 

To complement the eventbased surveillance, TESSy reporting allows for a long-term collection of key indicators. Data to TESSy can be uploaded retrospectively when more information becomes available but should be done as soon as feasible to avoid major reporting delays.

This reporting protocol describes data collection for zoonotic influenza viruses. With the data collected, the aim is to support situational risk assessment and trends over time.

For the reporting of case-based data, the record type INFLZOO should be used. Case-based data is the preferential record type for reporting confirmed cases to TESSy. Aggregate data on zoonotic influenza (number of tested samples and number of detected cases by NA and HA subtype) can be uploaded to INFLZOOAGGR. This record type should ideally be used mainly for reported testing data. 

If a country is not able to report to the case-based record type above, then numerator data can also be reported to this record type.

Aim

To support the timely and complete reporting on number of samples tested, number of detected cases and key information of zoonotic influenza cases

Objectives

• To collect data on number of tested people.

• To help assess the onset of the disease, confirmation of the subtype of infection and severity.

• To provide information on exposure, treatment and outcome.

• To provide additional contextual information to help understand the case identification.

• To analyse trends over time.

Record types

The following record types exist for reporting of zoonotic influenza virus in TESSy:

1. INFLZOO for reporting of case-based data of zoonotic influenza virus

2. INFLZOOAGGR for reporting of aggregated data of zoonotic influenza virus

Variables for each record type are outlined in the annex of this reporting protocol.

        (Continue . . . )

Among the changes in this update are expansions to the metadata and codes used in these reports.   A list of changes (which includes animal exposures, exposure activities, and consumption of raw or unpasteurized animal products) follows:

The ECDC continues to urge member countries to improve their surveillance, testing, and reporting of zoonotic or novel flu infections; in humans and in animals. 

Last January, in  ECDC: Avian Flu - Virus mutations and Response Strategies, we looked at two new European avian flu initiatives; 

Avian influenza: EU agencies track virus mutations and analyse response strategies

Preparedness, prevention and control related to zoonotic avian influenza

But as far as what is happening across much of the rest of the world, infectious disease reporting remains sparse, and - for varied economic and political reasons -  only seems to be getting worse (see From Here To Impunity).

A reminder that `no news' isn't necessarily `good news'. 

JID: HPAI H5N1 Subclade 2.3.4.4b Isolated from a European Grey Seal (Halichoerus grypus) is Highly Virulent in Ferrets

Gray Seals - Credit Wikipedia

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Up until 4 about years ago, HPAI H5N1 was considered mostly a disease of birds, with rare (but often serious) spillovers into humans, and very limited and/or sporadic detections in other mammals (primarily captive felines). 

The host range of H5N1 began to change in 2021-2022, with increased detections in foxes, skunks, and other carnivorous peridomestic mammals (see Ontario: CWHC Reports HPAI H5 Infection With Severe Neurological Signs In Wild Foxes (Vulpes vulpes)).

With these new mammalian host infections, we began to see signs of increased mammalian adaptions within the virus (amino acid changes, including HA-T143A, PB2-E627K,  PB2 D701N, PB2 Q591K, HA Q226L, etc.). 

A few examples:

Emerging Microbes & Inf.: Neurotropic HPAI H5N1 Viruses with Mammalian Adaptive Mutations in Free-living Mesocarnivores in Canada

Eurosurveillance: HPAI A(H5N1) Virus Infection in Foxes with PB2-M535I Novel Mammalian Adaptation - Northern Ireland, July 2023

Emerg. Microbes & Inf.: Marked Neurotropism and Potential Adaptation of H5N1 Clade 2.3.4.4.b Virus in Naturally Infected Domestic Cats

J. Gen. Virology: H5N1 2.3.4.4b: A Review of Mammalian Adaptations & Risk of Pandemic Emergence

Due to limited surveillance, testing, and reporting around the globe, we don't have a very good handle on how many different mammalian species have now been infected with clade 2.3.4.4b H5Nx. 

As the following FAO map (zoonotic avian flu reports since Oct 1, 2024) illustrates, semi-robust reporting of avian influenza is pretty much limited to the United States, Southern Canada, Europe, and some parts of Southeast Asia.

Vast swaths of Russia, China, South America, Africa and the Middle East rarely (if ever) report outbreaks, yet there is little doubt that (except for Australia/NZ) the virus is present in - at least in wild birds - most of countries of the world.

The USDA's list of Detections of Highly Pathogenic Avian Influenza in Mammals shows 46 different species, but this excludes livestock (cattle, pigs, goats, alpacas, mink, etc.) and is based primarily on passive surveillance of mostly peridomestic animals. 

The most recent ECDC/EFSA Avian influenza overview March - June 2025 lists more than 90 species (pgs. 28-32). But once again reporting and testing are limited.  

Identifying and tracking H5N1 in terrestrial animals is undoubtedly easier than in marine mammals, yet we've seen numerous reports of H5 infected pinnipeds (seals, sea lions, walruses), particularly from South America (see EID Journal: Mass Mortality of Sea Lions Caused by HPAI A(H5N1) Virus (Peru)).

In last January's Nature Reviews: The Threat of Avian Influenza H5N1 Looms Over Global Biodiversity the authors estimated the loss of hundreds of thousands of marine mammals (seals, sea lions, dolphins, etc.).

 

As the H5N1 virus expands its host range, it also gains access to alternative - and unpredictable - evolutionary pathways. While we focus primarily on HPAI in humans, dairy cows, poultry, and cats . . . we may very well be missing important milestones occurring in deer mice, camels, foxes, seals or other mammalian hosts.

All of which brings us to a study, published Saturday, in The Journal of Infectious Diseases by Guilfoyle et al. that finds that a newer HPAI H5N1 subclade 2.3.4.4b virus - isolated from a European grey seal in 2023 - is significantly more virulent in ferrets than is an older (2005) strain from Indonesia.

Researchers found that ferrets infected with the 2023 seal-derived H5N1 virus succumbed more rapidly than with the 2005 virus; exhibiting severe pneumonia, hypothermia, and histopathological changes in their respiratory tract and other organs. 

Aside from the increased pathogenicity (in ferrets) over the 2005 H5N1 strain, they report a couple of other notable findings.

• First, while fever is a common symptom in humans and ferrets infected with H5N1, ferrets infected with this seal-derived virus developed irreversible hypothermia prior to death. 
• Note: We saw reports of hypothermia in cats infected with H5N1 in Washington State earlier this year (link).
• In the past, mostly lab-infected mice have demonstrated hypothermia post H5N1 infection (see Animal Models for Influenza Research: Strengths and Weaknesses). 

• Second, this increased virulence occurred despite the absence of many of the amino acid changes (e.g. E627K, D701N, or S714R in the PB2 protein, and Q226L and G228S in the HA) known to promote mammalian adaptation.

I've posted the link, abstract, and some excerpts from this open access report.  Follow the link to read in its entirety.  I'll have a brief postscript after the break.

Highly Pathogenic Avian Influenza Virus A/H5N1 subclade 2.3.4.4 b isolated from a European grey seal (Halichoerus grypus) is highly virulent in ferrets  

Kate Guilfoyle , Monica Mirolo , Leon de Waal , Geert van Amerongen , Guido van der Net , Theresa Störk , Mara Sophie Lombardo , Wolfgang Baumgärtner , Ásgeir Bjarnason , Hekla Bryndís Jóhannsdóttir ... 

https://doi.org/10.1093/infdis/jiaf348

Published: 28 June 2025 

PDF

Abstract

Highly pathogenic avian influenza A viruses subtype H5N1 (HPAIV H5N1), subclade 2.3.4.4b infect multiple avian and mammalian species, posing a potential pandemic risk.

Here we describe the outcomes of infection of ferrets with a HPAIV H5N1 virus, isolated from a European grey seal in 2023, compared with an older HPAIV H5N1 (A/Indonesia/05/2005).

Overall, infection of ferrets with A/grey seal/Netherlands/302603/2023 caused more rapid mortality than infection of ferrets with A/Indonesia/05/2005. Animals developed severe pneumonia and irreversible hypothermia, associated with high levels of virus replication and histopathological changes in the respiratory tract and peripheral organs.

As animal models for severe avian influenza virus infections in humans play a key role in the development of intervention strategies against these infections, these findings highlight the importance of using 

       (SNIP)

It has been well documented in mammalian species that H5N1 viruses primarily infect the respiratory tract when inoculated intratracheally [17], yet lethal pathogenesis is also associated with virus replication in extra-respiratory organs [37].

In our study, ferrets infected with A/grey seal/NL/2023,displayed severe inflammation of liver and spleen that was characterised by a mild to moderate inflammation and diffuse areas of necrosis, with AIV NP antigen present in necrotic hepatocytes and splenocytes. Although virological analyses revealed slightly higher mean levels of replicating virus and RNA copies in the spleen of ferrets infected with A/grey seal/NL/2023 than in the ferrets infected with A/Indo/2005, these differences were not statistically significant.

Hypothermia and tachypnoea were detected prior to the death of three A/grey seal/NL/2023- infected ferrets.. In contrast, high yet stable body temperature and respiration rates were observed in ferrets infected with A/Indo/2005, corroborating previously published results in which all ferrets survived until at least 4 dpi with this virus [17,19, 23, 24].

In conclusion, our data show that intratracheal infection of ferrets with A/grey seal/NL/2023 causes accelerated mortality, as compared to intratracheal infection with A/Indonesia/05/2005. Collectively, our data supports the development and use of updated ferret models, to test preventive and therapeutic intervention strategies for human H5N1 infections. 

       (Continue . . . )
 

While ferrets are admittedly not a perfect analog for humans, they possess a remarkably human-like physiology and respiratory system, and are considered among the best small animal surrogates for influenza research. 

The fact that this 2023 seal-derived H5N1 virus is significantly more virulent in ferrets than the 2005 Indonesian strain is a concern.

It reminds us that just because the North American Bovine (B3.13 genotype) of H5N1 has produced mostly mild illness in humans, there are no guarantees that every strain that is brewing unseen in scores - perhaps hundreds - of new hosts around the world, will prove as benign. 

Cambodia Reports 2 More Human H5N1 Cases for 2025

 
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According to my count, the latest update from the ECDC, and FluTracker's list, Cambodia has reported (prior to today) 7 human H5N1 cases, and 5 deaths in 2025.  

Today they report 2 more cases from the same village as last week's case, which they describe as cases #9 & #10.  Whether this is a misprint, or there is another case as yet unannounced, remains to be seen. 

In any event, these two new cases were neighbors of last week's case, and are both reportedly in stable condition.  Both families reportedly had contact with sick or dying chickens.

Unlike the milder 2.3.4.4b clade seen in the United States, Europe, and much of the rest of the world, recent cases from Cambodia and Vietnam have stemmed from a resurgent older, and more virulent, clade (formerly clade 2.3.2.1c but recently redubbed as 2.3.2.1e).

The announcement (see screen shot below) was made overnight on the Cambodian MOH Facebook page. I've provided a translation (emphasis mine).

       (Translation)

Kingdom of Cambodia

Ministry of Health

Press Release

2 more cases of bird flu

The Ministry of Health of the Kingdom of Cambodia would like to inform the public that, following an active investigation to find suspected cases and contacts in Lek village, Daun Keo commune, Puok district, Siem Reap province, the village where the 41-year-old woman tested positive for bird flu, which was reported on June 23, 2025, two more cases of bird flu were found, in a 46-year-old woman and a 16-year-old boy, who were mother and child and were confirmed to be positive for the H5N1 bird flu virus by the National Institute of Public Health. These are the 9th and 10th cases for 2025. 

The two cases live approximately 20 meters away from the 41-year-old patient’s home. Currently, the health status of both patients is stable and they are being treated with Tamiflu with continued close monitoring. Investigations revealed that there were sick and dead chickens in the patient’s home, the neighbor’s home, and in the village. The patient had handled and touched sick and dead chickens and cooked them.

The emergency response teams of the national and sub-national ministries of health have been collaborating with the provincial agriculture departments and local authorities at all levels to actively investigate the outbreak of bird flu and respond according to technical methods and protocols, find sources of transmission in both animals and humans, and search for suspected cases and contacts to prevent further transmission in the community. They have also distributed Tamiflu to close contacts and conducted health education campaigns among residents in the affected villages.

The Ministry of Health would like to remind all citizens to always pay attention to and be vigilant about bird flu because H5N1 bird flu continues to threaten the health of our citizens. We would also like to inform you that if you have a fever, cough, sputum discharge, or difficulty breathing and have a history of contact with sick or dead chickens or ducks within 14 days before the start of the symptoms, do not go to gatherings or crowded places and seek consultation and treatment at the nearest health center or hospital immediately. Avoid delaying this, which puts you at high risk of eventual death.

        (Continue . . . )


Although Cambodia continues to do an admirable job of reporting hospitalized cases, it is entirely possible that some milder infections are going unreported. Severe or critical cases are far more likely to be hospitalized, tested, and confirmed as H5N1 positive.

While we are understandably focused on H5N1 clade 2.3.4.4b - clade 2.3.2.1e in Cambodia, the recently imported (ex India) clade 2.3.2.1a case in Australia, and > 90 H5N6 cases in China - remind us that HPAI H5 continues to evolve along multiple concurrent pathways.

Virus Research: A 15-year Study of Neuraminidase Mutations and the Increasing of S247N Mutation in Spain

 

Credit NIAID

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While current seasonal (and novel) influenza viruses remain largely susceptible to our limited antiviral armamentarium (primarily oseltamivir and other NAIs, and the newer Baloxavir), we are constantly looking for signs of increased resistance. 

A few recent reports include:

EID Journal: Influenza A(H1N1)pdm09 Virus with Reduced Susceptibility to Baloxavir, Japan, 2024

Eurosurveillance: An outbreak of A(H1N1)pdm09 Exhibiting Cross-resistance to Oseltamivir & Peramivir in an Elementary School in Japan, Sept 2024

EID Journal: Antiviral Susceptibility of Influenza A(H5N1) Clade 2.3.2.1c and 2.3.4.4b Viruses from Humans, 2023–2024

EID Journal: Cluster of Oseltamivir-Resistant & Antigenically Drifted Influenza A(H1N1)pdm09 Viruses, Texas, USA, January 2020, 

Although these detections have been limited, growing antiviral resistance is not an idle concern. Twice over the past 2 decades we've seen two frontline antivirals quickly loose effectiveness against seasonal influenza viruses. 

First in 2005, our preferred influenza anti-viral drug - Amantadine - suddenly lost effectiveness after decades of use (see MMWR Levels of Adamantane Resistance Among Influenza A (H3N2) Viruses and Interim Guidelines for Use of Antiviral Agents --- United States, 2005--06 Influenza Season).

Luckily, there was already an alternative available - Oseltamivir (aka `Tamiflu') - although it was far more expensive.

 

While occasional instances of Oseltamivir resistance had been observed, in nearly every case, it developed after a person was placed on the drug (i.e. `spontaneous mutations’).

Studies suggested that these resistant strains were `less biologically fit’, and were therefore unlikely to spread from human-to-human.

And that happy status quo held until `biologically fit'  highly resistant H1N1 viruses emerged in early 2008. By the end of that year - nearly all H1N1 viruses were resistant, forcing the CDC to issue major new guidance for the use of antivirals (see CIDRAP article With H1N1 resistance, CDC changes advice on flu drugs).

This resistance was primarily due to an H275Y mutation - where a single amino acid substitution (histidine (H) to tyrosine (Y)) occurs at the neuraminidase position 275 (Note: some scientists use 'N2 numbering' (H274Y)). 

It seemed as if antiviral crisis was inevitable, but a new swine-origin H1N1 virus - that happened to retain its sensitivity to Tamiflu - swooped in as a pandemic strain in the spring of 2009, supplanting the older resistant H1N1 virus.

Since then oseltamivir has remained effective against 99% of seasonal flu viruses, but over the past couple of years we've seen some new cracks in its veneer. 

Fifteen months ago we saw a worrisome report in The Lancet - Global Emergence of Neuraminidase Inhibitor-Resistant Influenza A(H1N1)pdm09 Viruses with I223V and S247N Mutations - which reported a much higher incidence of oseltamivir resistance among samples tested in Hong Kong in 2023 (along with a concurrent rise in GISAID sequences deposited since last summer).

Instead of the H275Y mutation which caused so much trouble in 2008, these viruses carried dual I223V/S247N mutations. 

While neither of these mutations are anywhere near as impactful as H275Y, they are believed able to work synergistically with other mutations (including H275Y) to dramatically impair antiviral effectiveness (see Viruses: Increase of Synergistic Secondary Antiviral Mutations in the Evolution of A(H1N1)pdm09 Influenza Virus Neuraminidases).

Last summer the authors of the above study wrote:

It seems likely that the viruses have reached the next stage in the evolution of prerequisite viruses that enable the emergence and spread of stable lineages of resistant viruses, in which the substitutions NA-I223V and NA-S247N may have been added in 2023–2024 after the appearance of the two permissive substitutions NA-V241I and NA-N369K in 2011.

If synergistic amino acid changes such as NA-I223V and NA-S247N spread globally, there is the risk that other NA mutations which may have previously caused only slight or moderate reductions in susceptibility could instead cumulatively decrease NAI susceptibility to levels that may be clinically significant and affect treatment efficacy [37]

A year ago, in EID Journal: Multicountry Spread of Influenza A(H1N1)pdm09 Viruses with Reduced Oseltamivir Inhibition, May 2023–February 2024, we saw a report which found this resistance signature has spread from Asia to Europe, and suggested it may be just as `biologically fit' as antiviral susceptible viruses.

All of which brings us to a new study, published this week in Virus Research, which reports a sharp increase in the NA-S247N mutation in seasonal flu viruses collected in Spain over the 2023-2024 flu season.

First the link, abstract, and a few excerpts from this open access report, after which I'll have a postscript. 

A 15-year study of neuraminidase mutations and the increasing of S247N mutation in Spain 

Iván Sanz-Muñoz, Alejandro Martín-Toribio, Adrián García-Concejo, Irene Arroyo-Hernantes, Marina Toquero-Asensio,  Javier Sánchez-Martínez , Carla Rodríguez-Crespo , Silvia Rojo-Rello , Marta Domínguez-Gil, Eduardo Tamayo-Gómez, Marta Hernández-Pérez , José M Eiros 

https://doi.org/10.1016/j.virusres.2025.199599

Highlights

• In a landscape of a very narrow arsenal of influenza antivirals, resistance mutations are a significant threat.

• Resistance mutations were present in 0.5-5% in A and B influenza viruses during the last 15 years.

• However, S247N resistance mutation in the NA gene sharply increased during 2023-2024 season.

• While this mutation does not confer strong resistance by itself, their fixation could increase the risk of resistance in the future if other resistance mutations appears or get fixed together with it.

Abstract

The therapeutic arsenal against influenza is extremely limited and resistance often arises due to the emergence of mutations, especially in the neuraminidase (NA) gene. This study aimed to evaluate the evolution of NA mutations over 15 years in Spain. To do so, we used the GISAID database from which we downloaded a total of 11,125 influenza A(H1N1)pdm09, A(H3N2), B/Victoria and B/Yamagata NA virus sequences, and analyzed the resistance mutations using FluSurver software.

Our results showed that the occurrence of NA resistance mutations remained constant in the four viruses during the 15 seasons evaluated, being around 0.5-5%. Most of the resistance was found in the A(H1N1)pdm09 subtype (around 70%), especially from the 2023-2024 season onwards, when a significant increase in the occurrence of S247N mutation was observed.

The occurrence of this type of mutation before 2022 was rare, but in the 2023-2024 season a total of 44 influenza viruses harboring S247N mutations were detected, while in the other years, only two cases were observed. Some studies have described a significant increase in this mutation over the past two seasons and although it appears to confer only slightly reduced inhibition to oseltamivir, its increase is noteworthy and should be a reason for increased their vigilance.

        (SNIP)

To summarize, the prevalence of antiviral drug resistance mutations against NA has remained stable in the influenza viruses analyzed in Spain over the last 15 years, 70% of them being detected in viruses of A(H1N1)pdm09 subtype.

Significantly, a remarkable increase of the S247N mutation has been observed in this subtype during the 2023-2024 influenza seasons. Although this mutation does not significantly reduce susceptibility to antiviral drugs, it may pose, in combination with other mutations such as H275Y, a real risk to the limited therapeutic arsenal currently available against this virus.

The available information shows that this mutation is stable in cell culture and does not negatively affect viral fitness. Therefore, these data should reinforce surveillance efforts through the genotyping of a relevant number of influenza samples each year by every country.

       (Continue . . . )

 

While this study only reviews data from Spain, it seems to align with other studies we've seen suggesting that the S247N mutation continues to spread globally. 

It will be of particular interest if we should see this trend has continued throughout the 2024-2025 flu season (and beyond). 

While this study deals with seasonal flu, these resistance mutations also increase the threat from novel flu viruses.  

Three months ago, St. Jude Researchers warned Current Antivirals Likely Less Effective Against Severe Infection Caused by Bird Flu in Cows’ Milk, and last February we learned from Emerg. Microbes & Inf: Oseltamivir Resistant H5N1 (Genotype D1.1) found On 8 Canadian Poultry Farms.

Although we've not seen any reports of H275Y in D1.1 samples collected in the United States, last November the CDC did report finding the above mentioned NA-S247N mutation in 3 poultry workers from Washington State, which they stated may slightly reduce the virus's susceptibility to antivirals. 

All of which serve as sobering reminders that evolution never stops - and while our pharmacological victories over bacteria, fungi, and viruses can be lifesaving -  they are often fleeting.