Vet. Research: E627V Mutation in PB2 Protein Promotes the Mammalian Adaptation of Novel H10N3 Avian Influenza Virus

 

#18,750

Among the more concerning mutations seen in avian influenza viruses is PB2-627K, which is associated with enhanced replication and pathogenicity in mammals. Fortunately, it doesn't provide the same replication benefits to wild birds and poultry, making it relatively uncommon. 

Another version of this mutation, however, PB2-637V, has recently made headlines for its ability to spread efficiently in poultry (see last year's Preprint: An Emerging PB2-627 Polymorphism Increases the Pandemic Potential of Avian Influenza).

The authors reported that this PB2-627V mutation not only maintained viral fitness in poultry, it facilitated the aerosol transmission of AIVs between ferrets. Raising concerns this mutation could go a long way in overcoming the `species barrier' between avian and mammalian-adapted influenza viruses.

Last summer, in Transboundary & Emerg. Dis.: H3 Avian Influenza Virus Isolated from China in 2021–2022 Showed the Emerging H3N8 Posed a Threat to Human Health, we learned this mutation turned up in a human infection with avian H3N8 in Hunan province. 

 The authors wrote:

The E627K mutation of PB2 is known to play a decisive role in the mammalian adaptation of AIVs [41, 42]. The A/Henan/4-10CNIC/2022(H3N8) virus contains the E627K mutation, whereas A/Changsha/1000/2022(H3N8) contains the E627V mutation, which has also been shown to increase the replication or virulence of AIVs in mammals [14, 34, 43]

Until about 10 years ago PB2-627V was a fairly rare mutation - and mostly seen in LPAI H9N2 viruses - but it has now been found in at least 10 avian flu subtypes, including a handful of H5N1 clade 2.3.4.4b samples (cite).

Since 2021 we've seen the spread of H10N3 in poultry in China, and at least 5 human infections. 

Six months ago Chinese researchers warned of its pandemic potential (see Vet. Microbiology: The novel H10N3 Avian Influenza Virus Acquired Airborne Transmission Among Chickens: An Increasing Threat to Public Health).

 

While the number of cases (and limited reporting) prevent us from determining a reasonable CFR (Case Fatality Rate), a recent study (see The Novel H10N3 Avian Influenza Virus Triggers Lethal Cytokine Storm . . .) suggests it has a high capacity for virulence in mammals.

Today, we have a new study published in Veterinary Research, which finds that PB2-626V is becoming increasingly common in AIV poultry isolates - including H10N3 - and that it significantly enhances mammalian adaptation while maintaining fitness in avian hosts.

While H5N1 currently gets the bulk of our attention, the emergence and spread of a `poultry-friendly' PB2-627V mammalian adaptation in China is a genuine concern.  There it has a plethora of AIV subtypes to play with, and plenty of opportunities to spillover into mammals. 

Due to its length, I've only posted some excerpts. Follow the link to read it in its entirety. 

E627V mutation in PB2 protein promotes the mammalian adaptation of novel H10N3 avian influenza virus

Meishan Song, Jianyu Liang, Sige Wang, Ruyi Gao, XiaologLu, Wenhao Yang, Yu Chen, Jingxia Ma,  Min Gu, Jiao Hu, Xiaowen Liu, Shunlin Hu, Xiaoquan Wang, Kaituo Liu & Xiufan Liu

Veterinary Research volume 56, Article number: 111 (2025)  

Abstract


Since 2021, the novel H10N3 has caused four cases of human infection in China, the most recent of which occurred in December 2024, posing a potential threat to public health. Our previous studies indicated that several avian H10N3 strains are highly pathogenic in mice and can be transmitted between mammals via respiratory droplets without prior adaptation. 

By analyzing the genome sequence, we found that these H10N3 viruses carry the PB2-E627V mutation, which is becoming increasingly common in several subtypes of avian influenza viruses (AIV); however, its mechanism in mammalian adaptation remains unclear. 

Using a reverse genetics system, we investigated the role of PB2-E627V in the adaptation of H10N3 to mammals and poultry. Our findings demonstrate that the PB2-E627V mutation is critical for the high pathogenicity of novel H10N3 in mice and its ability to be transmitted through the air among mammals.

Additionally, we found that the role of PB2-627 V in promoting AIV adaptation to mammals is comparable to that of PB2-627 K. More importantly, PB2-627 V appears to be equally suited to long-term persistence in poultry. Therefore, using PB2-627 V as a novel molecular marker to assess the epidemic potential of AIV is of great significance for preventing possible influenza pandemics in the future.      

(SNIP)

In recent years, the PB2-E627V mutation has appeared with high frequency in several subtypes of AIV capable of infecting humans, drawing widespread attention. In 2015, an H7N9 AIV strain with the PB2-E627V substitution was isolated in Hunan, and revealed that this mutation increases the virus’s replication efficiency in mouse organs, enhancing its pathogenicity in mice. Additional research indicates that the E627V mutation in the PB2 protein can increase the virulence of H9N2 AIV in mice [32], and enhance the transmissibility of H7N9 AIV in ferrets [33].

Recently, three cases of human infection with the novel H3N8 AIV were reported in China, and one of the human-derived strains, A/Changsha/1000, carries the PB2-627 V molecular marker. Pathogenicity experiments demonstrated that it could kill mice [52]. 

Our previous studies have shown that the avian-derived H10N3 viruses harboring the PB2-E627V mutation are highly pathogenic in mice and can be transmitted between guinea pigs via respiratory droplets without prior adaptation [13, 23]. In this study, we found that the H10N3 strains carrying PB2-627 V and the mammalian signature PB2-627 K exhibit similar pathogenicity in mice, and are at least 100 times more pathogenic than strains carrying the avian signature PB2-627E (Figure 1). 

Furthermore, the H10N3 strains with the molecular marker PB2-627 V are capable of spreading among guinea pigs through direct contact and aerosol transmission, whereas strains carrying the PB2-627E do not possess the ability to transmit among guinea pigs. Typically, PB2-627 V is considered an intermediate state of the E to K mutation [20]. However, in this study, H10N3 strains carrying PB2-627 V demonstrate a capacity for full adaptation to mammalian hosts comparable to that of PB2-627 K.

In addition, the PB2-E627V/K mutation does not impact the transmissibility of H10N3 in chickens, one key reason is that PB2-627E is non-essential for H10N3’s ability to spread in chickens, as other genomic sites can perform the same function too. This feature underscores the public health threat posed by H10N3 strains naturally carrying the PB2-627 V molecular marker, because poultry exposure is a major risk factor for human AIV infections. Furthermore, previous studies have demonstrated that PB2-627 V can be stably maintained in both avian and mammalian species [33].

(SNIP)

In summary, our findings demonstrate that the PB2-E627V mutation is key to the high pathogenicity of novel H10N3 in mice and its ability to be transmitted through the air in mammals. Additionally, the role of PB2-627 V in promoting AIV adaptation to mammals is comparable to that of PB2-627 K, and more importantly, PB2-627 V also appears to be equally suited to long-term persistence in poultry. Therefore, using PB2-627 V as a novel molecular marker to assess the epidemic potential of AIV is of great significance for preventing possible influenza pandemics in the future.

(Continue . . . )

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

 

#18,749

The sobering reality of the HPAI H5 avian flu threat is we are not just dealing with a single entity - one specific subtype/genotype circulating in one host species - but rather a growing array of similar viruses, spreading in numerous avian and mammalian hosts, and each on its own evolutionary path. 

The map above illustrates some of the better known spillover/adaptations, but vast regions of the world are blank, suggesting are either not looking for - or simply not reporting - local activity. 

Complicating matters, while HPAI H5 may be the most obvious pandemic threat today, it is simply one of many influenza A viruses we are watching.  In China, we've seen concerns raised over Swine H1Nx, Avian H3Nx and H9N2 viruses, while in Europe Swine H1Nx viruses are being closely watched. 

The CDC's IRAT list of zoonotic influenza A viruses with pandemic potential now lists 27 different viruses, of which just 12 belong to the H5 subtype. This list, however, is far from complete. 

As a segmented virus with 8 largely interchangeable parts, the flu virus is like a viral LEGO (TM) set which allows for the creation of a vast number of unique variants via reassortment.  And each subtype/genotype is also subject to amino acid changes (mutations) due to replication errors and host adaptation.

The emergence of new clades, subtypes, genotypes or variants can be associated with abrupt changes to the behavior (transmissibility, pathogenicity, host range, etc.) of the H5 virus. 

 Sometimes we get lucky, and it attenuates the virus's threat. Sometimes it enhances it. 

While we are far from knowing what every amino acid change does (particularly in concert with other changes), we do have a short list of known mutations of concern (e.g. PB2 E627K, PB2 D701N, PB2 Q591K, HA Q226L, etc.). 

While evolution is often depicted as a straight line, it isn't linear. It branches, meanders, and sometimes even regresses. 

Which is why we've seen HPAI H5 appear to be on the cusp of exploding before, only to see it lose its momentum, and virulence (Indonesia & Egypt were once both hotbeds of human infections).

Sometimes, nature has to go back to the drawing board. 

Despite attempts to generate risk analyses, limited surveillance and the complexity of viral evolution mean we aren't to the point of being able to predict what this virus will do next. But, today we have an excellent review of the recent changes in the H5 virus, and in its ability to infect mammalian hosts. 

 

One that focuses not only on the changes that have already been observed, but also on what the virus would likely need to do next to pose a greater pandemic threat. 

Due to its length, and technical nature, I'll just provide the link and a few excerpts.  Those who wish a deep dive into H5's recent evolution will want to read it in its entirety. 

Review Article
Open Access
H5N1 2.3.4.4b: a review of mammalian adaptations and risk of pandemic emergence 

Fernando Capelastegui1​ and Daniel H. Goldhill1​
 
Published: 04 June 2025 https://doi.org/10.1099/jgv.0.002109

PDF

 

 
ABSTRACT

Avian influenza viruses can cause severe disease when they spill over into mammalian and human hosts. H5N1 clade 2.3.4.4b has spread globally since 2021, decimating avian species, and has spilled over into mammalian species, causing sporadic infections and fatal outbreaks in sea lions, cats, mink and dairy cattle. Increased human cases of H5N1 are fuelling concern that H5N1 could soon adapt to become a new pandemic virus.

Adaptive mutations have emerged following spillover, which support H5N1 outbreaks in mammalian populations and include changes to the PB2 such as E627K, D701N, M631L and T271A. Further changes to haemagglutinin, altering binding preference to human-like α2,6 sialic acid receptors have yet to be seen. Here, we review the adaptations that have emerged in mammals throughout the 2.3.4.4b outbreak and the molecular mechanisms behind these mutations to assess the pandemic risk of this virus.

         (SNIP)

Our understanding of the mechanism behind adaptation allows us to predict whether a virus is adapting to human hosts and assess the risk it poses to public health. Deep mutational scanning coupled to the potential of AI models promises a future where we may have greater predictive power over which mutations and viruses pose the greatest threat. Further research should focus on key unknowns such as the determinants of severity across species and the importance of modes of transmission.

The prevalence of infection across so many species creates a perfect storm for exposure to new hosts and the potential for further adaptive mutations to emerge. There is also a significant risk of reassortment with other circulating influenza viruses, which may fast-track the acquisition of human adaptive mutations from seasonal lineages or other AIVs. Ultimately, time will tell if this virus evolves into a pandemic virus. However, we can be certain that limiting transmission and the opportunities avian viruses have to adapt is important to prevent existing and new mammalian epidemics.

          (Continue . . . )

 

JEGH: Al-Tawfiq & Memish On Recurrent MERS-CoV Transmission in Saudi Arabia

 

Credit Wikipedia

#18,748

Jaffar A. Al-Tawfiq and Ziad A. Memish - either writing together or separately - are probably the two best known and most prolific authors on the public health aspects of the Hajj - and since its emergence in  2012 - on the novel MERS coronavirus.

A partial list of my past blogs highlighting their work include:

AJIC: Intermittent Positive Testing For MERS-CoV

Evaluation of a Visual Triage for the Screening of MERS-CoV Patients

Frontiers Med.: MERS-CoV In 7 Pediatric Patients

ATS: Mass Gatherings And Lessons From The Hajj

 

This year's Hajj (June 4th - 9th) will see over 1.6 million religious pilgrims from > 180 countries attend, and then return to their respective countries.  As with all mass gathering events, the Hajj has the potential to amplify and disperse emerging and existing infectious diseases - sometimes on a global scale (see J, Epi & Global Health: Al-Tawfiq & Memish On Hajj Health Concerns).

Most infectious illnesses acquired during these mass gathering/migration events are fairly common; seasonal flu, pneumonia, measles, meningococcal disease, norovirus, and vector borne infections (Zika, CHKV, Dengue, Malaria, Yellow Fever, etc.) (see CDC's Traveler's Health Saudi Arabia).

But Saudi Arabia has also been the source of > 80% (n=2218) of all known MERS-CoV cases, and after a 5 year lull, we are seeing a noticeable uptick in human cases reported during the first 5 months of the year (see WHO: Saudi Arabia Reports 9 New MERS-CoV Cases). 

Today we have an open-access editorial, published this past week in the Journal of Epidemiology & Global Health, by Al-Tawfiq & Memish on the recent surge in cases in KSA, and the importance of healthcare preparedness and surveillance. 

This is  a relatively short review, but it reminds us that the threat from MERS-CoV has not gone away, and that asymptomatic (or mild cases) may contribute to the silent spread of the virus.  

I've only posted some excerpts, so follow the link to read the article in its entirety. I'll have a bit more after the break. 

Recurrent MERS-CoV Transmission in Saudi Arabia– Renewed Lessons in Healthcare Preparedness and Surveillance

Editorial Open access

Published: 02 June 2025

Volume 15, article number 77, (2025)

Jaffar A. Al-Tawfiq & Ziad A. Memish  

The World Health Organization (WHO) had recently announced on May 12, 2025, the reporting of nine new laboratory-confirmed cases of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Saudi Arabia [1]. This announcement is a sobering reminder that while the global community remains focused on emergent threats like avian influenza or post-pandemic resilience, endemic zoonotic diseases like MERS-CoV may continue to circulate, evolve, and exploit gaps in infection prevention.

MERS-CoV, first reported in 2012 [2], is a betacoronavirus endemic to the Arabian Peninsula. Dromedary camels remain the primary animal reservoir. Though zoonotic transmission dominates most spillover events, human-to-human transmission—particularly in healthcare settings—has triggered multiple nosocomial clusters, sometimes involving a large number of cases. Notably, outbreaks in 2014 (Saudi Arabia) and 2015 (South Korea) underscored MERS-CoV’s epidemic potential when detection, triage, and infection control measures faltered or other patients characteristics predominate [3]. The number of cases had decreased significantly in the last few years (Fig. 1), with the highest number of cases in 2014.

Between February and April 2025, nine laboratory-confirmed MERS-CoV cases were reported in Saudi Arabia, primarily from Riyadh. Two elderly, non-healthcare individuals died following symptomatic illness, while a third recovered. The remaining six cases, all healthcare workers aged 18–65, were linked to secondary exposure from one index case. Five of them were asymptomatic and not hospitalized, highlighting the risk of silent transmission (Table 1) [1]. This cluster underscores the critical need for early detection, contact tracing, and strict infection control measures, especially in healthcare settings. The first multi-hospital MERS-CoV outbreak was controlled with basic infection control measures [4].

          (SNIP)

MERS-CoV’s case-fatality rate remains disturbingly high—approximately 35%—making every missed opportunity for containment a gamble with lives. The clustering of cases in early 2025, involving asymptomatic transmission, healthcare settings, and camel contact, is not new—but that’s precisely the concern. We’ve been here before.

MERS-CoV persists due to multiple factors and the complex human-animal interactions and at times due to wanes in the absence of crisis headlines. The continues zoonotic risks should be further explored from various aspects to prevent the spillover and to reawaken global stakeholders to the realities of persistent zoonoses. Surveillance must be strengthened, IPC rigorously enforced, and frontline workers empowered with knowledge and protection.

(Continue . . . )

 

While MERS-CoV hasn't managed to acquire the kind of transmissibility that made COVID a household name - we've seen large MERS-CoV outbreaks - particularly in crowded hospitals (see  Ziad Memish: Two MERS-CoV Hospital Super Spreading Studies).

Coronaviruses are highly mutable, and have the potential to recombine into new variants, which raises concerns over the co-circulation of MERS-CoV along with COVID, and other coronaviruses (see Nature: CoV Recombination Potential & The Need For the Development of Pan-CoV Vaccines).

While MERS and SARS get most of our attention, over the years we've looked at a number of non-MERS/SARS coronaviruses with zoonotic potential that are often found in bats, swine and even cattle.  A few (of many) examples include:

• In 2014, in SECD: Another Emerging Coronavirus Threat - in the wake of several newly discovered coronaviruses detected in North American swine - we looked at growing concerns that some porcine-adapted coronaviruses might have zoonotic potential, given the similar physiology between our two species.  

• In the summer of 2017 we looked at paper published in the  EID Journal: A New Bat-HKU2–like Coronavirus in Swine, China, 2017, on the recent discovery of a new HKU2-like coronavirus in Chinese pigs showing symptoms of PED (Porcine Epidemic Diarrhea), which they tentatively named porcine enteric alphacoronavirus (PEAV). 

• Also that  summer, in a letter published in the Journal of Medical Virology, Chinese scientists made a case for the Potential Cross-Species Transmission Risks of Emerging Swine Enteric Coronavirus to Human Beings.
• Last year we looked at the concerns over the Circulation of Non-(MERS) Coronaviruses in Imported Camels in Saudi Arabia and a preprint on Human Cell adaptation of the Swine Acute Diarrhea Syndrome Coronavirus Spike Protein

• And just over two weeks ago, in J. of Infection: Novel Coronaviruses Identified in Livestock we looked at a warning from Chinese researchers on risks of livestock-borne coronaviruses.

Not so very long ago, influenza A was considered the primary viral pandemic threat to humanity. But after SARS in 2002-2003 followed by COVID in 2019, we've learned there are plenty of other legitimate contenders in the wild (see OFID: Viral Families with Pandemic Potential).

Despite this painfully gained knowledge, most countries have elected - for political or economic reasons - to scale back on their surveillance, reporting, and preparedness efforts. 

While I can't tell you what emerging disease will spark the next pandemic - or when - it is all but guaranteed to happen again.  And when it does, we'll regret every day we squandered not aggressively preparing for its arrival.

Virology Journal: Immediate PB2-E627K Amino Acid Substitution after Single Infection of Highly Pathogenic Avian Influenza H5N1 Clade 2.3.4.4b in Mice

 

Editor's note: I've been offline for much of the past 36 hours due to an internet problem, which was restored late yesterday afternoon.  

#18,747

It obviously isn't easy for a novel avian influenza virus to jump - and fully adapt - to a mammalian species, otherwise we'd be hip-deep in pandemic viruses all of the time.  Instead we usually see sporadic and tentative spillovers - often to what turn out to be dead-end hosts - and only rarely do we see ongoing transmission of the virus. 

But we need look no further than avian H3N2's successful spillover into dogs in South Korea in 2007 - the marine mammal mass mortality events of the past two years from H5N1 - or the multi-state spread of H5N1 to dairy cows -  to see examples of avian viruses getting a solid foothold in a mammalian species. 

Recent studies have turned up H5N1 in wild rats in Egypt - and H5N6 in Shews in China - raising uneasy questions about what other inroads these viruses might be making around the globe outside of our view. 

The assumption has long been that it would likely take long-chains of infections in a mammalian species for the virus to successfully adapt (see serial passage graphic below).  One-off, or dead-end, infections were viewed as posing a lower risk. 

 

Adaptive mutations following long chains of Infection

In these experiments, subsequent infections are done artificially, in order to overcome early transmission barriers. In the wild, most of the time these chains are unable to sustain transmission.

Unless, of course, a permissive mutation - like PB2-E627K - were to emerge in the index infection. 

PB2-627K, which is associated with enhanced replication and pathogenicity in mammals, is one of the most important mutations that H5Nx is thought to need in order to spread more efficiently in mammals  (see A rapid review of the avian influenza PB2 E627K mutation in human infection studies).

There are others, of course (PB2 D701N, PB2 Q591K, HA Q226L, etc.) - each providing the virus with unique advantages - but if you wanted to kickstart transmission, PB2-E627K would be at or near the top of  your list. 

All of which brings us to a study - published yesterday in the Virology Journal - which finds that H5N1  lab infected rats quickly developed the PB2-627K mutation and by day 6, the mutation was nearly fixed (60%) in the index animal (see graphic below).

Furthermore, all challenged mice succumbed to the (now nearly 100% fixed) infection, with significant viral loads in both their lungs and brains.  The authors wrote:

Notably, the PB2-E627K variant, initially present at 4% in the virus stock, was selected and reached near-fixation (~ 100%) in the lungs and brains by 6 days post-challenge and was subsequently transmitted. No other mammalian-adaptive mutations were identified, emphasizing the pivotal role of PB2-E627K in early stages of mammalian adaptation.

This is a fascinating (and detailed) open access study, and I've only posted the link, and a few excerpts.  You'll want to follow the link to read it in its entirety. 

Immediate PB2-E627K amino acid substitution after single infection of highly pathogenic avian influenza H5N1 clade 2.3.4.4b in mice

Brief Report

Published: 05 June 2025

Volume 22, article number 183, (2025)

Deok-Hwan Kim, Dong-Yeop Lee, Yeram Seo, Chang-Seon Song & Dong-Hun Lee 

Abstract

The highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b has rapidly disseminated globally, with mammalian infections reported in multiple species. Recent evidence of mammal-to-mammal transmission has heightened concerns about the virus’s potential adaptation to mammals. The polymerase basic 2 (PB2) protein E627K mutation appears to be of key importance for mammalian adaptation. 

We isolated an HPAI H5N1 clade 2.3.4.4b virus from wild birds in Korea with 96% E and 4% K at amino acid position 627 of PB2. To investigate the genomic characteristics of this clade regarding mammalian adaptation, we studied the replication and transmission of the H5N1 virus in mice. Two experiments with different challenge-to-contact ratios were conducted to assess transmission dynamics and mutation development.

In experiment 1, a 4:1 challenge-to-contact ratio resulted in 100% transmission among direct-contact mice, with all mice succumbing to the infection. In experiment 2, a 1:1 ratio yielded 50% transmission, with all challenged mice also succumbing. High viral loads were observed in the lungs and brains in both experiments, with viral titers increasing over time. 

Notably, the PB2-E627K variant, initially present at 4% in the virus stock, was selected and reached near-fixation (~ 100%) in the lungs and brains by 6 days post-challenge and was subsequently transmitted. No other mammalian-adaptive mutations were identified, emphasizing the pivotal role of PB2-E627K in early stages of mammalian adaptation. These findings highlight the need for continuous genomic monitoring to detect mammalian adaptation markers and assess interspecies transmission risks.

(SNIP)

Since the widespread dissemination of the clade 2.3.4.4b H5N1 virus after 2020, mammalian-adapted mutations in the PB2 protein have been reported in both mammals [19] and wild birds [20]. Studies conducted between 2020 and 2023 identified PB2-E627K or PB2-D701N mutations in 10 of 48 documented H5N1 infections in mammals [19].

Furthermore, from 2023 to 2024, seventeen cases of clade 2.3.4.4b H5N1 virus carrying PB2-E627K, K526R, or D701N mutations were detected in wild and domestic birds across eight European countries, indicating that birds may play a role in the dissemination of HPAI viruses with enhanced mammalian infectivity [20]. Among the 5,311 genome sequences of clade 2.3.4.4b viruses identified between 2003 and 2023, only 53 (1.0%) viruses had the PB2-E627K mutation. Notably, 48 of these 53 viruses were identified since 2021. 

Of these viruses, twenty-three 627K variants were from avian species, suggesting potential spillover of mammalian-adapted viruses to avian species [21]. Furthermore, a previous study demonstrated that the PB2-E627K mutation did not affect pathogenesis or transmission in ducks, indicating that the 627K variant can persist in avian species [22]. Mutations acquired within infected mammals, rather than direct transmission from wild ducks, could facilitate mammal-to-mammal transmission [23]. 

(Continue . . . )

Although this is obviously a significant finding, it must be tempered with the knowledge that PB2-E627K has been circulating (albeit at low levels) in HPAI in birds, and spilling over into other species, for years and it has yet to spark a pandemic.  

While a key component, PB2-E627K would likely need to team up with several other advantageous mutations before H5Nx could pose a global health threat.

The notion that we are `one mutation away' from a pandemic is more media hype than science. But anything that permits or extends the transmission of HPAI in mammals is a concern, particularly since so much of H5Nx's evolution occurs outside of our view. 

Nature: Genetic diversity of H9N2 avian influenza viruses in poultry across China and implications for zoonotic transmission

 

Range Of Endemic H9N2 Viruses

#18,746

While H5 and H7 viruses get the bulk of our attention - primarily because they often produce severe (sometimes fatal) disease in humans - there are a number of other `lesser' zoonotic influenza threats we keep close watch on as well. 

 Amongst them, LPAI H9N2 is probably the most evolutionarily agile.

It is has not only become ubiquitous in Asian and Middle Eastern poultry, it readily reassorts with other subtypes (see The Lancet: H9N2’s Role In Evolution Of Novel Avian Influenzas), and has been increasingly reported as spilling over into humans (see last month's HKCHP Reports 8 H9N2 cases).

Although most H9N2 infections have been mild, several deaths have been reported.  

A 2021 study (see J. Virus Erad.: Ineffective Control Of LPAI H9N2 By Inactivated Poultry Vaccines - China)) by researchers from Shanghai and the Netherlands found the current inactivated virus vaccines used in China against H9N2 to be no match for this rapidly evolving pathogen. 

They warned:

The failure of vaccination might be because of inefficient application, low dose, and low vaccination coverage (especially in the household sector).11,12 Moreover, the continuing transmission in combination with the intensive long-term usage of the inactivated virus vaccine may have led to antigenic changes leading to immune escape.

The CDC has identified 2 different lineages (A(H9N2) G1 and A(H9N2) Y280) as having some pandemic potential (see CDC IRAT SCORE) - and several candidate vaccines have been developed - but much of H9's evolution remains hidden. 

H9N2 is such a versatile virus, a variant of it has even been detected in Egyptian Fruit bats (see Preprint: The Bat-borne Influenza A Virus H9N2 Exhibits a Set of Unexpected Pre-pandemic Features).  

Last March, in Cell: Early-warning Signals and the Role of H9N2 in the Spillover of Avian Influenza Viruses, we took a deep dive into the evolving threat from H9N2.  Yesterday Nature published a study (see below) on the continued evolution of H9N2 in China that, alas, is behind a paywall. 

Genetic diversity of H9N2 avian influenza viruses in poultry across China and implications for zoonotic transmission 

Published: 03 June 2025(2025) 

Jing Yang, Juan Li, Ju Sun, Jiaming Li, Guanghua Fu, Tian Tian, Yongchun Yang, Xuancheng Lu, Shan Li, Lixia Wang, Jia Dong, Mingjia Wu, Yun Liu, Delong Li, Dongfang Hu, Hui Dong, Ruoyu Shang, Yanqing Wang, Kunpeng Yuan, Lin Ran, Honglei Sun, Wenxia Tian, Yu Huang, Jinhua Liu, … Yuhai Bi

Those that have access will certainly want to read the full report, but for the rest of us we have a press release that provides the gist.   First some excerpts (emphasis mine), after which I'll have a postscript.

Researchers uncover genetic keys to the increasing threat of H9N2 avian influenza

Peer-Reviewed Publication

Chinese Academy of Sciences Headquarters

Credit: BI Yuhai

A new study published in Nature Microbiology has uncovered significant genetic and antigenic diversity among H9N2 avian influenza viruses (AIVs) circulating in poultry across China, highlighting the growing public health risk posed by H9N2 AIVs.

Although H9N2—first identified in China in 1994—has been targeted by ongoing vaccination strategies, it has remained the dominant subtype in poultry. Its persistence, along with increasing reports of human infections in recent years, has become a growing public health concern.

Previously, the molecular basis for the virus' cross-species transmission and zoonotic potential remained largely unclear. Now, however, a collaborative team led by Prof. BI Yuhai and Prof. George F. Gao (GAO Fu) from the Institute of Microbiology of the Chinese Academy of Sciences, together with Prof. SHI Weifeng of Ruijin Hospital at the Shanghai Jiao Tong University School of Medicine, has conducted a comprehensive investigation into the virus' genetic evolution, antigenic variability, and adaptive mutations. Their findings offer crucial insights concerning the virus' molecular mechanism for mammalian adaptation and evasion of human MxA gene-mediated innate immune responses.

Since 2014, Prof. BI Yuhai has organized teams from the Center for Influenza Research and Early-warning (CASCIRE) to conduct continuous surveillance and early warning of AIVs in China and study cross-species transmission mechanisms of AIVs. Surveillance in live poultry markets from 2019 to 2023 revealed that the A/chicken/Beijing/1/94 (BJ94) lineage of H9N2 AIVs has consistently dominated in poultry.

To better understand its evolutionary trajectory, the team developed a novel clade classification system for BJ94 viruses based on genetic distances and phylogenetic relationships. They also launched an online classification platform to enable global researchers to track and study H9 AIV evolution.

Using this framework, they identified ten hemagglutinin (HA) sub-subclades currently co-circulating among poultry, each exhibiting distinct antigenic variations. These differences may explain why the existing vaccines have been unable to curb the epidemic of H9N2 AIVs.

Additionally, the researchers found a rising prevalence of key mutations associated with increased infectivity and pathogenicity in mammals.

• Between 2021 and 2023, 99.46% of H9N2 isolates carried the HA-L226 mutation linked to human receptor binding;
• 96.17% contained the NP-N52 mutation associated with resistance to the human MxA antiviral protein;
• and 32.61% had the PB2-V627 mutation known to enhance polymerase activity in human cells.

Experiments demonstrated that strains harboring these mutations preferentially bound to human-type receptors, replicated efficiently in human cells, and were capable of direct contact and aerosol transmission in guinea pigs and ferrets—key indicators of zoonotic potential.

These results highlight the heightened zoonotic risks of H9N2 AIVs. This study underscores the urgent need for enhanced surveillance, updated vaccine strategies, and a deeper understanding of avian influenza virus evolution to mitigate the growing threat of H9N2 to public health.

This work was supported by the National Key R&D Program of China and the National Natural Science Foundation of China for Distinguished Young Scholar.

Whether as a standalone zoonotic virus - or a co-conspirator with another subtype - LPAI H9N2 poses a significant public health threat.  It is often underestimated because it is not considered a `reportable' disease in poultry, and mild or moderate human cases are unlikely to be picked up by passive surveillance. 

While poultry and (potentially) livestock vaccination are being heavily touted to control H5N1, the experience with H9N2 reminds us that poultry vaccination alone hasn't always solved the problem. 

A safe and effective vaccination program (see UK Joint Taskforce Policy Paper: Vaccination of Birds Against HPAIV (bird flu)) requires the use of proven, and continually updated vaccines, along with regular testing (and if necessary, quarantining or culling) of vaccinated flocks.

Last April, in NPJ Vaccines: Impact of Inactivated Vaccine on Transmission and Evolution of H9N2 Avian Influenza Virus in Chickens. we looked at a Chinese study that warned that improper or inadequate use of inactivated vaccines have failed to prevent - or even reduce - H9N2 in China's poultry, and may well have driven viral evolution (including mammalian adaptations).

While on paper, an H9N2 pandemic doesn't look to be as fearsome as an H5 or H7 pan-flu, we've only limited experience with human infections with this subtype (< 160 cases), and past performance isn't always indicative of future results. 

The H9N2 virus is obviously still evolving, and deserves both our attention and our respect.

WHO Novel Flu Update Includes Two Previously Undisclosed H5N1 Cases From Bangladesh

 

#18.845

Overnight the WHO has published their latest Influenza at the human-animal interface report, which covers confirmed reports submitted by member nations from  23 April to 27 May 2025.  It does not, however, contain the fatal H5N1 case reported last week from Cambodia. 

While most of the cases mentioned (1 H5N1, 1 H10N3, and 8 H9N2) have already been covered in this blog, among them are also two previously undisclosed H5N1 cases from Bangladesh. 

Although India has reported 2 local  H5N1 cases (here & here) over the past 4 years (and appears to have exported 1 case to Australia in 2024), we've not seen a case reported from neighboring Bangladesh in nearly a decade.   

Surveillance and laboratory testing for this virus in this part of the world is often suboptimal, and so it is possible (perhaps even likely) that many cases have gone undetected. 

 The details from these two additional cases follow:

A(H5N1), Bangladesh 

A human infection with an H5 clade 2.3.2.1a A(H5N1) virus was detected in a sample collected from a child in Khulna Division in April 2025, who recovered from his illness. Genetic sequence data are available in GISAID (E EPI_ISL_19875512; submission date 18 May 2025; Institute of Epidemiology, Disease Control & Research (IEDCR); Virology - National Influenza Centre (NIC)). WHO was notified of this case on 4 May 2025.In March 2025, an avian influenza A(H5N1) outbreak was reported in poultry in the same district (Jessore) where the case resides. 7

A second human infection with an H5 clade 2.3.2.1a A(H5N1) virus was retrospectively detected in a sample collected from a child in Khulna Division in February 2025, who recovered from his illness, according to genetic sequence data available in GISAID (EPI_ISL_19882255; submission date 26 May 2025; Institute of Epidemiology, Disease Control & Research (IEDCR); Virology - National Influenza Centre (NIC)). WHO was notified of this case on 27 May 2025. 

 This report also provides brief updates on previously reported H5N1 and H10N3 cases from China:

A(H5N1), China 

On 10 May 2025, China notified WHO of one confirmed case of human infection with an avian influenza A(H5N1) virus in an adult traveling from Viet Nam that was detected through routine screening at the port of entry in China. The case was admitted to hospital in China on 7 April and had recovered at the time of notification. The likely source of exposure was domestic poultry at the case’s home. According to reports received by WOAH, various influenza A(H5) subtypes continue to be detected in wild and domestic birds in Africa, the Americas, Asia and Europe. Infections in non-human mammals are also reported, including in marine and terrestrial mammals. 8 A list of bird and mammalian species affected by HPAI A(H5) viruses is maintained by FAO.9  

A(H10N3), China 

On 10 May 2025, China notified WHO of one confirmed case of human infection with avian influenza A(H10N3) virus in an adult from Shaanxi province. The case developed symptoms on 13 April 2025, was admitted to hospital on 18 April with pneumonia and was improving at the time of notification. According to the notification, the case had exposure to backyard poultry. No additional cases have been reported among family members. Environmental samples collected from the backyard tested negative for influenza A(H10) virus.

In addition to these case updates, the WHO once again implores member nations to abide by the IHR regulations which require prompt notification of the WHO of all human infections caused by novel flu subtypes (see screenshot below).

While supposedly binding for all WHO member nations, the IHR is notoriously lacking in `teeth’. Nations agree to abide, but there is little recourse if they don’t follow through - a topic we looked at in depth in 2015 in Adding Accountability To The IHR.

Despite repeated prodding (see WHO Guidance: Surveillance for Human Infections with Avian Influenza A(‎H5)‎ Viruses), many nations continue to treat novel flu infections as more of an economic or political concern, than a public health threat. 

Which means that while lulls in the reporting of human cases are certainly welcome, they may not be telling the whole story.