Viruses: Zoonotic Implications of the Co-Circulation of Clade 2.3.4.4b and 2.3.2.1a H5N1 Avian Influenza Viruses in Nepal in 2023

 

#18,937

Not quite 3 years ago (Feb 2023), a long-dormant strain of HPAI H5 (clade 2.3.2.1c) began to spillover again into humans in Cambodia after a 9 year absence. Since then, 33 cases have been confirmed, with nearly half of those proving fatal. 

In contrast to the newer clade 2.3.4.4b viruses - which currently circulate across most of the globe - these older clades appear to produce more severe illness in humans. 

In April of 2024, the FAO released a statement on the Reassortment Between H5N1 Clade 2.3.4.4b & Clade 2.3.2.1c Viruses In Mekong Delta Region, which was later redesignated by the WHO as New H5 Clade 2.3.2.1e Infections in Cambodia & Vietnam. 

While clade 2.3.2.1c was thought to be on its way to obscurity -  a chance reassortment with the newer clade 2.3.4.4b appears to have breathed new life into it, allowing it to spread readily while retaining its old virulence. 

So far, clade 2.3.2.1e has only been detected in Cambodia and Vietnam, but today we learn of a similar reassortment - between a clade 2.3.2.1a and clade 2.3.4.4b in Nepal - which has produced another hybrid virus. 

While these two hybrids are different, they came via the same process; whereby an older, nearly extinct clade reassorts with the newer, 2.3.4.4b clade.  

While we've not seen the same surge in human cases in Nepal, this new reassortant reportedly has `. . . a high identity with 2.3.2.1a clade viruses identified in humans and cats in India in 2024–2025'. 

This reports notes several secondary mammalian-adaptation markers of interest in the reassortant virus (e.g., PB2 L89V, M1 N30D/T215A, NS1 P42S/D92E, HA V226I), but none fall into the `red flag' category (e.g. PB2 E627K/D701N, HA Q226L/G228S, etc.).

I've posted the abstract, and some excerpts below, but you'll want to follow the link to read it in its entirety.  I'll have a brief postscript after the break.

Zoonotic Implications of the Co-Circulation of Clade 2.3.4.4b and 2.3.2.1a H5N1 Avian Influenza Viruses in Nepal in 2023

Pragya Koirala 1, Manju Maharjan 2, Sharmila Chapagain 3, Barun K. Sharma 1,
Tirumala B. K. Settypalli 4, Charles E. Lamien 4 and William G. Dundon 4,*

Viruses 2025, 17(11), 1481; https://doi.org/10.3390/v17111481    

Abstract

Samples collected from two avian influenza outbreaks in Bagmati Province in central Nepal between January and March 2023 were positive for H5N1. Full genomes were generated for both viruses, which revealed that one of the viruses was very similar to clade 2.3.4.4b H5N1 identified in Bangladesh in 2021/2022.

The second virus was a reassortant H5N1 virus consisting of four genes (HA, NA, NP, and M) originating from a clade 2.3.2.1a H5N1 and the remaining four genes (NS, PB1, PB2, and PA) originating from a 2.3.4.4b H5N1. Notably, this second virus had a high identity with 2.3.2.1a clade viruses identified in humans and cats in India in 2024–2025.

These are the first full genome sequences of H5N1 avian influenza viruses from Nepal and given the recent human infections by 2.3.2.1a H5N1 viruses in the region, these data will be of interest to both public health and veterinary authorities. 

       (SNIP)

Results and Discussion

Epidemiological investigations identified two primary transmission routes of the viruses into the farms. The predominant route most likely involved indirect transmission via fomites, such as contaminated vehicles, feed, equipment, and human activity, which facilitated the spread of the virus into and between farms. A second potential transmission pathway was through contact with wild birds, which are known natural reservoirs of HPAI viruses. Nepal is a part of the Central Asian Flyway, which overlaps with the East Asian–Australasian Flyway that covers much of eastern Asia.

(SNIP)

Since its arrival in North and South America in 2022, clade 2.3.4.4b H5N1 viruses have now become the dominant subtype globally [15]. Recently, clade 2.3.4.4b viruses have been identified in Bangladesh and India [16,17]. The first clade 2.3.4.4b identified by Barman et al. (2023) in Bangladesh in 2021, and now referred to as genotype BD-1, was similar to H5N1 clade 2.3.4.4b viruses from Europe [16,18]. 

The authors conducted further surveillance and characterization in Bangladesh between January 2022 and November 2023 and identified three additional genotypes of clade 2.3.4.4b viruses (BD-2, BD-3, and BD-4), highlighting the diverse and dynamic nature of viruses in the region [18]. From the BLAST and phylogenetic analysis of A/chicken/Nepal/A146_079_80/2023, it was revealed that this virus belonged to the original genotype BD-1 identified in Bangladesh in 2021 (Supplementary file Figure S1).

In conclusion, the current study has identified both a clade 2.3.4.4b and a clade 2.3.2.1a/2.3.4.4b reassortant virus (Figure 3) in Nepal for the first time.

The clade 2.3.2.1a/2.3.4.4b reassortant virus was highly similar to viruses that have caused recent human fatalities in India, and so, these findings should encourage Nepalese authorities to increase surveillance and molecular characterization of AIVs present in their country. The study should also encourage further studies in order to better understand the impact of circulating avian influenza viruses and their zoonotic potential.

        (Continue . . . )

There are some limitations in this report, including:

• This study analyzed only two poultry isolates - taken from two different farms roughly 10 km apart in early 2023 - which tells us very little about prevalence or spread of variants the region.

• No phenotypic or antigenic testing was performed, so any mutation effects are inferred from past findings, not demonstrated with this reassort ant.

What this study suggests, however - particularly when combined with the recent events in Cambodia - is that we can't afford to ignore older clades of HPAI H5, just because a newer, `shinier' strain (like clade 2.3.4.4b) has emerged. 

The Lancet: Vascular and Inflammatory Diseases after COVID-19 Infection and Vaccination in Children and Young People in England

Credit ACIP/CDC

#18,936

Despite ample evidence to the contrary, much of the world seems to have decided that COVID is now a relatively benign seasonal respiratory virus, on par with the common cold or - in severe cases - the flu. 

The following is a partial list of studies we've covered over the past 90 days that would beg to differ. 

JAHA: Viral Infections and Risk of Cardiovascular Disease: Systematic Review and Meta‐Analysis

Nature: Viral Infections and the Risk of Neurodegenerative Diseases (Meta-Analysis & Systemic Review)

European Society of Cardiology: Major Consensus Statement Released on Long-Term Cardiovascular Impact of COVID Infection

CSIRO Pub: Impacts of Long COVID on Disability, Function and Quality of Life for Adults Living in Australia

EHJ: Accelerated Vascular Ageing After COVID-19 Infection: The CARTESIAN Study

Preprint: Incidence of Long COVID Following Reinfection with COVID-19

Despite this mountain of evidence, uptake of COVID vaccines has plummeted, mask-wearing has gone decidedly out of fashion, and most people no longer see the need to stay home when symptomatic. 

While some people will allow that COVID might be dangerous to older individuals, they continue to insist that the risk for young adults - and particularly children - is minimal.  

Today we've a major retrospective cohort study - using national health records for nearly 14 million people under 18 in England - to compare risks of vascular and inflammatory diseases after COVID-19 infection and following BNT162b2 vaccination.

While the risks for younger individuals from COVID is lower, it is not zero.

• This study found that COVID infected children and teens had a small, but higher risk of blood clots, heart inflammation, and rare inflammatory diseases - sometimes lasting up to a year post infection.

• Following vaccination, this study only found a short-term rise in mild heart inflammation. Overall, infection caused more, and longer-lasting problems, than vaccination.

I've posted the abstract below (follow the link to read it in its entirety), followed by a link to and an excerpt from the press release. 

Vascular and inflammatory diseases after COVID-19 infection and vaccination in children and young people in England: a retrospective, population-based cohort study using linked electronic health records
Alexia Sampri, PhDa,b as3293@medschl.cam.ac.uk ∙ Wen Shi, PhDa,b ∙ Thomas Bolton, PhDc ∙ Samantha Ip, PhDa,b,d,e ∙ Rochelle Knight, MScif,g ∙ Venexia Walker, PhDf,g,i ∙ et al. Show more

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Summary

Background

The rarity of severe diseases following COVID-19 infection balanced against rare COVID-19 vaccination-related adverse effects is an important consideration for vaccination policies. We aimed to assess the short-term and long-term risks of vascular and inflammatory diseases following first COVID-19 diagnosis and vaccination in children and young people.

Methods

In this retrospective, population-based cohort study, we analysed whole-population linked electronic health records for all individuals in England aged younger than 18 years, registered with a general practitioner, and with known age, sex, and region of residence, between Jan 1, 2020, and Dec 31, 2022. Outcomes were arterial thrombotic events, venous thrombotic events, thrombocytopenia, myocarditis or pericarditis, and inflammatory conditions. COVID-19 diagnosis was defined as the earliest record of a positive SARS-CoV-2 PCR or antigen test, or a COVID-19 diagnosis code in primary-care or secondary-care records; COVID-19 vaccination was defined as the earliest documented receipt of the BNT162b2 vaccine (the predominant vaccine during the study period). Adjusted hazard ratios (aHRs) for all outcomes were estimated by time since a first COVID-19 diagnosis during Jan 1, 2020–March 31, 2022 and by time since a first COVID-19 vaccination during Aug 6, 2021–Dec 31, 2022, adjusting for age, sex, ethnicity, region, deprivation, general practitioner contact frequency, and medication use.

Findings

Of 13 896 125 individuals younger than 18 years (6 784 260 [48·8%] female and 7 111 865 [51·2%] male; 9 979 420 [71·7%] White), 3 903 410 (28·1%) had a COVID-19 diagnosis. COVID-19 diagnosis (compared with no or before diagnosis) was associated with higher risk of arterial thromboembolism (aHR 2·33 [95% CI 1·20–4·51]), venous thromboembolism (4·90 [3·66–6·55]), thrombocytopenia (3·64 [2·21–6·00]), myocarditis or pericarditis (3·46 [2·06–5·80]), and inflammatory conditions (14·84 [11·01–19·99]) in the first week after diagnosis. Incidence declined in weeks 2–4, but remained elevated to beyond 12 months for venous thromboembolism (1·39 [1·14 –1·69]), thrombocytopenia (1·42 [1·01–2·00]), and myocarditis or pericarditis (1·42 [1·05–1·91]). Among 9 245 395 individuals aged between 5 and younger than 18 years who were eligible for vaccination (4 510 490 [48·8%] female and 4 734 905 [51·2%] male; 6 684 140 [72·3%] White), 3 407 560 (36·9%) received a first vaccine. COVID-19 vaccination (compared with no or before vaccination) was associated with elevated risk of myocarditis or pericarditis within the first 4 weeks after vaccination (1·84 [1·25–2·72]). The 6-month absolute excess risks for myocarditis or pericarditis were 2·24 (1·11–3·80) per 100 000 individuals after diagnosis versus before diagnosis or undiagnosed, and 0·85 (0·07–1·91) after vaccination versus before vaccination or unvaccinated.

Interpretation

Children and young people have higher risks of rare vascular and inflammatory diseases up to 12 months after a first COVID-19 diagnosis and higher risk of rare myocarditis or pericarditis up to 4 weeks after a first BNT162b2 vaccine, although the risk following vaccination is substantially lower than the risk following infection.

These findings are of great importance for national policy makers and caregivers considering vaccination consent for children, and support the public health strategy of COVID-19 vaccination in children and young people to mitigate the more frequent and persistent risks associated with SARS-CoV-2 infection.

Funding

Wellcome Trust, British Heart Foundation Data Science Centre, and Health Data Research UK.

        (Continue . . . )

Research finds higher rare risk of heart complications in children after COVID-19 infection than after vaccination

Health Data Research UK news release 

 

The study is the largest of its kind in this population, and is published in The Lancet Child and Adolescent Health. It was led by scientists at the Universities of Cambridge and Edinburgh, and University College London, with support from the BHF Data Science Centre at Health Data Research UK.

Principal author Dr Alexia Sampri, University of Cambridge, said:

“Our whole-population study during the pandemic showed that although these conditions were rare, children and young people were more likely to experience heart, vascular or inflammatory problems after a COVID-19 infection than after having the vaccine — and the risks after infection lasted much longer.”

The research team uncovered these findings by analysing linked electronic health records (EHRs) for nearly 14 million children in England under the age of 18 between 1 January 2020 and 31 December 2022, covering 98% of this population. During this period, 3.9 million children and young people had a first COVID-19 diagnosis. And 3.4 million had a first COVID-19 BNT162b2 (Pfizer–BioNTech) vaccine, the main vaccine used in 5-18-year-olds during the study period.

       (Continue . . . )

While all of this should be highly reassuring to parents considering the COVID vaccine for their kids, the reality is, most people will probably never see it.  

It isn't `click-baity' enough for social media, and frankly, it challenges our increasingly laissez-faire post-pandemic public health policies. 

But at least its out there, for anyone who cares to read it. 

China MOA Announces New Guidelines to Expedite Animal Vaccine Strain Approvals

#18,935

While I had not planned on doing two H9N2-centric blogs in a row, overnight China's MOA published a remarkable announcement - which tacitly admits that many of their current animal vaccines (including against H9N2) are inadequate and/or suboptimal -  and orders major regulatory changes in order to accelerate updates.

As we discussed yesterday in EM&I: Enhanced Replication of a Contemporary Avian Influenza A H9N2 Virus in Human Respiratory Organoids - despite near universal vaccination - H9N2 is poorly controlled in China's poultry, and it continues to spill over into humans. 

This is not a new revelation, as over 4 years ago we looked at J. Virus Erad.: Ineffective Control Of LPAI H9N2 By Inactivated Poultry Vaccines - China, which warned that their current inactivated vaccines were no match against this rapidly evolving pathogen.

Last April, in NPJ Vaccines: Impact of Inactivated Vaccine on Transmission and Evolution of H9N2 Avian Influenza Virus in Chickens, we saw evidence that not only had inactivated vaccines failed to prevent - or even reduce - H9N2 in China's poultry, they may have driven viral evolution (including mammalian adaptations).

This problem extends beyond H9N2, as 2014's EID Journal dispatch Subclinical Highly Pathogenic Avian Influenza Virus Infection among Vaccinated Chickens, China addressed similar concerns with HPAI H5 (bolding mine): 

HPAI mass vaccination played a crucial role in HPAI control in China. However, this study demonstrated multiple disadvantages of HPAI mass vaccination, which had been suspected (13,14). For example, this study showed that H5N1 subtype HPAI virus has evolved into multiple H5N2 genotypes, which are all likely vaccine-escape variants, suggesting that this virus can easily evolve into vaccine-escape variants.

This observation suggests that HPAI mass vaccination, which is highly effective in the beginning of an outbreak, may lose its effectiveness with time unless the vaccine strains are updated. Moreover, this study showed that vaccinated chicken flocks can be infected with vaccine-escape variants without signs of illness.

As the world contemplates moving towards more aggressive vaccination of poultry against avian influenza, it must also accept that once we begin, we need to devote considerable and continual time, energy, and resources in order to keep vaccines current.  

Otherwise, we risk making matters worse. 

The Chinese announcement (MOA No. 962) calls for major changes in the way that new vaccine updates are produced, and approved, in China. While the primary focus of this document is obviously LPAI H9N2, they list 27 animal pathogen vaccines in its appendix. 

The intent is to reduce the time it takes to develop, approve, and deploy new updates to animal vaccines; from several years to a matter of months.  This would almost certainly involve some trade-offs, particularly in the amount of pre-release testing of updates that would be required. 

Of course, continuing to use existing, ineffective vaccines, is problematic in its own right.

The glacial pace of updating agricultural vaccines is not just a problem in China, as we've seen similar issues around the globe, including:

• In 2012's Egypt: A Paltry Poultry Vaccine, researchers examined the effectiveness of six commercially available H5 poultry vaccines used in Egypt and found only one actually appeared to offer protection.

• Six years later, another study (see Sci. Reports: Efficacy Of AI Vaccines Against The H5N8 Virus in Egypt) found `. . . most of the commercial poultry H5 vaccines used in the present study were ineffective. . . '

• in 2023's WUR: 2 of 4 H5 Poultry Vaccines Tested Appear Effective Against H5N1, researchers in the Netherlands found ineffective vaccines were still being marketed.

Desperate farmers, beleaguered politicians, and worried epidemiologists are all looking for ways to reduce the burden of avian flu, and poultry vaccination is understandably an attractive option. 

But the devil is always in the details.  

Not only do we need effective (and continually updated) avian flu vaccines, we need ways to ensure they are being properly and consistently applied, along with greatly enhanced surveillance and testing of vaccinated flocks (looking for breakthrough infections), including quarantine and culling if necessary.

None of this will be cheap, or easy. But doing anything less risks making a bad situation far worse in the long run.

Sadly, our 20+ year track record of poultry vaccine stewardship, has not been encouraging. 

EM&I: Enhanced Replication of a Contemporary Avian Influenza A H9N2 Virus in Human Respiratory Organoids

Flu Virus binding to Receptor Cells – Credit CDC

#18,934

If there is any constant in the chaotic and highly mutable world of influenza A viruses, it is that these viral intruders are always evolving; either slowly - via antigenic drift - or more abruptly, through antigenic shift (reassortment). 

It is why, in 2013 we saw the unexpected emergence of a deadly (in humans) LPAI H7N9 virus in China, and in 2014, the debut of an HPAI H5N8 virus in South Korea, which would eventually outshine H5N1 for the rest of the decade. 

Over time, both viruses lost momentum.  H7N9, in large part due to an emergency poultry vaccine introduced in 2017, and H5N8, due to the emergence of a `new and improved' H5N1 subtype in 2020. 

A more recent example; 18 months ago we learned that the older 2.3.2.1c clade of H5N1 had reassorted with the newer clade 2.3.4.4b in the Mekong Delta, which has sparked > 2 dozen Cambodian human infections with a near 50% CFR. This new reassortment was subsequently reclassified as clade 2.3.2.1e. 

The point being, that anything we say about a given influenza A virus today is subject to change tomorrow.  

While much of our attention is focused on HPAI H5 right now, we also keep one eye on lesser threats, including LPAI H9N2, which is not only ubiquitous in poultry across much of Asia and Africa, it has increasingly been reported in humans. 

While H9N2 has a reputation of being a relatively mild viral infection - primarily reported in children - we've been changes in its epidemiology of late; including more adult infections (some seriously ill) reported in out of China.  

H9N2 also easily reassorts with, and often enhances, other novel influenza viruses (including H7N9, H5N1, and H5N6), making it an important viral co-conspirator (see Vet. Sci.: The Multifaceted Zoonotic Risk of H9N2 Avian Influenza).

But, as a pandemic threat, LPAI H9N2 doesn't get a lot of respect.  

It is a low-path virus in poultry, and not considered `reportable' to WOAH.  Some countries vaccinate poultry against it, but existing vaccines have not been very effective (see (see J. Virus Erad.: Ineffective Control Of LPAI H9N2 By Inactivated Poultry Vaccines - China).

In China, where the bulk of human cases have been reported, we've seen renewed interest the past couple of years, as the virus has slowly evolved towards a more mammalian-adapted virus. 

Last week, in China CDC Weekly: Epidemiological and Genetic Characterization of Three H9N2 Viruses Causing Human Infections, we looked at a local CDC investigation into 3 pediatric cases which were reported last April from Changsha City, Hunan Province, China.

Their report found a number of indicators of increased mammalian adaptation within the virus, including an enhanced ability to infect upper respiratory (α2,6-sialic acid) tract receptors, and a number of HA protein mutations, including; H191N, A198V, Q226L, and Q234L.

Which brings us to a new study, published yesterday in Emerging Microbes & Infections, which compares two H9N2 isolates (from 2024 and 1999) across several models, and finds today's virus far better adapted to human hosts. 

While only two virus isolates were compared, and this study does not test transmission dynamics beyond cell and organoid infectivity, this is a reminder that even presumed `minor threats' like H9N2 have the potential to evolve into something more formidable.  

I've posted the Abstract and a small excerpt, but you'll want to follow the link to read it in its entirety.   I'll have a small postscript after the break. 

Enhanced replication of a contemporary avian influenza A H9N2 virus in human respiratory organoids

Lin-Lei Chen, Jonathan Daniel Ip, Wan-Mui Chan, Stephanie Joy-Ann Lam,
Rhoda Cheuk-Ying Leung, Cyril Chik-Yan Yip, show all
Article: 2576574 | Received 15 May 2025, Accepted 14 Oct 2025, Accepted author version posted online: 16 Oct 2025, Published online: 03 Nov 2025
https://doi.org/10.1080/22221751.2025.2576574

ABSTRACT

H9N2 is currently the second most common avian influenza A virus subtype infecting humans. Monitoring viral phenotypic and genotypic adaptation to humans is crucial for risk assessment. Here, we compared the replication of an H9N2 human isolate collected in 2024 (A/HK/2346/2024) to a human isolate collected in 1999 (A/HK/1073/1999).

In Madin Darby canine kidney (MDCK) cells, A/HK/2346/2024 and A/HK/1073/1999 replicated to 8 and 5 log10 plaque-forming units (PFU) per ml, respectively. In both human nasal and lung organoids, A/HK/2346/2024 replicated to 6 log10 PFU/ml, but A/HK/1073/1999 failed to replicate in either organoid. The infection rates of both ciliated and non-ciliated cells and the ratios of infected 2,6/2,3 cells were higher for A/HK/2346/2024 than A/HK/1073/1999.

Apart from the mammalian adaptive substitutions that were present in the nasopharyngeal specimen collected on day 1 post-symptom onset (pso) (HA-D183N/D190 T/Q192R/Q226L; NA-del62-64; PB2-A588V/K702R; PB1-I368V; PA-K356R/S409N; M1-R95K), the mammalian-adaptive substitution PB2-D253N emerged de novo on day 7 pso.

Analysis of all human (n = 96) and avian influenza (n = 14,762) H9N2 deposited at GISAID showed the dominance of several human-adaptive substitutions in H9N2 strains collected from humans in recent years.

In summary, we demonstrated that a recent H9N2 virus is more adapted to humans, and is able to replicate to high titres in both upper and lower human respiratory tract which may confer higher person-to-person transmissibility and virulence. Our study underscores the importance of human organoid-based phenotypic monitoring and inter/intrahost genotypic monitoring for assessing the zoonotic risk of avian influenza viruses.

       (SNIP)     

H9N2 has been relatively neglected as a potential pandemic agent. Over the years, H9N2 has already acquired numerous mutations that facilitate adaptation to humans [35].

 In this study, we demonstrated the enhanced fitness of the contemporary H9N2 isolate in human nasal organoid and identified the emergence of human adaptive mutations that only appeared late during infection. Being a reservoir of internal genes for AIVs that cause severe human infections, H9N2 can significantly contribute to an AIV that causes a human pandemic. Having a low pathogenicity in birds, H9N2 can spread widely in wild birds without being noticed. In fact, H9N2 is the predominant AIV subtype detected among poultry [19]. 

Antigenic drift has allowed the virus to evade from vaccine-induced immunity [49]. With widespread co-circulation of H5N1 2.3.3.4b and H9N2 in wild birds [50], there is an increased chance of reassortment between these two strains. If an H5N1 2.3.4.4b, which already circulates widely in mammals, reassorts with an H9 virus, there is a high risk of an endemic or even a pandemic virus. Continual genotypic and phenotypic surveillance of H9N2, especially from human and mammalian infections, plays a pivotal role in the assessment of zoonotic risk of all AIVs [51].

(Continue . . . )

While H9N2 remains far from our biggest pandemic concern, the CDC has designated 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.

And many will be surprised to see that, in terms of risk of emergence, the H9N2 Y280 lineage is ranked higher than H5N1, while the G1 lineage is ranked only slightly lower.

Of course, something that is currently not even on our radar could emerge as the next big threat. Afterall, no one saw SARS-CoV-2 as a threat until it was already upon us, and H7N9 emerged with no warning. 

The only real questions are when the next pandemic will emerge, how bad it will be, and whether we'll be ready when it does. 

The first two are out of our control.  As for the third, we seem even less prepared today, than we were before the last pandemic hit.  

Pre-Print: A Cattle-derived Human H5N1 Isolate Suppresses Innate Immunity Despite Efficient Replication in Human Respiratory Organoids

#18,933

As the graphic above illustrates, over the past 20+ years we've seen a wide variance in the virulence - and CFR (case fatality rate) - of the H5N1 virus in countries around the globe. 

While 84% of confirmed Indonesian cases died, less than half that percentage succumbed in Egypt, and in Bangladesh, only one fatality out of 12 cases has been recorded. 

As we discussed more than a dozen years ago, in Differences In Virulence Between Closely Related H5N1 Strains, while there are likely many factors involved - including quality of, and delays in seeking medical care – it suggests that some HPAI H5N1 strains (clades, subclades, genotypes) are far more virulent than others.

Here in the United States, over the past couple of years, we've seen 70 confirmed and 7 probable H5N1 infections, and only 1 fatality. At the same time, in Cambodia nearly half of 33 recent cases (of a different clade; 2.3.2.1e) have died. 

While it may be obvious that some strains of H5N1 are deadlier than others, what isn't as apparent is what makes the difference.  We have seen some theories, however.

• In yesterday's mBio: Low levels of influenza H5N1 HA and NA antibodies in the human population are boosted by seasonal H1N1 infection but not by H3N2 infection or influenza vaccination, researchers suggest a similar NA gene in seasonal H1N1 may evoke some degree of immunity against H5N1. 

• It has also been suggested that the route of exposure (ocular or gastrointestinal) - or a lower viral load - may produce more localized, less severe infections.

• And unlike places like Cambodia, Egypt, and Indonesia - in the United States it is primarily adults occupationally exposed to H5N1 - and we already know that HPAI H5 has a predilection for infecting a younger cohort.

Of course, there may be multiple factors at work. 

Today, however, we have a preprint that offers another intriguing possibility; that the B3,13 genotype disables our innate immune response - suppressing cytokine production (and many symptoms) - but still replicates efficiently in human hosts. 

Using lab-created (influenza-naive) lung organoids, these researchers were able to study the response of the innate immune system.  How their results match up with humans typically saddled with a long and complex immune history remains to be seen. 

But they did find that the TX37-H5N1 human-bovine strain replicated quite efficiently in these human lung organoid tissues, but that the virus failed to trigger a robust immune response, which could help explain mild or asymptomatic infections. 

I've reproduced the abstract, and an excerpt from the discussion below. Follow the link to read it in its entirety.  I'll have a bit more when you return.

A cattle-derived human H5N1 isolate suppresses innate immunity despite efficient replication in human respiratory organoids
Shintaro Shichinohe, Hikaru Sugimoto, Masako Yamasaki, Rina Hashimoto, Tatsuru Morita, Daiki Kobayashi, Takahiro Hiono, Daisuke Motooka, Makoto Shimooka, Norikazu Isoda, Mai Thi Quynh Le, Ayato Takada, Yoshihiro Sakoda, Eiryo Kawakami,Kazuo Takayama, Tokiko Watanabe
doi: https://doi.org/10.1101/2025.11.02.684669
This article is a preprint and has not been certified by peer review

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Abstract

The H5N1 high pathogenicity avian influenza virus (HPAIV) of clade 2.3.4.4b, which spreads globally via wild birds, has become a major public health concern because it can infect a variety of mammals, including humans. In 2024, infection of dairy cattle with H5N1 HPAIV clade 2.3.4.4b was confirmed in the United States, and subsequent human cases were reported. Although these viruses are highly pathogenic in animal models, human infections have generally been mild, revealing a striking discrepancy. 

Here, we characterized the cattle-derived human H5N1 isolate A/Texas/37/2024 (TX37-H5N1) using three-dimensional human respiratory organoids derived from induced pluripotent stem (iPS) cells. Despite efficient replication, TX37-H5N1 induced minimal interferon and inflammatory cytokine responses.

 Bulk and single-cell RNA sequencing revealed reduced STAT1-mediated transcriptional activity in TX37-H5N1-infected organoids compared to the historic H5N1 human isolate A/Vietnam/1203/2004. These findings suggest that TX37-H5N1 fails to trigger the strong innate responses, including robust cytokine production, that are typically associated with severe H5N1 disease and are thought to contribute to cytokine storm-medicated pathogenesis. 

This attenuated response may help explain the discrepancy between the high pathogenicity of TX37-H5N1 in animal models and its mild clinical presentation in humans. While zoonotic influenza risk is often assessed using cell lines or animal models, our study highlights the value of using human respiratory organoids to evaluate human-specific virus-host interactions. This platform provides a complementary tool for assessing the risk of emerging avian influenza viruses.

        (SNIP)

Our study provides evidence that selective suppression of innate immunity may explain the mild clinical manifestations of B3.13 H5N1 viruses in humans and offers new perspectives for reconciling inconsistencies between animal models and clinical outcomes.

Viruses like TX37-H5N1 that replicate efficiently yet cause only mild disease in humans may evade detection if only traditional risk assessment strategies are employed (e.g., measuring LD₅₀ values and viral titers in mice and ferrets). 

Immune response profiling using human-derived organoids, as demonstrated here, offers a powerful complementary approach to capture human-specific innate immune responses that may not align with animal-model outcomes. Moreover, viruses with low-pathogenic but high-replication phenotypes pose unique challenges for public health. Such viruses may silently spread through symptomatic infection and prolonged viral shedding. 

While no sustained human-to-human transmission of B3.13 viruses has been reported to date, future mutations may alter transmissibility.

Because no single model can fully capture public health threats of emerging influenza viruses, an integrated framework is needed to evaluate viral replication efficiency, immune modulation, and transmissibility. Finally, the suppression of the STAT–IRF pathway identified in this study may offer new avenues for therapeutic intervention and have implications for pandemic preparedness and emerging infectious disease control. Future efforts should focus on characterizing host immune  responses in human cases and performing cross-strain comparisons to identify predictive markers of human pathogenicity.

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If we could depend upon H5N1 to only produce mild symptoms going forward, then we could all relax - deal with a brief bout of conjunctivitis or the sniffles - and get on with life. 

But we are seeing an entirely different picture with other mammalian species, and there are no guarantees this virus won't evolve into a more virulent strain in humans.

Even if the B3.13 genotype can't make that leap, there are hundreds of other HPAI H5 genotypes currently circulating around the globe, and it only takes one to hit the genetic jackpot to plunge the world back into a pandemic crisis. 

Unfortunately, mild or asymptomatic individuals are the least likely to be tested for the virus - meaning that if  HPAI H5 were spreading stealthily in the community - we could easily be none the wiser. 

For more on the challenges in tracking the spread of this virus in the community, you may wish to revisit NAS : Diagnostic Tools, Gaps, and Collaborative Pathways in Human H5N1 Detection (Rapid Expert Consultation).

 

mBio: Low levels of influenza H5N1 HA and NA antibodies in the human population are boosted by seasonal H1N1 infection but not by H3N2 infection or influenza vaccination

 
CDC Official List 

#18,932

Over the past 18 months we've seen numerous (mostly) mild - sometimes even subclinical - human infections with the bovine B3.13 genotype of H5N1. Unlike other subclades (e.g. 2.3.2.1e) and genotypes, this `bovine' variant (so far) appears to be less virulent in humans.

This, despite the initial CDC assessment (see July 14th, 2024 H5N1 Update):

CDC analyzed sera (blood) collected from people of all ages in all 10 HHS regions. Blood samples were collected during the 2022-2023 and 2021-2022 flu seasons. These samples were challenged with H5N1 virus to see whether there was an antibody reaction. Data from this study suggest that there is extremely low to no population immunity to clade 2.3.4.4b A(H5N1) viruses in the United States.

These studies were focused on the HA component of the H5N1 virus, but we've previously seen studies suggesting NA antibodies may play a role as well (see EID Journal: A(H5N1) NA Inhibition Antibodies in Healthy Adults after Exposure to Influenza A(H1N1)pdm09).

Since then, we've seen a number of studies suggesting that prior infection (or possibly vaccination) with the H1N1pdm virus may - due to H5N1 having a similar NA gene - covey some (unknown) degree of protection in humans.

 A few examples include:

Preprint: Cross-Reactive Human Antibody Responses to H5N1 Influenza Virus Neuraminidase are Shaped by Immune History

Preprint: Neuraminidase Imprinting and the Age-related Risk of Zoonotic Influenza

Two EID Journal Articles On Prior Immunity From A(H1N1)pdm09 Infection Against H5N1 (in Ferrets)

How much real-world protection past H1N1 exposure might offer is - or how long it lasts - is unknown, but it has been suggested it might provide an `edge' against severe disease; at least against some H5N1 variants.

While the notion that some degree of immunity to H5N1 might exist gets a lot of attention by the mainstream media, the reality is, it is likely to be quite small. But knowing what drives this immunity might help researchers create more effective vaccines against H5N1 in the future. 

All of which brings us to a new study, published on Friday in mBio, which uses blood samples from a relatively small cohort of JHH & JHMI employees (n=73) (vaccinated, unvaccinated, and some with recent H1N1 or H3N2 infection) to test for (binding & reactive) HA & NA antibodies against H5N1 (including Bovine B3.13). 

While you'll want to follow the link to read the report in its entirety, briefly they found:

• at baseline, they confirmed humans have low levels of antibodies targeting the H5N1 hemagglutinin (HA) and neuraminidase (NA) proteins

• NA antibodies, however, were more common and more reactive than HA antibodies

• Seasonal influenza vaccination did not appear to significantly boost those antibodies

• But recent H1N1 (but not H3N2) infection resulted in increased NA cross-reactive neutralizing antibodies against the Bovine B3.13 genotype of H5N1

• They don't appear to have directly tested other genotypes (like D1.1, D1.3) for comparison

While promising, the authors cautioned:

Since there are no established correlates of protection for A/H5N1 infection in the human population, we cannot make any conclusions about how these cross-reactive antibodies against bovine A/H5N1 might modulate infection or disease severity.

First the abstract, and a couple of brief excerpts, after which I'll return with brief postscript.

Low levels of influenza H5N1 HA and NA antibodies in the human population are boosted by seasonal H1N1 infection but not by H3N2 infection or influenza vaccination
Authors: Anne P. Werner , Cosette G. Schneider, Elgin H. Akin, Juliahna Hayes, Katherine Z. J. Fenstermacher , Richard E. Rothman, Lynda Coughlan , Andrew Pekosz  apekosz1@jhu.eduAuthors Info & Affiliations
https://doi.org/10.1128/mbio.02145-25

ABSTRACT

An increase in the number of human cases of influenza A/H5N1 infection in the USA has raised concerns about the pandemic potential of the virus. Pre-existing population immunity is a key determinant for risk assessment and pandemic potential for any virus.

Antibody responses against the bovine A/H5N1 hemagglutinin (HA) and neuraminidase (NA) proteins were measured among a population of influenza-vaccinated or influenza-infected individuals. Modest titers of bovine A/H5N1 HA-binding antibodies and low to undetectable neutralizing antibody titers were detected in a cohort of 73 individuals.

Conversely, bovine A/H5N1 NA-binding and neuraminidase-inhibiting antibody titers were comparable to those against a human A/H1N1 NA at baseline. Seasonal influenza vaccination failed to significantly increase antibody titers against both HA and NA glycoproteins of bovine A/H5N1.

Recent infection with human A/H1N1 but not A/H3N2 viruses induced significant increases in bovine A/H5N1-neutralizing antibody, as well as increases in NA-binding and NA-inhibiting antibodies to bovine A/H5N1 NA.

While the degree of protection afforded by these A/H5N1 cross-reactive antibodies is not known, incorporating NA or enhancing current seasonal vaccine formulations to increase NA-specific antibody titers may increase antibody breadth and protection against both seasonal and pandemic influenza viruses.

IMPORTANCE

A/H5N1 influenza A viruses continue to pose a pandemic threat to humans. Recent infection of dairy cattle and poultry with A/H5N1 in the USA has magnified that concern. We determined the level of antibodies that recognize A/H5N1 hemagglutinin (HA) and neuraminidase (NA) proteins in a population in Baltimore, MD. We show that while low levels of H5 HA-binding and A/H5N1-neutralizing antibodies are present, there is a significantly stronger recognition of bovine N1 NA. Vaccines that target the N1 NA protein may induce protective antibody responses in humans due to the presence of cross-reactive human N1 NA antibodies.

(SNIP)

We show evidence that supports changing current seasonal vaccine formulations to either include greater NA content or to manipulate immunogen design to increase immunofocusing toward immunosubdominant domains of IAV HA and NA (25, 66, 77, 78).

The work presented here reiterates the importance of NA as a conserved antigen between human seasonal viruses and A/H5N1 HPAIs and underscores the need for investigation of NA-mediated antibody responses and their role in protective immunity. NA-centered vaccine design would enable robust boosting of cross-reactive N1 antibodies and may serve as a more feasible approach to increasing population-level pre-existing antibodies to A/H5N1 compared to HA-focused vaccines. 

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While all of this could lead to more NA-centered vaccine designs, which might instill better population immunity against a potential H5N1 pandemic, none of this is likely to happen overnight. 

• In 2010 Dr. Klaus Stohr, former head of the World Health Organization's global influenza program, floated the idea (see The Prime Of Our Lives) of including potential future pandemic strains in yearly seasonal flu shots to create some degree of pre-existing immunity.

• In a similar vein, in 2011 (see Nature: A Preemptive H2N2 Vaccine Strike?), researchers concerned over the possible return of H2N2 (last seen in the 1960s), suggested including an H2N2 strain in the yearly jab.

Those ideas never gained traction, and in all likelihood would have faced massive resistance from a vaccine-wary public. Tweaking the NA content of the seasonal flu shot would likely garner far less backlash, but its effectiveness remains unknown. 

This study, however, does provide new avenues for exploration.  And that may eventually lead to more effective vaccines in the down the road. 

But in the short term, if H5N1 (or any other respiratory virus) decides to embark on a world tour, your best friend will likely be your stash of N95/KN95 masks in your first aid supplies.