#19,157
When we talk about the dangers of the H5Nx virus, it is with the understanding that there are currently several genetically distinct subclades of the virus circulating around the globe (2.3.4.4b, 2.3.2.1a, 2.3.2.1e, etc.), spanning numerous subtypes (H5N1, H5N2, H5N5, H5N6, H5N8, etc.) and literally hundreds of genotypes.
Human infections may be mild - as we've typically seen with the North American `Bovine' B.13 genotype of H5N1 (clade 2.3.4.4b) - or more severe - as we've seen with the clade 2.3.2.1e H5N1 viruses (40% CFR) found in Cambodia.Fortunately, these HPAI viruses are not all created equal. Most only infect birds, while some have evolved enough to spillover into mammals, while fewer still have shown the ability to infect humans.
Even when you compare nearly identical viruses (same subclade, same subtype, and same genotype) some have proved more virulent, or more transmissible, than its close relatives.
Which is why we often find ourselves focusing on tiny amino acid changes in the influenza genome - including PB2 mutations like E627K, D701N, Q591K, and M631L and HA mutations like Q226L and E190D - which may favor mammalian adaptation.
But new ones (see Sci. Adv.: PB2 and NP of North American H5N1 Virus Drive Immune Cell Replication and Systemic Infections) continue to be discovered.
The impact of these mutations can vary between virus strains, and can be enhanced or attenuated by other amino acid changes (some known, others yet to be discovered) that may occur elsewhere in the genome.
For years, researchers have looked at the PB2-E627K mutation as possibly being the most important, but we've a preprint today that finds - at least in one specific H5N1 D1.1 strain - that the PB2 D701N mutation appears to drive virus transmissibility in ferrets.
First the link, abstract, and the discussion from the preprint. I'll have a bit more after the break.
Variable transmission efficiency of mammalian origin HPAI D1.1 H5N1 strains in ferrets
Grace E. Quirk, Michelle N. Vu, Valerie Le Sage, Kaitlyn Bushfield-Thomason, Hanh Dung Nguyen, Seema S. Lakdawala
doi: https://doi.org/10.64898/2026.05.07.722809
This article is a preprint and has not been certified by peer review
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Abstract
Highly pathogenic avian influenza H5N1 2.3.4.4b genotype D1.1 lineage continues to predominate in the United States wild bird population and has spilled over into dairy cattle three independent times. To assess the transmission risk of this sublineage, we performed direct-contact transmission experiments for three distinct D1.1 strains in ferrets.Two of these strains were isolated from humans and one from a lethal cat infection. We found that only one human isolate (A/NV/10/2025) was able to transmit efficiently between ferrets. Compared to the other strains, this isolate harbored the mammalian adaptive PB2 D701N mutation, suggesting this mutation may be critical for D1.1 transmission as opposed to the PB2 E627K substitution present in the lethal cat isolate.Based on these data we conclude that the transmission fitness of D1.1 strains is modest but that special attention should be paid to emergence of adaptation at the PB2 701 position.
(SNIP)
Discussion
In this study we describe transmission fitness of three D1.1 genotype 2.3.4.4b clade H5N196 viruses. Two of the three strains, A/WA/255/2024 and A/Cat/TX/009022-007/2025, had low to no spread to cohoused ferrets. Reports by the CDC using a related D1.1 strain A/WA/239/202498 reported low transmission efficiency by direct contact to 1/3 recipient ferrets [9].
In contrast, A/NV/10/2025 spread to all three cohoused animals. Additionally, recipient ferrets naturally infected with A/NV/10/2025 reached humane endpoint criteria prior to the scheduled end of the study, suggesting that not only is this virus transmissible, but it can result in severe natural infection.
The viruses in this study were chosen because of their mammalian origin and their unique PB2 mammalian adaptive residues. A/NV/10/2025 has a classical PB2 mammalian adaptive mutation at position 701, in contrast to the 627 residue present in the A/Cat/TX/009022-007/2025106 strain.
While we cannot exclude the impact of other mutations in present in A/NV/10/2025 (Table107S2) compared to A/Cat/TX/009022-007/2025 and A/WA/255/2024, it is highly likely that mammalian adaptation of residue 701 is playing a role in the forward transmission potential of this strain.
Interestingly, only 1% of all D1.1 strains curated by Nextstrain [10] contain a PB2110 D701N mutation, but 7% of spillovers into mammalian species contain this mutation [3]. While PB2 E627K mutation is more prevalent in mammalian spillover viruses at 35%, this mammalian adaptation in D1.1 has not been related to forward transmission of the virus in ferrets in our study or others [9].
Our findings suggest that while less common in initial mammalian spillover events, PB2 D701N may increase the risk of onward transmission. Targeted surveillance of D1.1 strains for the acquisition of PB2 D701N in animals at the human-animal interface may identify priority variants for pandemic risk assessment.
We've looked at the impact of the PB2-D701N mutation often in the past, and while considered to be an important mammalian-adaptation marker in both H5 and H7 subtypes, it has not been a reliable predictor of transmissibility or virulence across the board.
But for the H5N1 D1.1 virus (and perhaps others), the presence of the PB2-D701N mutation may be a useful early warning sign.
I would note that just two days ago, in Sci Adv: Mammary and Respiratory Infection of Sheep with H5Nx clade 2.3.4.4b Viruses with Milk-mediated Transmission to Lambs, that one of the mutations seen in H5N1 infected sheep was PB2-D701N.
Deep sequencing of milk samples from mammary glands, oral swabs, and lung tissues revealed the emergence of viral variants distinct from the consensus sequence generated from the challenge D1.1 virus.
Notably, the left mammary gland of a D1.1-infected sheep appeared to select for the variant PB2-701N, while the right gland retained mixed residues of PB2-627 (E/K) and PB2-701 (D/N).
Another indication that while the emergence of a pandemic strain of H5N1 may be a long shot, Nature's laboratory is open 24/7, and it continues to tinker.
