Pathfinder
Editor, Senior Moderator
- Article
- Open access
- Published: 24 December 2025
- Anastasia S. Panova,
- Andrey S. Gudymo,
- Natalia P. Kolosova,
- Alexey V. Danilenko,
- Kiunnei N. Shadrinova,
- Natalia V. Danilchenko,
- Olga N. Perfilieva,
- Anastasia A. Moiseeva,
- Elena I. Danilenko,
- Galina S. Onkhonova,
- Natalia I. Goncharova,
- Svetlana V. Svyatchenko,
- Natalia N. Vasiltsova,
- Marina L. Egorova &
- Vasiliy Yu. Marchenko
- 1964 Accesses
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The global spread of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses has recently extended to include diverse mammalian species, raising new concerns about pandemic risk. In 2023, this clade was first detected in Russian marine mammals during a mass mortality event among northern fur seals in the Far East. Genetic analyses revealed the causative viruses to belong to genotype A3 of European origin, which is known to have circulated in wild birds across the Far East since 2022. Notably, these isolates harbor the mammalian-adaptive substitutions PB2-K482R and NP-N319K—mutations previously linked to enhanced virulence in non-H5 avian influenza viruses, but whose impact on A(H5N1) clade 2.3.4.4b viruses remained to be characterized. The heightened virulence of A3 genotype viruses is confirmed by data obtained via a mouse model. However, despite these adaptive changes, ferret transmission models showed no evidence of airborne transmission of the fur seal-derived virus. Our findings indicate that while PB2-K482R and NP-N319K may contribute to increased mammalian pathogenicity, they do not significantly increase the efficiency of respiratory transmission—a key prerequisite for human pandemic potential. Although suggesting a limited immediate pandemic threat from this A3 genotype, these results underscore the critical need for continued surveillance and functional assessment of emerging mammalian-adaptive mutations in circulating A(H5N1) viruses.
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Discussion
The global expansion of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses into diverse mammalian hosts since 2022 has raised significant concerns regarding zoonotic potential and pandemic risk[SUP]34[/SUP]. Although such spillover events are frequently accompanied by the acquisition of mammalian-adaptive mutations, the contribution of specific genetic changes—particularly in the context of emerging genotypes—remains incompletely understood. Reassortment among clade 2.3.4.4b viruses has generated multiple genotypes having varying phenotypic properties[SUP]2[/SUP]. Virulence and transmission may be critically influenced by the interplay between adaptive substitutions and genomic background.
In 2023, A(H5N1) clade 2.3.4.4b viruses of genotype A3 were isolated from northern fur seals (Callorhinus ursinus) in the Russian Far East during a mass mortality event. Genetically similar A3 viruses were concurrently detected in wild birds on Sakhalin Island, strongly implicating avian species as the likely source of infection. This genotype joins others—such as A2, B1.2, B3.2, and D1.1—identified in marine mammals across the Americas during 2022–2023 (GISAID), underscoring the widespread circulation of reassorted A(H5N1) variants in non-avian hosts.
We used a murine model to demonstrate that A3 genotype viruses exhibit enhanced virulence compared to both a closely related genotype G10 virus (A/turkey/Tyumen/81-96V/2021) and genotype BB viruses, which have caused extensive outbreaks in gulls and other birds across Europe and western Russia. The heightened pathogenicity of A3 viruses correlates with the presence of two key mammalian-adaptive substitutions: PB2-K482R and NP-N319K. PB2-K482R has been shown to increase virulence in A(H7N9)[SUP]32[/SUP] and mouse-adapted seasonal influenza viruses[SUP]35,36[/SUP], while NP-N319K enhances viral polymerase activity in mammalian cells by strengthening NP binding to importin-α1 and -α7, thereby facilitating nuclear import—particularly when co-occurring with PB2 mammalian-adapting mutations[SUP]37,38[/SUP]. Structural studies indicate that NP residue 319 lies within a domain that interacts directly with PB2, supporting the potential for synergistic effects between these proteins[SUP]39,40[/SUP]. Notably, a previous study associated an A(H5N1) isolate from Russia with the same seal outbreak[SUP]41[/SUP] that harbored the PB2-E627K substitution, along with PB2-K482R and NP-N319K AASs characteristic of the A3 genotype. This further underscores the propensity of marine mammal-derived influenza viruses to accumulate multiple adaptive markers associated with mammalian replication. Additionally, the A3 viruses in our study carried substitutions in NS1 and NEP/NS2 previously linked to increased virulence, suggesting that genome-wide synergistic interactions may further modulate pathogenicity. Despite the potentially important role of NP-N319K and PB2-K482R mutations in mammalian virulence, they remain understudied in contemporary A(H5N1) 2.3.4.4b viruses, and their functional roles need to be investigated through reverse genetics or other functional characterization approaches.
In light of previous studies, our findings underscore the capacity of A(H5N1) clade 2.3.4.4b viruses to rapidly acquire combinations of mutations—through reassortment or selection of adaptive variants during mammalian infection—that enhance virulence in mammalian hosts. Prior studies have shown that specific PB2 mutations associated with increased virulence may also enhance the transmission of avian influenza viruses, including clade 2.3.4.4b A(H5N1), in mammalian models[SUP]13,18,27,28,29[/SUP]. However, enhanced virulence does not necessarily confer efficient airborne transmission. To evaluate this critical parameter, we assessed the airborne transmission of the fur seal-derived A3 virus (A/fur seal/Sakhalin/399-1V/2023) in a ferret model using a validated dynamic aerobiology chamber (TIEGEL ELC 04–60).
Despite high and sustained viral loads in nasal washes of donor ferrets over five days post-infection, no airborne transmission to naïve contact animals was observed under controlled environmental conditions (20 °C, 30% RH). Under identical experimental conditions and using the same transmission system in our laboratory, efficient airborne transmission was previously demonstrated for pandemic A(H1N1)pdm09 and swine-origin A(H3N2) viruses, limited airborne transmission for 2.3.4.4b A(H5N1) carrying PB2-E627K, and no transmission for investigated H5 clades 2.3.4.4b and 2.3.2.1c viruses[SUP]13,42[/SUP].
The lack of transmission for the A(H5N1) virus in this study is likely multifactorial. First, the A3 virus retains strong preference for avian-type α2,3-linked sialic acid receptors, whereas the upper respiratory tract of ferrets predominantly expresses human-type α2,6-linked receptors—a key barrier to efficient airborne spread. Second, although viral replication in donors was robust, titers and infectivity were lower than those achieved by a PB2-E627K—bearing A(H5N1) strain that itself showed only limited transmission under identical conditions[SUP]13[/SUP]. Third, the absence of other known adaptation markers—such as HA mutations that lower the pH of fusion or enhance binding to α2,6 receptors—may further restrict transmission potential[SUP]43[/SUP].
Our study has certain limitations. The donor-recipient exposure period was limited to 5 h per day due to technical constraints of the chamber, though this duration has proven sufficient to detect transmission in positive-control experiments. Only female ferrets were used; while sex-based differences in influenza immunity have been observed[SUP]44[/SUP], their impact on airborne transmission remains uncertain. Nevertheless, the experimental conditions used in our study closely mirror those used in benchmark transmission studies to enhance comparability.
From a pandemic risk perspective, the World Health Organization’s Tool for Influenza Pandemic Risk Assessment (TIPRA) identifies mammalian adaptation, disease severity, and transmission as key determinants of pandemic potential[SUP]30[/SUP]. Although the A3 genotype harbors mammalian-adaptive mutations PB2-K482R and NP-N319K and exhibits high virulence, it shows no detectable airborne transmission in the ferret model. These findings indicate that, despite enhanced pathogenicity due to its genetic features, the virus lacks the capacity for efficient respiratory transmission in mammals, thereby representing low pandemic risk.
To date, no human infections with A3 genotype viruses have been reported (GISAID); however, the ongoing circulation of this genotype in wild and domestic birds across the Russian Far East and geographically neighboring regions—including other countries—poses a persistent risk of spillover into mammals, including humans.
In conclusion, our data indicate that currently circulating A3 genotype A(H5N1) viruses, despite exhibiting enhanced virulence in mammals, pose a limited immediate pandemic threat owing to their inability to transmit efficiently via the airborne route. Nevertheless, their capacity to acquire mammalian-adaptive mutations during spillover events into marine mammals underscores the dynamic evolutionary potential of clade 2.3.4.4b viruses. Sustained, integrated surveillance—complemented by functional characterization of emerging variants—is therefore essential for monitoring the accumulation of mammalian-adaptive genetic features and enabling the timely detection of avian influenza virus variants with heightened pandemic potential.
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https://www.nature.com/articles/s41598-025-28032-3