Microbiol Spectr . Decoding non-human mammalian adaptive signatures of 2.3.4.4b H5N1 to assess its human adaptive potential

[tetano]

Microbiol Spectr


. 2025 Aug 11:e0094825.
doi: 10.1128/spectrum.00948-25. Online ahead of print. Decoding non-human mammalian adaptive signatures of 2.3.4.4b H5N1 to assess its human adaptive potential

Ranjana Nataraj[SUP] 1 [/SUP], Avinash Karkada Ashok[SUP] 1 [/SUP], Ayushi Amin Dey[SUP] 1 [/SUP], Sannula Kesavardhana[SUP] 1 [/SUP]



Affiliations

• PMID: 40788041
• DOI: 10.1128/spectrum.00948-25

Free article Abstract

The 2.3.4.4b clade highly pathogenic avian influenza H5N1 infected diverse non-human mammalian species, gained mammal-to-mammal transmission potential, and caused sporadic human infections. However, whether non-human mammals enable the human adaptation of 2.3.4.4b H5N1 to establish human infections is unclear. Gain-of-function research restrictions may hinder the assessment of 2.3.4.4b H5N1 human adaptations. Here, we tracked the evolution of 2.3.4.4b H5N1 that infected non-human mammals and evaluated their ability to gain human adaptations. The non-human mammal 2.3.4.4b H5N1 partly acquired classical human-adapting mutations, which are identical to the residues of H1N1pdm09 and seasonal human H3N2 viruses, while showing a few species-specific adaptations that might be potential barriers for successful human infections. The polymerase complex proteins, PA and PB2, acquired human adaptations in non-human mammals, with fox-infected viruses showing more positive selection in the polymerase complex. The human-adapting Q591K/R substitution in PB2 appeared only in the 2.3.4.4b clade but not in previously circulating H5N1 strains. Despite minimal changes in hemagglutinin (HA), A160T and T199I mutations near the receptor binding site of HA in dairy cattle viruses indicate the rapid HA glycan surface evolution affecting virus entry and immune evasion. The unbiased quantitative assessment of virus adaptations indicated that 2.3.4.4b H5N1 circulating in bears, cattle, dolphins, and foxes might show better human adaptive potential. Thus, 2.3.4.4b H5N1 appears to be acquiring human adaptations due to natural selection pressure in non-human mammals. Overall, our study delineates human adaptation and infection risk of specific non-human mammalian circulating 2.3.4.4b H5N1 strains.IMPORTANCEThe 2.3.4.4b clade H5N1 virus emerged as a panzootic strain, leading to the unprecedented deaths of domestic and wild birds and diverse non-human mammalian species. Intriguingly, the 2.3.4.4b H5N1 transmitted to diverse mammalian species and gained mammal-to-mammal transmission, suggesting its pandemic potential. The H5N1 outbreaks in dairy cattle and sea lions are devastating, and they contributed to sporadic human infections. This indicates the ability of non-human mammal hosts, like dairy cattle, as potential sources for human transmission. However, the signatures of non-human mammal adaptations of 2.3.4.4b H5N1 and how these adaptations drive the human adaptive potential of 2.3.4.4b H5N1 are unclear. In this study, we show the specific molecular patterns of H5N1 proteins that determine species-specific adaptations in non-human mammals. We identified that 2.3.4.4b H5N1 circulating in non-human mammals is rapidly evolving with critical adaptations in PA, PB2, and HA and gaining human adaptive potential in specific non-human mammalian species.

Keywords: 2.3.4.4b H5N1; HPAIs; avian influenza; hemagglutinin; host-directed evolution; human adaptation; non-human mammals; panzootic virus; virus evolution; virus polymerase complex.

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[Commonground]

Tracing the evolution of the H5N1 virus

​12 August 2025​
REFERENCE: above, Post #1
Nataraj R, Ashok AK, Dey AA, Kesavardhana S, Decoding non-human mammalian adaptive signatures of 2.3.4.4b H5N1 to assess its human adaptive potential, Microbiology Spectrum (2025).
https://journals.asm.org/doi/10.1128/spectrum.00948-25​



In recent years, there has been growing concern over the H5N1 influenza virus. It was first identified in birds three decades ago and has now gradually found its way to humans. H5N1 is a strain of the influenza virus harbouring type 5 haemagglutinin (H5) and type 1 neuraminidase (N1) surface proteins, which help in viral entry and spread, respectively.

Researchers led by Kesavardhana Sannula, Assistant Professor in the Department of Biochemistry, Indian Institute of Science (IISc) have now discovered that the currently circulating 2.3.4.4b clade of H5N1 has specific mutations in its genome that increase its human adaptive potential. Clade represents a group of organisms having a common ancestor.

“The 2.3.4.4b clade has infected many mammalian species and is adapting to [non-human] mammals, which is a concern for human adaptation,” says Kesavardhana. “The clade is panzootic, causing unprecedented mortality in birds and mammals, along with several sporadic human infections.”

When the influenza virus enters a new organism, it can develop genetic mutations. This helps the virus adapt to the new host. The researchers were trying to decode whether the 2.3.4.4b clade was evolving to produce crucial adaptations in its proteins that allow it to infect humans. They also wanted to decipher which host animals can potentially accelerate this adaptation, giving the virus a leg up in scaling the evolutionary ladder.

Kesavardhana’s team took a computational approach and analysed 7,000 protein sequences of 2.3.4.4b H5N1 found in birds, 820 sequences from non-human mammals, and 35,000 human H1N1 and H3N2 sequences, in order to identify which amino acids are under selection pressure – rapidly changing. They used multiple sequence alignment (a tool to identify similar regions in multiple proteins), constructed phylogenetic trees (which represent how species have diverged from their common ancestor over time) and annotated specific variations in all the proteins of H5N1 infecting non-human mammals and humans.

The team found an increased number of mutations specifically in the viral polymerase complex (PA, PB2), nucleoproteins, and haemagglutinin (HA) proteins. Once they identified these mutations, the team classified them depending on whether the mutations can help the virus spread from non-human mammals to humans (adaptive) or simply survive in the non-human host (barrier). Finally, they developed a simple mathematical approach and estimated the human adaptive potential for the 2.3.4.4b clade.

The team was also able to pinpoint animals that would be likely to harbour virus strains with the highest human adaptive potential. Interestingly, viruses that can adapt to fox hosts seemed to have higher adaptive potential than cattle-adapted strains. “It is very surprising,” Kesavardhana says.

Based on their findings, the researchers suggest that enhanced and proactive surveillance measures need to be implemented.

“This clade is acquiring the same key mutations that pandemic human influenza strains possess, which could be a growing risk,” says Ranjana Nataraj, Project Associate at the Department of Biochemistry and the study’s first author.
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https://journals.asm.org/doi/10.1128/spectrum.00948-25