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Nature CommunicationsMicrobiologyPeer-reviewed journal article

Polymerase mutations underlie early adaptation of H5N1 influenza virus to dairy cattle and other mammals.

Vidhi DholakiaiD, Jessica L. QuantrilliD, Samuel A. S. RichardsoniD, Nunticha PankaewiD, Maryn D. BrowniD, Jiayun YangiD, Fernando CapelasteguiiD, Tereza MasonouiD, Katie‐Marie CaseiD, Jila Ajeian, Maximillian N. J. WoodalliD, Callum MagilliD, Graham FreimanisiD, Amy Mccarron, Ecco StalleriD, Carol SheppardiD, Ian Hay BrowniD, Pablo R. MurciaiD, Claire Mary SmithiD, Munir IqbaliD, Paul DigardiD, William BarclayiD, Rute Maria PintoiD, Thomas P. PeacockiD, Daniel H. GoldhilliD

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Abstract

In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA1. As of mid-2025 the epidemic is ongoing, resulting in spillbacks into poultry, wild birds and other mammals including humans2. Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells and tissues, as well as cells of other mammalian species including humans and pigs. We find evidence of several mammalian adaptations gained early in the evolution of these viruses in cattle including PB2 M631L, which is found in all cattle sequences, and PA K497R, which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication. We show that this mutation adapts the polymerase to better interact with bovine ANP32 proteins, particularly ANP32A, and thereby enhances virus replication in bovine mammary systems and primary human airway cultures. Importantly, we show that ongoing evolution during 2024 and 2025 in the PB2 gene, including E627K and a convergently arising D740N substitution, further increase polymerase activity and virus replication in a range of mammalian cells. Thus, the continued circulation of H5N1 in dairy cattle not only allows virus adaption improving replicative ability in cattle, but also increases the risk of zoonotic spillover.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (14 Aug 2025) · CC BY 4.0

Cattle B3.13 H5N1 polymerase gained PB2 M631L and PA K497R, boosting replication in bovine and human cells via better bovine ANP32A use; later PB2 E627K and D740N add activity.

Avian influenza viruses usually replicate poorly in mammals, and the classic PB2 adaptations (E627K, Q591R/K, D701N) were absent from the initial cattle viruses. The paper identifies which polymerase changes did enable efficient replication in cattle and tracks further changes arising during the ongoing outbreak.

Key results

  1. ShownAll cattle viruses form one monophyletic group and share PB2 M631L, E362G and PA L219I. PA K497R marks the largest clade, and the first human case carried different mutations (PB2 E627K, PA K142E).

    Concatenated-protein phylogeny of n=902 sequences (GISAID, to 1 Dec 2024); the K497R clade is about 95% of cattle sequences as of December 2024. · Phylogenetic analysis of the cattle H5N1…; Phylogenetics

  2. ShownIn bovine minireplicon assays, PB2 M631L is the main driver of polymerase activity, and PA K497R adds a smaller, cumulative effect. Both have minimal effect in chicken DF-1 cells.

    Removing M631L and K497R lowered activity about 100-fold; adding back M631L alone gave about 50-fold and K497R alone about 10-fold. DF-1 effects were under 2-fold. Mean of N=3 independent repeats. · Activity of bovine H5N1 polymerase in mammalian…; Figure legends

  3. ShownIn infectious virus, M631L alone or with K497R raises replication in bovine explants and cells and in human lung lines. In primary human nasal cultures the precursor (no M631L or K497R) grew worse than wild type. Wild type (with K497R) beats M631L alone; avian cells are unaffected.

    Over 10-fold higher titres at 24 and 48 h in bovine mammary explants (3 donor cattle) vs the minimal avian-like precursor; wild type beat M631L alone at 48 and 72 h. In primary human nasal cultures from 3 donors, the precursor virus had lower titres than wild type at 72 h. · PB2 M631L, and to a lesser extent PA K497R…; Figure legends

  4. ShownM631L adapts the polymerase to bovine ANP32 proteins, with bovine ANP32A used better than ANP32B. A split-luciferase assay shows stronger polymerase binding to ANP32A, consistent with the functional preference.

    In ANP32A/B/E triple-knockout human eHAP cells, avian polymerases were supported only by chicken ANP32A, while cattle/Texas used bovine and human ANP32A/B. Bovine ANP32A beat ANP32B for all polymerases tested; split-luciferase N=4. · Mutations in the polymerase of the cattle H5N1…; Figure legends

  5. ShownLater PB2 changes increase mammalian polymerase activity without helping in avian cells. E627K on top of M631L is strongest. D740N, which arose independently at least three times, gives a smaller and less consistent gain.

    D740N raised minireplicon signal about 4-fold in bovine, about 2-fold in human and 1.2-fold in swine cells. In primary nasal cultures from 3 donors, D740N varied between donors and was not significantly different from wild type. · More recent mutations in cattle sublineages…; Discussion

Methods, data and reproducibility

Cattle H5N1 polymerase mutations were identified by phylogenetics of public sequences, then tested by reverting or adding them in minireplicon reporter assays and in recombinant viruses. Replication was measured in cell lines, bovine explants and primary human nasal cultures; ANP32 usage was tested with knockout-cell complementation and split-luciferase binding.

  • GISAID influenza sequences (cattle, US wild avian Sep 2023 to Mar 2024, and the first human case) used for the phylogeny · Influenza A virus (H5N1) · n = n=902 sequences in the cleaned alignment · List of GISAID accessions is in the GitHub repository
  • Bovine ex vivo mammary gland explants · cattle · n = 3 donor cattle, 3 independent experiments
  • Primary human nasal epithelial cultures at air-liquid interface · human · n = 3 adult donors
  • Cell-line minireplicon and infection assays (MAC-T, BAT II, 293T, A549, MRC5, Calu-3, ST, DF-1, CLEC213, CCL-141) · cattle, human, swine, chicken, duck · n = N=3 independent repeats (minireplicons); curves from one representative of N=3
  • Whole-genome sequencing of virus after replication in nasal cultures · Influenza A virus (H5N1) · n = 2 donors, 72 h
  • Structure used to map adaptive mutations (replicase dimer with human ANP32B) · Influenza A virus with human ANP32B · PDB 8R1J
Methods & tools
MAFFT v7.490, pytrimal, IQ-TREE2 (ModelFinder, FLAVI+F+I+R2), GISAID, Geneious Prime, Dual-luciferase minireplicon assay, Split Gaussia luciferase assay, Reverse genetics (PR8 2:6 reassortants and full H5N1), PDB 8R1J, Python 3.12.2, R 4.4.1
Software & environment
MAFFT v7.490, IQ-TREE2 with the FLAVI+F+I+R2 model, Python 3.12.2, R 4.4.1.
Reproducibility gaps
Sequences come from GISAID; the accession list is in the GitHub repository. The github.com/Flu1/bovinePolI link holds the bovine PolI reporter plasmid map. No raw data repository is named for the assay measurements.
Limitations (4)
  • How PB2 M631L enables use of mammalian ANP32 proteins is unresolved, although the data suggest enhanced interaction. · stated by the authors
  • Why M631L rather than the stronger E627K emerged in cattle is unclear. D740N effects were variable and subtle, and not consistently captured by the assays. · stated by the authors
  • All evidence comes from cell lines, explants, primary cultures and reporter assays. No in vivo infection or transmission experiments are reported, so effects on cattle infection or human risk are inferred. · noted in this summary
  • Several infection curves come from one representative repeat with technical replicates. Many comparisons are against engineered reversion mutants, and some conclusions rest on non-significant trends. · noted in this summary

Read this if you track H5N1 mammalian adaptation and want reconstructed-ancestor evidence that PB2 M631L and PA K497R drove cattle replication through ANP32A.

Citations
47
FWCI
128.10
Field percentile
top 1%

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