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Emerging Microbes & InfectionsMicrobiologyPeer-reviewed journal article

The Haemagglutinin Gene of Bovine Origin H5N1 Influenza Viruses Currently Retains an Avian Influenza Virus phenotype.

Jiayun YangiD, Mehnaz QureshiiD, Reddy Kolli, Thomas P. PeacockiD, Jean-Rémy SadeyeniD, Toby CarteriD, Samuel RichardsoniD, Rebecca DainesiD, William BarclayiD, Ian Hay BrowniD, Munir IqbaliD

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Abstract

Clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) has caused a panzootic affecting all continents except Australia, expanding its host range to several mammalian species. In March 2024, H5N1 HPAIV was first detected in dairy cattle and goats in the United States. Since then, over 230 dairy farms across 14 states have tested positive, with zoonotic infections reported among dairy workers. This raises concerns about the virus undergoing evolutionary changes in cattle that could enhance its zoonotic potential. The Influenza glycoprotein haemagglutinin (HA) facilitates entry into host cells through receptor binding and pH-induced fusion with cellular membranes. Adaptive changes in HA modulate virus-host cell interactions. This study compared the HA genes of cattle and goat H5N1 viruses with the dominant avian-origin clade 2.3.4.4b H5N1 in the United Kingdom, focusing on receptor binding, pH fusion, and thermostability. All the tested H5N1 viruses showed binding exclusively to avian-like receptors, with a pH fusion of 5.9, outside the pH range associated with efficient human airborne transmissibility (pH 5.0 to 5.5). We further investigated the impact of emerging HA substitutions seen in the ongoing cattle outbreaks, but saw little phenotypic difference, with continued exclusive binding to avian-like receptor analogues and pHs of fusion above 5.8. This suggests that the HA genes from the cattle and goat outbreaks do not pose an enhanced threat compared to circulating avian viruses. However, given the rapid evolution of H5 viruses, continuous monitoring and updated risk assessments remain essential to understanding virus zoonotic and pandemic risks.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (27 Sept 2024) · CC BY 4.0

Haemagglutinin of US cattle and goat H5N1 binds only avian-type receptors and fuses at pH 5.9; seven emerging cattle mutations did not switch receptor preference.

Clade 2.3.4.4b H5N1 is now circulating in dairy cattle, raising concern that HA could adapt toward human receptor binding or a lower fusion pH. This work profiles cattle and goat HAs and seven emerging cattle mutants against UK avian HAs for receptor binding, fusion pH and thermostability.

Key results

  1. ShownCattle (Texas) and goat (Minnesota) HAs bound the avian receptor analogue 3SLN only, with no 6SLN binding, matching two UK chicken-origin clade 2.3.4.4b HAs; the goat HA bound 3SLN more strongly than a UK HA.

    Bio-layer interferometry: TX-Cattle, MN-Goat, Sct477/21 and Eng598/22 bound 3SLN only. Controls: Eng195/09 bound 6SLN only; AH1/13 bound both. MN-Goat had 17-fold stronger 3SLN binding than Sct477/21. · HA genes from cattle and goat H5N1 viruses…; Figure legends

  2. ShownCattle and goat HAs triggered fusion at pH 5.9, slightly higher than the UK viruses at 5.8, and all fall above the pH 5.0 to 5.5 range linked to human airborne transmissibility. The goat HA was the most heat stable.

    Syncytium assay: pH 5.9 (TX-Cattle, MN-Goat) vs 5.8 (Sct477/21, Eng598/22); human airborne range is pH 5.0 to 5.5. HAU unchanged at 50°C, abolished at 56.1°C. At 51.9°C, MN-Goat retained roughly 4 times the HAU of TX-Cattle and Eng598/22 (three independent experiments). · Abstract; HA genes from cattle and goat H5N1 viruses…; Figure legends

  3. ShownNone of seven HA head-domain mutations emerging in cattle sequences (single changes in the TX-Cattle HA) produced 6SLN binding, though avian-receptor affinity shifted widely.

    Seven mutations were selected from 490 sequences. All mutants bound 3SLN only. Versus WT TX-Cattle, 3SLN binding rose 6-, 28-, 4- and 84-fold (T143A, D171N, I178V, Q234R) and fell 3-, 199- and 5-fold (Q154L, A172T, P174Q). · The emerging HA mutants from cattle H5N1 HPAIV…; Figure legends

  4. ShownMutant fusion pH stayed at or above 5.8, and three mutants were less thermostable than WT.

    Six mutants (T143A, D171N, A172T, P174Q, I178V, Q234R) fused at pH 5.9 like WT TX-Cattle; Q154L fused at pH 5.8. A172T, P174Q and I178V had lower HAU than WT at 50.7°C and 51.9°C; the other four matched WT. · The emerging HA mutants retained a fusion pH…; Discussion

  5. SuggestedCattle and goat HAs were judged antigenically similar to UK viruses using chicken antisera, though HI titres dropped against the heterologous viruses.

    HI titres of TX-Cattle and MN-Goat were reduced by about 3 log2 relative to homologous titres against Sct477/21 and Eng598/22 antisera. Virus neutralisation titres in MDCK cells showed no significant change. · HA genes from cattle and goat H5N1 viruses…; Figure legends

Methods, data and reproducibility

Reverse-genetics viruses carrying test HAs (monobasic cleavage site, PR8 internal genes) were compared by bio-layer interferometry for 3SLN/6SLN binding, syncytium formation for fusion pH, heat-treated haemagglutination for thermostability, and HI/VNT for antigenicity.

  • HA/NA sequences of TX-Cattle, MN-Goat, Sct477/21 and Eng598/22 H5N1 viruses (sequence details in Table S1) · Influenza A virus H5N1 (cattle, goat, chicken origin) · n = 4 test viruses
  • Bovine H5N1 sequences used to pick seven emerging HA mutations · Influenza A virus H5N1 (dairy cattle, USA) · n = 490 sequences
  • Assay control viruses: pandemic 2009 H1N1 Eng195/09 and zoonotic H7N9 AH1/13 · Influenza A virus (H1N1, H7N9) · n = 2 viruses
  • H5 HA crystal structure used for HA structure modelling and visualisation · Influenza A virus H5 · PDB 4JUL
  • Chicken antisera against Sct477/21 and Eng598/22 for HI and VNT · Chicken (Gallus gallus)
Methods & tools
Bio-layer interferometry (Octet R8), 3SLN and 6SLN receptor analogues, Reverse genetics with PR8 backbone, QuikChange II site-directed mutagenesis, Vero syncytium formation assay, Thermostability haemagglutination assay, Haemagglutination inhibition assay, Virus neutralisation test (MDCK), SWISS-MODEL, PyMol, GraphPad Prism 10.0, GISAID
Data & accessions
https://www.rcsb.org/
Reagents & environment
GraphPad Prism 10.0 for statistics; PyMol version 4.6 for structure visualisation; SWISS-MODEL for HA modelling.
Reproducibility gaps
No analysis code is stated. HA sequences come from GISAID (submitters in Table S1) with no accessions printed; the 490 bovine sequences are only attributed to the Sequence Read Archive generally.
Limitations (4)
  • The authors state that it is unresolved whether further HA mutations will emerge as the virus keeps spreading among dairy cattle and adapting to bovine hosts. · stated by the authors
  • The authors note that a New Mexico cattle study reported dual receptor binding by solid-phase ELISA, and suggest the discrepancy with their data could reflect methodological differences. · stated by the authors
  • The panel used reverse-genetics viruses with PR8 internal genes and a monobasic cleavage site, tested in vitro with simple 3SLN/6SLN analogues. Mutants were tested singly in one TX-Cattle HA, so combinations, other genes and in vivo transmission are untested. · noted in this summary
  • Fusion pH was read as the highest pH showing syncytia in 0.1 pH steps, with no replicate statistics shown for it. Receptor-binding replicates are not described, and the HI and VNT antigenicity results rest on antisera against two UK viruses. · noted in this summary

Read this if you assess H5N1 zoonotic risk and want receptor-binding, fusion pH and thermostability data for bovine and goat HAs and their emerging mutants.

Citations
38
FWCI
23.40
Field percentile
top 1%

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