Bovine H5N1 influenza shows potential for human adaptation through key mutations

By Vijay Kumar MalesuReviewed by Lily Ramsey, LLMDec 9 2024

Study reveals how specific mutations in the H5N1 virus enhance its ability to bind human receptors, underscoring the need for ongoing surveillance of emerging strains.

Study: A single mutation in bovine influenza H5N1 hemagglutinin switches specificity to human receptors. Image Credit: RaffMaster/Shutterstock.com

In a recent study published in Science, a group of researchers analyzed receptor-binding adaptations in bovine H5N1 hemagglutinin (HA) linked to human infection and transmission potential.

Background

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in 1996, its spread across continents has led to diverse clades and significant cross-species infections. In late 2021, clade 2.3.4.4b appeared in North America, infecting avian species, mammals, and humans.

By 2024, outbreaks in United States (U.S.) dairy herds resulted in the first human case of bovine H5N1 infection, raising concerns about interspecies transmission.

Genetic analysis revealed potential HA receptor-binding adaptations facilitating human infections, though no human-to-human transmission has been reported. Historical evidence of receptor-binding shifts highlights the risk of pandemics, necessitating further research on H5N1 evolution globally.

About the study

The HA ectodomain sequence of the influenza A/Texas/37/2024 H5N1 virus was obtained from the Global Initiative on Sharing All Influenza Data (GISAID) and subcloned into the pFastbac-1 vector with a secreted signal peptide, trimerization domain, thrombin site, and His-tag.

Recombinant baculoviruses were generated using a Bac-to-Bac system in Sf9 cells. Soluble HA was expressed in High Five cells with a multiplicity of infection (MOI) of 5-10. The protein was purified via metal-affinity and size-exclusion chromatography, then digested with trypsin for use in structural studies and binding assays.

Crystallization utilized sitting-drop vapor diffusion with optimized conditions for apo-HA and mutant HA. Crystals were soaked with avian (Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LSTa)) or human (Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc (LSTc)) receptor analogs.

Diffraction data collected at synchrotron facilities were solved via molecular replacement and refined using PHENIX. Receptor specificity was evaluated using surface plasmon resonance (SPR), enzyme-linked immunosorbent assays (ELISA), and glycan microarrays.

Biotinylated glycans representing avian and human receptors were synthesized for assays, with binding affinities calculated using Biacore software.

Study results

The wild-type (WT) bovine Texas H5 HA exhibited a strong avian receptor preference in binding assays, as demonstrated by SPR, ELISA, and glycan array analyses.

The WT HA bound avidly to α2-3 sialosides (avian-type receptors) with a dissociation constant (K_D) of 138 nM but showed no detectable binding to α2-6 sialosides (human-type receptors). These results were consistent across all methods, confirming the avian-type specificity of the Texas H5 HA.

A Gln226Leu mutation in the receptor-binding site (RBS) of Texas H5 HA caused a complete switch in specificity from α2-3 to α2-6 sialosides, reflecting a shift to human receptor preference. The Gln226Leu mutant demonstrated stronger binding to biantennary α2-6 sialosides compared to linear forms. Additionally, this mutant exhibited a greater affinity for α2-6 sialosides than the HA of the 2009 H1N1 pandemic strain, highlighting its enhanced specificity for human-type receptors.

Structural analyses revealed that the Gln226Leu mutation positioned leucine to form van der Waals interactions with α2-6 receptors, providing a molecular basis for the specificity switch.

Further mutations were introduced to explore their effects on receptor binding. The addition of Gly228Ser to the Gln226Leu mutant, mimicking human H2 and H3 virus adaptations, did not significantly alter receptor specificity beyond that of the Gln226Leu mutation alone.

Mutations such as Gly225Asp and Glu190Asp, which are hallmark residues in H1 human receptor-binding transitions, failed to induce a similar switch in the Texas H5 HA.

Combining Asn224Lys with Gln226Leu further enhanced binding to α2-6 sialosides, particularly in extended and biantennary forms, while introducing only weak binding to α2-3 sialosides.

However, other mutations, including those targeting positions 193 and 227, did not significantly improve human receptor binding when combined with Gln226Leu. Notably, these mutants generally retained strong binding to biantennary glycans.

Crystal structures of Texas H5 HA in complex with avian and human receptor analogs provided detailed insights into the molecular interactions underlying receptor specificity. The WT HA complex with LSTa revealed hydrogen bonding with Gln226, critical for avian receptor preference.

In contrast, the Leu226 mutant complex with LSTc showed altered interactions, including new contacts mediated by Asn193 and Lys156, aligning with its specificity for human-type receptors.

Conclusions

To summarize, avian influenza H5N1 viruses significantly affect poultry production and pose a pandemic threat through sporadic infections in humans and mammals. Since 2021, clade 2.3.4.4b H5 viruses have spread widely, infecting wild birds, poultry, dairy cows, and various mammals, including humans.

Despite the predominance of avian-type receptor specificity in bovine H5 HA, a study demonstrated that a single Gln226Leu mutation can switch receptor binding to human-type receptors.

While weak, this binding mirrors the affinity of some transmissible human influenza viruses, such as the 2009 H1N1 pandemic strain. Additional mutations, like Asn224Lys, enhance binding to human receptors, raising concerns about zoonotic adaptation.