In 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA resulting in spillbacks into poultry, wild birds and other mammals including humans. 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 mammals including humans. We find evidence of several mammalian adaptations 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. We show that ongoing evolution 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, circulation of H5N1 in dairy cattle allows virus adaption improving replicative ability in cattle and poses a continued risk of zoonotic spillover.
The emergence and sustained spread of H5N1 in US dairy cattle since 2024 have demonstrated that highly pathogenic avian influenza (HPAI) is capable of establishing long-term transmission in livestock. Genomic surveillance has clarified national patterns of spatial diffusion, interspecies transmission, and viral evolution, but critical data gaps remain that impede efforts to track virus movements in real time, identify modes of transmission, and inform control efforts.
Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance due to end-joining repair is a significant challenge to the deployment of these systems. The use of multiple sgRNAs has the potential to solve this issue. Here we perform experimental crosses to assess the homing and cutting efficiency of both classical (e.g. four adjacent sgRNAs all in one construct) and additive (e.g. separate constructs each expressing a single sgRNA) multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We find resistance alleles at one sgRNA target site can be mitigated by the presence of the additional sgRNAs with either strategy, and do not significantly reduce the homing efficiency for either strategy, validating their effectiveness. Further modelling using parameters derived from the strains generated indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.
BACKGROUND:Seasonal influenza causes significant morbidity and mortality annually. In 2025, the genetically divergent A/H3N2 K subclade (J.2.4.1) emerged with substantial haemagglutinin mutations. However, despite suggested antigenic escape, UK vaccine effectiveness estimates and epidemiological data demonstrated a relatively normal influenza season across 2025-26. We examined neutralising antibody responses in human cohorts to investigate existing and vaccine-induced immunity to K clade viruses. METHODS:We characterised the antigenic relationships of a selection of A/H3N2 viruses spanning recent evolution including a subclade K virus using antigenic cartography, followed by serological antibody profiling of four human cohorts from the United Kingdom and Norway using microneutralisation (MN) and haemagglutination inhibition (HAI) assays. FINDINGS:Antigenic cartography from single-infection ferret antisera suggests significant antigenic drift from the vaccine strains. MN and HAI titres from 243 individuals across 4 human cohorts (ages 1-105 years) were measured for comparison. The 2025/26 Northern Hemisphere seasonal inactivated egg-derived trivalent influenza vaccine (eTIV, with J.2 A/H3N2) significantly boosted MN and HAI titres against all A/H3N2 viruses tested, including a subclade K virus (p < 0.001). Furthermore, serological profiles of cohorts stratified by age groups (≤5, >5-≤15, >20-≤25, >25-<60, and ≥60) showed pre-existing reactivity against the emergent subclade K viruses, with minimal inter-age variation, suggesting there was not an immunity gap within particular age groups. INTERPRETATION:The 2025/26 seasonal inactivated eTIV vaccine effectively boosted neutralising titres despite substantial genetic and antigenic drift. Human serological profiling should be included in risk assessments and continued surveillance. FUNDING:The Francis Crick Institute with core funding from Cancer Research UK, UK Medical Research Council, and Wellcome Trust; UK Research and Innovation and UK Medical Research Council; National Institute for Health Research University College London Hospitals Biomedical Research Centre; UK Health Security Agency; Norwegian Institute of Public Health.
A standard method in phylogenetic reconstruction for representing variation in substitution rates between sites in the genome is the discrete Gamma model (DGM). Relative rates are assumed to be distributed according to a discretised Gamma distribution, where the probabilities that a site is included in each class are equal. Here, we identify a serious bias in the branch lengths of reconstructed phylogenies when the DGM is used, with the magnitude of the effect varying with the number of sequences in the alignment. We demonstrate the existence of the bias, using both simulated datasets and real HIV-1 sequences; in both cases branch lengths are overestimated. The phenomenon is exacerbated by increasing the number of discrete rate categories, is only very slightly mitigated by the addition of an invariant sites category, and happens regardless of the software package used for reconstruction. We show that the alternative "FreeRate" model, which assumes no parametric distribution and allows the class probabilities to vary, is not subject to the issue. We further establish that the reason for the behaviour is the equal size of the class probabilities in the discretisation, not simply the fact that a continuous distribution has been discretised. We explore the mathematics of the phenomenon, showing how maximum likelihood branch lengths under the DGM may differ from the true ones used to generate the tree, and how the magnitude of this difference is equal to the departure of the mean maximum likelihood substitution rate across all sites in the genome from 1. We recommend that the DGM be retired from general use. While FreeRate is an immediately available replacement, it is known to be difficult to fit, and thus there is scope for innovation in rate heterogeneity models.
