Research Projects

Continuous surveillance of H5N1 virus in avian, cattle and other species is necessary to track the potential of the virus to acquire human-type receptor specificity that would increase the human health risk and its emergence as a pandemic virus. In the spring of 2024, an unprecedented outbreak of highly pathogenic avian influenza H5N1 (HPAI H5N1) virus has occurred into cattle [1, 2]. This virus can spread within cattle herds, cause infections in poultry and cats, and can spill over into humans, collectively raising concern on public health risk worldwide [2,3]. This HPAI H5N1 possesses features that may facilitate infection and transmission among mammals. The HPAI H5N1 viruses isolated from cattle are phylogenetically related to H5N1 viruses from wild birds [4,5]. These viruses belong to the HA clade 2.3.4.4b and were recently found in late 2021. Given the high pathogenicity, ease of air borne transmissibility and lack of vaccines against this HPAI H5N1, raised alarm for potential pandemic of an influenza virus [6, 7]. Therefore, understanding this HPAI H5N1 influenza virus cellular entry mechanism is outmost important to fast forward vaccine developments and antiviral treatments. The HPAI H5N1 entry is mediated by the mushroom like trimeric envelope hemagglutinin (HA) glycoprotein [8]. HA is comprised of two functional subunits responsible for host receptor binding (HA1 subunit) and fusion between the viral and host cellular membrane (HA2 subunit) [9-10]. Canonical avian influenza A viruses preferentially bind to α2,3-linked sialic acids, whereas human influenza A viruses preferentially bind to the α2,6-linked sialic acids. Strikingly, The HPAI-H5N1 virus has been able to infect cattle and human, indicating that the this H5N1 virus may have the ability to bind both the α2,3 and α2,6-linked sialic acids to cells in the upper respiratory tract of humans [1]. This dual receptor binding specificity of HPAI-H5N1 delineates a unique feature to this emerging H5N1 virus [1-4]. The functional characteristics of hemagglutinin (HA) glycoprotein of HPAI H5N1 viruses for its high infectivity is currently unknown. What is the receptor specificity and conformational landscape spectrum of the novel H5N1 HA. What is the structural flexibility of the receptor binding domains (RBD) of HA1 subunit and how the conformational landscape changes upon HA1 domain binding with α2,6 linked sialic acids compared to the binding with α2,3 linked sialic acids is currently unknown. How the HA1 conformational change propagates among neighbouring HA1 subunits in a single HA trimer of HPAI H5N1. Still less is understood about how the receptor mediated HA1 uncaging dynamics are coupled to the triggering of large-scale conformational dynamics of fusion machine HA2 domain for mediating membrane fusion for viral entry. Finally, if any broadly neutralizing influenza antibody (bNab) can neutralize the H5N1 HA mediated infectivity, is currently unknown. Hence, a detailed knowledge of the receptor specificity, conformational landscape of HA, driving fusion is lacking, which is required to define the accessibility of epitopes targeted by neutralizing antibodies for engineering improved vaccine candidates. To address this knowledge gap, we will establish array of combinatorial biochemical assays and single molecule fluorescence resonance energy transfer (smFRET) imaging assay set up, to directly visualize the HA glycoprotein dynamics on the HPAI H5N1 virion surface during membrane fusion for cellular entry in real time. Our single molecule imaging techniques will provide unprecedented details of HA protein structural dynamics for its receptor specificity and the mechanism of cellular entry of HPAI H5N1 viruses. Our smFRET imaging will capture the prefusion intermediates structure of HA on pathway to the fusion. These prefusion HA intermediates will be very important for rational stem-helix based immunogen and antibody design against HPAI H5N1 influenza viruses.