Periodic Reporting for period 2 - Bat Flu (Deciphering the unconventional receptor binding and modulation activity of bat influenza A viruses)
Período documentado: 2022-05-01 hasta 2023-10-31
In recent years, bats received increasing attention as hosts for several emerging zoonotic viruses, including Marburg, Ebola, SARS and MERS coronaviruses. Paradoxically, however, bats have long been neglected as a potential reservoir for influenza A viruses (IAVs). Extensive surveillance studies in 2009 and 2010 identified novel IAV genomes of the subtypes H17N10 and H18N11 in Central and South American fruit bats. Despite a high degree of functional and structural homology to previously known conventional IAVs, these bat-derived IAV subtypes demonstrate several unprecedented characteristics. For example, unlike conventional IAVs, which infect cells by using canonical sialic acid receptors, bat-derived IAVs utilize major histocompatibility complex class II (MHC-II) molecules for cell entry. Furthermore, we observed that N11 downregulates surface expression of MHC-II, suggesting that it potentially harbors a receptor-destroying function. Most surprisingly, bat IAV could replicate to even higher titers in the absence of functional NA, a capability that has never been observed among influenza viruses. We therefore hypothesize that the surface glycoproteins of bat IAVs possess a structural plasticity that is much broader than that of conventional IAVs. In light of the critical importance of the surface proteins for cross-species transmission of IAV, the goal of this ERC-funded Project BatFlu is to probe this plasticity, first by determining the mode of interaction between H17/H18 and MHC-II and elucidating the mechanism of N10/N11-dependent downregulation of MHC-II. The insights from these studies will have a major impact on our understanding of influenza virus tropism and the ability of bat-IAVs to spill-over into the human population.
The characterization of the virus binding site within MHC-II had been complicated by the fact that classical biochemical approaches failed to visualize the interaction. We pursued a different approach generating chimeras between the human MHC-II HLA-DR, which functions as a viral receptor and the non-classical human MHC-II HLA-DM, which does not promote viral entry. With this method, we could identify amino acids that are essential for viral infection. Importantly, these amino acids are highly conserved among diverse mammalian species, explaining the broad range of MHC-II molecules that can serve as viral entry receptor.
In order to establish a robust binding assay for the viral hemagglutinin and MHC-II, which might pave the ground for the resolution of this interaction by crystallography or Cryo-EM, we expressed and purified high-quality H18 and MHC-II, respectively, and can now show a direct interaction of these two complexes by two independent biochemical binding methods.
We speculated that bat-derived IAV would induce clustering of MHC-II molecules on the host cell surface to provide sufficient avidity for viral entry. In order to challenge this hypothesis, we generated MHC-II molecules, which are labeled with a photoswitchable fluorophore. Using these labeled MHC-II molecules we can now show by high resolution microscopy that indeed the attachment of bad-derived IAV triggers MHC-II clustering and subsequent uptake.
In order to establish a robust binding assay for the viral hemagglutinin and MHC-II, which might pave the ground for the resolution of this interaction by crystallography or Cryo-EM, we expressed and purified high-quality H18 and MHC-II, respectively, and can now show a direct interaction of these two complexes by two independent biochemical binding methods.
We speculated that bat-derived IAV would induce clustering of MHC-II molecules on the host cell surface to provide sufficient avidity for viral entry. In order to challenge this hypothesis, we generated MHC-II molecules, which are labeled with a photoswitchable fluorophore. Using these labeled MHC-II molecules we can now show by high resolution microscopy that indeed the attachment of bad-derived IAV triggers MHC-II clustering and subsequent uptake.
Although we previously demonstrated that the bat-derived IAVs H17N10 and H18N11 utilize major histocompatibility complex class II (MHC-II) molecules for cell entry, the cellular tropism, i.e. the target cells within an infected organism, remained unclear. This is important to know since functional studies of N11 seem to be cell type dependent. As part of the “ERC Advanced Grant BatFLu”, we performed cutting-edge single-cell RNA sequencing of organs from H18N11-infected Jamaican fruit bats, the natural reservoir of bat IAVs. Single-cell RNA sequencing is a relatively new next-generation sequencing approach that allows antibody-independent characterization of cell types and the study of biological processes in heterogeneous cell populations. As a result, we were able to generate, for the first time, a comprehensive single-cell atlas of the Jamaican fruit bat intestine and mesentery, the target organs of H18N11. Specifically, we identified intestinal and mesenteric leukocytes as the major cell type in which H18N11 is replicating and studied the imposed immune response to infection. With this study we provide fundamental resources for studying various aspects of Jamaican fruit bats innnate and adaptive immune responses and lay the foundation for comparative immunology studies with other species.
Following the discovery of H17N10 and H18N11 in New World bat species, a distinct H9N2 IAV was isolated from Egyptian fruit bats in the Nile Delta region. In contrast to the New World bat IAVs, the bat H9N2 shares many characteristics of conventional IAVs, including the sialic acid receptor specificity required for cell entry. To address whether bat H9N2 is of zoonotic concern, we, together with our collaboration partners evaluated (i) the infection and transmission potential in ferrets, a commonly used animal model to mirror infection of humans, (ii) the ability to replicate in human lung explants, (iii) the escape from human MxA, a crucial innate antiviral factor for zoonotic IAVs, and (iv) antigenic novelty to the human population. This study demonstrates that bat H9N2 meets key characteristics of pre-pandemic IAVs and is available as a preprint (https://www.researchgate.net/publication/370972286_The_bat-borne_influenza_A_virus_H9N2_exhibits_a_set_of_unexpected_pre-pandemic_features).
This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 882631).
Following the discovery of H17N10 and H18N11 in New World bat species, a distinct H9N2 IAV was isolated from Egyptian fruit bats in the Nile Delta region. In contrast to the New World bat IAVs, the bat H9N2 shares many characteristics of conventional IAVs, including the sialic acid receptor specificity required for cell entry. To address whether bat H9N2 is of zoonotic concern, we, together with our collaboration partners evaluated (i) the infection and transmission potential in ferrets, a commonly used animal model to mirror infection of humans, (ii) the ability to replicate in human lung explants, (iii) the escape from human MxA, a crucial innate antiviral factor for zoonotic IAVs, and (iv) antigenic novelty to the human population. This study demonstrates that bat H9N2 meets key characteristics of pre-pandemic IAVs and is available as a preprint (https://www.researchgate.net/publication/370972286_The_bat-borne_influenza_A_virus_H9N2_exhibits_a_set_of_unexpected_pre-pandemic_features).
This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 882631).