The project combined experimental virology, cell biology, computational analyses, and viral evolution to systematically study how animal viruses enter human cells. To do this safely and at large scale, the project used viral pseudotypes, which are harmless viruses engineered to display only the surface proteins, or receptor-binding proteins (RBPs), of animal viruses. These pseudotypes cannot replicate or cause disease, but they allow researchers to test whether a given animal viral RBP can mediate entry into a human cell. Using this approach, RBPs from more than one hundred wildlife viruses were tested across a diverse panel of over fifty human cell types, generating a large functional map of virus–cell entry compatibility. This unprecedented dataset enabled direct comparison of human cell entry capacity across viral families and revealed substantial variation in entry breadth between groups of viruses. For example, RBPs from arenaviruses and hantaviruses were often able to enter a wide range of human cell types, suggesting broad compatibility with human entry factors. In contrast, most coronaviruses showed much narrower entry profiles, with only a few capable of entering specific human cells. Statistical and phylogenetic analyses were used to identify viral traits associated with human cell entry, while machine-learning approaches were developed to predict RBP-mediated infectivity patterns based on viral and cellular features.
Building on this large functional screen, the project next focused on identifying the human cellular factors that determine whether animal viruses can enter human cells. This was achieved by combining infectivity data with comprehensive gene expression profiles of the human cell lines, allowing systematic identification of cellular proteins that promote or restrict viral entry. This approach confirmed the role of some known viral receptors but also revealed that, in many cases, entry could not be explained by previously described receptors alone, suggesting the importance of alternative entry factors or restriction mechanisms. Using targeted genetic and functional experiments, the project validated several of these factors and led to the discovery of a previously unknown cellular receptor used by the porcine coronavirus PHEV (porcine haemagglutinating encephalomyelitis virus). This receptor, dipeptidase 1 (DPEP1), was characterised in depth through complementary genetic, biochemical, structural, and virological analyses, providing a detailed molecular understanding of how this virus attaches to and enters its target cells. Interestingly, DPEP1 was used specifically by PHEV and other related viruses, such as the human coronavirus OC43, could not use DPEP1 as a receptor. In parallel, the project systematically investigated the role of widely shared cellular components, such as glycans present on the surface of human cells. These analyses revealed a dual role for such molecules: some, including specific sulfated sugars, act as essential entry factors for certain virus families, while others, such as sialic acids, can inhibit the entry of some viruses and may therefore function as natural protective barriers.
Finally, the project used experimental evolution to examine how selected animal viruses adapt to human cells over time. By repeatedly propagating viruses in human cells, the project identified specific viral mutations that increased entry efficiency or enabled viruses to overcome cellular restrictions. Together, these results provide direct experimental evidence that animal viruses can rapidly evolve improved compatibility with human cells, illuminating concrete evolutionary pathways through which zoonotic potential can emerge.
