Periodic Reporting for period 1 - EmerVir (Experimental assessment of wildlife viruses emergence potential through systematic characterization of human cell tropism)
Période du rapport: 2023-11-15 au 2025-11-14
Emerging viral diseases such as COVID-19, Ebola, Zika, or avian influenza have demonstrated the profound health, societal, and economic impacts that viruses can have. While large-scale sequencing efforts continue to uncover thousands of viruses circulating in wildlife, a critical gap remains: for most of these viruses, it is unknown whether they can infect human cells at all and, if they can, which mechanisms and host factors enable replication in human cells. This lack of functional information severely limits our capacity to assess zoonotic risk, prioritise surveillance efforts, and design early intervention strategies. Among the viruses described in wildlife, enveloped RNA viruses represent a particularly high threat to humans, as the majority of known human-infective viruses belong to this group. Viral entry into host cells is a decisive early step in infection and a key determinant of host range, yet it remains poorly characterised for the vast majority of animal viruses. The overall objective of the EmerVir project was therefore to establish a systematic, high-throughput experimental framework to evaluate the ability of wildlife enveloped RNA viruses to enter human cells, identify the cellular factors that govern this process, and determine how viral evolution can modulate human cell tropism. By generating large-scale functional data across multiple viral families and human cell types, the project aimed to provide new conceptual and practical tools for pandemic preparedness.
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.
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.
The EmerVir project substantially advanced the state of the art in virology by providing the first large-scale functional assessment of the ability of wildlife viruses to enter human cells. A key outcome is the demonstration that, for many animal viruses, entry into human cells is not a major barrier to zoonotic transmission, challenging a long-standing assumption in the field. This finding indicates that later stages of infection, such as viral replication, immune evasion, and transmission, may play a more decisive role in determining whether animal viruses can successfully infect humans. In addition, the project revealed a remarkable diversity of cellular entry strategies, including the identification of a previously unknown receptor for a porcine coronavirus and the demonstration that closely related viruses can rely on distinct host factors to infect cells. These results highlight the evolutionary plasticity of virus-host interactions at the entry level and underscore the limitations of predicting zoonotic risk based solely on viral sequence similarity.
Beyond fundamental insights, the project delivers tangible tools and resources with broad applicability. The high-throughput experimental platform, large infectivity datasets, and predictive machine-learning models developed during the project provide a robust foundation for prioritising newly discovered viruses for surveillance and experimental follow-up. The identification of host entry factors and natural barriers to infection opens new avenues for antiviral research and host-targeted intervention strategies. Finally, experimental evolution experiments demonstrate that animal viruses can rapidly acquire mutations that enhance compatibility with human cells, offering concrete examples of evolutionary pathways through which zoonotic potential may emerge and providing valuable information for wildlife surveillance efforts.
Beyond fundamental insights, the project delivers tangible tools and resources with broad applicability. The high-throughput experimental platform, large infectivity datasets, and predictive machine-learning models developed during the project provide a robust foundation for prioritising newly discovered viruses for surveillance and experimental follow-up. The identification of host entry factors and natural barriers to infection opens new avenues for antiviral research and host-targeted intervention strategies. Finally, experimental evolution experiments demonstrate that animal viruses can rapidly acquire mutations that enhance compatibility with human cells, offering concrete examples of evolutionary pathways through which zoonotic potential may emerge and providing valuable information for wildlife surveillance efforts.