Periodic Reporting for period 2 - EVADER (Experimental Virology for Assessing Disease Emergence Risks)
Periodo di rendicontazione: 2023-11-01 al 2025-04-30
The objective of the EVADER project is to investigate the emergence of viral pathogens in a laboratory setting. The transmission of wildlife viruses to humans is a complex process that requires a multidisciplinary approach, encompassing the fields of ecology, evolution, social sciences, virology, immunology, and molecular biology. The project's primary focus is on the molecular aspects of viral emergence. Performing experiments with such viruses is challenging for two main reasons. Firstly, many of these viruses are only known from their sequences as they have never been isolated. Secondly, there are significant biosafety risks involved. These limitations can be overcome by recapitulating in the laboratory specific aspects of the viral infection cycle. We focus on receptor-binding proteins (RBP), which are responsible for viral entry into cells and therefore play a crucial role in viral infection. A well-known example of an RBP is the spike protein of the coronavirus. The project studies different spike proteins, but also RBPs from other viral families. To achieve this objective, we use viral pseudotypes, which are constructs of standard viral vectors carrying the RBPs of interest. We are investigating whether wildlife virus RPBs can mediate entry into human cells, exploring whether these RBPs could evolve to become more human-infectious, trying to understand the molecular mechanisms involved in viral entry, and promoting the development of new RBP-targeting antivirals. By addressing these goals, we expect to gain a deeper understanding of how viruses jump between host species and to improve our pandemic preparedness.
The initial objective of the project was to create a large number of viral pseudotypes that express the RBPs of various animal viruses and evaluate their capacity to infect human cells. To this end, over 100 RBP genes from 14 viral families have been synthesized, used for producing pseudotypes and tested in more than 50 different human cell lines, resulting in >5000 RBP-cell combinations. A machine learning model has been developed to predict which viruses may enter human cells, and host factors involved in viral entry have been identified. The focus of this research has been on enveloped RNA viruses, which demonstrate the greatest potential for emergence and are more frequently amenable to pseudotyping. Subsequently, experimental evolution has been employed to examine the potential for RBPs to enhance their infectivity in human cells. A variety of methodologies have been investigated for this purpose, with a particular focus on coronavirus spike proteins. The experiments have identified specific mutations that increase RBP efficiency, contingent on factors such as cell type, receptor availability, entry route, and temperature. To further advance our comprehension of viral entry determinants, a systematic analysis of documented virus receptors has been conducted, and cellular proteins with a heightened propensity for serving as virus receptors have been identified. A novel coronavirus receptor has been identified and characterised, and the tools required for the discovery of new receptors are being implemented. Finally, laboratory techniques for the development of entry inhibitors, such as phage display, are the subject of ongoing work.
The pseudotype infectivity dataset represents the largest of its kind to be published to date, shedding new light on the internalization of animal viruses in human cells. This, in turn, opens up new opportunities for the prediction of viral emergence. The findings indicate that the process of viral entry represents a relatively weak barrier to the viral zoonosis, suggesting that post-entry mechanisms may play a more significant role in restricting viral infection. However, bat coronaviruses represent a notable exception to this general pattern, as the majority of their RBPs were unable to mediate infection of human cells, yet they have been responsible for a number of significant emerging diseases. Therefore, coronaviruses may encounter different obstacles to cross-species transmission compared to other viral families. Furthermore, the project has yielded innovative methodologies for the identification of cellular proteins with a heightened propensity to serve as virus receptors, culminating in the discovery of a novel coronavirus receptor. It is anticipated that the second phase of the project will reveal new host factors that determine viral entry and will advance our understanding of the evolution of emergent viruses. It is anticipated that this research will provide novel tools for the development of antivirals targeting viral entry.