Virus evolution and host responses in positive-sense RNA viruses

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The last year of work in the QVEU has seen significant progress in three of long-term projects in the lab. First, in work led by Visiting Fellow Dr. Nathania Dabilla, we have continued to develop single-cell sequencing approaches to observe the heterogeneous and dynamic process of infection in the complex selective environment in vivo and in vitro. This has focused primarily on development of our technology, single-cell RNAseq-enabled Acquisition of RNA and Consensus haplotypes linking individual genotypes to host transcriptomes (SEARCHLIGHT), a paradigm-changing technique to describe the dynamics of viral quasispecies. This approach opens new doors which fundamentally change our ability to understand how viral populations evolve. We published our initial study with this technology in 2024, and have spent this year making significant improvements to the technology, and applying it to new viral systems, and in new model systems, including organoid models of human infection. Secondly, in work led by Visiting Fellow Dr. William Bakhache, we have applied our mutational scanning studies to better understand the evolutionary. Our approach, based on the Deep Insertional and Mutational Protein Library Engineering (DIMPLE) technology, allows us to engineer complete libraries of insertions, deletions, and coding substitutions into every position of a protein. We are currently characterizing our first of these libraries focused on the capsid region of the human enterovirus, EV-A71. We are using this information to better understand the long and short-term evolutionary constraints on viral evolution. This year, we published two studies in this area, one in Nature Microbiology, describing the evolutionary constraints on Enterovirus A71 (EV-A71), and another on the use of this technology to produce a toolkit of useful reagents for EV-A71 research in Antiviral Research. We are currently applying this technology to West Nile Virus, a flavivirus, to understand its evolution across its diverse host and vector species. Finally, in work led by Jack Dorman, a PhD student in the Unit, we have established a computational and experimental pipeline to reconstruct the evolutionary histories and ancestral sequences of viral proteins. Our current studies are focused on the reconstruction of the historical evolution of structural proteins of flaviviruses, including the ancestors of dengue, Zika, and West Nile Virus. We have reconstructed and synthesized the ancestral sequences of these viral species and are currently developing biochemical and cell biological assays to characterize the function of these reconstructed ancestral proteins. This work has and will continue to clarify how viral pathogens emerge, focusing on the influence of protein stability on viral protein evolvability.