Location: Arthropod-borne Animal Diseases Research
Project Number: 3020-32000-019-002-N
Project Type: Non-Funded Cooperative Agreement
Start Date: Aug 1, 2022
End Date: Dec 31, 2024
Objective:
Virological research has increasingly included investigations on the role that virus particles, including defective virus particles, can have on disease transmission, evolution, and severity. We propose to investigate the emergence, transmission, and impact on the innate immunity of defective viral fragments in the cell line of a natural arthropod host to address these questions and their implications for human and animal health.
Defective viral genomes (DVGs) are truncated genomes that emerge as a byproduct of virus infection. DVGs can only replicate in the presence of full-length genomes by exploiting the resources of the standard virus. DVGs interfere with the replication of virus, co-transmit, and over multiple generations may enter a co-evolutionary arms race. As DVGs emerge and increase, they inhibit wildtype virus which selects for variants that can replicate uninhibited until the process repeats. DVGs can also elicit immune responses that can reduce viral load. Once considered an artifact of laboratory experiments with cell lines infected with virus at high ratio of infectious particles to cells, DVGs have now been identified in patients with human respiratory syncytial virus, dengue, and H1N1 among others. There is growing evidence that DVGs play a role in the severity of infections and there are therapeutic DVGs being designed and explored for SARS-COV-2 and dengue virus.
Many of the emerging infectious diseases of human concern are zoonotic and arboviral in nature. However, often little is known about the pathogenesis of these diseases in their vectors hosts and how that might impact spillover to humans. Vesicular stomatitis virus (VSV), a zoonotic disease that spreads from insect vectors (e.g., culicoides sonorensis) to mammalian species, is a good model for more severe zoonotic diseases. An unknown aspect of viral infections is how the evolutionary interactions of DVGs and wildtype virus and the innate immune pathways triggered by DVGs in initial vectors will impact the severity and spread of zoonotic viruses to humans.
We aim to: (i) elucidate how expression of genes associated with innate immune responses impacts emergence and enrichment of DVGs during viral culture, and (ii) characterize co-evolution between DVGs and standard wildtype virus over multiple generations. To address concerns that DVG emergence may differ significantly in infections from lab-adapted VSV when compared to wild type VSV, we further aim to compare the one-step growth curves and the emergence and coevolution of DVGs in lab-adapted VSV vs. a VSV isolate from the 2019 VSV outbreak.
Approach:
W8 cell lines from culicoides sonorensis will be infected with either the lab adapted VSV culture or the VSV 2019 outbreak isolate and then be serially passaged for 10 generations. To compare the pathology of the lab adapted VSV vs. 2019 VSV field isolate, one step growth curves and plaque assays of both will be done. We expect to observe, via RNA sequencing of each passage and analysis of standard viral genomes and the DVGs, DVG emergence, changing fitness levels of DVGs, and oscillations of standard and DVGs as they co-evolve. We also anticipate upregulation of innate immunity genes as DVGs emerge, enrich, and adapt. We expect that DVG emergence will be observed in both cultures. Our findings will be a first step toward a deeper understanding of how co-evolution and innate immunity gene regulation in an arthropod vector will affect zoonotic viral infections and how that may impact spillover to humans. The Yin lab is well suited for this project because of the years of experience with research of both defective interfering genomes and VSV.
