| Description |
As pathogens adapt to new hosts, they also evolve increased virulence; however, the factors that contribute to virulence evolution have been difficult to explain or predict. Two factors that lead to virulence evolution are the genetic diversity of the infecting pathogen population and of the host population, which have opposite effects on virulence. One potential source of pathogen diversity are multiple infections of the same pathogen originating from more than one host. Despite several theoretical explorations on how multiple infections might affect virulence evolution trajectory, no experiments had been conducted that directly test these predictions in a vertebrate host-pathogen system. Here, by serially passaging viruses from different numbers of murine hosts, we test hypotheses on the effect of multiple infections on the virulence evolution of influenza A. We also test one-time multiple infections with independently evolved viral lineages and manipulate the genotypes of the donor hosts that contribute to multiple infections. Pathogen adaptation to specific host genotypes is associated with antagonistic pleiotropic effects once the virus infects novel host genotypes. To test how host population diversity affects influenza virulence and virulence evolution, we also performed a series of experiments utilizing viral lines adapted to specific host genotypes to infect familiar and unfamiliar hosts. Lastly, we have found that a widely used experimental infection method - infections with lung homogenate supernatant - can lead to discrepant virulence measurements due to associated host-specific virulence factors. To test this, we performed several infection experiments aimed at understanding the magnitude and nature of these effects. Our results demonstrate that multiple infections increase the rates of virulence evolution, as measured by weight loss and mortality. We also show that a single multiple infection event can recapitulate gains in virulence obtained through continuous multiple infections across serial passage, particularly in infections of new host genotypes or where viral lines adapted to distinct host genotypes are combined. However, we did not observe tradeoffs associated with infections of novel host genotypes. Lastly, we have found that host factors associated with lung homogenate can influence virulence measurements, and have developed a cell-based viral purification protocol that eliminates host virulence factors. |
