| Description |
The methodology for passaging influenza A virus in the mouse model has widely been established. However, traditional infection methods involving the inoculation of lung homogenates fail to account for the possible effects of host-associated factors on measures of virulence and in the virulence evolution of the virus. To test these effects, we performed a series of experiments utilizing single-origin virus to compare lung homogenate inoculations to infections with bronchoalveolar lavage fluid or with homogenate purified through a custom tissue culture-based technique. To assess whether host-associated factors cause allogeneic effects, virus adapted to a specific mouse genotype was used to infect both the familiar and novel host genotypes, either via lung homogenate or purified virus infections. Finally, to test whether host-associated factors affect the virulence evolution of influenza A virus, we also compare the virulence of virus serially passaged via lung homogenates to virus passaged via bronchoalveolar lavages. Our results indicate that there are no major differences in virulence measurements when influenza virus contained in homogenate versus bronchoalveolar lavage or cell culture supernatant is used to infect same-genotype hosts. There is also no major effect on viral virulence evolution. However, there is a strong allogeneic effect and associated increase in virulence when virus in lung homogenates is used to infect novel host genotypes. Infection assays and sequencing of pre- and post-purification viral populations demonstrate that our novel cell-based purification method is effective at eliminating allogeneic effects caused by host-associated factors in lung homogenate with negligible effects on the genetic composition of the viral populations. |
