Specific mutations in H5N1 mainly impact the magnitude and velocity of the host response in mice

Tchitchek, Nicolas; Eisfeld, Amie J; Tisoncik-Go, Jennifer; Josset, Laurence; Gralinski, Lisa E; Bécavin, Christophe; Tilton, Susan C; Webb-Robertson, Bobbie-Jo; Ferris, Martin T; Totura, Allison L; Li, Chengjun; Neumann, Gabriele; Metz, Thomas O; Smith, Richard D; Waters, Katrina M; Baric, Ralph; Kawaoka, Yoshihiro; Katze, Michael G

All experiments using live H5N1 and H1N1 viruses were performed in an animal enhanced biosafety level 3 (ABSL3 and ABSL3+) containment laboratories at the University of Wisconsin-Madison and University of North Carolina, approved for use by the United States (US) Centers for Disease Control (CDC) and the US Department of Agriculture. All animal experiments and procedures were approved by the University of Wisconsin-Madison School of Veterinary Medicine Animal Care and Use Committee and by the University of North Carolina Institutional Animal Care and Use Committee. Twenty-week-old female C57BL/6J mice (Jackson Laboratories) were anesthetized by isoflurane inhalation or i.p. ketamine/xylazine injection and mock-infected or infected with 10 2 , 10 3 , 10 4 , 10 5 or 10 6 Plaque Forming Units (PFU) of virus, as indicated in the text, figures and figure legends. Viruses were diluted in Minimum Essential Medium (MEM) containing 0.3% BSA, and the same medium without virus was used for mock infections. Three to five mice from both mock-infected and infected groups were euthanized at days 1, 2, 4 and 7 post-infection, and lungs were harvested and divided into multiple lobes for virus titration, RNA extraction or protein extraction. For each wildtype or mutant virus, infections were performed in independent experiments, and the same lung lobe was collected for each analysis method from every mouse. Importantly, we did not detect any differences in the amount of virus recovered from any lung lobe, indicating there was no bias in the distribution of viral infection in the mouse lungs (Additional file 8: Figure S5 ). Individual or group mouse body weights were collected on a daily basis to monitor the disease course, and mice were humanely euthanized upon reaching the experimental endpoint (i.e., sample collection or severe clinical symptoms).