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

The different viruses can be ranked based on their pathogenicity as determined by Median Lethal Dose (MLD) values in 6-week-old BALB/c mice ( Figure 1A ). The VN1203-WT (MLD < 1 PFU) and the VN1203-PB1F2del (MLD = 3.2 PFU) viruses were associated with the highest level of pathogenicity. The CA04-WT (MLD = 630,957 PFU; previously determined [22] ) and the VN1203-HAavir (MLD = 316,228 PFU) viruses were associated with the lowest level of pathogenicity. The VN1203-NS1trunc (MLD = 631 PFU) and the VN1203-PB2627E (MLD = 6,310 PFU) viruses showed an intermediate level of pathogenicity. Morbidity associated with infection, determined by mouse body weight loss ( Figure 1B ), viral replication quantified by viral titration (Figure 1C ), viral messenger RNA ( Figure 1D) , and viral genomic RNA ( Figure 1E ) measured by quantitative RT-PCR were used to characterize the viruses in our study. These data were collected in the same experiment in which mouse lung tissues were collected for global transcriptome and proteome profiling. Over the 7-day time course, VN1203-WT and all of the mutant viruseswith the exception of VN1203-PB2627Eexhibited similar, rapid weight loss culminating in a 20-30% reduction from initial body weights by 5-7 days post-infection ( Figure 1B ). In contrast, both VN1203-PB2627E and CA04-WT displayed a more reduced rate of weight loss, which peaked at~17% on day 7 post-infection. None of the mice survived to day 7 for the VN1203-WT infection at 10 4 PFU. Virus titers were roughly distributed from low to high according to virus pathogenicity, with CA04-WT exhibiting the lowest mean titer in all four time-points and VN1203-WT exhibiting the highest ( Figure 1C ; see Additional file 2: Figure S2 for statistical comparisons). The one exception was titers observed in VN1203-HAavir infections, which rivaled that of VN1203-WT in all time-points. Both viral messenger RNA and genomic RNA production levels exhibited early (day 1) segregation into two groups, with VN1203-PB2627E and WT-CA04 displaying a 1-1.5 log 10 -fold reduction in expression compared to all the other viruses ( Figure 1D and E and Additional file 2: Figure S2 ). Viral RNA expression levels remained partially segregated on day 2, and by day 4, expression levels were more similar for all viruses in the panel.