Increased Infectivity of H5N1 Influenza Virus Figure 7. Viral growth capacity in the mouse upper respiratory tract. Mice were infected i.n. at doses of 2.5, 3.5, or 4.5 log10 TCID50/animal of VN1203wtNS or VN1203wtNS-K58I virus. The presence of the virus was determined in 10% w/v nasal tissue homogenates by TCID50 assay 60 h p.i. The lower limit of detection for the TCID50 titer is 1.5 log10 TCID50/ml indicated by the horizontal dashed line. The number of animals infected/total number is indicated above the bars. Error bars represent the standard deviation. doi:10.1371/journal.pone.0018577.g007 Mutation HA2 58KRI contributes to increased virus immunogenicity in mice In a previous study, we noticed that vaccine candidate VN1203DNS1 did not induce any HAI antibodies in mice in contrast to macaques and ferrets, although still providing protection against challenge viruses [20]. Therefore, we were interested in whether modified VN1203DNS1-K58I virus is able to evoke functional HAI or neutralizing antibodies in a mouse model. A single i.n. vaccination with the VN1203DNS1 virus induced detectable HAI titers only in 2 out of 6 animals Figure 8. Viral infectivity in the mouse respiratory tract. Mice were infected i.n. with 6 log10 TCID50/animal of VN1203DNS1 or VN1203DNS1- K58I virus or mock-infected with PBS. Nasal, tracheal, and lung tissues were examined 12 h p.i. for the presence of virus infected cells by the immunostaining of influenza NP. doi:10.1371/journal.pone.0018577.g008 PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e18577