Increased Infectivity of H5N1 Influenza Virus Figure 4. Infectivity of viruses at different pH values in tissue culture. (A) Virus VN1203DNS1 or VN1203DNS1-K58I was mixed with MES buffer at indicated pH values and used for infection of Vero cells at moi 2. Influenza NP was visualized by immunostaining after incubating for 5 h. (B) MDCK cells were infected with VN1203DNS1 or VN1203DNS1-K58I viruses at indicated pH values. The number of infected cells was determined after the immunostaining of viral NP (5 h p.i.) by flow cytometry. The indicated numbers show the percentage of infected cells. (C) Vero or MDCK cells were infected with seasonal human influenza virus BN/59/07 (H1N1) at indicated pH values. Virus infectivity was analyzed analogously by immunostaining or by flow cytometry. doi:10.1371/journal.pone.0018577.g004 times. A similar tendency was observed in Vero, MDCK, and A549 cells (Fig. 6). Therefore, a mutation decreasing the pH threshold of the HA conformational change may affect the growth of H5N1 viruses in continuous cell lines. Mutation HA2 58KRI increases virus infectivity in mice To assess the relevance of the in vitro findings in animals, we compared the virus infectivity in mice. First, we measured the MID50 of viruses with mutant or original HA. The animals were infected with VN1203wtNS or VN1203wtNS-K58I viruses in a dose ranging from 2.5 to 4.5 log10 TCID50/animal. Nasal tissues were collected 60 h after i.n. administration and the tissue homogenates were investigated for the presence of infectious virus by TCID50 assay. The mutant virus VN1203wtNS-K58I had an approximately 25-fold lower MID50 (MID50 2.9 log10) compared to that found for the original virus (MID50 4.3 log10) (Fig. 7). This result was consistent in two independent experiments. To compare the extent of the uptake of the original and mutated viruses, the animals were infected i.n. with live attenuated replication deficient VN1203DNS1 or VN1203DNS1-K58I vaccine candidates to ensure single round infection at a dose of 6 log10 TCID50/animal. A qualitative assessment of infected foci was carried out by the immunohistochemistry of the sections obtained from the nasal mucosa, trachea, and lung tissues stained with anti- influenza NP primary antibody. The mutant VN1203DNS1-K58I virus increased the extent of the infection of the alveolar regions of lungs, trachea and nasal mucosa, where more foci of the infection were seen for all 5 immunized animals when compared to the original VN1203DNS1 virus (Fig. 8). Thus, the introduced mutation promoted more intensive viral uptake in the mouse respiratory tract. PLoS ONE | www.plosone.org 5 April 2011 | Volume 6 | Issue 4 | e18577