(GMT = 6.3), whereas the administration of the mutant virus VN1203DNS1-K58I induced a response in 5 animals (GMT = 25.4) (Fig. 9A). An MNA test also revealed higher titers of neutralizing antibodies induced by VN1203DNS1-K58I (GMT = 57) virus than that induced by VN1203DNS1 (GMT = 27.9) (Fig. 9B). The significant superior induction of specific antibodies by the mutated vaccine virus was confirmed in an ELISA, where the IgG GMT evoked by VN1203DNS1 was 3225 and by VN1203DNS1-K58I was 8127 (Fig. 9C). The measurement of specific IgA antibodies in pooled nasal washes of immunized mice indicated fourfold more antibodies after immunization with the mutant virus (Fig. 9D). Thus, a single immunization of mice with the VN1203DNS1-K58I virus, containing the stability improved HA, induced a significantly higher immune response as reflected by increased serum and mucosal antibody titers. Despite the observed differences in immunogenicity, both VN1203DNS1 and VN1203DNS1-K58I viruses completely pre- vented the replication of the homologous challenge virus VN1203wtNS in the lungs of 3/5 and 4/5 animals, respectively, taken 3 days post immunization, (data not shown) showing no statistically significant difference. Discussion The effective spread of influenza viruses in the human population may require a certain level of environmental stability Increased Infectivity of H5N1 Influenza Virus of the virus particles depending on a pathway of their transmission. Three modes of infection with influenza viruses in people have been postulated, including aerosol transmission, infection with large droplets, and self-inoculation of the nasal mucosa by contaminated hands [26]. Although, these routes of infection are not mutually exclusive, effective infection via the nasal route could be dependent on the pH stability of the virus particles due to the mechanisms of the fast acidification of human nasal mucosal surfaces [22]. Similar to the previous observation [27], we found that human epidemic influenza viruses, irrespective of their subtype (H1, H1v, H3), are more resistant to physical factors such as low pH (5.4–5.6) or elevated temperature exposure than highly pathogenic H5N1 influenza strains. It should be mentioned that the pH stability phenotype might be abrogated upon passaging in tissue culture [28]. Therefore, the virus isolates of the early passage level were used in this study. Contrary to human strains, HPAIVs and their derivates with a modified HA cleavage site were not infectious at pH#5.6. We hypothesize that this relatively low pH stability of HPAIVs viruses might be a limiting factor for infectivity in the mammalian upper respiratory tract particularly in the nasal compartment. To prove the hypothesis we introduced a known stabilizing mutation 58KRI into HA2 subunit of the VN1203 H5N1 virus and compared the properties of the obtained viruses in vitro and in vivo. We revealed that the in vitro parameters, such as the pH threshold Figure 9. Antibody titers after single immunization of mice. Mice were vaccinated i.n. with 6 log10 TCID50/animal of VN1203DNS1 or VN1203DNS1-K58I virus. Sera (collected 4 weeks p.i.) were analyzed for HAI (A), neutralizing (B) and for total IgG (C) antibodies measured by ELISA. The titers of the individual animals (symbols) and the geometric mean titers (horizontal lines) are indicated. An undetectable antibody titer was assigned a value of 4 (HAI; A) or 8 (MNA; B). (D) Pools of nasal washes (collected 4 weeks p.i.) were examined for the presence of homologous IgA antibodies. * indicates p,0.05 determined by a t-Students test. doi:10.1371/journal.pone.0018577.g009 PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e18577