Increased Infectivity of H5N1 Influenza Virus Figure 1. Infectivity of human and highly pathogenic avian viruses at an acidic pH in HNEpCs. Primary Human Nasal Epithelial cells were infected with human (A) epidemic BN/59/07 (H1N1), VI/25/07 (H3N2), SP/14/10 (H1N1v) or avian (B) highly pathogenic viruses VN/1203 (H5N1) and TH/01/04 (H5N1) at the indicated pH values. Influenza NP protein was visualized by immunostaining after incubating for 5 h. doi:10.1371/journal.pone.0018577.g001 The reassortant virus containing mutation HA2 58KRI and the deletion of the NS1 ORF was generated by the eight-plasmid rescue method in Vero cells analogously to the previously described VN1203DNS1 virus [20] and was named VN1203DNS1-K58I. Both viruses comprised an HA with a polybasic cleavage site modified in a trypsin dependent manner [25]. In addition, a corresponding pair of reassortants with a complete NS segment was generated in the same way and named VN1203wtNS and VN1203wtNS-K58I, respectively. Mutation HA2 58KRI decreases the pH threshold of HA fusion and increases the virus resistance to acidic pH and high temperature To prove the impact of the introduced mutation on the pH optimum of HA fusion activity, we analyzed the reassortants VN1203DNS1 and VN1203DNS1-K58I in a fusion assay. The original virus had the capacity to trigger a fusion at pH values#5.6, whereas the same fusion rate for the mutant virus was observed at pH#5.3 (Fig. 2). It is noteworthy that the mutant H5N1 virus VN1203DNS1-K58I showed the same activation profile of HA as observed for the human seasonal virus A/ Solomon Island/03/06 (H1N1) (SL/03/06). The result of the HA fusion assay was confirmed by the atomic force microscopy (AFM) technique allowing visualization of viral Figure 2. Hemolytic activity of influenza viruses as a function of pH. Viruses VN1203DNS1, VN1203DNS1-K58I, and seasonal A/SL/03/ 06 (H1N1) standardized to 128 HA units were mixed with 1% suspension of human erythrocytes at 0uC and pH 7.4 for 1 h and then the buffer was replaced with MES buffer at various pH values. After incubation at 37uC for 1 h hemolysis representing the fusion activity was determined spectrophotometrically at 405 nm. Error bars represent the standard deviation from triplicate experiments. doi:10.1371/journal.pone.0018577.g002 PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e18577