Histopathology Groups of five 6 to 8 weeks old female BALB/c mice (Charles River) were intranasally (i.n.) infected under ether narcosis with 50 ml of VN1203DNS1 or VN1203DNS1-K58I viruses at a dose of 6 log10 TCID50/animal. An additional group of four mice was mock treated i.n. with 50 ml of PBS. 12 h p.i. mice were sacrificed and tissues of the respiratory tract were fixed in 7% formalin and paraffin embedded. Sections of the nasal epithelium, the trachea and lungs were prepared and influenza infected cells were detected with influenza anti-NP monoclonal antibody (1:5000 in PBS; Millipore) followed by immunoperoxidase visualization (standard protocol). Sections were counterstained with hemalum. This animal study was approved by the Austrian regulatory authorities (MA58/000351/2009/13). Immunogenicity and protective efficacy in mice Groups of 6 to 8 week old out bred female mice were immunized i.n. under narcosis once with 50 ml of VN1203DNS1 or VN1203DNS1-K58I virus at a dose of 6 log10 TCID50/animal. The control group was treated with PBS. 28 days post treatment, sera and nasal washes were taken and analyzed for the presence of vaccine strain specific antibodies by HAI assay, MNA, or ELISA. To obtain nasal secretions, salivation was induced by i.p. injection of 0.1 mg pilocarpine-HCl (Sigma). Concurrent small amounts of nasal secretions in the nostrils were immediately absorbed with the aid of sterile wicks and eluted in 50 ml of ice-cold sterile PBS for 4 h. Group-specific pools were made and stored at 220uC. Treated animals were challenged on day 28 p.i. with 50 ml of VN1203wtNS (4 log10 TCID50/animal) virus under ether narcosis. On day 3 post challenge, 3 mice from each group were euthanized; the lung tissues were collected. The viral load was determined in a 10% w/v tissue homogenate by TCID50 assay on Vero cells. Detection of serum antibody titer by a hemagglutination inhibition assay (HAI) Sera from the mice immunized with VN1203DNS1 or VN1203DNS1-K58I or mock-treated were diluted 1:4 with Receptor Destroying Enzyme (RDE; Denka Seiken, Japan) and incubated at 37uC o.n. Thereafter, samples were inactivated at 56uC for 30 min and serial twofold dilutions were prepared. 25 ml/well of the standardized antigen (4 HAU/25 ml) were added. After incubating 1 h at RT, 50 ml of 1% horse erythrocytes (+ 1% BSA, Sigma) were added and the plates were incubated at RT for 1 h. Detection of neutralizing antibody titer by a microneutralization assay (MNA) MNA was performed as previously described [20]. Briefly, serial twofold dilutions of RDE-pre-treated sera were combined with 50 ml of a standardized viral suspension (100 TCID50/50 ml) and incubated for 2 h at 37uC. Vero cells were added and incubated for 20 h, washed, and acetone fixed. An influenza A virus NP- specific monoclonal antibody conjugated with HRP (107L-Px, 4 mg/ml, Dr. E. Vareckova, Slovak Academy of Sciences) diluted in a blocking buffer (PBS containing 0.5% I-Block and 0.1% Tween-20) was added for 1 h. After adding the substrate (TMB, KPL), the average absorption at 450 nm (A450) was determined for the control wells of virus-infected (VC) and uninfected (CC) cells and the neutralizing endpoint (NEP) was determined by using a 50% specific signal calculation. Increased Infectivity of H5N1 Influenza Virus NEP ~ ½ðaverage A450 of VC wellsÞ { ðaverage A450 of CC wellsÞ 2 zðaverage A450 of CC wellsÞ The endpoint titer was expressed as the reciprocal of the highest dilution of serum with an A450 value less than NEP. Detection of vaccine specific serum IgG and mucosal IgA by ELISA A modified enzyme-linked immunosorbent assay (ELISA) protocol was performed as described previously [48,49]. Briefly, UV-inactivated VN1203DNS1 virus (adjusted to 40 HAU/well in carbonate buffer; pH 9.6) was used as a coating antigen. After washing and blocking serially twofold diluted samples (serum for IgG ELISA or group specific pools of nasal washes for mucosal IgA ELISA) were added and incubated at RT for 1.5 h. H5- specific IgG or IgA antibodies were detected with goat anti-mouse IgG conjugated to HRP (0.25 mg/ml; KPL) or goat anti-mouse IgA conjugated with AP (0.25 mg/ml; Rockland Immunochemi- cals). Amount of IgG was determined by using TMB (KPL) substrate and level of IgA by Lumi-Phos Plus substrate (Aureon Biosystems). The cutoff values were defined as the mean value of the negative control samples plus 3 standard deviations. H5- specific IgG as well as IgA were presented in log2 titer. Supporting Information Figure S1 Virus stability to low pH determined by atomic force microscopy (AFM). (A) Supported bilayer lipid membrane (sBLM) on mica: phase and topography image. Topography image of VN1203DNS1-K58I virus adsorbed on sBLM at pH 6.5. (B) Phase images of VN1203DNS1 and VN1203DNS1-K58I viruses on sBLM at pH 6.5–5.8–5.0. The bright regions in the phase images correspond to the liquid disordered domains in the lipid bilayer. (C) Change of the area ratio of the bright and dark regions on the phase images of the two different viruses. The error bars represent the standard deviation from five measurements. (TIF) Text S1 The stability of the viruses VN1203DNS1 and VN1203DNS1-K58I to low pH analyzed by AFM. (DOC) Acknowledgments We would like to thank Nicole Ferstl and Andrea Triendl for their excellent technical assistance. We would also like to thank Dr. Horst Fischer (CHORI, Oakland) for his helpful discussions and reviewing the manuscript. Moreover, we would like to acknowledge Dr. E. Vareckova of the Slovak Academy of Sciences for providing the influenza A anti-NP antibody. Author Contributions Conceived and designed the experiments: BMK AE MW SN RZ PH OK TM JC MM JR. Performed the experiments: BMK ER TR BK AM NW SS BF OB AI MK JH JG. Analyzed the data: BMK AE RZ PH TM JR. Contributed reagents/materials/analysis tools: ER TR SS RZ PH. Wrote the paper: BMK AE JR OB AI. PLoS ONE | www.plosone.org 11 April 2011 | Volume 6 | Issue 4 | e18577