(Invitrogen) supplemented with 10% FBS and 2 mM L-glutamine. For the latter the dishes, were coated with 10 mg/ml BSA (Sigma), 30 mg/ml bovine collagen type I (Promocell), and 10 mg/ml human fibronectin (BD Pharmingen) in Ham’s F12 medium (HyClone). The carcinoma human alveolar basal epithelial A549 cells (obtained from J. Seipelt, Austria) were maintained in MEM supplemented with 10% FBS and 2 mM L-glutamine. Human Nasal Epithelial Cells (HNEpCs) were provided by PromoCell (Germany) and cultivated according to the manufacture’s procedure. Viruses The human influenza viruses A/Solomon Island/3/2006 (H1N1; NIBSC) (SL/03/06) and A/Brisbane/59/2007 (H1N1; NIBSC) (BN/59/07) were propagated in the allantoic cavity of 9- to 11-days old embryonated hen’s eggs at 37uC. Allantoic fluids were collected 48 h post infection (p.i.) and clarified by centrifugation. The primary influenza isolate A/Vienna/25/07 (H3N2) (VI/25/07) (MDCK - derived, A/Wisconsin/67/05-like, HA and GenBank accession numbers are JF340081 and JF340082 respectively) was provided by the Austrian National Reference Centre for Influenza (Institute of Virology, Vienna, Austria). The primary influenza isolate A/St.Petersburg/14/10 (H1N1v) (SP/14/10, MDCK – derived, HA and GenBank accession numbers are JF340083 and JF340084) was provided by Influenza Research Institute (Saint Petersburg, Russia). Avian influenza viruses A/Vietnam/1203/04 (H5N1) (VN1203) (MDCK - derived, passage 3) and A/Thailand/01/04 (H5N1) (TH/01/04) (MDCK - derived, passage 2) were provided by the Institute for Medicinal Virology, Johann Wolfgang Goethe University Frankfurt (Frankfurt, Germany). Viral stocks were produced in MDCK cells in SFM supplemented with 5 mg/ml porcine trypsin (Sigma) cultivated at 37uC and 5% CO2. The generation of the reassortant virus VN1203DNS1 was described previously [20]. Briefly, the 5:3 reassortant viruses contain HA, NA, and M from VN1203 (H5N1) and the internal protein genes from the WHO influenza vaccine strain IVR-116 (H1N1). The HA polybasic cleavage site as well as the NS1 open reading frame were deleted. The virus VN1203DNS1-K58I differs from VN1203DNS1 by a single mutation in the HA2 58KRI introduced by site directed mutagenesis (Stratagene). In addition, a corresponding couple of viruses was constructed as 5:3 reassortants deriving the HA with a modified cleavage site as described above, the M and the of influenza virus VN1203 in combination with all other genes including the complete NS segment of the IVR-116 strain and named VN1203wtNS or VN1203wtNS-K58I (comprising the HA2 58KRI mutation). For virus stocks, Vero cells were inoculated at a moi of 0.001 in SFM or MES buffer (100 mM MES, 150 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2; pH 5.6) and cultivated at 37uC and 5% CO2 in SFM supplemented with 0.25 mg/ml Amphotericin B (Amph B; Bristol-Myers Squibb) and 5 mg/ml porcine trypsin (Sigma). Infectious virus titers were determined in Vero cells by calculating the 50% tissue culture infectious doses per ml (TCID50/ml) according to Reed and Muench [46]. Fusion assay Viruses standardized to 128 HA units (HAU; determined following standard procedure) per 50 ml were diluted 1:4 in a 1% suspension of human erythrocytes (Siemens) and incubated on ice for 1 h to allow virus binding. Then, the mixtures were pelleted at 72 g and the supernatants were removed. 100 ml of MES buffer at various pH values (from 5.0 to 6.0) were added, followed by incubating at 37uC for 1 h. After centrifugation (72 g), 50 ml of supernatant were transferred to 96-well plates and the amount of Increased Infectivity of H5N1 Influenza Virus hemoglobin released by virus-cell fusion induced hemolysis was determined by the measurement of optical density at 405 nm. Reported results are the means 6 standard deviations (SD) of 3 replicates at the indicated pH value. Effect of various pH values or lysosomotropic reagents on virus infectivity in vitro Semi-confluent Vero cells were infected with virus at moi 2. Virus inoculum was prepared in MES buffer (0.25 mg/ml Amph B) at the indicated pH value or in SFM supplemented with 0.25 mg/ml Amph B and chloroquine (Sigma) or NH4Cl at indicated concentrations. It was applied to the cells at 37uC and 5% CO2 for 30 min. Then, the inoculum was replaced with SFM and 5 h p.i. cells were fixed with 4% para-formaldehyde, permeabilized with 1% Triton X100 (in PBS) and blocked (PBS + 1% BSA). Infected cells were stained with influenza anti- nucleoprotein (NP) monoclonal antibody (Millipore, 1:5000 in PBS + 1% BSA) followed by Alexa Fluor 488 conjugated anti- mouse antibody (Invitrogen, 1:1000 in PBS + 1% BSA). Images were taken on an Olympus CKX41 Fluorescence Microscope with connected Olympus camera system E330. Flow cytometry Confluent MDCK cells were infected with virus at moi 2 in MES buffer (0.25 mg/ml Amph B) at the indicated pH values or mock-infected. 5 h p.i. cells were detached with 0.2% trypsin– EDTA (Cellgro), fixed with Fix/Perm buffer (BD Cytofix/ Cytoperm Plus Kit), blocked in PBS (+1% FBS) overnight (o.n.), permeabilized and stained with the FITC-labelled mAb against NP (Imagen, UK). The percentage of NP-positive cells was determined by flow cytometry using an Epics Elite XL-MCL flow cytometer and EXPO 32 software (Coulter Immunotech, F). Virus growth kinetics To determine the growth kinetics, four different cells lines (Vero, MDCK, A549, and HBE cells) were infected with VN1203wtNS or VN1203wtNS-K58I at moi 0.001 in SFM (+ 0.25 mg/ml Amph B). After incubating at 37uC and 5% CO2 for 30 min, the inoculum was removed and the cells were maintained at 37uC 5% CO2 in SFM supplemented with 4 mM L-glutamine, 0.25 mg/ml Amph B, and 5 mg/ml porcine trypsin (Sigma). Supernatants were collected 16, 24, 48, and 72 h p.i. and virus titers were determined by TCID50 assay on Vero cells. Infectivity in mice Animal experiments were conducted in accordance with the Declaration of Helsinki and approved by National Authorities: One mouse study was approved by the Austrian regulatory authorities (MA58/000351/2009/13). The other two studies were done under the Russian Influenza Research Institutes Ethics Committee that is registered at the US Department of Health and Human Services under the IORG Number IORG0004322 Rsch inst of Influenza IRB # 1. Groups of 6 to 8 weeks old out bred female mice were infected i.n. without narcosis with 20 ml of preparations of VN1203wtNS or VN1203wtNS-K58I virus at a dose of 4.5, 3.5, or 2.5 log10 TCID50/animal. 60 h p.i. nasal tissues were collected and the presence of infectious virus in 10% w/v tissue homogenates [in SFM supplemented with 1% antibiotic-mix (Invitrogen), 25 mg/ml gentamicin (Invitrogen)] was determined by TCID50 assay on Vero cells. Mouse infectious dose 50 (MID50) titers were calculated by using the method of Kerber and expressed as the TCID50 value corresponding to 1 MID50 [47]. PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e18577