MDCK cells were maintained in Dulbeccos Modified Eagle Medium (DMEM; Life Technologies,

USA) containing 10% Fetal GW-572016 supplier Bovine Serum (FBS; Life Technologies, USA). 293 T were maintained in Opti-MEMI (Life Technologies, USA) containing 5% FBS. After 48 h the transfected supernatants were collected and virus titers were determined by standard hemagglutination assays. The sequences were confirmed using a specific set of universal primers as described previously (21). Viruses were propagated in 10 day old specific pathogen free embroyonated chicken eggs at 37°C. The tissue culture infectious dose 50 (TCID50) of reassortant virus was then calculated by the Muench-Reed method (1938). Table 1 HI and neutralization (VN) titer of 62 and 98 (200 ug/ml) against different H7 Virus Subtype HI titer VN titer     (Mab 62, 98) (Mab 62, 98) PF-3084014 clinical trial A/Chicken/Malaysia/94* H7N1 256, 256 640, 640 A/Canada/rv504/04 H7N3 128,256 320, 640 A/quail/Aichi/4/09 H7N6 64, 64 80,

80 A/duck/Hokkaido/1/10 H7N7 128, 256 320, 640 A/Netherlands/219/03 H7N7 256, 256 640, 1280 A/Shanghai/1/13* H7N9 64, 128 160, 320 A/Puerto Rico/8/34 H1N1 <8, <8 <20, <20 A/TLL51/Singapore/09 H1N1 Selleck Vorinostat <8, <8 <20, <20 A/duck/Nanchang/4-184/2000 H2N9 <8, <8 <20, <20 A/Chicken/Malaysia/02* H3N2 <8, <8 <20, <20 A/Chicken/Malaysia/92* H4N1 <8, <8 <20, <20 A/Vietnam/VN1203/03 H5N1 <8, <8 <20, <20 A/Shorebird/DE/12/04 H6N8 <8, <8 <20, <20 A/duck/Yangzhou/02/05 H8N4 <8, <8 <20, <20 A/chicken/Malaysia/98*

H9N2 <8, <8 <20, <20 A/mandarin duck/Malaysia/98* H10N5 <8, <8 <20, <20 A/pintail/Alberta/84/2000 H11N9 <8, <8 <20, <20 A/pintail/Alberta/49/03 H12N5 <8, <8 <20, <20 A/gull/Maryland/704/1977 H13N6 <8, <8 <20, <20 HI titer below 8 and VN titer below 20 indicated negative activity. *: wild type virus. Production and characterization of Mab BALB/c mice were immunized twice subcutaneously at intervals of 2 weeks with BEI (binary ethylenimine) inactivated H7N1 (A/Chicken/Malaysia/94) and adjuvant (SEPPIC, France). Mice were boosted with the same Phloretin viral antigen, 3 days before the fusion of splenocytes with SP2/0 cells [15]. The fused cells were seeded in 96-well plates, and their supernatants were screened by immunofluorescence assays as described below. The hybridomas that produced the Mabs were cloned by limiting dilution at least three times. The positive Mabs were tested for their hemagglutination inhibition activity as described below. Immunoglobulins from selected positive Mabs were isotyped using a commercial isotyping kit (Amersham Bioscience, England) as described in the manufacturer’s protocol.