, St. Louis, MO) to remove contaminating amounts kinase inhibitor Rucaparib of endotoxins [30]. Each batch was tested for unspecific stimulation of splenocytes before use. Enzyme-linked immunosorbent assay (ELISA), Western blot and Immunofluorescence analysis (IFA) Indirect ELISA (total Ig) was performed using microtiter plates (NUNC-immuno? MaxiSorp, Nalgene Nunc International, Rochester, NY) coated with 3 ��g/ml of purified recombinant N protein as previously described [31]. Wells lacking the primary antibody were used to establish the background levels and negative or pre-immune sera were used to determine unspecific binding. Western blot was performed using E. coli extracts containing the complete N protein or truncated variants thereof (N1, N2, N3, N1/2, N2/3). The separated proteins were transferred to Immobilon TMP transfer membranes (type PVDF, Millipore Co.

, USA). Membranes containing the antigens were incubated with serum samples from individual mice at dilution 1:600 in parallel with internal controls, either an anti-V5 antibody (Invitrogen) diluted 1:5000 or a mouse anti-poly-histidine antibody (ZYMED? Laboratories, S. San Francisco, CA) diluted 1:3000. A horseradish peroxidase (HRP) conjugated rabbit anti-mouse Ig antibody (DacoCytomation, Glostrup, Denmark) diluted 1:2000 was used as secondary antibody. The antibody-antigen complexes were visualised with enhanced chemiluminescence (ECL, Amersham Bioscience, Uppsala, Sweden). The blotting and incubation procedures have previously been described in detail [28].

For IFA, BHK-21 cells were grown on cover slips and infected with ZH548 at MOI 1, or transfected with cDNA constructs using FuGene? reagent according to the manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland). At 36 h p.i. or 48 h post transfection the cells were fixed with 3% paraformaldehyde in PBS (for anti-glycoprotein antibody detection) or methanol (for anti-N antibody detection). Labelling was performed with mouse sera diluted 1:200, followed by visualisation with an Alexa Fluor? 488 (Molecular probes, Invitrogen) secondary antibody at dilution 1:5000. The expression of the antigens was verified using an anti-V5 antibody (Invitrogen) diluted 1:5000, positive sera from previously infected mice or monoclonal antibodies directed against the GN and GC proteins, kindly provided by Dr. George Ludwig (USAMRIID, Fort Detrick, MD) at predetermined dilutions.

Lymphocyte proliferation test The lymphocyte proliferation assay was performed as described earlier [32]. Briefly, spleen cells of five mice vaccinated with cDNA encoding the full length N protein of RVFV were prepared in RPMI 1640 (GIBCO, Invitrogen) supplemented with 5% FCS, 2 mM sodium pyruvat, 2.5 �� 10-5 M ��-Mercaptoethanol Batimastat and 50 ��g/ml gentamicin sulphate. After washing the spleen cells three times in cell culture media by centrifugation at 600 �� g, the lymphocytes were resuspended to 4 �� 105 cells/ml.