1/100) from sentinel chickens were used for the analysis. The virus proteins were separated by10% polyacrylamide gel electrophoresis under reducing conditions until the dye front had run off the bottom of the gel. A Biorad semi-dry blotter was used to transfer the protein bands from the gel onto the Hybond-P PVDF transfer membrane. After transfer the membrane was blocked in 5% milk powder (in TBS and 0.05% Tween 20) for 1 hour at room temperature. The blot was then cut into identical strips (approximately 6 mm wide) which were individually treated with a chicken serum diluted 1/100 to test for antibodies to WNV. The strips were washed in TBS/Tween 20 three times before addition of 1:20,000 dilution of Rabbit anti Chicken conjugated with alkaline phosphatase (Sigma) for 1 hour at room temperature. The strips were washed three times in TBS/Tween 20 then once in 0.1 M Tris pH9.6 before addition of the BCIP/NBT liquid substrate system (Sigma).
Indirect immunofluorescence microscopy
This was performed on chicken sera (diluted 1:100 or 1:1000 in PBS). Each diluted serum was added to acetone-fixed WNV-infected Vero cells on glass coverslips. After incubation for 1 hour at 37°C the cells were washed in warm PBS for 30 minutes. Rabbit anti-chicken FITC (Sigma) diluted to 1:400 was then added and after incubation for 1 hour at 37°C, the coverslips were washed in warm PBS and water before mounting in DABCO/Glycerol/PBS pH8.6, on microscope slides. Each monolayer was examined for virus-specific immunofluorescence under a UV light microscope.
The author(s) declare that they have no competing interests.
AB carried out all the immunoassays and data processing and helped draft the manuscript, AD designed, set up and carried out the sentinel study, EAG conceived and co-ordinated the study, supervised the research and drafted the manuscript. All authors have read and approved the submitted manuscript.
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