nd serum samples collected at 9 weeks of age.
Plaque reduction neutralization tests
These tests were carried out as described previously  and are based on the WHO standard method. Briefly, each heat-inactivated (56°C for 30 minutes) serum sample was diluted serially in twofold stages. These were mixed in equal volume with 50 plaque-forming units of either WNV-Is, WNV-DAK, USUV or SINV. The mixtures were incubated overnight at 4°C. Each mixture was then placed on a monolayer of SW13 cells in 24-well Petri-plates and incubated for 60 mins at room temperature. 1 ml of overlay medium (RPMI-1640 with Hepes buffer, 1% foetal bovine serum, penicillin, streptomycin and 1% SeaPlaque Agarose) was added to each well and allowed to set at room temperature, then the plates were incubated at 37°C until plaques were identifiable in control wells. The monolayers were fixed in 10% formol-saline and stained with 0.1% naphthalene black stain. Serum neutralization titres were estimated as the highest dilution causing at least 50% reduction of plaque numbers. Titres less than 1/10 were considered to be negative.
Purification of WNV
The supernatant medium collected from 10 × 175 cm2 plastic tissue culture bottles was clarified by centrifugation at 5000 g for 30 mins and the virus was then precipitated from this clarified medium by the addition of 7% polyethylene glycol and 0.4 M NaCl. After stirring overnight at 4°C, the virus was sedimented by centrifugation at 5000 g for 1 hour. The pellets were resuspended in PBS and layered onto 15–60% (w/w) sucrose gradients prepared in Tris-EDTA buffer pH7.4. The gradients were spun at 90,000 g for 3 hours and the tube was then fractionated by upward displacement. Each fraction was tested for the presence of viral antigens by western blotting (see below). The sample in the 60% sucrose fraction produced a very distinct band of viral envelope (E) protein as deduced using a monoclonal antibody known to bind to WNV-E protein (see Results).
Gradient-purified West Nile virus antigen and sera (diluted 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.
Journal of Vector Ecology 1999, 24:1-39. PubMed Abstract
Emerging Infectious Diseases 2006, 12:549-555. PubMed Abstract
Murgue B, Z