s. These include the aerosol and faecal/oral routes, transmission via direct physical contact or maternal milk, and through contaminated water. It is also clear that WNV can persist in vertebrate hosts for months if not years without inducing obvious clinical symptoms [5,14-21]. It seems likely that these properties provide WNV with the tools to circulate silently in many regions of the world and this may explain our observations of seroconversion in sentinel chickens in the UK. It is also important to emphasize that similar studies using sera from sequentially bled sentinel chickens in Italy, known to circulate WNV but with no associated disease, have been carried out and will report similar findings to those reported herein (manuscript submitted for publication).
Our observations support and extend the findings of others that although mosquitoes are important vectors in disease transmission, other modes of transmission and persistence may also be important in the transmission and circulation of WNV and other arboviruses. We now need to understand why in most cases, WNV can disperse very successfully without causing overt disease but in other situations it can cause significant epidemic outbreaks involving substantial morbidity and mortality.
Materials and methods
Three groups of chickens were hatched in early April, mid May and mid June 2004 respectively on a farm in Cambridgeshire and reared outdoors. Individual sera were collected from birds at various ages from 4 days to 20 weeks. The last samples (20 weeks) were collected at the end of October 2004, when outdoor temperatures had dropped sufficiently to reduce insect-biting activity in the UK to relatively low levels. Groups of these animals were monitored periodically for the presence, in the sera, of neutralizing antibodies to WNV, USUV and SINV. For obvious technical reasons, only very small quantities of serum were obtainable from the very young chicks, limiting the scope of their investigation. Another group of chickens was hatched and reared indoors, and 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 