 the fluorescent infected cells. Fig. 3 illustrates the typical appearance of groups of WNV-infected fluorescent cells produced by PRNT-positive chicken antisera. In Fig. 3, non-infected cells are not stained by the antibody. Moreover, PRNT-negative control sera produced no fluorescence (not shown).
Figure 3. Indirect immunofluorescence microscopy performed on chicken sera with a neutralization titre of 1/80 (diluted 1:1000 in PBS) on West Nile virus infected Vero cells (Bar 50μm).
Attempts to isolate infectious virus from seropositive chickens
The sera from 46-day old, and older chickens (a total of 46 sera) and five 10% brain suspensions harvested from chickens that were seropositive in a WNV-PRNT-analysis were inoculated directly onto monolayers of SW13 cells which were then incubated at 37°C for 14 days. The supernatant medium from each sample was then inoculated onto fresh monolayers of SW13 cells and these were incubated for a further 14 days. Each monolayer from the first inoculation and subsequently each monolayer from the second inoculation was tested for the presence of flavivirus antigens using a flavivirus pan-specific monoclonal antibody (MAb 813), followed by fluorescein-conjugated mouse antiglobulin, as described previously . Although some monolayers deteriorated during the incubation period, suggesting that cytopathic effects (cpe) were developing, we were unable to demonstrate the presence of an infectious flavivirus in any of the tested samples either by indirect immunofluorescence microscopy using flavivirus-group-reactive MAb 813 or by RT-PCR using flavivirus-group-reactive primers . Moreover, the mild cpe that was observed in some cultures was not observed during subsequent passage of harvested material, ruling out the possibility of a different cytopathic arbovirus being isolated.
Although it was not possible to obtain sequential samples of serum from each animal the PRNT studies with groups of newly hatched, juvenile and young adult chickens produced evidence that these animals had been exposed either to infectious WNV or a very closely related virus during the summer of 2004. The supplementary positive results obtained by immunoblotting and immunofluorescence microscopy also support this conclusion by demonstrating specific immune responses against the WNV envelope protein. Many of the newly hatched chicks had antibodies that neutralized WNV and to a lesser extent USUV and SINV. It is well known that maternal antibodies are concentrated in the fertile egg and that the quantity of these antibodies declines rapidly in the newly hatched chick . Our PRNT results are totally consistent with these known observations and they demonstrate that WNV, USUV and SINV (at least), or closely related viruses, must have circulated on the farm in the previous year. The decline in antibody prevalence during the first few weeks after hatching is also consistent with the idea that WNV is unlikely to have been circulating significantly during the first three or four months of the year, i.e. late winter and early spring. The detection of a significant increase in the numbers of serologically positive chickens from July onwards can probably be explained most appropriately as due to this being the time immediately after the arrival of migratory birds from Africa, Europe and the Middle East and also being the warmest time of the year when mosquitoes would be relatively active and therefore capable of transmitting arboviruses, even in England. Some chickens seroconverted even though they had been kept indoors for most of their lives. However, the ventilation system for the building in which they were housed is positive and not filtered inwards, moreover, adjoining rooms contained wild birds, inferring that the chickens could have been exposed to aerosols containing virus. In addition to virus transmission by blood transfusion and organ transplantation, there is now compelling evidence that arboviruses such as WNV may be transmitted between vertebrates using a variety of mechanisms other than direct transmission by arthropod