hen tested at a 1/100 dilution. These tracks corresponded to sera with neutralization titres of 1/10 (tracks 3 and 4), 1/20 (tracks 5 and 6), 1/40 (tracks 7 and 8), and 1/80 (tracks 9 and 10). Track 1 contained a positive control hyperimmune mouse antiserum against WNV and Track 2 contained a negative control hyperimmune antiserum raised against SINV (both positive and negative control sera were diluted 1/500). Chicken sera that failed to neutralize WNV were negative in immunoblots when tested at 1/100 dilution. The bands at 45, 42 and 36 kDa in track 9 of Fig 2 probably correspond to breakdown products of the E protein. They were only detected by chicken sera with the highest neutralization titres (tracks 9 and 10 of Fig 2). It is also important to note that separate immunoblots that employed non-purified WNV-infected cell lysates as substrate, and the 1/40 or 1/80 neutralization positive chicken sera, performed exactly as published previously , i.e., these neutralization-positive sera highlighted the viral E, NS1, NS3 and NS5 proteins, confirming that WNV must have replicated in the chickens to elicit immune responses against the non-structural viral proteins.
Figure 2. Western blot using gradient-purified West Nile virus antigen and sera (diluted 1/100) from sentinel chickens. Tracks 1 and 2, hyperimmune mouse serum prepared against WNV (positive control) or SINV (negative control) respectively; tracks 3 to 10 pairs of chicken sera that produced neutralization titres of 1/10, 1/20, 1/40, and 1/80 respectively.
Indirect immunofluorescence tests
Indirect immunofluorescence microscopy was carried out using a PRNT-positive chicken serum (track 9 from Fig. 2) diluted 1/100 in PBS as described previously . Vero cells infected with WNV for 48 hours at 37°C were washed in PBS and fixed in acetone. The diluted chicken serum was allowed to react with the acetone-fixed infected cells for 1 hour at 37°C before the cells were washed in PBS. Fluorescein-conjugated anti-chicken serum diluted 1/400 in PBS was used to identify 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 exp