 tested against each of the four viruses and grouped according to age at time of sampling. All chicken sera were coded and all tests were carried out on these coded sera. The codes were revealed only after the results had been presented. The percentages of positive sera recorded at each antibody dilution are shown using a colour scheme; White <1/10; Green 1/10; Yellow 1/20; Orange 1/40; Red 1/80.
Interestingly, thirteen chicks sampled at 9 weeks post-hatching had been kept indoors for the entire period since hatching. Nevertheless, specific antibody responses to WNV in particular were detected in these chicks, viz., 12/13, and 6/13, for WNV-DAK and WNV-Is respectively and 1/13, and 3/13 for USUV and SINV respectively. However, whilst these chicks had been kept indoors, the airflows to their rooms came directly from the outside without isolation by filtration.
Western blot analysis
Western blot analysis of sera was performed, to confirm the presence of antibodies to viral envelope glycoprotein, 7% w/v polyethylene-glycol-precipitated virus was purified by centrifugation at 100,000 g on a continuous sucrose gradient (15% to 60% w/w sucrose in Tris buffer at pH 7.4). Serial fractions were collected from the sucrose gradient and subjected to immunoblotting using a high titre hyperimmune mouse antiserum prepared against WNV. Whilst many fractions contained large quantities of the recognized structural WNV proteins, the fraction collected from the 60% sucrose cushion produced a very strong and relatively clean band at 51kDa on the western blot, corresponding to the viral envelope (E) protein of WNV as confirmed (data not shown but equivalent appearance to track 1 of Fig 2) using an E protein-specific monoclonal antibody (MAb) designated MAb 528 . WNV-neutralization positive chicken sera were tested for the presence of E protein-specific antibodies using the gradient-purified fraction obtained from the 60% sucrose cushion (Fig. 2). As can be seen, tracks 3 to 10 produced increasingly intense labelling of the E protein when 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