ion of two strains of WNV maximized the data as we have previously demonstrated differences in sensitivity to neutralization of virus infectivity between different strains of WNV . As shown in Fig. 1, the sera from 6/10 and 8/10 of the four-day old chicks neutralized WNV-DAK and WN-Is respectively, presumably reflecting the presence of maternal antibody in the hatched chicks. For USUV, 5/10 newly hatched chick sera contained detectable neutralizing antibody but they were not necessarily the same chicks that produced antibody against WNV, demonstrating the specificity of the neutralization test. However, by the time the chicks were 10 days old, the proportion of maternally derived neutralization positive sera against the two strains of WNV and against USUV had dropped to 2/20, 0/20 and 2/20 respectively and at days 21 and 46 the figures remained low, ie 3/20, 0/20 and 0/20 at day 21, but by day 46 the figures showed evidence of increasing, ie 1/10, 3/10 and 2/10. In the case of SINV, 4/10 four-day old chicks were positive. This figure then dropped to 4/20 ten-day old chicks and 0/20 chicks by day 21 and was still zero at day 46. From this time onwards, the proportion of WNV positive sera increased noticeably, until by October 8/8, and 7/8 of the sera were positive for WN-DAK and WN-Is respectively. In many cases the titres of these sera were noticeably higher than those recorded in previous months. In contrast, the proportions of seropositive chickens for USUV and SINV remained lower than those observed for WNV, once again demonstrating that the PRNT can discriminate between WNV and USUV. It is important to note that the major increases in WNV-antibody positive sera were detected in samples collected from late July to the end of September, regardless of the date of hatching of the chicks. Moreover, the results in Fig. 1 emphasise the importance of using a highly neutralization sensitive strain of virus, in this case the WN-DAK strain.
Figure 1. Neutralization results (PRNT50) obtained for all sentinel chicken sera 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 w