We previously reported evidence of West Nile virus (WNV) circulation in UK birds, probably introduced by migratory birds from overseas. We now demonstrate WNV-specific seroconversion in sentinel chickens raised on an English farm. Maternal neutralizing antibodies to WNV in hatchlings declined within three weeks. During the following months, healthy chickens developed WNV neutralizing antibodies that were confirmed by immunoblotting and indirect immunofluorescence tests using WNV antigens. The proportion of seropositive chickens was higher for WNV than for Usutu virus or Sindbis virus. Attempts to isolate infectious virus or to detect viral RNA in the sera, failed.
West Nile virus (WNV) and Usutu virus (USUV) are antigenically closely related mosquito-borne members of the genus Flavivirus. Sindbis virus (SINV) is an unrelated mosquito-borne member of the genus Alphavirus. These
Results and discussion
Plaque reduction neutralization tests on sentinel chicken sera
All sera were tested for the presence of virus-specific neutralizing antibodies by plaque reduction neutralization tests (PRNT50) against two strains of WNV, a strain isolated from Israel (WN-Is) and a highly neutralization-sensitive strain isolated in the Central African Republic (WN-DAK). For these tests the sera were diluted in twofold steps from 1/10 dilution, the minimum possible, owing to the limited volume of serum. The World Health Organization (WHO) standard method based on 50% plaque reduction was employed to detect positive virus-neutralizing sera. Following the WHO recommendations, the highest dilution of serum that produced 50% reduction of plaque numbers (estimated 50 plaques per dish in control dishes) was taken as the endpoint for individual sera. In addition we also included USUV and SINV in this analysis because it extended the range of viruses analysed and also served as a form of internal control for virus-specificity. The results of plaque reduction neutralization tests (PRNT50) on the individual sera are presented in Fig 1. The inclusion 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