Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus

Abstract

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.

FIG. 1.

Comparison of anti-NS1 antibody responses in sera of AI-vaccinated and AI virus-infected chickens. Western blots show seroconversions to positivity for antibodies to the GST-NS1 (A) and the MBP-NS1 (B) fusion proteins of chicken sera (pool of four to five serum samples) collected 4 weeks after infection or vaccination with the H5 or the H7 subtype of AI virus. One group of chickens received an i.n. infection with TW/68 (LPAI) virus, followed by an i.n. boost with TO/66 (HPAI) virus 4 weeks later (B, lane 6). Chickens immunized with the commercial (H7N2) or gradient-purified (H5N9) inactivated vaccines received three s.c. inoculations (A, lane 5; B, lanes 4 and 5). Sera from sham-vaccinated or normal chickens were included as controls (A, lane 2; B, lane 1). The GST-NS1 fusion protein was detected by immunoblotting with chicken anti-GST antibody (A, lane 1).