Characterization and Control Strategies of Low Pathogenic Avian Influenza Virus H9N2

The avian influenza virus, subtype H9N2 subtype, defined as having a low pathogenicity, causes extensive economical losses in commercial flocks, probably due to management and synergism with other pathogens. AIV H9N2 was first identified in Israel in the year 2000, and since then it became endemic and widespread in Israel. Control by vaccination of commercial flocks with an inactivated vaccine has been introduced since 2007. In face of the continuous H9N2 outbreaks, and the application of the vaccination policy, we aimed in the present study to provide a method of differentiating naturally infected from vaccinated animals (DIVA). The aim of the assay would be detect only antibodies created by a de-novo infection, since the inactivated vaccine virus is not reproducing, and might provide a simple tool for mass detection of novel infections of commercial flocks. To fulfill the overall aim, the project was designed to include four operational objectives: 1. Evaluation of the genetic evolution of AIV in Israel; 2. Assessment of the diagnostic value of an NS1 ELISA; 3. NS1 ELISA as evaluation criteria for measuring the efficacy of vaccination against H9N2 AIV; 4. Development of an AIV H9 subtype specific ELISA systems.  Major conclusion and implications drawn from the project were: 1. A continuous genetic change occurred in the collection of H9N2 isolates, and new introductions were identified. It was shown thatthe differences between the HA proteins of viruses used for vaccine productionand local fieldisolatesincreasedin parallelwith the durationand intensity ofvaccine use, therefore, developing a differential assay for the vaccine and the wild type viruses was the project main aim.  2. To assess the diagnostic value of an NS1 ELISA we first performed experimental infection trials using representative viruses of all introductions, and used the sera and recombinant NS1 antigens of the same viruses in homologous and heterologous NS1 ELISA combination. The NS1 ELISA was evidently reactive in all combinations, and did not discriminate significantly between different groups.   3. However, several major drawbacks of the NS1 ELISA were recognized: a) The evaluation of the vaccination effect in challenged birds, showed that the level of the NS1 antibodies dropped due to the vaccination-dependent virus level drop; b) the applicability of the NS1-ELISA was verified on sera of commercial flocks and found to be unusable due to physico-chemical composition of the sera and the recombinant antigen, c) commercial sera showed non-reactivity that might be caused by many factors, including vaccination, uncertainty regarding the infection time, and possibly low antigen avidity, d) NS1 elevated antibody levels for less than 2 months in SPF chicks. Due to the above mentioned reasons we do not recommend the application of the DIVA NS1 ELISA assay for monitoring and differentiation AIV H9N2 naturally-infected from vaccinated commercial birds.