Avian Influenza Virus Isolation and Propagation in Chicken Eggs

The avian influenza (AI) virus is usually isolated and propagated by inoculating either swab or tissue samples from infected birds into the chorioallantoic sac of embryonating chicken eggs. This is the accepted method, but occasionally an isolation may only be successful when inoculated either into the yolk sac or onto the chorioallantoic membrane of embryonating chicken eggs. Chorioallantoic fluid is harvested from eggs with dead or dying embryos and is tested for the presence of hemagglutinating antigen. If hemagglutination-positive, this indicates that the isolate may be the AI virus. The presence of the AI virus may be confirmed by either an agar gel immunodiffusion (AGID) assay, RT-PCR specific for AI virus, or a commercially available immunoassay kit specific for type A influenza. Instructions for AI virus primary isolation and propagation, preparing antigen for an AGID test, setting up an AGID test, and interpreting results are given.