Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Take an influenza virus suspension, containing both wild-type and mutant strains.

A viral envelope glycoprotein termed hemagglutinin, or HA, binds to sialic acid on the host cell surface, mediating viral entry.

Add a neutralizing antibody in decreasing concentrations that binds to HA, blocking the sialic acid binding site.

Mutant strains, carrying mutations in the HA that help avoid antibody neutralization, are termed escape variants.

Inject the mixture into pathogen-free embryonated chicken eggs, delivering the viruses into the allantoic fluid, and incubate.

Antibody-neutralized viruses cannot enter the cells of the chorioallantoic membrane. Non-neutralized mutant viruses enter the cells, replicate, and are released into the allantoic fluid.

Harvest the virus population-containing allantoic fluid.

Introduce an HA-specific antibody in decreasing concentrations, which neutralizes the other strains except the escape variants.

Introduce chicken red blood cells or RBCs. Non-neutralized escape variants bind to the cells, causing the clumping of RBCs — termed hemagglutination, confirming escape variant generation.