Reverse Genetics of H9N2 Influenza Virus

Using standard reverse genetics an H9N2 virus of the G1 lineage, A/chicken/Pakistan/UDL-01/2008 (UDL1/08), was rescued from bidirectional reverse genetics plasmids as previously described47 (link). All other H9N2, H5N1 or H7N1 viruses (including stocks of UDL1/08 and its escape mutants used for downstream antigenic analysis) used were rescued using the HA and NA of the named viral strain with the remaining gene segments from A/Puerto Rico/8/1934 (PR8). Multiple, independently rescued virus stocks of UDL1/08 were propagated in 10 day old specific pathogen free (SPF) embryonated chicken eggs and titrated by plaque assay or TCID₅₀ on MDCKs. To guarantee the genetic purity of all the escape mutants used for downstream antigenic analysis identified amino acid substitution in HA were reintroduced into the reverse genetics plasmids by site directed mutagenesis and mutants were reconstituted through virus rescue as H9N2:PR8 2:6 recombinants. Full length sequencing (see below) of the HA and NA gene segments of all reconstituted escape mutants was performed to verify no additional mutations or reversions appeared after rescue of the reconstituted escape mutants. Viruses were purified by ultracentrifugation through a continuous 30–60% w/v sucrose gradient.
MDCK cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37^oC, 5% CO₂. Human embryonic kidney 293T (293T) cells were maintained in DMEM containing 2% FBS and 1 mM sodium pyruvate at 37^oC, 5% CO₂.