(A) Schematic diagram NS antigenomic RNA (positive sense, cRNA) depicting the WT H1N1pdm and the attenuating NS mutations designed to create NS-LAIV candidates. NS1 protein is directly translated from the full length mRNA and is shown on the top of the gene; NEP protein is translated from spliced mRNA and is illustrated below the gene. Selected amino acid positions are labeled for NS1 and NEP. LAIV candidates NS1-73, NS1-126 express truncated NS1 but intact NEP; NSΔ5 expresses NS1 and NEP proteins each having a five amino acid in-frame deletion. (B) Genomic amplification of the WT and NS-LAIV candidates. The viral genomes were amplified by M-RTPCR and the length of the NS vRNA amplicons was examined by subsequent agarose gel electrophoresis and staining with ethidium bromide. Lane 1, WT; Lane 2, NS1-73; Lane 3, NS1-126; Lane 4, NSΔ5; lane MW, 1Kb+ DNA ladder (Invitrogen). The viral gene segment amplified is listed to the left and length of DNA marker ladder is shown on the right.