Fig. 3

Next-generation sequencing (NGS) analysis of RNAs present within wild-type or HA(−)virions. a SDS-PAGE of purified wild-type and HA(−) virions treated with or without dithiothreitol (DTT) followed by Oriole staining. MW molecular weight marker. b Read coverage over the influenza virus genome. RNAs were extracted from purified virions and subjected to NGS analysis. Read sets from the wild-type and HA(−) viruses were mapped onto the influenza A virus genome and 18S and 28S rRNA sequences. Coverage depth is indicated as a ratio (coverage depth per nucleotide/total nucleotides read). The read sets of wild-type and HA(−) viruses are shown in the upper and lower columns, respectively. Nucleotide positions are indicated in the cRNA-sense for each vRNA. c An equal amount (5 ng) of RNA was subjected to northern blot analysis. The HA, NA, and NS vRNAs, 18S rRNA, and three different 28S rRNA (representing nucleotide positions 1081–2042, 3541–4679, and 1081–4679) specific riboprobes were used for detection. Intracellular RNAs from uninfected MDCK cells were used as a control. d Purified wild-type virions, HA(−) virions, and cell lysates were subjected to western blot analysis using anti-RPS3, anti-RPS5, and anti-RPSL7 antibodies and an anti-whole influenza virion polyclonal antibody