• The increased sensitivities afforded by EPX-mAb demonstrate that increases in lung infiltrating eosinophils and evidence of eosinophil degranulation are surprisingly characteristic features of biopsies from ALI patients relative to control subjects • Assessments of lung biopsies from ALI patients following EPX-mAb immunohistochemical staining may provide a potential basis to identify a subset of thẽ 40% of ALI patients who will not survive their hospitalization. Figure 4 EPX-mAb immunohistochemistry provides a quantitatively significant strategy to distinguish acute lung injury patients that survive their hospitalization vs. those patients that did not survive. Sections from acute lung injury patients were subjected to EPX-mAb immunohistochemistry prior to evaluation for evidence of eosinophil degranulation as described in the Materials amd Methods and the legend of Figure 3 . Eosinophil degranulation scores were determined by individual investigators (n = 3) as the average numerical score resulting from the examination of 10 randomly selected high powered fields (hpf -400x); investigators were blinded to both the clinical outcome and the scores of the fellow evaluator. The scatter plots presented represent values for each individual ALI patient stratified based on hospital survival. Patient eosinophil degranulation values are expressed as the mean of the average eosinophil degranulation score from all three evaluators. The error bars associated with each patient data point is the SEM linked with the mean value derived from each of the three evaluators. The mean for each cohort is presented as a horizontal bar. *p < 0.01 Overlapping signals for translational regulation and packaging of influenza A virus segment 2 Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1–4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1–4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging. Influenza A virus (IAV) is a major pathogen, capable of infecting a number of species including humans, birds, swine and horses. Its genome is contained on eight segments of negative sense viral RNA (vRNA), individually complexed with the trimeric viral polymerase (PB2, PB1 and PA) and nucleoprotein (NP) to form ribonucleoprotein (RNP) particles (1) . On infection, the RNPs migrate to the nucleus where the polymerase initially transcribes the vRNA templates to produce mRNA, and later replicates the genome using positive sense cRNA intermediates (2) . Subsequently, new vRNAs are exported from the nucleus (as RNPs) and packaged into progeny virus particles at the plasma membrane. As each segment encodes at least one essential gene product, a viable virus particle must contain one copy of each segment, which is facilitated via specific cis-acting packaging signals present in the terminal non-coding and coding regions of each vRNA (3) . IAV strains also show considerable variation in pathogenicity, and the molecular mechanisms underlying this have not been fully elucidated. Segment 2 encodes PB1, the core component of the viral polymerase, which has been linked to inter-strain differences in pathogenicity and host range (4, 5) . However, the single mRNA species transcribed from the segment also encodes two further proteins that are non-essential for virus replication: PB1-F2 and PB1-N40 (6, 7) . PB1-F2 is encoded by the+1 open reading frame (ORF) relative to PB1 and is initiated from AUG4 ( Figure 1A ). Depending on virus strain, the PB1-F2 ORF is up to 90 codons long, but in many viruses (including the recent pandemic H1N1 virus, where the gene is effectively absent), is truncated to variable extents by one or more stop codons (8, 9) . PB1-F2 polypeptides of 79 amino acids or longer can localize to mitochondria and the protein has been associated with pro-apoptotic and pro-inflammatory effects (6, (9) (10) (11) (12) . A proportion of the protein also localizes to the nucleus where it interacts with PB1 and may influence polymerase activity (13, 14) . In some strains of virus, manipulating the expression or sequence of PB1-F2 altered replication and/or pathogenicity, leading to its identification as a virulence factor (6, 10, (14) (15) (16) (17) . However, in many cases, the presence or absence of an intact PB1-F2 ORF had little or no impact on virus replication in vitro or in vivo (7, 8, 17, 18) . Overall the contribution the protein makes to IAV pathogenesis is imperfectly understood.