AUG5 may be maintained either because methionine 40 is essential for PB1 function and/or because expression of N40 supplies a selective advantage in vivo, for reasons as yet unknown. However, an isoleucine change at position 40 does not obviously inhibit PB1 transcriptase activity or inhibit virus growth in vitro or in eggs (6,7), perhaps favouring the latter hypothesis. AUG4 is presumably conserved at least in part to allow expression of PB1-F2, although it is less obvious what maintains it in the large number of viruses (9,57) that do not possess an intact F2 ORF. It does not seem to contribute to a packaging signal, so one possibility is that it is retained as a 'ribosome sink' to prevent overexpression of N40. AUG3 and the stop codon for sORF2 also seem likely to be conserved for a regulatory role: depressing PB1-F2 synthesis and/or permitting N40 expression. Since neither PB1-F2 or PB1-N40 is required for virus replication in cell culture, elucidating which (if either) of these roles is more important for maintaining virus fitness (as well as the wider question of their function in virus pathogenicity) will require either animal experiments and/or more sophisticated model systems for virus replication in vitro. Understanding the mechanisms that underlie F2 and N40 expression informs the design of virus mutants that could answer these questions.  Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood. Acute and chronic infection, especially that induced by Gramnegative bacteria is associated with increased risk of thrombosis and atherosclerotic disease [1, 2, 3, 4, 5] . Little is known about the underlying cellular mechanisms responsible for these risks. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is an antigen which initiates inflammation and innate immune responses by interacting with Toll-like receptor 4 (TLR4). TLR4 is expressed on the surface of cells, including leukocytes and platelets [6, 7, 8] . Under physiological conditions, platelets and leukocytes circulate in quiescent state and do not interact with each other. However, once activated under pathophysiological conditions such as those associated with infection, platelets change shape, secrete prothrombogenic inflammatory and cellular adhesion molecules from alpha-and densegranules which cause the platelets to adhere to each other or to leukocytes and/or vascular endothelium [9, 10, 11, 12] . The physiological consequences of stimuli associated with infection, like LPS stimulation, are acute but can be sustained. For example, half-life of platelets was shortened and the activation state of newly formed platelets from bone marrow megakaryocytes increased within seven days following a single acute intravenous injection of LPS in mice [13, 14] . However, cellular events, specifically those occurring among blood elements, contributing to the shortened half-life and increased activation state of platelets remains to be clarified.