The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI ؊ mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI ؊ mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI ؊ virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2␣. WT SV5 was a poor activator of the eIF-2␣ kinase protein kinase R (PKR). By contrast, the P/V-CPI ؊ mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI ؊ mutant was restored to ϳ50% that of control HeLa cells. By contrast, the rates of P/V-CPI ؊ viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5-and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI ؊ mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V-or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI ؊ mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

Objective. To assess the impact of an advanced cardiac life support (ACLS) simulation on pharmacy student confidence and knowledge. Design. Third-year pharmacy students participated in a simulation experience that consisted of team roles training, high-fidelity ACLS simulations, and debriefing. Students completed a pre/postsimulation confidence and knowledge assessment. Assessment. Overall, student knowledge assessment scores and student confidence scores improved significantly. Student confidence and knowledge changes from baseline were not significantly correlated. Conversely, a significant, weak positive correlation between presimulation studying and both presimulation confidence and presimulation knowledge was discovered. Conclusions. Overall, student confidence and knowledge assessment scores in ACLS significantly improved from baseline; however, student confidence and knowledge were not significantly correlated.

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell (DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 (SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixedculture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8؉ and CD4 ؉ allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.Dendritic cells (DC) are important professional antigenpresenting cells that are capable of recognizing microbial products and promoting innate and adaptive immune responses (5, 38). Upon sensing viral components, DC are triggered to undergo a conversion from a phenotype termed immature into a form that is capable of activating naïve-T-cell functions, such as proliferation, cytokine secretion, and cytolytic activity (5, 38). These DC activation events include increased cell surface expression of costimulatory molecules, such as CD40, CD80, and CD86, as well as increased capacity to secrete immunomodulatory cytokines, such as interleukin-12 (IL-12), IL-6, and tumor necrosis factor alpha (TNF-␣) (5, 14, 38). As such, the ability of DC to be activated in response to infection has important consequences for development of immunity against viruses. Here we describe engineering the paramyxovirus simian virus 5 (SV5) to be more effective at activating human DC to stimulate T-cell function in vitro.Many viruses are poor activators of DC, with infected immature DC showing only limited production of cytokines or upregulation of costimulatory molecules (28). Examples include herpes simplex virus, cytomegalovirus (CMV), vaccinia virus, measles virus (MeV), influenza virus, Lassa fever virus, poliovirus, and Ebola virus (4,9,17,18,20,36,43). This lack of activation of infected DC can result from virus-induced suppression of signaling pathways. Fo...

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