Involvement of Ceramide in the Propagation of Japanese Encephalitis Virus ▿

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH- and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl β-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C6-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64 ▿

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation

Cardiac Autonomic Nervous System Activity during Slow Breathing in Supine Position

The purpose of this study is to clarify cardiac autonomic nervous system activity during slow breathing exercises in a supine position. Eighteen healthy young males were participated. Heart rate variability was measured for 5 minutes at rest, 5 minutes at slow breathing, and then 5 minutes at rest. As a result, the LF/HF ratio increased with slow breathing, but HF value did not change. We suggest that the increased LF/HF ratio may be due to increased airway resistance. Cardiac autonomic nervous system activity during slow breathing in the supine position was revealed

Baculovirus Induces Type I Interferon Production through Toll-Like Receptor-Dependent and -Independent Pathways in a Cell-Type-Specific Manner▿

Autographa californica nuclear polyhedrosis virus (AcNPV) is a double-stranded-DNA virus that is pathogenic to insects. AcNPV was shown to induce an innate immune response in mammalian immune cells and to confer protection of mice from lethal viral infection. In this study, we have shown that production of type I interferon (IFN) by AcNPV in murine plasmacytoid dendritic cells (pDCs) and non-pDCs, such as peritoneal macrophages and splenic CD11c+ DCs, was mediated by Toll-like receptor (TLR)-dependent and -independent pathways, respectively. IFN regulatory factor 7 (IRF7) was shown to play a crucial role in the production of type I IFN by AcNPV not only in immune cells in vitro but also in vivo. In mouse embryonic fibroblasts (MEFs), AcNPV produced IFN-β and IFN-inducible chemokines through TLR-independent and IRF3-dependent pathways, in contrast to the TLR-dependent and IRF3/IRF7-independent production of proinflammatory cytokines. Although production of IFN-β and IFN-inducible chemokines was severely impaired in IFN promoter-stimulator 1 (IPS-1)-deficient MEFs upon infection with vesicular stomatitis virus, AcNPV produced substantial amounts of the cytokines in IPS-1-deficient MEFs. These results suggest that a novel signaling pathway(s) other than TLR- and IPS-1-dependent pathways participates in the production of type I IFN in response to AcNPV infection