Genome-Wide Mutagenesis Reveals That ORF7 Is a Novel VZV Skin-Tropic Factor

The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles

Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms

Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses

Plaines de grandes cultures du Calvados et de l'Orne : Prospection aérienne

Bilan scientifique DRAC Basse-Normandie2 p

Plaines de grandes cultures du Calvados et de l'Orne : Prospection aérienne

Bilan scientifique DRAC Basse-Normandie2 p

Micro-patterning of NdFeB and SmCo magnet films for integration into Micro- Electro-Mechanical-Systems

The integration of high performance RE-TM (NdFeB and SmCo) hard magnetic films into Micro-Electro-Mechanical-Systems (MEMS) requires their patterning at the micron scale. In this paper we report on the applicability of standard micro-fabrication steps (film deposition onto topographically patterned substrates, wet etching and planarization) to the patterning of 5 µm thick RE-TM films. While NdFeB comprehensively fills micron scaled trenches in patterned substrates, SmCo deposits are characterized by poor filling of the trench corners, which poses a problem for further processing by planarization. The hal-00438119, version 1- 2 Dec 2009 magnetic hysteresis loops of both the NdFeB and SmCo patterned films are comparable to those of non-patterned films prepared under the same deposition/annealing conditions. A micron-scaled multipole magnetic field pattern is directly produced by the unidirectional magnetization of the patterned films. NdFeB and SmCo show similar behavior when wet etched in an amorphous state: etch rates of approximately 1.25µm/minute and vertical side walls which may be attributed to a large lateral over-etch of typically 20 µm. Chemical-Mechanical Planarization (CMP) produced material removal rates of 0.5-3µm/min fo