nsP4 is a major determinant of Alphavirus replicase activity and template selectivity

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis-active sequences. Here, we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123, while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s, while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV, or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. </p

Cross-utilisation of template RNAs by alphavirus replicases

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.</p

Peripapillary intrachoroidal cavitation at the crossroads of peripapillary myopic changes

AIM: To analyze the prevalence of peripapillary intra-choroidal cavitation (PICC) in eyes with gamma peripapillary atrophy (γPPA), in eyes with peripapillary staphyloma (PPS) and in those combining γPPA and PPS and to analyze border tissue discontinuity in PICC. METHODS: This prospective cross-sectional non interventional study included highly myopic eyes. Non-highly myopic eyes were used as control. Radial and linear scans centered on the optic nerve head were performed using spectral-domain optical coherence tomography. Variables were analyzed along the twelve hourly optical coherence tomography sections in both eyes of each subject. RESULTS: A total of 667 eyes of 334 subjects were included: 229 (34.3%) highly myopic eyes and 438 (65.7%) non highly myopic eyes. The mean age of the highly myopic group was 48.99±17.81y. PICC was found in a total of 40 eyes and in 13.2% (29/220) of highly myopic eyes. PICC was found in 10.4% (40/386) of eyes with γPPA, in 20.5% (40/195) of eyes with PPS and in 22.7% (40/176) of those combining γPPA and PPS. All the eyes with PICC showed the co-existence of γPPA and PPS whereas none of the eyes presenting only one of these entities exhibited PICC. A border tissue discontinuity in the γPPA area was found in all eyes with PICC. CONCLUSION: We confirm the presence of a border tissue discontinuity in the γPPA area of all eyes with PICC. These findings suggest the involvement of mechanical factors in the pathogenesis of PICC which may contribute to PICC-related visual field defects

The role of DNA microarrays in Toxoplasma gondii research, the causative agent of ocular toxoplasmosis

Ocular toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, is the leading cause of retinochoroiditis. Toxoplasma is an obligate intracellular pathogen that replicates within a parasitophorous vacuole. Infections are initiated by digestion of parasites deposited in cat feces or in undercooked meat. Parasites then disseminate to target tissues that include the retina where they then develop into long-lived asymptomatic tissue cysts. Occasionally, cysts reactivate and growth of newly emerged parasites must be controlled by the host’s immune system or disease will occur. The mechanisms by which Toxoplasma grows within its host cell, encysts, and interacts with the host’s immune system are important questions. Here, we will discuss how the use of DNA microarrays in transcriptional profiling, genotyping, and epigenetic experiments has impacted our understanding of these processes. Finally, we will discuss how these advances relate to ocular toxoplasmosis and how future research on ocular toxoplasmosis can benefit from DNA microarrays

Controlled mixing in a dynamically structured, pulsed fluidized bed

Vibration and pulsation of the inlet flow are two ways to enhance the performance of fluidized bed reactors and dryers by homogenizing mixing [1]. Furthermore, these perturbations could create a coherent, and sometimes responsive, macroscopic flow structure. However, mixing and solid-gas-contact patterns in such structured systems are still poorly known. In this work we discuss the circulation and mixing behavior in dynamically structured, pulsed beds, and compare them with unstructured pulsed beds and traditionally operated beds, without the use of pulsation. There is scant knowledge on lateral particle mixing in pulsed or vibrated beds. Pulsation and vibration both improve flowability by breaking up long force chains and clusters that restrict flow; therefore, one expects dispersion to improve under an oscillatory flow with the same average superficial velocity. However, a dynamically structured bubbling bed emerges in a different regime. The bed fluctuates around the minimum fluidization conditions and the response to cycles of fluidization and collapse results in a denser system than both the operation at constant flow or a traditional (unstructured) use of pulsation. In an ordered state, bubbles self-organize into a regular triangular lattice [2], but it is unclear how the lattice drives overall solid circulation, mixing and gas-solid contact. In this contribution we study structured and unstructured bubbling beds of Geldart B particles for various oscillating frequencies experimentally (Figure 1a), and quantify the associated degree of mixing with an effective lateral dispersion coefficient extracted from CFD-DEM simulations, validated in a quasi-2D system (Figure 1b). The mixing and solid circulation in a dynamically structured system exhibits major quantitative and qualitative differences. The organization of bubbles into a pattern contains the solids into a series of mixing compartments that rearrange every pulse. Unlike in a traditional operation, solid dispersion can no longer be described as a diffusive process. It is dominated by advection. A constant exchange rate between mixing compartments establishes a long-range lateral circulation, which depends on the bed dimensions. Solid mixing remains confined to a single type of loop fixed around each compartment and is superimposed to short-range velocity fluctuations caused by the oscillation. The scale of the mixing loops is constant and independent of the bed dimensions (Figure 1c), but it can be externally controlled by the amplitude and frequency of the gas flow oscillations. Here, we report exchange rates between the phases and across compartments, along with the values for an effective lateral dispersion coefficient in dynamically structured beds of Geldart B particles. This information can be used to inform surrogate models and enable the design and scale-up of larger devices that, besides homogenization, present unique advantages, namely scalability and external control. By modulating changes in gas velocity, one can fix the volume of the mixing compartments and, via external control, tailor transport rates and the stresses sustained by the powder. The ability to mix at very low gas velocities in a homogeneous and controllable way opens new ways to tackle heat and mass transfer management, as well as keep elutriation and attrition in check, which are particularly relevant issues for highly exothermic processes, or thermally sensitive and high-value materials

Solids lateral mixing and compartmentalization in dynamically structured gas–solid fluidized beds

An adequate use of gas pulsation can create an ordered, dynamically structured bubble flow in a bed of Geldart B particles. A structured bed is more homogeneous, responds to external control and is scalable. While earlier studies have focused on describing the self-organization of the gas bubbles, the solid mixing and gas-solid contact patterns have remained unclear. In this work, the solids circulation and mixing behavior in structured and unstructured beds at various pulsation frequencies are compared with a traditional fluidized bed operation. The degree of lateral mixing is hereby quantified through an effective lateral dispersion coefficient extracted from CFD-DEM (discrete element modelling) simulations in a thin fluidized bed system. Mixing shows major quantitative and qualitative differences amongst the investigated cases. The coordinated motion of the gas bubbles wraps the solid flow into a series of compartments with minimal interaction, whereby effective lateral dispersion coefficients are an order of magnitude lower than in an unstructured operation. More importantly, unlike a traditional bed, dispersion in a structured bed is driven by advection and is no longer a diffusive process. Compartmentalization decouples the time scales of micro- and macromixing. Every pulse, the compartments rearrange dynamically, causing a level of local axial mixing that is scale-independent. While further work is necessary to fully understand the compartmentalization at a larger scale, the circulation described here indicates that a dynamically structured bed can provide a tight control of mixing at low gas velocities and a narrower distribution of stresses in the solid phase compared to traditional devices

Analysis of Peripapillary Intrachoroidal Cavitation and Myopic Peripapillary Distortions in Polar Regions by Optical Coherence Tomography

info:eu-repo/semantics/publishe