Tectonic evolution of Variscan Iberia: Gondwana–Laurussia

An integrated interpretation of the late Paleozoic structural and geochronological record of the Iberian Massif is presented and discussed under the perspective of a Gondwana-Laurussia collision giving way to the Variscan orogen. Compressional and extensional structures developed during the building of the Variscan orogenic crust of Iberia are linked together into major tectonic events operating at lithosphere scale. A review of the tectonometamorphic and magmatic evolution of the IberianMassif reveals backs and forths in the overall conver- gence between Gondwana and Laurussia during theamalgamation of Pangea in late Paleozoic times. Stages dom- inated by lithosphere compression are characterized by subduction, both oceanic and continental, development of magmatic arcs, (over- and under-) thrusting of continental lithosphere, and folding. Variscan convergence re- sulted in the eventual transference of a large allochthonous set of peri-Gondwanan terranes, the Iberian Allochthon, onto the Gondwana mainland. The Iberian Allochthon bears the imprint of previous interaction be- tween Gondwana and Laurussia, including their juxtaposition after the closure of the Rheic Ocean in Lower De- vonian times. Stages governed by lithosphere extension are featured by the opening of two short-lived oceanic basins that dissected previous Variscan orogenic crust, first in the Lower-Middle Devonian, following the closure of the Rheic Ocean, and then in the early Carboniferous, following the emplacement of the peri-Gondwanan allochthon. An additional, major intra-orogenic extensional event in the early-middle Carboniferous dismem- bered the Iberian Allochthon into individual thrust stacks separated by extensional faults and domes. Lateral tec- tonics played an important role through the Variscan orogenesis, especially during the creation of new tectonic blocks separated by intracontinental strike-slip shear zones in the late stages of continental convergence

ADMINISTRACIÓ D'EMPRESES I ORGANITZACIÓ DE LA PRODUCCIÓ (Examen 2n Quadr.)

Grup mat

ADMINISTRACIÓ D'EMPRESES I ORGANITZACIÓ DE LA PRODUCCIÓ (Examen 2n Quadr.)

Grup mat

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV

Secondary RNA structure prediction of Domain II and Domain VI of the IRES in Brunenders and CAVA using the MFOLD program developed by M. Zuker.

<p>Circled nucleotides (at positions 133, 142, 146, and 163 in domain II and at positions 597, 609 in domain VI) represent nucleotide changes between CAVA and Brunenders. The last remaining CAVA IRES mutation (nt579) lies outside of any IRES domains and in the spacer region between Domains V and VI. After serial passage at increasing temperature (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005483#ppat.1005483.g006" target="_blank">Fig 6</a>) nucleotide 127 (indicated by square box) mutated in both passaging experiments (n = 2) from U to C, forming a C–G base pair with CAVA mutation nt 163. The CAVA mutations induce a change in predicted secondary structure and increased free energy (ΔG) for domain II, whilst for domain VI only the free energy is affected.</p

<i>In vivo</i> immunogenicity of the CAVA vaccine strains as compared to cIPV.

<p>Groups of ten (n = 10) rats were immunized with a full dose (FD: 100% 40:8:32 DU/dose or 150% 60:12:48 DU/dose) or a 1:2, 1:4 or 1:16 dilution of the full dose. Poliovirus type 1, 2 and 3-specific neutralizing antibody titers were determined by Sabin Virus Neutralizing Assay at day 21 post immunization. Each dot represents one individual animal; the connected line represents the geometric mean at each dose. Relative potency estimates and 95% confidence intervals of the difference between the CAVA vaccine strain and cIPV reference based on the number of seroconverting animals are depicted in the table, horizontal dotted line represents the seroconversion limit for each assay.</p

Multiple replication kinetics of infections in PER.C6 cells with a cell density of 10⁷ cells/ml at an MOI of 1–2, at 30°C and 37°C, harvested at 0–48 hours post infection.

<p>Panel A) Average and standard deviation of two (n = 2) replication kinetic curves of the Brunenders strain versus 3 selected clones (clone A, B and C) derived after passage with impaired growth at 37°C. Panel B) Average and standard deviation of three (n = 3) independent infections of Brunenders and the CAVA backbone, which contained all mutations from Clones A, B and C combined. Panel C) Average and standard deviation of three (n = 3) independent infections of the Brunenders strain versus the CAVA vaccine strains (CAVA-1 Mahoney, CAVA-2 MEF-1 and CAVA-3 Saukett).</p

Reversion of the temperature sensitive phenotype by stepwise increase of the infection temperature.

<p>Serial passage was performed using CAVA-1 Mahoney in suspension PER.C6 cells infected at a cell density of 10⁷ cells/ml at low MOI (0.01) and harvested at 3–4 days post infection. Temperature was gradually increased (33–37°C) or temperature was kept constant at 30°C for the control viruses. Panel A depicts the viral titers at each passage when titrated and incubated at 30°C or 37°C for the two independent experiments (n = 2). Panel B lists the reverting CAVA mutations of the viruses at passage number 6 where the nucleotide number refers to the position from the start of the viral genome.</p

Schematic overview of the viruses described here and their incorporated mutations.

<p>Black vertical lines represent the synonymous CAVA mutations whilst red vertical lines represent non-synonymous CAVA mutations, dispersed over the poliovirus genome; a detailed description of the individual mutations is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005483#ppat.1005483.s005" target="_blank">S1 Table</a>. 5’UTR = 5’ Untranslated Region, 3’UTR = 3’Untranslated Region.</p

Electron micrographs of two representative cells per panel after infection of suspension PER.C6 cells with a cell density of 10⁷ cells/ml at an MOI of 1, harvested between 24–48 hours post infection.

<p>Panel A) PBS (Mock) infected cells, Panel B) Mahoney infection at 37°C, Panel C) CAVA-1 Mahoney at 37°C, Panel D) CAVA-1 Mahoney at 30°C.</p