Structure probing of the MNV termination-reinitiation signal.

<p>RNA derived by transcription of p2luc-MNV-T3/<i>BamHI</i> with T3 RNA polymerase was 5′ end-labelled with [γ-³³P]-ATP and subjected to limited RNase or chemical cleavage using structure-specific probes. Sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH-) and the position of known RNase U2 and T1 cuts, determined empirically. Products were analysed on a 10% acrylamide/7M urea gel containing formamide. Data was also collected from 6% and 15% gels (gels not shown). Enzymatic structure probing was with RNases T1, U2, CL3 and CV1. Uniquely cleaved nucleotides were identified by their absence in untreated control lanes (0). The number of units of enzyme added to each reaction is indicated. Chemical structure probing was with imidazole (I, hours) or lead acetate (Pb; mM concentration in reaction). The water lane (W) represents RNA which was dissolved in water, incubated for four hours and processed in parallel to the imidazole-treated sample. The sequence of the probed RNA and the inferred secondary structure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008390#pone-0008390-g004" target="_blank">Figure 4</a>.</p

Minimal sequence requirements for MNV termination-reinitiation.

<p>A) Schematic of the p2luc-MNV reporter mRNA. The termination-reinitiation region (203 nt upstream and 52 nt downstream of the <b>UA<u>A</u></b><u>UG</u> motif) was cloned into the <i>SalI</i> and <i>BamHI</i> sites of the p2luc reporter plasmid. <i>HpaI</i> run-off transcripts for <i>in vitro</i> translation were generated using T7 RNA polymerase. The location of the T3 promoter present in the structure mapping construct p2luc-MNV-T3 is indicated. B) Deletion analysis of MNV termination-reinitiation. A series of p2luc-MNV variants were prepared with stepwise, in-frame deletions from the 5′ end of the inserted viral sequence. The wild-type (wt), premature stop (ps) and deletion mutant plasmids were linearised with <i>HpaI</i> and run-off transcripts translated in Flexi® RRL at a final RNA concentration of 50 µg/ml in the presence of [³⁵S]-methionine and 140 mM added KCl. The products were resolved by 12% SDS-PAGE and visualised by autoradiography. The number of nucleotides of viral sequence remaining up to the AUG start codon of the MNV ORF is shown below the gel. The product of the full-length or truncated versions of the rlucVP1 ORF (predicted size of MNVwt is 42 kDa) is marked rluc, and the VP2fluc product (predicted size, 62 kDa) is marked fluc. The MNV ps rluc product is the shortest (predicted size, 33 kDa). RRF denotes the relative reinitiation frequency in comparison to MNVwt (set at 100). The figure in brackets represents the ratio of the intensity of the fluc and rluc products (adjusted for methionine content and expressed as a percentage) for the MNVwt mRNA.</p

Comparison of caliciviral termination-reinitiation signals and 5′ flanking regions.

<p>The termination-reinitiation signal of influenza BM2 is also shown, as is a putative signal in the cellular gene glutamic acid decarboxylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008390#pone.0008390-Szabo1" target="_blank">[33]</a>. Confirmed and potential Motif 1 sequences are highlighted in pink and the stop-start window in blue. Potential base-pairing interactions flanking Motif 1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008390#pone.0008390-Luttermann2" target="_blank">[18]</a> are indicated in grey (or underlined in the case of the glutamic acid decarboxylase gene). Within the murine noroviruses, in reference to EU004666, base changes are highlighted in green. Abbreviations used: EBHSV, European brown hare syndrome virus; RHDV, rabbit hemorrhagic disease virus; VESV, vesicular exanthema of swine virus; FCV, feline calicivirus; SMSV, San Miguel sealion virus.</p

Mikrosare elektriko baten analisia eta diseinua La Muelan

[EU]Dokumentu honen xedea irakurleari Korronte Alternoko (AC) mikrosareen elementu nagusiei buruzko deskribapen eta analisia eskaintzea da. Gainera, garatu eta azaldu egiten da mota horretako mikrosare baten diseinua eta simulazioa inplementazio erreal bat gauzatzeko La Muelan, Espainia. Guzti honekin bilatzen dena da DER-etan eta komunikazio teknologia aurreratuenetan oinarritutako AC mikrosarea soluzio eraginkor eta ingurugiroarekiko jasangarriatzat aurkeztea, eta alternatiba guztietatik bereiztea bere hainbat onurengatik.[ES]El prop osito de este documento es proporcionar al lector una descripci on y an alisis completos de los principales componentes de una microrred aislada de corriente alterna (AC), as como el dise~no y simulaci on b asicos del mencionado tipo de microrred para una posible implementaci on en la localidad de La Muela, Espa~na. Con todo esto se pretende resaltar por sus m ultiples bene cios las microrredes AC basadas en DER y en la m as avanzada tecnolog a de la comunicaci on como la mejor soluci on entre todas las alternativas posibles para ofrecer un suministro de energ a electrica e caz y medioambientalmente sostenible.[EN]Analysis and design of an electrical microgrid in La Muela. Abstract: This paper aims to provide the reader a complete description and analysis of the main features of an Alternating Current (AC) o grid microgrid, as well as making a basic design and simulation of that kind of microgrid for a possible implementation in La Muela, Spain. It is also intended to highlight for its many bene ts the DER- and most advanced communication technology-based AC microgrids as the best solution among all the possible alternatives to o er an e ective and environmentally sustainable electric power supply

Effect of edeine on termination-reinitiation.

<p>Reporter mRNAs containing the termination reinitiation signals of FCV (panel A), BM2 (panel B) and MNV (Panel C) were translated in Flexi® RRL at a final RNA concentration of 50 µg/ml in the presence of [³⁵S]-methionine and 140 mM added KCl. At the indicated time points (min), an aliquot was removed, edeine added to 5 µM, and the sample reincubated for a total of 60 min. The translation products were resolved by SDS-PAGE on 12% gels and visualised by autoradiography. Identical experiments were performed in which cycloheximide replaced edeine (data not shown). In the cycloheximide experiments, it was found that in all cases, no termination-reinitiation product was evident until the 7.5 min time point, when only a trace was visible. The 7.5 min time point in the edeine gels is emboldened to reflect this. The relative levels of the rluc and fluc bands was determined by densitometry and in Panel D, the Rluc/Fluc ratio is plotted against the time of edeine addition for the three mRNAs.</p

Identification of Immune and Viral Correlates of Norovirus Protective Immunity through Comparative Study of Intra-Cluster Norovirus Strains

<div><p>Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity. While these are seemingly discordant observations, they may in fact reflect virus strain-, cluster-, or genogroup-specific differences in protective immunity induction. Here, we report that highly genetically related intra-cluster murine norovirus strains differ dramatically in their ability to induce a protective immune response: Primary MNV-3 infection induced robust and cross-reactive protection, whereas primary MNV-1 infection induced modest homotypic and no heterotypic protection. In addition to this fundamental observation that intra-cluster norovirus strains display remarkable differences in protective immunity induction, we report three additional important observations relevant to norovirus:host interactions. First, antibody and CD4⁺ T cells are essential to controlling secondary norovirus infections. Second, the viral minor structural protein VP2 regulates the maturation of antigen presenting cells and protective immunity induction in a virus strain-specific manner, pointing to a mechanism by which MNV-1 may prevent the stimulation of memory immune responses. Third, VF1-mediated regulation of cytokine induction also correlates with protective immunity induction. Thus, two highly genetically-related norovirus strains displayed striking differences in induction of protective immune responses, strongly suggesting that the interpretation of norovirus immunity and vaccine studies must consider potential virus strain-specific effects. Moreover, we have identified immune (antibody and CD4⁺ T cells) and viral (VP2 and possibly VF1) correlates of norovirus protective immunity. These findings have significant implications for our understanding of norovirus immunity during primary infections as well as the development of new norovirus vaccines.</p></div

B cells and CD4⁺ T cells, but not CD8⁺ T cells and IFN-γ, are essential for MNV-3 protective immunity.

<p><b>A</b>) Groups of mice of the indicated knockout strains (n = 7 mice per condition tested in at least two independent experiments) were inoculated with mock inoculum (black bars) or 10⁴ TCID₅₀ units of MNV-3 (grey bars). Six weeks later, all mice were challenged with 10⁷ TCID₅₀ units of MNV-3. One day later, animals were perfused, the indicated organs harvested, and viral burden determined by plaque assay. The data for all mice in each group were averaged. Limits of detection are indicated by dashed lines. The mock→MNV-3 infection group was compared to the MNV-3→ infection group for each mouse strain for statistical analysis. <b>B</b>) The mock→MNV-3 (1°) infection viral titers were divided by the MNV-3→ (2°) infection titers for the mice presented in panel A to determine the fold-reduction in titers as a quantitative measure of protective immunity. <b>C</b>) The same experiment was carried out for MNV-1; shown only are the fold-reductions in viral titers.</p

MNV-3 elicits protection from severe disease in the absence of type I interferon signaling.

<p><b>A</b>) Groups of mice lacking the type I interferon receptor (IFNAR^−/−; a minimum of 5 mice per condition) were inoculated with mock inoculum, 10⁴ or 10⁷ TCID₅₀ units of MNV-1 or MNV-3 by the peroral route. The percentage of mice surviving infection was calculated daily. All surviving mice were weighed daily and the weights compared to day 0 weights to calculate a relative weight. <b>B</b>) Groups of IFNAR^−/− mice (n = 3–5) were inoculated with either mock inoculum or 5×10³ TCID₅₀ units of MNV-3. Six weeks later, mice were challenged with 10⁷ TCID₅₀ units of MNV-3 or 10⁶ TCID₅₀ units of MNV-1 and monitored for survival and weight loss, as described above. <b>C</b>) Groups of IFNAR^−/− mice (n = 3) were inoculated with mock inoculum (1° mock; grey bars) or 5×10³ TCID₅₀ units of MNV-3 (1° MNV-3; black bars) by the peroral route. Six weeks later, mice were infected with 10⁷ TCID₅₀ units of MNV-1 or MNV-3 (2° challenge virus displayed on the x-axis). One day following 2° challenge, animals were perfused, the indicated organs harvested, and viral burden determined by plaque assay. Limits of detection are indicated by dashed lines. MLNs = mesenteric lymph nodes. Groups of mice receiving mock versus MNV-3-1° infection and the same 2° challenge virus were compared for statistical purposes.</p

MNV-3 initiates replication faster than MNV-1.

<p><b>A</b>) RAW 264.7 cells were infected with MNV-1 or MNV-3 at MOI 5. Supernatant was collected from two independent wells at the indicated hours post-infection (hpi) and virus titers determined using TCID₅₀ assay. The entire experiment was repeated three times and data from all experiments are averaged. The limit of detection of the assay is indicated by a dashed line. <b>B</b>) Infected cells from the same cultures used for panel A were lysed and viral proteins were detected by western blotting using the indicated antibodies. These data are representative of duplicate samples tested from each of three independent experiments. <b>C</b>) 1.5×10⁵ HEK-293T cells were transfected with 0.4, 2, 10 or 50 ng of purified MNV-1 or MNV-3 genomic RNA. The virus titers at the indicated hpi were determined using TCID₅₀ assay. Data for all replicates are averaged. Data for MNV-1 and MNV-3 at each time point were compared for statistical purposes.</p

Recombinant ORF3-swap viruses replicate comparably to parental viruses.

<p><b>A</b>) RAW 264.7 cells were infected with recombinant MNV-1, MNV-3, MNV-1.3VP2, or MNV3.1VP2 at MOI 0.05 or MOI 5 and growth curves were carried out as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003592#ppat-1003592-g006" target="_blank">Figure 6A</a>. Data from four independent experiments, with duplicate wells per condition tested in each experiment, are averaged. <b>B</b>) RAW 264.7 cells were infected with the indicated virus at MOI 5 for 0 or 24 hpi and cell lysates analyzed for viral proteins using western blotting. Data are representative of duplicate samples tested from each of three independent experiments.</p