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Purified SV40 virions were disrupted by sodium dodecyl sulfate (SDS), β-mercaptoethanol and heat, and the proteins analyzed in polyacrylamide gels in the presence of SDS. Six different polypeptide chains were found. Agregation of proteins was ruled out by treatment with 8 M urea. The major protein had a molecular weight (M. W.) of approximately 45, 000, two other proteins M. W.'s of 35, 000 and 25, 000 respectively. In addition, three minor components were found with M. W.'s of approximately 18, 000, 14, 000 and 10, 000.

Ordinarily, sedimentation analysis of RNA preparations is carried out in simple aqueous solutions. Recently, numerous investigators have carried out experiments where RNA's in cytoplasmic extracts from various types of cells have been analyzed by sedimentation through gradients of sucrose. The implicit assumption has been made that the presence of the cytoplasmic extract does not in itself affect the rate of sedimentation of the RNA. However, we have recently found that some component of HeLa cell cytoplasmic extracts causes a 1.5-2-fold increase in the sedimentation rate of a number of species of RNA. Since this result bears on the interpretation of many types of experiments, we will report the phenomenon at this time, even though we know little about its mechanism.

Bocquet Henri, Girard Marc. Retombées radioactives consécutives aux expériences russes. Mesures de l’Iode 131 dans la thyroïde. In: Bulletin de l'Académie Vétérinaire de France tome 114 n°10, 1961. pp. 395-404.

Goret Pierre, Brion Abel, Fontaine M., Fontaine M., Pilet Charles, Girard Marc, Girard M. Nouvelles recherches expérimentales sur le virus de la rhino-amygdalite contagieuse du chien. II - Pouvoir antigène et immunigène. In: Bulletin de l'Académie Vétérinaire de France tome 112 n°10, 1959. pp. 635-648.

Summary Poliovirus replicative intermediate (R.I.) was purified by repeated precipitations at high ionic strength and exclusion chromatography on agarose. Treatment of the purified material with limited amounts of ribonuclease yielded RNase-resistant R.I. cores, which were purified by gradient centrifugation. The cores were melted by treatment with dimethylsulfoxide, centrifuged through sucrose gradients, and the resulting molecules analyzed by hybridization with purified virus RNA. The results show 90 p. cent of the R.I. cores to consist of one intact complementary strand (minus strand) attached through hydrogen bonding to fragments of virus strands (plus strands). They support the idea of a semiconservative model for virus RNA replication. Purified poliovirus R.I. was examined under the electron microscope. The presence of numerous branched molecules was observed, in agreement with the proposed model for the architecture of R.I. The length of the backbones and the number of forks of 41 of these branched molecules were determined. Length varied from 2.0 to 2.70 μm, in agreement with the 2.37 μm previously reported for poliovirus RNA. The number or forks per molecule varied from 0 to 7.

Purified poliovirus single- and double-stranded ribonucleic acids (RNA) were examined by electron microscopy. The length of both molecules was found to be 2.37 μm. The uncorrected sedimentation coefficient for single-stranded RNA is 33 S , as compared to 27 S for the RNA of tobacco mosaic virus. It is calculated from these results that the molecular weight of the sodium form of poliovirus is 2.6 × 10 6 daltons.

Bocquet Henri, Girard O., Nicol Louis, Girard Marc, Renaudat C. Recherches sur les effets des rayons γ du colbat 60 sur la formation, l’utilisation et la conservation de l’antitoxine tétanique chez le cobaye. In: Bulletin de l'Académie Vétérinaire de France tome 115 n°8, 1962. pp. 325-335.

Publisher Summary This chapter describes the hot phenol-sodium dodecyl sulfate technique of extraction of RNA to release RNA from cultured animal cells, both virus infected and uninfected. The method involves three successive extractions with phenol at 60° and pH 5 in the presence of sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). Because of the SDS and elevated temperature, species of RNA are released in the aqueous phase. Proteins are extracted into the interface and phenol phase; DNA likewise is removed from the aqueous phase. The recovery of RNA is usually about 70%, and SDS and EDTA help prevent degradation, respectively, by nucleases and divalent ions, while also guarding against aggregation. The technique is described for the isolation of RNA from 50-100 million cells (approximately 1 mg of RNA). It is applied with considerable success to the extraction of RNA from HeLa cells, L cells, thyroid and liver, T4 infected E. coli , poliovirus, reovirus, Rous sarcoma virus, and so on. In each of these cases, high molecular weight RNA is yielded in an apparently undegraded state and reasonably free from proteins and DNA.

Goret Pierre, Fontaine M., Brion Abel, Pilet Ch., Girard Marc, Legrand P., Sicard H., Renaudat C. Effets de chlorhydrate de tétracycline administré par différentes voies sur la prévention et le traitement de la pneumonie à virus. In: Bulletin de l'Académie Vétérinaire de France tome 113 n°5, 1960. pp. 303-304.

Goret Pierre, Pilet Charles, Girard Marc. Souches «atypiques» ou «variantes» du virus de la Peste porcine isolées en France. In: Bulletin de l'Académie Vétérinaire de France tome 112 n°10, 1959. pp. 657-674.

Fontaine M., Fontaine M.-P., Goret Pierre, Brion Abel, Pilet Ch., Girard Marc, Alloui P. Une enzootie de pneumonie à virus du porc dans la région parisienne. Isolement et essai d'identification du virus en cause. In: Bulletin de l'Académie Vétérinaire de France tome 111 n°8, 1958. pp. 451-471.

Urban sprawl is a widely recognized phenomenon in many major cities worldwide and is a significant land use planning and management issue. This process has many impacts on the ecological function and structure of the landscape. In this article, we analyze the effects of urban sprawl on the ecological patterns and processes in the Montreal Metropolitan Region (MMR) between 1966 and 2010. The dispersed sprawl of low-density urban areas within the territory during this period sharply increased the fragmentation of the territory, isolating the few remaining natural spaces and decreasing their ecological connectivity and, ultimately, biodiversity. The results obtained clearly show that land-use changes that occurred in the MMR have caused profound changes in landscape properties, both structurally and functionally, and especially from 1981 to 2010. In 1966, around 45% of the land had a high or very high level of connectivity, and almost 38% in 1981. By 2010 only 6.5% of the landscape was connected and 73% of the territory possessed no or low connectivity.

Sequences from the principal neutralization domain of human immunodeficiency virus type 1 (HIV-1) strain LAI or RF have been expressed in antigenic site 1 of the capsid of the Sabin strain of poliovirus type 1. A number of the resulting chimeras were viable. Viable variants bearing mutations within the insertion site spontaneously arose from several nonviable chimeras. In general, these mutations result in a decrease in positive charge in the substituted antigenic site 1. Two of the chimeras were genetically stable and have been further characterized. Both chimeras were neutralized by various HIV-1 neutralizing antibodies. In rabbits, both chimeras produced high levels of antibodies which react with HIV-1 gp120/160 in immunoprecipitation and enzyme-linked immunosorbent assays. One of the chimeras (HIV-1LAI) produced a significant but weak HIV-1 neutralizing response.

Jokubaitis, Vilija G./0000-0002-3942-4340; Oreja-Guevara, Celia/0000-0002-9221-5716; Lugaresi, Alessandra/0000-0003-2902-5589; Slee, Mark/0000-0003-4323-2453; Horakova, Dana/0000-0003-1915-0036; Havrdova, Eva Kubala/0000-0002-9543-4359; Sanchez Menoyo, Jose Luis/0000-0003-2634-8294; Prat, Alexandre/0000-0001-6188-0580; Kalincik, Tomas/0000-0003-3778-1376; Butzkueven, Helmut/0000-0003-3940-8727; pucci, eugenio/0000-0001-7606-7330; van Pesch, Vincent/0000-0003-2885-9004; Trojano, Maria/0000-0002-6329-8946 WOS: 000365135700024 PubMed: 26359291 Prevention of irreversible disability is currently the most important goal of disease modifying therapy for multiple sclerosis. The disability outcomes used in most clinical trials rely on progression of Expanded Disability Status Scale score confirmed over 3 or 6 months. However, sensitivity and stability of this metric has not been extensively evaluated. Using the global MSBase cohort study, we evaluated 48 criteria of disability progression, testing three definitions of baseline disability, two definitions of progression magnitude, two definitions of long-term irreversibility and four definitions of event confirmation period. The study outcomes comprised the rates of detected progression events per 10 years and the proportions of the recorded events persistent at later time points. To evaluate the ratio of progression frequency and stability for each criterion, we calculated the proportion of events persistent over the five subsequent years once progression was achieved. Finally, we evaluated the clinical and demographic determinants characterising progression events and, for those that regressed back to baseline, determinants of their subsequent regression. The study population consisted of 16 636 patients with the minimum of three recorded disability scores, totalling 112 584 patient-years. The progression rates varied between 0.41 and 1.14 events per 10 years, with the length of required confirmation interval as the most important determinant of the observed variance. The concordance among all tested progression criteria was only 17.3%. Regression of disability occurred in 11-34% of the progression events over the five subsequent years. The most important determinant of progression stability was the length of the confirmation period. For the most accurate set of the progression criteria, the proportions of 3-, 6-, 12- or 24-month confirmed events persistent over 5 years reached 70%, 74%, 80% and 89%, respectively. Regression post progression was more common in younger patients, relapsing-remitting disease course, and after a smaller change in disability, and was inflated by higher visit frequency. These results suggest that the disability outcomes based on 3-6-month confirmed disability progression overestimate the accumulation of permanent disability by up to 30%. This could lead to spurious results in short-term clinical trials, and the issue may be magnified further in cohorts consisting predominantly of younger patients and patients with relapsing-remitting disease. Extension of the required confirmation period increases the persistence of progression events. NHMRC Early Career FellowshipNational Health and Medical Research Council of Australia [1071124]; NHMRC Practitioner FellowshipNational Health and Medical Research Council of Australia [1080518]; NHMRCNational Health and Medical Research Council of Australia [1083539, 1032484]; NHMRC Centre for Research ExcellenceNational Health and Medical Research Council of Australia [1001216]; MSBase Foundation; Merck SeronoMerck SeronoMerck & Company; BiogenBiogen; Novartis Pharma; Bayer-ScheringBayer AG; Sanofi-AventisSanofi-Aventis; BioCSL The work was supported by the NHMRC Early Career Fellowship (1071124), NHMRC Practitioner Fellowship (1080518), NHMRC Project Grants (1083539 and 1032484), NHMRC Centre for Research Excellence (Grant ID 1001216) and the MSBase Foundation. The MSBase Foundation is a not-for-profit organization that receives support from Merck Serono, Biogen, Novartis Pharma, Bayer-Schering, Sanofi-Aventis and BioCSL. The study was conducted separately and apart from the guidance of the sponsors.

Summary Aedes aegypti C17 cell cultures were infected with yellow fever virus and labelled with 35 S-methionine. The cell membranes were prepared and their proteins solubilized using a mixture of non-ionic detergents. The solubilized proteins were fractionated by chromatography on hydroxylapatite. Two peaks of viral antigens were eluted using a phosphate molarity gradient. Analysis by polyacrylamide gel electrophoresis showed that the fractions from the first peak, which eluted at about 0.14 M phosphate, contained a 52,000-dalton polypeptide and a 46,000-dalton polypeptide, corresponding to the virion envelope glycoprotein V3 and to the non-structural viral protein NV3, respectively. The fractions from the second peak, which eluted at about 0.25 M phosphate, contained a 48,000-dalton polypeptide, probably corresponding to the viral protein Z. The antigenic specificity of the two peaks was analysed using an enzyme-linked immunosorbent assay. The fractions containing polypeptide V3 (0.14 M phosphate eluate) exhibited both group and type specificity, whereas those containing polypeptide Z (0.25 M phosphate eluate) exhibited type specificity only. The use of this material as a type specific antigen for yellow fever is therefore recommended.

Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.

International audience; We previously described the generation of a set of mutations into a cDNA of poliovirus type 1 in the myristoylation signal of the capsid polypeptide VP4 (D. Marc, G. Drugeon, A.-L. Haenni, M. Girard, and S. van der Werf, EMBO J. 8:2661, 1989). Genomic transcripts synthesized in vitro from the mutated cDNAs were found to be noninfectious upon transfection of permissive cells, and this property correlated with the lack of VP0 myristoylation in vivo. In the study presented here, we analyzed the assembly intermediates that could be recovered from cells transfected with the mutated transcripts. We found that 14S pentamers could still assemble to a certain extent with an unmyristoylated VP0. Furthermore, viral particles sedimenting at 150S and containing capsid polypeptides VP1 to VP4 and virus-specific RNA were detected in the transfected cells. However, these mature virions were less abundant than those recovered after transfection with an infectious transcript, and they were devoid of infectivity. The results suggest that VP0 myristoylation plays a role in the late steps of poliovirus assembly and that the myristate moiety of VP4 may be required in the early steps of poliovirus infection.

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International audience

International audience