A single amino acid substitution in the mRNA capping enzyme λ2 of a mammalian orthoreovirus mutant increases interferon sensitivity.

In the last few years, the development of a plasmid-based reverse genetics system for mammalian reovirus has allowed the production and characterization of mutant viruses. This could be especially significant in the optimization of reovirus strains for virotherapeutic applications, either as gene vectors or oncolytic viruses. The genome of a mutant virus exhibiting increased sensitivity to interferon was completely sequenced and compared with its parental virus. Viruses corresponding to either the parental or mutant viruses were then rescued by reverse genetics and shown to exhibit the expected phenotypes. Systematic rescue of different viruses harboring either of the four parental genes in a mutant virus backbone, or reciprocally, indicated that a single amino acid substitution in one of λ2 methyltransferase domains is the major determinant of the difference in interferon sensitivity between these two viruses.

Further characterization and determination of the single amino acid change in the tsI138 reovirus thermosensitive mutant

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the bio- logical properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the l1 protein. In the present study, this mu- tant was further studied as a possible tool to establish the role of the putative l1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

Transient high level mammalian reovirus replication in a bat epithelial cell line occurs without cytopathic effect

Mammalian reoviruses exhibit a large host range and infected cells are generally killed; however, most studies examined only a few cell types and host species, and are probably not representative of all possible interactions between virus and host cell. Many questions thus remain concerning the nature of cellular factors that affect viral replication and cell death. In the present work, it was observed that replication of the classical mammalian reovirus serotype 3 Dearing in a bat epithelial cell line, Tb1.Lu, does not result in cell lysis and is rapidly reduced to very low levels. Prior uncoating of virions by chymotrypsin treatment, to generate infectious subviral particles, increased the initial level of infection but without any significant effect on further viral replication or cell survival. Infected cells remain resistant to virus reinfection and secrete an antiviral factor, most likely interferon, that is protective against the unrelated encephalomyocarditis virus. Although, the transformed status of a cell is believed to promote reovirus replication and viral “oncolysis”, resistant Tb1.Lu cells exhibit a classical phenotype of transformed cells by forming colonies in semisolid soft agar medium. Further transduction of Tb.Lu cells with a constitutively-active Ras oncogene does not seem cell growth or reovirus effect on these cells. Infected Tb1.Lu cells can produce low-level of infectious virus for a long time without any apparent effect, although these cells are resistant to reinfection. The results suggest that Tb1.Lu cells can mount an unusual antiviral response. Specific properties of bat cells may thus be in part responsible for the ability of the animals to act as reservoirs for viruses in general and for novel reoviruses in particular. Their peculiar resistance to cell lysis also makes Tb1.Lu cells an attractive model to study the cellular and viral factors that determine the ability of reovirus to replicate and destroy infected cells.

Amino acid substitutions in σ1 and μ1 outer capsid proteins are selected during mammalian reovirus adaptation to Vero cells

Establishment of viral persistence in cell culture has previously led to the selection of mammalian reovirus mutants, although very few of those have been characterized in details. In the present study, reovirus was adapted to Vero cells that, in contrast to classically-used L929 cells, are inefficient in supporting the early steps of reovirus uncoating and are also unable to produce interferon as an antiviral response once infection occurs. The Vero cell-adapted reovirus exhibits amino acids substitutions in both the σ1 and μ1 proteins. This contrasts with uncoating mutants from persistently-infected L929 cells, and various other cell types, that generally harbor amino acids substitutions in the σ3 outer capsid protein. The Vero cell-adapted virus remained sensitive to an inhibitor of lysosomal proteases; furthermore, in the absence of selective pressure for its maintenance, t he virus has partially lost its ability to resist interferon. The positions of the amino acids substitutions on the known protein structures suggest an effect on binding of the viral σ1 protein to the cell surface and on μ1 disassembly from the outer capsid.

Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

Addition of exogenous peptide sequences on viral capsids is a powerful approach to study the process of viral infection or to retarget viruses toward defined cell types. Until recently, it was not possible to manipulate the genome of mammalian reovirus and this was an obstacle to the addition of exogenous sequence tags onto the capsid of a replicating virus. This obstacle has now been overcome by the advent of the plasmid-based reverse genetics system. In the present study, reverse genetics was used to introduce different exogenous peptides, up to 40 amino acids long, at the carboxyl-terminal end of the σ1 outer capsid protein. The tagged viruses obtained were infectious, produce plaques of similar size, and could be easily propagated at hight titers. However, attempts to introduce a 750 nucleotides-long sequence failed, even when it was added after the stop codon, suggesting a possible size limitation at the nucleic acid level.

Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

In a recent study, the serotype 3 Dearing strain of mammalian orthoreovirus was adapted to Vero cells; cells that exhibit a limited ability to support the early steps of reovirus uncoating and are unable to produce interferon as an antiviral response upon infection. The Vero cell-adapted virus (VeroAV) exhibits amino acids substitutions in both the σ1 and μ1 outer capsid proteins but no changes in the σ3 protein. Accordingly, the virus was shown not to behave as a classical uncoating mutant. In the present study, an increased ability of the virus to bind at the Vero cell surface was observed and is likely associated with an increased ability to bind onto cell-surface sialic acid residues. In addition, the kinetics of μ1 disassembly from the virions appears to be altered. The plasmid-based reverse genetics approach confirmed the importance of σ1 amino acids substitutions in VeroAV's ability to efficiently infect Vero cells, although μ1 co-adaptation appears necessary to optimize viral infection. This approach of combining in vitro selection of reoviruses with reverse genetics to identify pertinent amino acids substitutions appears promising in the context of eventual reovirus modification to increase its potential as an oncolytic virus.

O ensino de ciências naturais nos anos iniciais: concepções e práticas pedagógicas dos docentes em formação pelo PARFOR/Pedagogia/UFPA

O estudo apresentou como objetivo geral compreender o Ensino de Ciências Naturais em escolas públicas da região metropolitana de Belém, a partir das concepções e das práticas pedagógicas dos docentes em formação pelo PARFOR que atuam nos anos iniciais do Ensino Fundamental. Os objetivos específicos se resumem em analisar as concepções de ciências naturais dos docentes em formação que atuam nos anos iniciais; descrever as práticas pedagógicas que são adotadas pelos docentes ao ensinarem Ciências Naturais e avaliar se há influência dos Parâmetros Curriculares Nacionais de Ciências Naturais na concepção e na prática pedagógica adotadas por estes professores em formação (que são alunos do PARFOR/UFPA). Como objeto de estudo, centrou-se no Ensino de Ciências em escolas públicas da região metropolitana de Belém, a partir da perspectiva dos docentes em formação no curso de Pedagogia da UFPA. Trata-se de uma pesquisa de caráter qualitativo. A técnica de coleta de dados foi feita por meio de análise documental e de campo, cuja amostra composta por 20 professores da rede pública de ensino que atualmente estão em formação pelo PARFOR no Curso de Licenciatura em Pedagogia do Campus de Belém-Pa. Na coleta de dados, utilizou-se documentos oficiais como os PCN de Ciências Naturais, Relatório de Gestão PARFOR/UFPA e o Projeto Político Pedagógico do Curso de Pedagogia/PARFOR. Aplicou-se um questionário com perguntas abertas e fechadas para investigar as variáveis: socioeconômica, formação profissional, mercado de trabalho, concepções de ciências e práticas pedagógicas. Adotou-se a análise de dados de conteúdo. Por fim, nas considerações finais discorreu-se sobre os aspectos centrais e relevantes da pesquisa, apresentando os resultados e análises mais significativos. O lócus da pesquisa ocorreu no Instituto de Ciências da Educação – Faculdade de Educação – Campus Silveira Neto – Belém-Pará- Brasil. Acredita-se que esse estudo subsidiará outras pesquisas sobre o Ensino de Ciências nos anos iniciais, pois nas bases de dados dos eventos e revistas indexadas da área em questão, ainda não existe nenhum trabalho que reporte a esta temática aqui no estado do Pará. Poderá, também, servir como fonte de pesquisa para outros estudos que surgirem na linha de Educação: Currículo, Epistemologia e História.

Quartärbotanische Untersuchungen zur Vegetationsgeschichte der Alpe d'Essertse (Hérémence, Wallis)

Afin d'étudier l'histoire de la végétation de l'Alpe d'Essertse, des sondages ont été effectués dans le Gouillé Rion, un étang situé à 2343 m d'altitude. Les grains de pollen contenus dans le sédiment lacustre ont été analysés palynologiquement. Le diagramme pollinique montre qu'après le retrait des glaciers vers 13000 BP (Before Present), l'Alpe d'Essertse fut colonisée par une végétation alpine et une végétation d'éboulis. Entre 9500 et 3600 BP le mélèze (Larix decidua) et l'arole (Pinus cembra) formaient une forêt qui atteignait au moins 2343 m. A partir de 5000 BP la forêt s'ouvrit et la limite de la forêt commença à s'abaisser. Des buissons d'aune vert (Alnus viridis) remplacèrent peut à peu la forêt. Entre 1700 et 900 BP seulement, cette végétation apparentée aux forêts fit place aux prés et pâturages. Seul l'utilisation d'autres méthodes permet d'estimer la limite d'altitude maximale atteinte par la forêt au cours de l'holocène: pour l'Alpe d'Essertse des charbons trouvés dans le sol, ainsi que des bioséquences pédologiques suggèrent une limite de la forêt maximale entre 2400 et 2450 m d'altitude.