RESUMO
Novel viruses might be responsible for numerous disease cases with unknown etiology. In this study, we screened 1800 nasopharyngeal samples from adult outpatients with respiratory disease symptoms and healthy individuals. We employed a reverse transcription (RT)-PCR assay and CODEHOP-based primers (CT12-mCODEHOP) previously developed to recognize known and unknown corona- and toroviruses. The CT12-mCODEHOP assay detected 42.0 % (29/69) of samples positive for human coronaviruses (HCoV), including HCoV-229 (1/16), HCoV-NL63 (9/17), and HCoV-OC43 (19/36), and additionally HCoV-HKU1 (3), which was not targeted by the diagnostic real-time PCR assays. No other coronaviruses were identified in the analyzed samples.
Assuntos
Coronavirus/isolamento & purificação , Primers do DNA/genética , Nasofaringe/virologia , Infecções Respiratórias/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coronavirus/classificação , Coronavirus/genética , Humanos , Infecções Respiratórias/diagnósticoRESUMO
On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pre-T cell receptor (pre-TCR), which orchestrates differentiation, cell proliferation, and survival. The exact mechanism of pre-TCR-mediated suppression of T cell death remains unclear and controversial. Here, we identify Bim and Bid, 2 members of the BH3-only group of the BCL2 family, as important regulators of pre-T cell death. Both factors are highly expressed in proapoptotic thymocytes and their expression is suppressed on signaling through the pre-TCR. Their expression is directly regulated by the transcription factors FoxO3a and p53. Bid expression and p53 activity are related to the ongoing rearrangement of the TCR loci and induced DNA damage responses. Bim expression and FoxO3a nuclear translocation are directly controlled by the pre-TCR by means of its downstream kinase Akt/PKB. Interestingly, deletion of either gene on a pre-TCR(-/-) background rescues survival, but fails to induce further progenitor differentiation uncoupling the 2 processes.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/imunologia , Células Progenitoras Linfoides/imunologia , Proteínas de Membrana/imunologia , Proteínas Proto-Oncogênicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína 11 Semelhante a Bcl-2 , Morte Celular/genética , Morte Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Dano ao DNA/genética , Dano ao DNA/imunologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Rearranjo Gênico do Linfócito T/genética , Rearranjo Gênico do Linfócito T/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Locos de Características Quantitativas/genética , Locos de Características Quantitativas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10(2) to 10(6) and from 10(5) to 10(9) RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10(3) folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.