Genetic Characterization of an Insect-Specific Flavivirus Isolated from Culex Theileri Mosquitoes Collected in Southern Portugal

We describe the full genetic characterization of an insect-specific flavivirus (ISF) from Culex theileri (Theobald) mosquitoes collected in Portugal. This represents the first isolation and full characterization of an ISF from Portuguese mosquitoes. The virus, designated CTFV, for Culex theileri flavivirus, was isolated in the C6/36 Stegomyia albopicta (=Aedes albopictus) cell line, and failed to replicate in vertebrate (Vero) cells in common with other ISFs. The CTFV genome encodes a single polyprotein with 3357 residues showing all the features expected for those of flaviviruses. Phylogenetic analyses based on all ISF sequences available to date, place CTFV among Culex-associated flaviviruses, grouping with recently published NS5 partial sequences documented from mosquitoes collected in the Iberian Peninsula, and with Quang Binh virus (isolated in Vietnam) as a close relative. No CTFV sequences were found integrated in their host’s genome using a range of specific PCR primers designed to the prM/E, NS3, and NS5 region.

Regulatory Cells, Cytokine Pattern and Clinical Risk Factors for Asthma in Infants and Young Children with Recurrent Wheeze

Several risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. OBJECTIVES: This study was designed to assess and compare the immunological status during the first 2 years in steroid-naive young children with >or= three episodes of physician-confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age-matched healthy controls (n=30). METHODS: Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) T cells and their cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN-gamma, TGF-beta and IL-10), CTLA-4 and Foxp3 mRNA expression were evaluated (real-time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12-myristate 13-acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). RESULTS: Flow cytometry results showed a significant reduction in the absolute number of CD4(+)CD25(high) and the absolute and percentage numbers of CD4(+)CD25(+)CTLA-4(+) in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4(+)CD25(+) (P=0.01) and CD4(+)CD25(high) (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA-4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA-4 (P=0.03) and IFN-gamma (P=0.04) expression was diminished in wheezy children compared with healthy children. High-risk children had lower expression of IFN-gamma (P=0.03) compared with low-risk and healthy children and lower expression of CTLA-4 (P=0.01) compared with healthy children. CONCLUSIONS: Although our findings suggest that some immunological parameters are impaired in children with recurrent wheeze, particularly with a high risk for asthma, further studies are needed in order to assess their potential as surrogate predictor factors for asthma in early life.