A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

Prenatal alcohol exposure increases TNFalpha-induced cytotoxicity in primary astrocytes.

We examined the effect of prenatal alcohol exposure (PAE) on tumor necrosis factor-alpha-(TNFalpha) induced cell death in primary astrocyte cultures. Flow cytometry revealed that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFalpha when compared to astrocytes prepared from pair-fed and chow-fed controls. In a number of cell types, TNFalpha regulates cell growth or death, in part, by the hydrolysis of sphingomyelin to ceramide and sphingosine-1-phosphate (SPP). Using a 3-(4. 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxic assay we found that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFalpha, sphingomyelinase (SMase), and C(2)- and C(6)-ceramide. The increasing cellular concentrations of SPP, a sphingolipid metabolic that induces cell growth, protected the cells from TNFalpha-induced cell death. N, N-dimethylsphingosine (DMS), which inhibits SPP production, and N-oleoylethanolamine, which inhibits acid ceramidases, increased TNFalpha-induced cytotoxicity in astrocytes prepared from PAE rats. These studies suggest that PAE shifts the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNFalpha.