Differential Oxidative Stress Induced by Dengue Virus in Monocytes from Human Neonates, Adult and Elderly Individuals

<div><p>Changes in immune response during lifespan of man are well known. These changes involve decreased neonatal and elderly immune response. In addition, it has been shown a relationship between immune and oxidative mechanisms, suggesting that altered immune response could be associated to altered oxidative response. Increased expression of nitric oxide (NO) has been documented in dengue and in monocyte cultures infected with different types of dengue virus. However, there is no information about the age-dependent NO oxidative response in humans infected by dengue virus. In this study, monocyte cultures from neonatal, elderly and adult individuals (n = 10 each group) were infected with different dengue virus types (DENV- 1 to 4) and oxidative/antioxidative responses and apoptosis were measured at days 1 and 3 of culture. Increased production of NO, lipid peroxidation and enzymatic and nonenzymatic anti-oxidative responses in dengue infected monocyte cultures were observed. However, neonatal and elderly monocytes had lower values of studied parameters when compared to those in adult-derived cultures. Apoptosis was present in infected monocytes with higher values at day 3 of culture. This reduced oxidant/antioxidant response of neonatal and elderly monocytes could be relevant in the pathogenesis of dengue disease.</p></div

Induction of nitric oxide (NO) by different DENV types and LPS according to monocyte source.

<p>Decreased NO values in neonatal and elderly monocyte cultures infected with all DENV types compared to adult monocyte cultures were observed at days 1 and 3. These responses were similar to those observed with LPS stimulation.</p

Induction of apoptosis (TUNEL) by different DENV types and LPS according to monocyte source.

<p>Increased apoptosis in LPS or infected-monocyte cultures, with higher values at day 3 were observed.</p

Lipid peroxidation (MDA) induced by different DENV types and LPS according to monocyte source.

<p>Lower values of MDA in neonatal and elderly monocyte cultures than those in adult cultures were observed at days 1 and 3. These responses were similar to those observed with LPS stimulation.</p

Index elderly or adult values/neonatal values.

<p>In general, values from neonatal cultures were lower than those observed in elderly and adult monocyte cultures in dengue infections (A, B, C, E) with the highest values in adult cultures; except for SOD activity with higher values in elderly cultures (D). These data represent the mean ± SD of the values from all DENV types.</p

Induction of superoxide dismutase (SOD) activity by different DENV types and LPS according to monocyte source.

<p>Decreased activity in neonatal monocyte cultures infected with all DENV types compared to adult and elderly monocyte cultures were observed at days 1 and 3.</p

Induction of catalase activity by different DENV types and LPS according to monocyte source.

<p>Decreased values in neonatal and elderly monocyte cultures infected with all DENV types compared to adult monocyte cultures were observed at days 1 and 3. These responses were similar to those observed with LPS stimulation.</p

Reduced glutathione (GSH) content in dengue virus or LPS treated monocyte cultures according to monocyte source.

<p>Decreased values in neonatal and elderly monocyte cultures infected with all DENV types compared to adult monocyte cultures were observed at days 1 and 3. These responses were similar to those observed with LPS stimulation.</p

Evolutionary Dynamics of West Nile Virus in the United States, 1999–2011: Phylogeny, Selection Pressure and Evolutionary Time-Scale Analysis

<div><p>West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006–2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999–2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006–2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10⁻⁴ substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies.</p></div

Genetic Variability of West Nile Virus in U.S. Blood Donors from the 2012 Epidemic Season

<div><p>West Nile virus (WNV) is an arbovirus maintained in nature in a bird-mosquito enzootic cycle which can also infect other vertebrates including humans. WNV is now endemic in the United States (U.S.), causing yearly outbreaks that have resulted in an estimated total of 4–5 million human infections. Over 41,700 cases of West Nile disease, including 18,810 neuroinvasive cases and 1,765 deaths, were reported to the CDC between 1999 and 2014. In 2012, the second largest West Nile outbreak in the U.S. was reported, which caused 5,674 cases and 286 deaths. WNV continues to evolve, and three major WNV lineage I genotypes (NY99, WN02, and SW/WN03) have been described in the U.S. since introduction of the virus in 1999. We report here the WNV sequences obtained from 19 human samples acquired during the 2012 U.S. outbreak and our examination of the evolutionary dynamics in WNV isolates sequenced from 1999–2012. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses. Selection pressure analyses were performed with the HyPhy package using the Datamonkey web-server. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. Thirteen of the 19 completely sequenced isolates from 10 U.S. states were genetically similar, sharing up to 55 nucleotide mutations and 4 amino acid substitutions when compared with the prototype isolate WN-NY99. Overall, these analyses showed that following a brief contraction in 2008–2009, WNV genetic divergence in the U.S. continued to increase in 2012, and that closely related variants were found across a broad geographic range of the U.S., coincident with the second-largest WNV outbreak in U.S. history.</p></div