Pre-vaccination screening strategies for the use of the CYD-TDV dengue vaccine: A meeting report.

The first licensed dengue vaccine, CYD-TDV (Dengvaxia) is efficacious in seropositive individuals, but increases the risk for severe dengue in seronegative persons about two years after administration of the first dose. For countries considering the introduction of Dengvaxia, WHO recommends a pre-vaccination screening strategy whereby only persons with evidence of a past dengue infection would be vaccinated. Policy-makers need to consider the risk-benefit of vaccination strategies based on such screening tests, the optimal age to introduce the vaccine, communication and implementation strategies. To address these questions, the Global Dengue and Aedes-transmitted diseases Consortium (GDAC) organized a 3-day workshop in January 2019 with country representatives from Asia and Latin America. The meeting discussions highlighted many challenges in introducing Dengvaxia, in terms of screening test characteristics, costs of such tests combined with a 3-dose schedule, logistics, achieving high coverage rates, vaccine confidence and communication; more challenges than for any other vaccine introduction programme. A screening test would require a high specificity to minimize individual risk, and at the same time high sensitivity to maximize individual and population benefit. The underlying seroprevalence dependent positive predictive value is the best indicator for an acceptable safety profile of a pre-vaccination screening strategy. The working groups discussed many possible implementation strategies. Addressing the bottlenecks in school-based vaccine introduction for Dengvaxia will also benefit other vaccines such as HPV and booster doses for tetanus and pertussis. Levels of public trust are highly variable and context specific, and understanding of population perceptions and concerns is essential to tailor interventions, monitor and mitigate risks.

Complete Genome Sequences of Zika Virus Strains Isolated from the Blood of Patients in Thailand in 2014 and the Philippines in 2012.

Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, Zika virus/Homo sapiens-tc/THA/2014/SV0127-14 and Zika virus/H. sapiens-tc/PHL/2012/CPC-0740, isolated from the blood of patients collected in Thailand, 2014, and the Philippines, 2012, respectively. Sequencing and phylogenetic analysis showed that both strains belong to the Asian lineage.

Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.

Viral subpopulation diversity in influenza virus isolates compared to clinical specimens.

BACKGROUND: Influenza virus (IFV) isolates obtained from mammalian cell cultures are valuable reagents used for vaccine production, antigenic characterization, laboratory assays, and epidemiological and evolutionary studies. Complete genomic comparison of IFV isolates with their original clinical specimens provides insight into cell culture-driven genomic changes which may sequentially alter the virus phenotype. OBJECTIVES: The genome of the viral isolates and of the viruses in the clinical specimens was examined by deep sequencing in order to determine nucleotide heterogeneity (measured number of variances or numbers of mixed bases) as a marker for IFV population diversity. STUDY DESIGN: Clinical respiratory specimens were collected between July and October 2012 and identified by RT-PCR as positive for influenza A H3N2 or H1N1, or influenza B. The viruses in the clinical specimens were amplified using mammalian cell culture. Next generation sequencing (NGS) was used to investigate genomic differences between IFV isolates and their corresponding clinical specimens. RESULTS: There was less nucleotide heterogeneity in 5 of 6 viral isolates compared to the corresponding clinical specimens, especially for influenza B. A phylogenetic analysis of the hemagglutinin (HA) gene consensus sequences obtained from deep and Sanger sequencing showed that the viral isolates and their corresponding clinical specimens contained the same IFV strains with less than 5% pair-wise genetic distance. CONCLUSION: The IFV sequence data analysis detected a substantial decrease in nucleotide heterogeneity from clinical specimens to viral cultures in 5 out of 6 investigated cases.

Effects of type of carbohydrate supplementation to lush pasture on microbial fermentation in continuous culture.

Eight single-flow continuous culture fermenters were used to study the effects of the type of energy source on ruminal N utilization from high quality pasture. The four dietary treatments included high quality grass and legume pasture alone (50:50; wt/wt), pasture plus soybean hulls, pasture plus beet pulp, and pasture plus corn. Diets supplemented with additional sources of energy (soybean hulls, beet pulp, and corn) were isocaloric but differed in the type and rate of carbohydrate fermentation. Energy supplements constituted 45% of the total dietary dry matter and were fed twice daily at 12-h intervals in place of pasture, which is characteristic of grain feeding at milking when animals are in a grazing situation. Energy supplementation reduced pH, NH3 N flow, and NH3 N concentration and increased bacterial N flow (as a percentage of N intake). The supplementation of corn and soybean hulls resulted in the highest microbial N flow (as a percentage of N intake). Corn had a tendency to reduce fiber digestion because of excessively low NH3 N concentrations. Beet pulp was similar to corn in that it decreased NH3 N concentrations. Supplementation of soybean hulls resulted in a more synchronized fermentation, greater volatile fatty acid production, and greater fiber digestion. Nitrogen utilization by microbes was maximized by supplementation with soybean hulls or corn twice a day. With diets based on pasture, it may be more important to improve bacterial N flow and bacterial utilization of N than to maximize the efficiency of bacterial protein synthesis because better utilization of N by ruminal microorganisms results in higher bacterial N flow and higher fiber digestion.

Effects of Saccharomyces cerevisiae and Aspergillus oryzae cultures on ruminal fermentation in dairy cows.

Four lactating Holstein cows, fitted with ruminal and duodenal cannulas, were used in a 4 x 4 Latin square design to examine the effects of supplemental yeast (Saccharomyces cerevisiae) and fungal (Aspergillus oryzae) cultures on ruminal fermentation, microbial populations, and nutrient supply to the small intestine. Cows were fed a basal diet comprising 32.5% corn silage, 17.5% alfalfa hay, 35.3% corn grain, 12.7% soybean meal, and 2% vitamin and mineral mixture on a DM basis. Treatments were arranged in a 2 x 2 factorial as follows: 1) basal diet, 2) basal diet plus 57 g/d of yeast culture, 3) basal diet plus 3 g/d of fungal culture, and 4) basal diet plus 57 g/d of yeast culture and 3 g/d of fungal culture. Ruminal pH, ammonia N concentration, and total VFA concentration were similar among treatments. Molar percentages of ruminal isoacids were lower for cows fed a mixture of yeast and fungal culture than for cows fed yeast or fungal culture alone. Yeast culture increased ruminal OM and CP digestion and decreased OM and N flow to the duodenum. Fiber digestion in the rumen was similar among treatments. Fungal culture stimulated proteolytic and cellulolytic bacterial counts. Proteolytic bacterial counts were also stimulated by yeast culture. Results from this experiment demonstrated that yeast and fungal cultures could influence ruminal fermentation and microbial populations.

Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP.

Bacillus anthracis exotoxins mediate most of the symptomatology of severe anthrax. In addition to a clinical syndrome reminiscent of septic shock, which may be mediated by cytokines produced by macrophages stimulated with lethal toxin, infected patients show profound edema at sites of infection. Edema is mediated by edema toxin (ET), which comprises of a binding molecule, protective antigen, and an active moiety, edema factor, which possesses intrinsic adenylyl cyclase activity. Intracellular cyclic AMP (cAMP) regulates the production of several cytokines that modulate edema formation and play important roles in host defense against invading bacteria. To determine whether ET enhanced the accumulation of cAMP in monocytes and thereby influenced cytokine production, we cultured human monocytes with endotoxin (lipopolysaccharide [LPS]) and dilutions of ET and determined the levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) in culture supernatant fluids. We further estimated cytokine-specific mRNA accumulation in monocytes by reverse transcription PCR and examined intracellular cAMP concentrations following treatment with ET. ET and LPS each induced monocytes to secrete comparable amounts of IL-6. ET did not inhibit and in most experiments modestly enhanced LPS-induced IL-6 production. In contrast to this stimulatory effect on IL-6 production, ET induced little or no TNF-alpha production. Moreover, ET profoundly inhibited LPS-induced TNF-alpha synthesis. These regulatory phenomena were also observed at the mRNA level in association with dose-related enhancement of intracellular cAMP in ET-treated monocytes. Monocytes treated with dibutyryl cAMP, an active analog of cAMP, produced cytokines in a pattern identical to that of cells treated with ET. The disruption of cytokine networks as a consequence of unregulated, ET-induced cAMP accumulation in human monocytes may impair cellular antimicrobial responses and contribute to clinical signs and symptoms.

Strand breaks in DNA induced by a thiol/Fe(III)/O2 mixed-function oxidase system and its protection by a yeast antioxidant protein.

Strand breaks can be produced in pBluescript plasmid DNA and calf thymus DNA by a mixed-function oxidase (MFO) system comprised of Fe3+, O2, and dithiothreitol as an electron donor. Superoxide dismutase does not block this damage whereas a 27-KDa yeast antioxidant protein specifically inhibits strand breaks in DNA induced by the dithiothreitol MFO system. In contrast, this protein does not inhibit strand breaks in DNA induced by an ascorbate MFO system although catalase inhibits damage in DNA caused by both MFO systems. Based on the specificity of this protein, we propose that the antioxidant protein functions as a sulfur radical scavenger.