A High-Throughput Yellow Fever Neutralization Assay.

Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.

3'-Deoxy phosphoramidate dinucleosides as improved inhibitors of hepatitis C virus subgenomic replicon and NS5B polymerase activity.

Phosphoramidate dinucleosides named "GC 3'-OH" series, carrying various phosphoramidate linkages have been previously reported as hepatitis C virus (HCV) inhibitors. To enhance the efficacy of these dinucleotides, we synthesized a novel "GC 3'-H" series as potential chain terminators. We showed that their inhibition potency is strongly increased by the introduction of novel neutral and bis-negatively charged phosphoramidate side chains. Their inhibitory effect on HCV NS5B polymerase was evaluated in vitro and in HCV subgenomic replicon containing Huh-6 cells. As expected, 3'-H compounds are more potent than their 3'-OH counterparts to inhibit HCV polymerase activity. The most potent inhibitor, a 5'-phosphorylated dinucleotide bearing a bis-negatively charged amino side chain (7), exhibits an IC(50) value of 8 µM in vitro and EC(50) value of 2.6 µM in the HCV subgenomic replicon system. A molecular structure model is presented to propose an interpretation of the gain afforded by the of 3'-H-cytidine modification.