Real-time monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C protease potency by fluorescence resonance energy transfer.

A real-time assay system that allows monitoring of intracellular human enterovirus (HEV) protease activity was established using the principle of fluorescence resonance energy transfer (FRET). It was accomplished by engineering cells to constitutively express a genetically encoded FRET probe. The FRET-based probe was designed to contain an enterovirus 71 3C protease (3C(pro)) cleavage motif flanked by the FRET pair composed of green fluorescent protein 2 and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by fluorescence microscopy, and the disruption of FRET was readily monitored upon HEV infection. The level of the repressed FRET was proportional to the input virus titer and the infection duration as measured by the fluorometric method. The FRET biosensor cell line was also responsive to other related HEV serotypes, but not to the phylogenetically distant herpes simplex virus, which was confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for the determination of antiviral susceptibility, as the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their susceptibilities to a 3C(pro) inhibitor, rupintrivir, were all accurately determined. In summary, this novel FRET-based system is a means for rapid detection, quantification, and drug susceptibility testing for HEVs, with potential for the development of a high-throughput screening assay.

Can melamine levels in 1-spot overnight urine specimens predict the total previous 24-hour melamine excretion level in school children?

BACKGROUND: We investigated whether melamine concentrations in 1-spot overnight urine sample can represent the previous 8- and 24-h total urinary melamine excretions in school children. The relationship between urinary melamine levels and several clinical biomarkers of early renal injury such as microalbumin and N-acetyl-beta-D-glucosaminidase (NAG) in urine was also examined. METHODS: School children, aged 6-10 y, and their parents who were healthy and lived closely to Kaohsiung Medical University-affiliated hospitals were recruited. All study children had the first 1-spot overnight urine sample collected on the Sunday morning (the first day) immediately when they woke up, and then all the subsequent urine samples continued to be collected until the first 1-spot overnight urine sample on the morning of the next day (Monday, the second day). Two first 1-spot overnight urine samples from their parents on the same Sunday and Monday mornings were also collected. This protocol was completed in the July and August of 2011. All urine samples were measured for melamine, biomarkers of early renal injury, and creatinine. RESULTS: There were 7 girls and 16 boys in this study. Except for one missing urine sample from 1-spot overnight urine sample on the morning of the second day, melamine levels in the rest of urine samples among the study children were all detectable. The median melamine levels of 1-spot overnight urine samples on the first and second day mornings were 0.93 and 1.73 µg/mmol of creatinine respectively. We found that melamine concentrations of 1-spot overnight urine samples on the second day morning were highly correlated with the previous 8- and 24-h total melamine excretions in urine (r=0.936, p<0.001, n=21 and r=0.616, p<0.001, n=21 respectively). Good correlation of 1-spot overnight urine sample on the first and second day mornings was also found (r=0.619, p=0.003, n=21). In contrast, there were no significant correlations of 24-h total urinary melamine and 24-h total urinary microalbumin and NAG excretions (r=-0.221, p=0.319, n=22 and r=0.084, p=0.710, n=22). CONCLUSION: Melamine levels in 1-spot overnight urine sample can predict the previous 8- and 24-h total melamine excretions in urine. Since melamine exposure levels in these school children were relatively low, its association with clinical biomarkers of early renal injury was not found. A future study is necessary to increase the sample size and to find the more sensitive preclinical biomarkers of renal injury to link with low melamine exposure in children in the community.