An Extracorporeal Plasma Filtration Column with Specific Binding to Dengue Virions.

INTRODUCTION: Dengue infection is a significant public health concern that no specific treatment is available. Extracorporeal plasmapheresis or plasma filtration is a treatment option for severe cases with complications. However, the commercial adsorption devices mainly contained size-exclusive porous beads to adsorb the plasma proteins nonselectively. METHODS: We developed a 1:50 simulated circuit for dengue virus-specific adsorption using a flavivirus-specific (4G2) antibody entrapped into the alginate bead. RESULTS: The reduction ratios of the viral titer after 3 h of continuous run were 63.00 ± 1.21%, and 93.97 ± 1.27% measured by reverse transcription qPCR, and plaque titration, respectively. No specific adsorption was observed with Enterovirus A71 or Escherichia coli bacteria. CONCLUSION: This study is a proof-of-concept for the potential use of a dengue virus-specific adsorption column in the 1:50 simulated circuit. The system could be applied to various clinical platforms by substituting target-specific antibodies.

Machine-learning-based automated quantification machine for virus plaque assay counting.

The plaque assay is a standard quantification system in virology for verifying infectious particles. One of the complex steps of plaque assay is the counting of the number of viral plaques in multiwell plates to study and evaluate viruses. Manual counting plaques are time-consuming and subjective. There is a need to reduce the workload in plaque counting and for a machine to read virus plaque assay; thus, herein, we developed a machine-learning (ML)-based automated quantification machine for viral plaque counting. The machine consists of two major systems: hardware for image acquisition and ML-based software for image viral plaque counting. The hardware is relatively simple to set up, affordable, portable, and automatically acquires a single image or multiple images from a multiwell plate for users. For a 96-well plate, the machine could capture and display all images in less than 1 min. The software is implemented by K-mean clustering using ML and unsupervised learning algorithms to help users and reduce the number of setup parameters for counting and is evaluated using 96-well plates of dengue virus. Bland-Altman analysis indicates that more than 95% of the measurement error is in the upper and lower boundaries [±2 standard deviation]. Also, gage repeatability and reproducibility analysis showed that the machine is capable of applications. Moreover, the average correct measurements by the machine are 85.8%. The ML-based automated quantification machine effectively quantifies the number of viral plaques.

Simplified dengue virus microwell plaque assay using an automated quantification program.

The plaque assay is essential for virion quantitation but the classic protocol requires considerable efforts. A simplified dengue 96-well plaque assay with automated quantitation program is an alternative to access the level of infectious virus. Dengue plaque assay was simplified using LLC/MK2 cells and virus mixing simultaneously before semisolid addition. Results were obtained using a flatbed scanner and analysis by the self-written program optimized to manual reads. The newly developed microwell system was accurate to the standard assay because 19 independent titrations from all subtypes obtained from both systems differed less than a log10 p.f.u./ml with no significance (p>0.05) with good correlation (R2=0.9058). Coefficient of variations within and between assays, indicating assay reliability and repeatability, were 19.29%, and 12.50%, respectively. This method serves various experimental designs in drug discovery that requires viral titers assessment. Effective concentrations (EC90) results showed no significant difference between 24- and 96-well assays (p>0.05). Compound screening for potential antivirals and clinical isolate titrations were successfully arranged. The method contains distinguished features including protocol simplicity, less reagent consumption in microwell format, convenient and affordable data acquisition and analysis system.