Automated real time detection of Arboviruses in culture

Abstract

Background: Major methods of isolating and identifying arboviruses viruses from human and vector populations are tissue culture – based. Viral growth in culture is monitored by the detection of cytopathic effects (CPE) by microscopy. Some viruses do not cause visible CPE or have delayed CPE and can easily be missed because the assay is subjective and tedious. Host cell anomalies can mask or mimic true CPE. Cell culture assays are also limited by the difficulty in identifying novel or multiple infections, the high cost of culture reagents, and the ability to perform adequate quality controls.

Objective: The automated XCELLigence system (Roche) uses plates lined with electrodes to continuously measure changes in electric impedance in cultured cells. This system was tested to validate its utility in addressing the challenges associated with cell-culture based assays.

Methodology: Viruses were inoculated into VERO (green monkey kidney) cells in 96-well or 24-well plates and incubated at 37°C in a humidified CO₂ incubator. Cells were monitored for CPE using the XCELLigence system in real-time or daily by microscopy. For the neutralization test viruses were serially diluted and combined with -positive serum, incubated for 30 minutes, inoculated onto VERO cells and monitored after using plaque assays after 3 days or the xCELLigence system.

Results: On xCELLigence®, CPE was detected earlier than by microscopic with a signature proliferation profile that could be distinguished from cellular anomalies and other virus profiles and required only ~5 minutes of staff time daily to monitor. The automated methods for virus detection and neutralization gave more consistent and reliable results compared to the conventional assay, had internal quality control capabilities, and minimized reagent use significantly.

Conclusions: Automated monitoring of virus cultures improves reproducibility and early virus detection, reduces staff time and error, provides quality control for cell lines and allows for virus quantification.