Novel, Real-Time Cell Analysis for Measuring Viral Cytopathogenesis and the Efficacy of Neutralizing Antibodies to the 2009 Influenza A (H1N1) Virus

Abstract

A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×102 to 5.50×107 copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together, this new CPE assay format provided label-free and high-throughput measurement of viral growth and the effect of neutralizing antibodies, illustrating its potential in influenza vaccine studies.

Human Cytokinome Analysis for Interferon Response

“…The IFN biosensor assay is based on real-time monitoring of the change in electric impedance allowing sensing of cellular activity and has been used in several studies in a virology setting (19,20). It allowed us to develop an IFN assay that made detailed observations, such as the effective dose at any given time point, e.g., the ED 30 , ED 50 , and ED 95 , and the time until CPE at any desired percentage of intact cells.…”

“…A label-free cell-based assay (ACEA Biosystems, San Diego, CA) was employed to monitor virus-induced cellular changes in real time (19,20). Cells were seeded in electrode strip-coated 96-well electrode-integrated microplates at a density of 3 ϫ 10 5 cells per well.…”

Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

“…The factors influencing the impedance (referred to as the cell index [CI]) are the number of cells seeded in the well, the way they interact, the quality of interaction of the cells with the microelectrodes, and the overall morphology of the cells (13). The use of the RTCA technology has already been reported for monitoring the cytopathic effects induced by a series of viruses belonging to different families as well as for the determination of antibody-neutralizing activity (14)(15)(16)(17)(18).…”

Real-time replication of swine vesicular disease virus (SVDV) in cell culture systems in vitro

“…The system of real-time cell analysis was also used for monitoring CPE in a Madin-Darby canine kidney (MDCK) cell culture infected with strains of A/H1N1 influenza virus (12), as well as for titration of the virus from the Poxviridae family in a human embryo kidney 293 cell culture (13).…”

“…CPE is a basis for studies using cell cultures. A novel method for the real-time assay of in vitro cells is real-time cell analysis (RTCA) (4,5,9,10,11,14), also applied to diagnosis of viral diseases (2,3,12,13), and it creates a possibility for improvement in that area. The aim of this experiment was to evaluate the applicability of RTCA in CPE detection in SVDV-infected cell cultures.…”