Technical report | Monitoring of Viral-Induced Cell Death Using Real-Time Cell Analysis
The TCID50 is a measure of infectious viral titre in a sample. Traditionally this assay has been carried out using a microscopy based assay. This assay is highly subjective and relies on the correct identification of cytopathic effect (CPE) by eye. To ascertain whether the xCELLigence real-time cell analysis (RTCA) platform could be used to deliver a more objective and robust measure of TCID50, a comparative study of the two methods was carried out. Two viruses were analysed, Gan Gan and Holmes Jungle, members of family Bunyaviridae and Rhabdoviridae respectively. The RTCA method gave results very close to the traditional method and was significantly quicker and easier to analyse. The RTCA method also has the potential to provide additional useful information about the viral infection of cells.
To develop a de novo pathogen identification capability, the virology team in Land Division has been utilising next-generation DNA sequencing to sequence and characterise novel Australian viruses. Due to advances in the speed, throughput and cost of next generation sequencing, this technology can be applied for the identification of viruses and other pathogens from complex samples, such as clinical and environmental samples. DST is currently developing methods to apply this technology to identify viruses directly from serum. A proof of concept experiment using serum samples spiked with known viruses will aid in determining appropriate procedures and detection limits. To proceed with this project, it is essential to determine the concentration of the viral stocks that will be used to spike the samples.
Two viruses previously sequenced and characterised by DST Group have been selected for this project. The first, Gan Gan virus (GGV; family Bunyaviridae) has been associated with an acute endemic polyarthritis-like illness in humans. The second, Holmes Jungle virus (HOJV; family Rhabdoviridae) has been detected in hospitalised patients. One measure of virus concentration is through the determination of the 50% tissue-culture infectious-dose (TCID50). This measure describes the amount of virus that is required to produce an infection morphology, the cytopathic effect (CPE), in 50% of the population of cultured cells. Traditionally this work is carried out using an end point dilution assay; infecting cultured cells with serially diluted aliquots of virus which, after a fixed period of time, are scored for CPE by manual observation. This assay is highly subjective and relies on the correct identification of cytopathic effect (CPE) by eye.
In this study, the xCELLigence real-time cell analysis (RTCA) platform was evaluated as to whether it could be used to deliver a more objective and robust measure of TCID50. The xCELLigence platform measures the electrical signal exerted by cultured cells grown on a microelectronic biosensor on the base of a 96-well cell culture plate. Using this instrument, it is possible to dynamically monitor attributes such as cell attachment, proliferation and morphology. By measuring the change in impedance produced by infected cells over time, a process such as the formation of CPE can be observed.
A comparative study of the two methods was carried out. The RTCA method gave results very similar to the traditional method and was significantly quicker and easier to analyse. The RTCA method also has the potential to provide other information about the viral infection of cells, for example, the time-line of infection and viral specificity assays. It may also be highly useful for viruses which do not grow well in cell culture or do not produce easily discernible CPE.