Abstract
In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its applications in viral replication and viral-cell interactions, as well as its capacity to quantify proteins (qFCM). In the present study, we have designed and evaluated a qFCM procedure for the in vitro analysis and quantification of fish viral proteins, using the infectious pancreatic necrosis virus (IPNV) as a model. We have also tested its use for viral titration and adapted the MARIS (method for analysing RNA following intracellular sorting) method for simultaneous quantification of viral RNA expression in infected cells. The procedure has proved to be repeatable and reproducible to an acceptable level, although to ensure reproducibility, the repetition of standard curves is inevitable. Regarding its use for viral quantification, a direct relationship (by a second-degree polynomial regression) between viral titres and Molecules of Equivalent Soluble Fluorochrome (MESF) was observed. Finally, the results support the use of this technology, not only for virus quantification, but also to study viral replication from a quantitative approach.
Highlights
Since its first development, flow cytometry (FCM) has become a reliable tool to study virus-cell interactions and, among its applications, the quantification of cellular antigens gained popularity in the early 80s [1,2]
We designed a preliminary study to evaluate the reliability of quantitative flow cytometry (qFCM) to study viral replication, using Infectious pancreatic necrosis virus (IPNV) as a model
Prior to assessing the use of qFCM for the quantification of viral components, we first performed a preliminary test of the procedure to ensure its reliability and performance to discriminate between viral concentrations, and we have confirmed the correlation between viral doses (MOI) and fluorescence (MFI), and between these and the number of RNA copies in the positive cells
Summary
Flow cytometry (FCM) has become a reliable tool to study virus-cell interactions and, among its applications, the quantification of cellular antigens gained popularity in the early 80s [1,2]. This methodology, known as quantitative flow cytometry (qFCM), is defined as “the calibrated measurement of fluorescence intensity from labelled particles to determine the actual number of fluorescent ligands labelling each particle” [3]. The causative agent, the Infectious pancreatic necrosis virus (IPNV), is an Aquabirnavirus, belonging to the family
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