## 4 Discussion  
In addition to its use simply for viral detection, flow cytometry (FCM) has also been employed for studying the replication of fish viruses such as IHNV [17], _Viral nervous necrosis virus_[18], picscine orthoreovirus [19; 21], and even for coinfections between IPNV and fish rhabdomyures [20]. However, its use for quantitative purposes (qFCM) has never been validated for these viruses. Therefore, we designed a preliminary study to evaluate the reliability of qFCM to study viral replication, using IPNV as a model. In addition, we have adapted the FACS methodology described by [PERSON] and co-workers [27] for the quantification of viral replication in the positive sorted cells. In the present study, 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. In addition, one of the main issues regarding the validation of a quantitative procedure is to ensure  
Figure 4: Quantification of VP2 protein production and RNA synthesis. The kinetics of RNA synthesis (**A**) and VP2 protein production (**B**) were studied in the first 24 h of viral replication in BF-2 cells. In addition, the correlation between both has been assessed (**C**). Each data corresponds to a single data point. its repeatability and reproducibility. In this study, both parameters were first tested on the standard beads (which are used as reference to transform AFI into MESF), and they were demonstrated to be reliable based on the CV values below 10%--in all cases--and on the significant correlation values (R2 \(>\) 0.99). In the case of the IPNV infected cells (FITC\({}^{+}\)), repeatability and reproducibility were also high (CV \(\leq\) 7.2%) in terms of MESF. However, in terms of MFI, the CV corresponding to reproducibility was slightly high (11.5%), probably due to the low repeatability observed on day 2 (CV = 17.4%: data not shown). Therefore, if a qFCM assay must be repeated on different days, the beads standard curve must be simultaneously repeated, in order to reduce the effect of the dispersion introduced by the system. On the other hand, because in general terms the procedure has been confirmed to be repeatable, the application of replicas is not needed when serial dilutions (or time course) are employed, since regression can be applied to confirm the reliability of the data. Otherwise--for single assays--the use of three replicas is mandatory. The procedure was then tested for viral quantification, compared to the traditional methods by pfu and endpoint dilution. The results clearly demonstrated that for the quantification of virus, the use of MFI values from FITC\({}^{+}\) events is appropriate given that a direct relationship between viral titres and MESF was observed. However, we must point out that such a relationship did not correspond to a linear regression, but to a second-degree polynomial function, mainly due to the lowest viral titres. To this regard, the 24 h incubation time applied without overlaying the monolayer (to avoid the progeny viruses spreading to primarily uninfected cells) cause secondary infection to contribute to the MFI data, providing higher values than expected at the lowest MOIs. And, in addition, the lower the MOI, the higher the effect on the MFI data. Another analysis of the data showed that the qFCM methodology was able to detect 2.6 \(\times\) 10\({}^{1}\) pfu/mL (equivalent to 1 \(\times\) 10\({}^{1}\) TCID\({}_{50}\)/mL), a viral concentration which could not be re-isolated in cell culture. This result is similar to that reported by [PERSON] et al. (2016), who stated that FCM was more that 10-fold more sensitive that pfu and isolation in cell culture.