## 3 Results
### Quantification of the VP2 Protein Production and RNA Synthesis  
To assess the use of this methodology to study the production of viral proteins and RNA synthesis during the replication of a virus, a time course experiment was designed to test the production of the VP2 protein in IPNV BF2 infected cells throughout the course of the in vitro infection. For that purpose, the absolute fluorescence per cell was quantified by qFCM testing FITC\({}^{+}\) events; additionally, all sorted FITC\({}^{+}\) (around 10\({}^{5}\) positive cells) were pelleted and subjected to RNA extraction and quantification by RT-qPCR to evaluate the RNA synthesis.  
RNA production raised from around 10\({}^{2}\) copies per cell at 0 h p.i. (653.9 copies per positive cells; Table 4) to almost 3 \(\times\) 10\({}^{5}\) copies/cell 12 h p.i., coinciding with the peak of VP2 production. Then it experimented a small increase until 24 h p.i. (up to 3.7 \(\times\) 10\({}^{5}\) copies/cell) (Table 4 and Figure 4A). The intracellular VP2 fluorescent signal rose from 115073 MFI at 0 h p.i. to 1321944.9 at 24 h p.i., corresponding to an increase from 6.3 to 51.9% positive cells. As shown in Figure 4B, a peak of VP2 production was detected at 12 h p.i., coinciding with the maximum level of viral RNA (corresponding to the VP2 sequence) detected in the cells. Furthermore, a significant (R\({}^{2}\) = 0.9514) second degree polynomial regression between RNA synthesis and VP2 protein production was observed (Figure 4C). In addition, we applied a Pearson correlation test to the FITC\({}^{+}\) data, confirming that the increase of VP2 fluorescence positively correlates (Pearson's r = 0.8913) with the RNA detected in the sorted positive cells. Although the linearity was weak, correlation was significant (\(\alpha\) = 0.05), with \(p\) (two-tailed) = 0.0061, and the 95% confidence interval from 0.4201 to 0.9839.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Time p.i. 1** & **\% + Cells 2** & **MESF 3** & **MFI 4** & **RNA/Cell 5** & **RNA/*Cells 6** \\ \hline
0 h & 6.3 & 18,179 & 115,073.1 & 103.3 & 653.9 \\
4 h & 11.0 & 19,647 & 216,117.0 & 615.5 & 6770.6 \\
8 h & 26.8 & 34,673 & 929,236.4 & 24,000.5 & 643,213.7 \\
12 h & 43.7 & 34,206 & 1,494,802.2 & 297,121.5 & 12,984,209.8 \\
16 h & 32.6 & 25,698 & 837,754.8 & 319,035.6 & 10,400,561.0 \\
20 h & 38.5 & 23,221 & 894,008.5 & 250,950.8 & 9,661,604.2 \\
24 h & 51.9 & 25,471 & 1,321,944.9 & 370,657.1 & 19,237,102.3 \\ \hline \hline \end{tabular}  
* Time (in hours) post infection of cell monolayers; 2 Percentage of positive cells; 3 MESF, Molecules of Equivalent Soluble Fluorochrome units, normalized with respect to the negative control (C-MOCK, Mock infected cells); 4 MFI, Mean Fluorescence Intensity of the infected cells, calculated multiplying the MESF data by the corresponding percentages of infected cells; 5 Number of RNA copies per cell determined by RT-qPCR using in vitro transcribed viral RNA as standard; 6 Number of RNA copies per all positive infected cells (considering the percentage of positive cells). Each data corresponds to a single data point.  
\end{table}
Table 4: Time course experiment: VP2 production and RNA synthesis.  
Figure 4: \(Cont.\)