Quantitative flow cytometry to measure viral production using infectious pancreatic necrosis virus as a model: a preliminary study

applied sciences Article Quantitative Flow Cytometry to Measure Viral Production Using Infectious Pancreatic Necrosis Virus as a Model: A Preliminary Study Diego Vázquez, Carmen López-Vázquez, JoséG. Olveira , Isabel Bandín and Carlos P. Dopazo * Instituto de Acuicultura, Departamento de Microbiología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] (D.V.); [email protected] (C.L.-V.); [email protected] (J.G.O.); [email protected] (I.B.) *Correspondence: [email protected]; Tel.: +34-8818-16048 Received: 6 September 2018; Accepted: 22 September 2018; Published: 26 September 2018   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. Keywords: qFCM; IPNV; viral replication 1. Introduction 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 ]. 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 ]. Different clinical studies using qFCM have been published [ 4 ], including applications in viral research, like monitoring the multiplicity of infection of a virus during vaccine production, the evaluation of protein heterogeneity in the Herpes simplex virus, or the evaluation of cell antiviral response to Rhinovirus [5–7]. FCM has also been extensively used in fish virology, for the screening of antibody libraries [ 8 ], for the detection of different viruses such as IPNV [ 9 – 13 ], the Lymphocystis virus [ 14 ], the Infectious haematopoietic necrosis virus (IHNV) [ 15 ] or the Iridovirus [ 16 ], and in viral replication studies [ 17 – 21 ]. However, to our knowledge, its use for real quantitative purposes has never been reported. Infectious pancreatic necrosis is an important disease affecting salmonid aquaculture. The causative agent, the Infectious pancreatic necrosis virus (IPNV), is an Aquabirnavirus, belonging to the family Birnaviridae. Members of this family have a genome composed of two segments of double-stranded RNA (named A and B), and a naked icosahedral single-shelled capsid. Segment A encodes a 105 KDa Appl. Sci. 2018,8, 1734; doi:10.3390/app8101734 www.mdpi.com/journal/applsci Appl. Sci. 2018,8, 1734 2 of 14 pVP2-NS-VP3 polyprotein, which is cotranslationally processed and cleaved into the structural VP2 and VP3 viral proteins, and the NS viral protease (also known as VP4) [ 22 ]. This segment also encodes a 17 KDa non-structural protein called VP5 whose biological function still needs to be determined [ 23 ]. Segment B encodes VP1, the viral RNA-dependent RNA-polymerase (RdRp) [ 24 ]. Since VP2 elicits neutralizing antibodies and carries serotype-specific and group-specific antigenic determinants, this protein is important for the development of subunit vaccines, for diagnosis and for serological typing. Different applications of FCM have been employed for this virus, both for the detection of the virus in different cells and for in vitro and in vivo viral replication studies; although in some of those studies FCM was used to determine the level of production of certain viral proteins, a quantitative approach of this technique has yet to be validated [17,20,25,26]. The objective of the present study has been the design and evaluation of a qFCM procedure for the analysis and quantification of VP2 proteins expressed in in vitro infected bluegill fry (BF-2) cells. We have also adapted the method for analysing RNA following intracellular sorting (MARIS) reported by Hrvatin et al. [ 27 ] for the simultaneous quantification of the viral RNA expression in infected cells. 2. Materials and Methods 2.1. Virus and Cell Line Employed For this study, the IPNV West Buxton (WB) strain (ATCC VR-877) has been employed. The BF-2 (Bluefin gill) cell line (ECACC # 00021712) was used for viral propagation. Cells were maintained at 20 ◦ C in 25 cm 2 flasks with Eagle’s Minimum Essential Medium (EMEM, Gibco, Thermofisher, Bilbao, Spain) supplemented with 10% foetal bovine serum (FBS) (Lonza, Madrid, Spain), penicillin (100 IU/mL) and streptomycin (0.1 mg/mL) (Lonza, Madrid, Spain). For viral propagation and for the FCM assays described below, monolayers with around 80% confluence were inoculated with the virus at a multiplicity of infection (MOI) of 0.1–0.01 (or lower, in some experiments). After 1 h at 15 ◦ C, the remaining inoculum was withdrawn, and the monolayers covered with fresh 2% EMEM (EMEM supplemented with 2% FBS and antibiotics) and incubated at 15 ◦ C either until cytopathic effect (CPE) became extensive for the propagation of the virus, or during the corresponding time indicated for each assay. 2.2. Titration by Plaque and Endpoint Dilution Assays For plaque assay titration, 6-well plates with 80% confluent BF-2 monolayer were employed. Ten-fold dilutions of the virus were inoculated in the wells (3 replicas per dilution, 200 µ L per replica) and, after 1 h of adsorption at room temperature, the remaining inoculum was removed, and the monolayers overlaid with 2 mL of 1.2% low melting agarose (Pronadisa, Madrid, Spain) in 2%-EMEM. After 5 days of incubation at 15 ◦ C, the monolayers were fixed for 1 h with a fixative solution composed of 25% formalin, 10% ethanol and 5% acetic acid, in phosphate-buffered saline (PBS). After fixation, the agarose overlay was removed, and the monolayers were stained with 1 mL of 5% crystal violet (Sigma, Madrid, Spain) in PBS. The wells were washed, the plaques counted, and the titre expressed as plaque forming units per millilitre (pfu/mL). For the endpoint dilution titration assay, 96-well microtitre plates with BF-2 cells in 2%-EMEM were employed. Ten-fold dilutions of the virus were inoculated in the wells (3 replicas per dilution, 100 µ L per replica) and the plates incubated at 15 ◦ C for 1 week and examined for the presence of CPE. The titres were determined as tissue culture infection dose per millilitre (TCID50/mL) [28]. 2.3. MARIS Staining and Fluorescence Activated Cell Sorting (FACS) The MARIS staining procedure and FACS acquisition of positive infected cells [ 27 ] was adapted and optimized to sort IPNV infected BF-2 cells, using a monoclonal antibody against the VP2 protein and indirect labelling with a FITC conjugate. After viral infection in 25 cm 2 flasks, the cells were Appl. Sci. 2018,8, 1734 3 of 14 dispersed using PBS and vigorous pipetting to detach them from the flask. After centrifugation at 500 × gfor 30 min, the supernatant was withdrawn, and the cells were fixed and permeabilized for 30 min at 4 ◦ C with 4% paraformaldehyde (PFA, Panreac, Barcelona, Spain) and 0.1% saponin (Sigma-Aldrich, Madrid, Spain) solution in PBS supplemented with 1:100 RiboLock RNase inhibitor (Thermo Scientific, Bilbao, Spain). The cells were then centrifuged 5 min at 500 × gand washed twice with a washing buffer: PBS supplemented with 0.2% bovine serum albumin (BSA), 0.1% saponin and 1:1000 RiboLock RNase inhibitor. Incubation with primary monoclonal antibody (anti-IPNV_VP2, IBT systems GmbH, Ertinger, Germany) was carried out in stain buffer (PBS, 1% BSA, 0.1% saponin and 1:100 RiboLock RNase inhibitor; Sigma-Aldrich, Madrid, Spain) during 60 min at room temperature. The cells were washed twice in washing buffer followed by secondary antibody staining (FITC conjugated goat anti-mouse antibody, Sigma-Aldrich, Madrid, Spain) for 45 min at room temperature in staining buffer. The cells were washed twice in wash buffer and resuspended in sort buffer containing 0.5% BSA and 1:1000 RiboLock RNase inhibitor in PBS. The sorting procedure was performed on a FACSAria (BD Biosciences, Madrid, Spain) using FACS Diva software. 2.4. Quantitative Flow Cytometry (qFCM) Assessment Fluorescence data was exported to FlowJo Vx software and analysed in terms of forward light scattering (FSC) and side-light scattering (SSC) to gate cells from background noise. Single events were discriminated from cell aggregate gating events in plots of forward-light scattering area (FSC-A) against forward-light scattering signal height (FSC-H). Fluorescence parameters (in terms of arbitrary fluorescence intensity; AFI) were standardised in Molecules of Equivalent Soluble Fluorochrome (MESF) units by means of type IIIb standard microsphere beads [ 29 ] using a QuantumTM MESF Kit (Bangs Laboratories, Inc., Burlington, ON, Canada). Standard beads were resuspended in the same sort buffer in order to avoid pH-based differences between standards and samples. The standard beads were analysed on the same day of the cell samples analysis, according to the indications of the supplier. The AFI geometric mean of each bead population was recorded and the calibration curve resulting from the linear regression of the geometric mean channel number versus the expected MESF values of the corresponding beads provided a slope of MESF per fluorescence channels using the QuickCal v2.3 (Bangs Laboratories, Inc., Burlington, ON, Canada). The Mean of Fluorescence Intensity (MFI) was then calculated as MESF × percentage of infected cells. Once the threshold limit between positives and negatives was determined using negative controls (mock infected cells), positive cells (against IPNV VP2) were sorted and collected in sort buffer filled tubes. 2.5. RNA Isolation The collected VP2-IPNV positive cells were pelleted by centrifugation for 30 min at 500 × gand 4 ◦ C. The supernatant was discarded and total RNA isolated using the kit Recover All Total Nucleic Acid Isolation (Ambion, Madrid, Spain), following the modified protocol described by Hrvatin et al., (2014). The concentration of the extracted RNA was quantified with a ND-1000 spectrophotometer (Nanodrop Technologies, Inc., Wilmington, CA, USA) and its quality was evaluated from the ratios A260/280 and A260/A230 as described by Sambrook et al. [30]. 2.6. Quantitative RT-PCR We have used a previously optimized reverse transcription-quantitative PCR (RT-qPCR) method for the absolute quantification of IPNV, using the RNA isolated from sorted cells. The RNA was transcribed to cDNA using SuperScript III (Invitrogen, Merelbeque, Belgium) for a 20 µ L reaction volume. Briefly: 9 µ L of RNA were mixed with 1 µ L of random primers (Invitrogen, Merelbeque, Belgium), heated to 99 ◦ C for 5 min and immediately cooled down to 4 ◦ C. Then, 4 µ L (5 × ) of First Strand Buffer, 1 µ L dithiothreitol (DTT) (0.1 M), 1 µ L of dNTPs (10 mM), 3.8 µ L H 2 O and 0.2 µ L Super Script III RT polymerase were added per reaction. The thermal profile continued with 10 min at 25 ◦ C, 50 min at 50 ◦ C and 5 min at 85 ◦ C. Finally, the cDNA was maintained at 4 ◦ C until use or stored at Appl. Sci. 2018,8, 1734 4 of 14 − 20 ◦ C. Real time PCR amplifications were performed in a Bio-Rad CFX96 (Bio-Rad Laboratories, Inc., Madrid, Spain) using 10 µ L of 2x SYBR ® Green Supermix (iQ SYBR ® Green Supermix, Thermofisher, Bilbao, Spain), 500 nM of each primer (PP_WB 2370F, 5 0 -CAAGTTTGGCAGGCTCATCAG-3; PP_WB 2614R, 5 0 -CGTAGTCCTCGTACTCTTCTCC-3) and 2 µ L of cDNA in a 20 µ L final reaction, with the following thermal profile: 95 ◦ C for 3 min, followed by 42 cycles of 95 ◦ C for 15 s and 60 ◦ C for 30 s, and a melting curve analysis of 55 ◦ C to 95 ◦ C with an increment of 0.2 ◦ C for 10 s. Absolute quantification was performed using in vitro transcribed RNA standard from a cloned plasmid with a 364 bp fragment from RNA region 2317–2681 of reference strain WB [31]. 2.7. Preliminary Optimization of the Assay and Evaluation of qFCM Reliability In a preliminary approach, serial dilutions of the virus were inoculated in BF-2 cells and, after 24 h incubation, processed for qFCM as described above. After the threshold limit between positive and negative events was established using the mock infected cells, positive events with different levels of fluorescence were observed. The correlation between the MOI employed and the MFI was analysed by means of linear regression using Prism V5.0 (GraphPad Software, Inc., La Jolla, CA, USA). In addition, 100,000 cells were sorted for further RNA isolation and qPCR analysis, in order to test the method for recovering viral RNA from fixed BF2 positive cells described above. 2.8. Repeatability and Reproducibility of Quantitative Flow Cytometry Measurements To assess the reliability of the qFCM in terms of repeatability and reproducibility of the fluorescent measurements, replicate assays were performed on three non-consecutive days (3 simultaneous replicas per day), using BF-2 infected cells (at a MOI of 0.1) and the standard beads as described before. Afterwards, the fluorescence data in terms of AFI, MESF and MFI were subjected to analysis of the coefficient of variation (CV) values. 2.9. Comparative Evaluation of Correlation between qFCM and Traditional Titration Methods Ten-fold viral dilutions were inoculated in 25 cm 2 flasks with semiconfluent BF-2 cells (approximate density 5 × 10 5 cells/cm 2 ). Adsorption was carried out at room temperature and, after 60 min, the inoculum was withdrawn and replaced by fresh 2%-EMEM. Infected cultures were kept during 24 h at 15 ◦ C, and then the cells from each flask were collected and the fluorescence parameters quantified as described above. The same virus sample was titrated by two different procedures: plaque assay and end point dilution as described. 2.10. Assessment of VP2 Protein Expression during the IPNV Time Course Infection Ten 25 cm 2 cultured flasks were seeded with the same density of BF2 cells from a single 150 cm 2 confluent monolayer. When the subcultured monolayers were semiconfluent, the virus was inoculated at a MOI of 0.1–0.01 and after 1 h of adsorption the monolayers were covered with 2%-EMEM and incubated at 15 ◦ C for a maximum of 24 h. The cells were collected at the following post-infection (p.i.) times: 0 h p.i. (collected right after adsorption) and 4, 8, 12, 16, 20 and 24 h p.i. Mock infected cells (negative control of infection) were also collected at the end of the experiment. The cells were disaggregated, fixed and permeabilized as described above. The samples were maintained at 4 ◦ C in washing buffer until all cells were collected, in order to incubate all the samples with the antibodies at once. Fluorescence values were analysed and recorded and then the positive cells were collected, and the RNA isolated as described previously. RNA copies (corresponding to the VP2 sequence) per IPNV infected cell were quantified and correlated with the VP2 fluorescence values in the MFI units. For this purpose, the Pearson correlation test was performed using GraphPad Prism version 5.00 (GraphPad Software, Inc., La Jolla, CA, USA). Appl. Sci. 2018,8, 1734 5 of 14 3. Results 3.1. Optimization of the Procedure and Assessment of the Reliability of the Data For the optimization of the procedure, and to evaluate its ability to discriminate between different viral concentrations, four multiplicities of infection (MOI) were tested (from 0.1 to 0.0001), and the correlation between the viral doses and the different parameters was assessed by regression analysis. As shown in Table 1, the percentage of infected cells decreased with the MOI and both parameters correlated significantly (R 2 = 0.9882; Figure 1A). Using the obtained MESF data and the percentages of positive cells, the corresponding MFI values were calculated, which as expected also showed a significant correlation with the infected cells (R2= 0.9682; Figure 1B). Appl. Sci. 2018, 8, x FOR PEER REVIEW 5 of 16 analysis. As shown in Table 1, the percentage of infected cells decreased with the MOI and both parameters correlated significantly (R2 = 0.9882; Figure 1A). Using the obtained MESF data and the percentages of positive cells, the corresponding MFI values were calculated, which as expected also showed a significant correlation with the infected cells (R2 = 0.9682; Figure 1B). Figure 1. Correlation between multiplicity of infection (MOI), infected cells, Mean of Fluorescence Intensity (MFI) and RNA molecules. Each data corresponds to a single data point. (A) Regression between MOI and percentage of infected cells; (B) Regression between MOI and MFI; (C) Regression between MFI and viral RNA copies per cell; (D) Regression between MFI and viral RNA copies per positive cells. In graphs with 2 equations, the first one corresponds to a regression line equation (and R2 value) using the values shown on both axes; the second one corresponds to a regression line equation (and R2 value) using logarithm of the values shown on both axes. Dashed lines: regression curves. In addition, the viral RNA was extracted from a number of positive cells and quantified by RT- qPCR to determine the number of RNA copies per cell. The total number of copies (per 100 cells) was calculated multiplying that value by the percentage of infected cells (Table 1). As shown in Figure 1C,D, a clear correlation was observed between either parameters (RNA copies per cell and per all positive cells) and the MFI values, following a second-degree regression with R2 values 0.9734 and 0.9998, respectively. Table 1. Preliminary assays to evaluate the performance of the method for discrimination between different virus concentrations. Fluorescence Parameters Viral RNA Sample % +Cells 1 AFI 2 MESF 3 MFI 4 Per Cell 5 Per All +Cells 6 FITC+ MOI 0.1 65.40 1025 373,696 24,439,718.4 402,807 26,343,577.8 MOI 0.01 8.81 34,962 919,698 8,102,539.4 38,615 340,198.2 MOI 0.001 1.82 4537 2 , 386 , 761 4 , 343 , 905.0 33 , 104 60 , 249.3 MOI 0.0001 0.12 211,266 6,912,796 829,535.5 21,675 2601.0 1 % +Cells: Percentage of positive cells; 2 AFI: arbitrary fluorescence intensity units (geometric mean values); 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 Figure 1. Correlation between multiplicity of infection (MOI), infected cells, Mean of Fluorescence Intensity (MFI) and RNA molecules. Each data corresponds to a single data point. ( A ) Regression between MOI and percentage of infected cells; ( B ) Regression between MOI and MFI; ( C ) Regression between MFI and viral RNA copies per cell; ( D ) Regression between MFI and viral RNA copies per positive cells. In graphs with 2 equations, the first one corresponds to a regression line equation (and R 2 value) using the values shown on both axes; the second one corresponds to a regression line equation (and R2value) using logarithm of the values shown on both axes. Dashed lines: regression curves. Table 1. Preliminary assays to evaluate the performance of the method for discrimination between different virus concentrations. Fluorescence Parameters Viral RNA Sample %+Cells 1AFI 2MESF 3MFI 4Per Cell 5Per All +Cells 6 FITC+ MOI 0.1 65.40 1025 373,696 24,439,718.4 402,807 26,343,577.8 MOI 0.01 8.81 34,962 919,698 8,102,539.4 38,615 340,198.2 MOI 0.001 1.82 4537 2,386,761 4,343,905.0 33,104 60,249.3 MOI 0.0001 0.12 211,266 6,912,796 829,535.5 21,675 2601.0 1 % + Cells: Percentage of positive cells; 2 AFI: arbitrary fluorescence intensity units (geometric mean values); 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. Each data corresponds to a single data point. Appl. Sci. 2018,8, 1734 6 of 14 In addition, the viral RNA was extracted from a number of positive cells and quantified by RT-qPCR to determine the number of RNA copies per cell. The total number of copies (per 100 cells) was calculated multiplying that value by the percentage of infected cells (Table 1). As shown in Figure 1C,D, a clear correlation was observed between either parameters (RNA copies per cell and per all positive cells) and the MFI values, following a second-degree regression with R 2 values 0.9734 and 0.9998, respectively. 3.2. Repeatability and Reproducibility To assess reliability in terms of repeatability and reproducibility, the procedure was tested with BF-2 infected cells (24 h p.i.), using three replicas to evaluate repeatability, and the assay was repeated in three different days to test reproducibility. Since the MESF values of the normalized beads are provided by the manufacturer as standard units, their repeatability and reproducibility were evaluated using the observed AFI values. As shown in Table 2, the CV values corresponding to both repeatability and reproducibility were always below 10% (on all 3 days). In addition, the reliability of the standard curves was demonstrated by the high correlation values (R 2 > 0.99) of the regression lines between the expected MESF (as indicated by the manufacturer) and the observed AFI values (Figure 2). In addition, the repeatability of the three fluorescence parameters (AFI, MESF, and MFI) of the IPNV infected cells was high, with CV values ≤ 7.2% in all cases. The same result was observed in the reproducibility of the AFI and MESF data; although, in the case of MFI the CV corresponding to reproducibility was slightly higher (CV = 11.5%; Table 2). Appl. Sci. 2018, 8, x FOR PEER REVIEW 6 of 16 per cell determined by RT-qPCR using in vitro transcribed viral RNA as standard; 6 Number of RNA copies per all positive infected cells. Each data corresponds to a single data point. 3.2. Repeatability and Reproducibility To assess reliability in terms of repeatability and reproducibility, the procedure was tested with BF-2 infected cells (24 h p.i.), using three replicas to evaluate repeatability, and the assay was repeated in three different days to test reproducibility. Since the MESF values of the normalized beads are provided by the manufacturer as standard units, their repeatability and reproducibility were evaluated using the observed AFI values. As shown in Table 2, the CV values corresponding to both repeatability and reproducibility were always below 10% (on all 3 days). In addition, the reliability of the standard curves was demonstrated by the high correlation values (R2 > 0.99) of the regression lines between the expected MESF (as indicated by the manufacturer) and the observed AFI values (Figure 2). In addition, the repeatability of the three fluorescence parameters (AFI, MESF, and MFI) of the IPNV infected cells was high, with CV values ≤ 7.2% in all cases. The same result was observed in the reproducibility of the AFI and MESF data; although, in the case of MFI the CV corresponding to reproducibility was slightly higher (CV = 11.5%; Table 2). Figure 2. Repeatability and reproducibility. Linear regression between the observed fluorescence (AFI) and the expected MESF reported by the manufacturer of the beads employed as standard. 3.3. Reliability of the qFCM for Viral Titration To evaluate the procedure for viral quantification, qFCM was applied using ten-fold dilutions of the virus (titrated by two methods: plaque forming units and endpoint dilution), and the results are shown in Table 3. Surprisingly, the MESF values corresponding to the lowest MOIs (0.00001 and 0.000001) were higher than the previous ones; however, this was corrected when the percentage of infected cells at each MOI was considered to calculate the MFI values. Figure 2. Repeatability and reproducibility. Linear regression between the observed fluorescence (AFI) and the expected MESF reported by the manufacturer of the beads employed as standard. 3.3. Reliability of the qFCM for Viral Titration To evaluate the procedure for viral quantification, qFCM was applied using ten-fold dilutions of the virus (titrated by two methods: plaque forming units and endpoint dilution), and the results are shown in Table 3. Surprisingly, the MESF values corresponding to the lowest MOIs (0.00001 and 0.000001) were higher than the previous ones; however, this was corrected when the percentage of infected cells at each MOI was considered to calculate the MFI values. Appl. Sci. 2018,8, 1734 7 of 14 Table 2. Repeatability and Reproducibility. Repeatability Day 1 AFI MESF Replica 1 Replica 2 Replica 3 Mean SD CV Replica 1 Replica 2 Replica 3 Mean SD CV Beads B1 15.07 15.14 15.39 15.20 0.17 1.1 1929 1929 0 0 B2 36.10 36.30 37.39 36.60 0.70 1.9 8750 8750 0 0 B3 127.85 127.91 134.23 130.00 3.67 2.8 34,864 34,864 0 0 B4 406.90 425.17 463.92 432.00 29.12 6.7 127,311 127,311 0 0 B5 1197.54 1219.18 1325.49 1247.40 68.49 5.5 421,992 421,992 0 0 Samples Blank 102.50 105.80 112.70 107.00 5.20 4.9 23,579.62 24,483.96 26,391.86 24,818.48 1435.66 5.8 C-MOCK 140.20 131.50 145.40 139.03 7.02 5.1 34,205.24 31,699.19 35,717.23 33,873.88 2029.41 6.0 FITC +1647.40 1656.80 1795.20 1699.80 82.75 4.9 638,252.09 642,579.82 706,821.61 662,551.17 38,400.90 5.8 Repeatability Day 2 AFI MESF Replica 1 Replica 2 Replica 3 Mean SD CV Replica 1 Replica 2 Replica 3 Mean SD CV Beads B1 18.41 17.05 17.64 17.70 0.68 3.9 1929 1929 0 0 B2 38.67 36.72 38.01 37.80 0.99 2.6 8750 8750 0 0 B3 152.00 129.09 117.91 335.00 7.91 5.9 34,864 34,864 0 0 B4 457.93 487.65 449.49 465.02 20.04 4.3 127,311 127,311 0 0 B5 1495.67 1410.55 1263.89 1390.04 117.24 8.4 421,992 421,992 0 0 Samples Blank 96.51 95.59 90.93 94.30 2.99 3.2 20,011.7 19,785.44 18,645.63 19,480.93 732.18 3.8 C-MOCK 178.71 145.90 130.96 151.90 24.43 16.1 41,586.38 32,686.35 28,751.93 34,341.55 6575.37 19.1 FITC +1847.76 1949.35 1761.12 1852.70 133.10 7.2 665,827.32 709,507.84 628,507.58 668,087.58 40,337.94 6.0 Repeatability Day 3 AFI MESF Replica 1 Replica 2 Replica 3 Mean SD CV Replica 1 Replica 2 Replica 3 Mean SD CV Beads B1 16.69 16.28 15.34 16.10 0.69 4.3 1929 1929 0 0 B2 41.19 38.93 37.48 39.20 1.87 4.8 8750 8750 0 0 B3 145.51 130.46 128.15 135.04 10.00 7.4 34,864 34,864 0 0 B4 490.08 543.75 434.09 489.31 44.84 9.2 127,311 127,311 0 0 B5 1560.61 1544.34 1314.78 1473.24 137.47 9.3 421,992 421,992 0 0 Appl. Sci. 2018,8, 1734 8 of 14 Table 2. Cont. Samples Blank 135.17 135.82 128.07 133.00 4.3 3.2 29,451.79 29,615.92 27,667.32 28,911.68 1080.77 3.7 C-MOCK 184.52 186.45 169.11 180.01 9.50 5.3 42,236.19 42,748.36 38,177.84 46.032.39 2504.07 6.1 FITC +1980.12 1906.03 1818.85 1901.70 80.72 4.2 660,066.13 631,542.06 598,203.17 629,937.12 30,962.69 4.9 Reproducibility Day 1 AFI MESF Beads AFI MESF AFI MESF AFI MESF Mean SD CV Mean SD CV B1 15.2 1929.0 17.7 1929.0 16.1 1929.0 16.33 1.3 7.8 1929.00 0.00 0.00 B2 36.6 8750.0 37.8 8750.0 39.2 8750.0 37.87 1.3 3.4 8750.00 0.00 0.00 B3 130.0 34,864.0 133.0 34,864.0 135.0 34,864.0 132.67 2.5 1.9 34,864.00 0.00 0.00 B4 432.0 127,311.0 465.0 127,311.0 489.0 127,311.0 462.00 28.8 6.2 127,311.00 0.00 0.00 B5 1247.4 421,992.0 1390.0 421,992.0 1473.0 421,992.0 1370.23 114.2 8.3 421,992.00 0.00 0.00 Blank 107.00 24,818.48 94.34 19,480.93 133.02 28,911.68 111.45 19.7 17.7 24,403.69 4729.0 19.4 C-MOCK 139.03 33,818.88 151.86 34,341.55 180.03 41,054.13 156.97 21.0 13.4 34,215.67 4017.3 11.7 FITC+1699.80 662,551.17 1852.74 668,087.58 1901.67 629,937.12 1818.07 105.3 5.8 635,525.29 20,614.7 3.2 (MFI) (24,050,607.6) (25,387,328.0) (29,859,019.5) (26,432,318.4) (3,041,943) (11.5) AFI, arbitrary fluorescence intensity units (geometric mean values); MESF, Molecules of Equivalent Soluble Fluorochrome units; SD, Standard deviation; CV, Coefficient of variation (SD/average ×100); C-MOCK, Negative control (Mock infected cells); MFI, Mean Fluorescence Intensity of the infected cells. Table 3. Reliability of the qFCM for viral titration. Inoculated Virus 1Assay 1 Assay 2 MOI TCID50/mL pfu/mL %+Cells 2AFI 3MESF 4MFI 5%+Cells 2AFI MESF MFI 0.1 1×1062.6 ×10666.4 7901 206,112 13,685,836.8 82.6 5761 241,276 19,929,423.9 0.01 1×1052.6 ×10554.1 6641 173,314 9,376,287.4 67.8 4154 166,502 11,288,862.9 0.001 1×1042.6 ×10413.3 6967 181,801 2,417,953.3 23.7 1655 58,628.7 1,389,500.3 0.0001 1×1032.6 ×10317.6 6344 165,581 2,914,225.6 18.6 1395 48,297.5 898,333.3 0.00001 1×1022.6 ×1021.87 13,615 324,712 607,211.4 2.63 874 28,418.9 74,741.8 0.000001 1×1012.6 ×1011.97 11,188 291,618 574,487.5 2.76 963 31,723 87,555.6 1 Titrated by endpoint dilution (TCID 50 ) and plaque forming units (pfu); 2 % + Cells, Percentage of positive cells; 3 AFI, arbitrary fluorescence intensity units (geometric mean values); 4 MESF, Molecules of Equivalent Soluble Fluorochrome units, normalized with respect to the negative control (C-MOCK, Mock infected cells); 5 MFI, Mean Fluorescence Intensity of the infected cells, calculated multiplying the MESF data by the corresponding percentages of infected cells. Each data corresponds to a single data point. Appl. Sci. 2018,8, 1734 9 of 14 On the other hand, a significant correlation (R 2 = 0.9559, assay 1; R 2 = 0.9577, assay 2) was observed between the viral titre and the percentage of infected cells, by a second-degree polynomial regression (y = 2.8979x 2− 6.7267x + 5466, assay 1; y = 3.3423x 2− 6.2588x + 4.229, assay 2) (data not shown). More importantly, a clear correlation between viral titres and the MFI values was observed, also by a second-degree polynomial regression (Figure 3). Appl. Sci. 2018, 8, x FOR PEER REVIEW 10 of 16 On the other hand, a significant correlation (R2 = 0.9559, assay 1; R2 = 0.9577, assay 2) was observed between the viral titre and the percentage of infected cells, by a second-degree polynomial regression (y = 2.8979x2 − 6.7267x + 5466, assay 1; y = 3.3423x2 − 6.2588x + 4.229, assay 2) (data not shown). More importantly, a clear correlation between viral titres and the MFI values was observed, also by a second-degree polynomial regression (Figure 3). Figure 3. Reliability of qFCM for viral titration. Second degree polynomial regression between the viral titre (in pfu and TCID50) and fluorescence (in MFI). Each data corresponds to a single data point. Finally, to compare the limit of detection of qFCM with that of the traditional method involving virus isolation, the same viral dilutions employed in qFCM were subjected to re-isolation in BF-2, observing that re-isolation was not possible from the last dilution (corresponding to 101 TCID50/mL or 2.6 × 101 pfu/mL). Figure 3. Reliability of qFCM for viral titration. Second degree polynomial regression between the viral titre (in pfu and TCID 50 ) and fluorescence (in MFI). Each data corresponds to a single data point. Finally, to compare the limit of detection of qFCM with that of the traditional method involving virus isolation, the same viral dilutions employed in qFCM were subjected to re-isolation in BF-2, observing that re-isolation was not possible from the last dilution (corresponding to 10 1 TCID 50 /mL or 2.6 ×101pfu/mL).