Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR

Standard curves of RT-qPCR assays on viral RNA

Linear regression analyses were performed by plotting the cycle threshold (Ct) values against the logarithm of the PFU of HAV or TCID₅₀ (50% tissue culture infectious dose) of RV (SA11 and Wa) with RT-qPCR assays A, B and C corresponding to the RNA target. The mean parameters of the standard curves were as follows: standard curves respectively obtained with HAV assays A, B and C showed efficiencies of 100.00%, 95.93%, and 104.83% and regression coefficients of 0.999, 0.997, 0.996; standard curves respectively obtained with RV assays A, B and C showed efficiencies of 90.93%, 94.03%, and 94.23% and regression coefficients of 0.993, 0.986, 0.976 with Wa; standard curves respectively obtained with RV assays A, B and C showed efficiencies of 78.83%, 76.53%, and 85.50% and regression coefficients of 0.989, 0.984, 0.989 with SA11.

Evaluation of dyes-RT-qPCR assays on viral RNA

Lastly, optimal PMA / EMA concentrations were determined on viral RNA samples after dye treatment including photoactivation and purification steps. The effects of dye (concentrations of 10 to 200 μM) were determined by measuring the decrease in RNA quantification by RT-qPCR (Table 1C). PMA at 50 μM enabled the highest reduction of the RT-qPCR signal for HAV RNA (− 3.16 log₁₀) and PMA at 100 and 200 μM respectively enabled the highest reductions of the RT-qPCR signal for RV (SA11) (− 3.58 log₁₀) and RV (Wa) (− 2.52 log₁₀). EMA was still found to be less efficient than PMA treatment for all the viral RNA tested. These data showed that PMA and EMA are able to bind to viral RNA upon photoactivation making the RNA unavailable for amplification by RT-qPCR, although excess dye concentrations can inhibit RT-qPCR assays. The effectiveness of PMA and EMA treatments depends on the type of dye, the concentration of the dye and the viral RNA type, although PMA was found to be the most effective dye for the three viral RNA tested.

Optimization of pretreatment combining dyes and surfactants before RT-qPCR assays for the selective detection of infectious viruses

Kinetic thermal viral inactivation curves and impact of RT-qPCR assays

The heat sensitivity of HAV and RV (Wa, SA11) at four temperatures (37°C, 68°C, 72°C, 80°C) was analyzed by kinetic evaluation of the loss of infectivity in cell culture compared with the loss of genomic titer in RT-qPCR assays with or without pre-treatment. Stable secondary structures may facilitate the covalent binding of PMA / EMA to viral RNA rendering the RNA undetectable by RT-qPCR. Moreover, amplicon length may influence the effectiveness of these assays. Three RT-qPCR assays were assayed for each viral target to explore the impact of the amplified genomic region on the success of the pre-treatment-RT-qPCR assays in detecting the infectious viruses. The log₁₀ reduction detection limits of the cell culture technique were −4 log₁₀ PFU of HAV, -5.5 log₁₀ TCID₅₀ of RV (Wa) and −3.5 log₁₀ TCID₅₀ of RV (SA11). For describing all the inactivation curves, the log-linear + tail model was found to be the most appropriate. Figures 1 and 2 show the values of the parameters of Equation (2) that characterized the fate of the HAV and RV strain levels respectively according to the four different temperatures, and to the three methods of quantification of the virus titer, i.e. RT-qPCR and pre-treatment RT-qPCR depending on the three different RT-qPCR assays used and the infectious titer.

For HAV, the values of S_i,0 were not different from zero, which means that the EMA IGEPAL CA-630 treatment did not affect virus quantification with regard to the RT-qPCR method. At 37°C, the level of HAV remained constant regardless of the method used. For other temperatures, k_max, which is the inactivation rate, increased with temperature. Using a Bigelow-type model, relating D-value to temperature, this increase corresponds to z-values ranging from 17°C to 22°C for EMA-IGEPAL CA-630 -RT-qPCR and infectious titration methods and of 44°C for the RT-qPCR method, regardless of the RT-qPCR assay. Confidence intervals of S_i,res indicate that the fraction of virus that can survive thermal treatment differs depending on the titration method used and the temperature. With EMA-IGEPAL CA-630 - RT-qPCR and RT-qPCR assay C, the S_2,res value is approximately −1.6 log_10, which means that 1 virus out of 40 is quantifiable after 20 min of treatment regardless of the temperature. With EMA-IGEPAL CA-630 - RT-qPCR and RT-qPCR assay A or B, between 1 virus out 200 and 1 virus out of 6000 is still quantifiable after treatment at 68°C and 80°C (with S_2,res ranged between −2.3 log₁₀ and −3.8 log₁₀). For RT-qPCR, S_1,res are much higher than S_2,res, but the difference between RT-qPCR assays A and B and RT-qPCR assay C was also observed for RT-qPCR. For the infectious titration method, S_3,res is around −3.5 log_10, close to the values obtained with EMA-IGEPAL CA-630 - RT-qPCR associated with RT-qPCR assays A and B.

For RV strains, the values of S_2,0 were lower than zero, which means that the EMA / PMA treatment affected virus quantification with regard to the RT-qPCR method. Indeed, the reduction of the concentration of infectious virus due to the monoazide pre-treatment was about of −0.5 log₁₀ by using RT-qPCR assay A and is ranged from −1.2 log₁₀ to −2.5 log₁₀ by using RT-qPCR assays B and C. These reduction levels were the same for both RV strains. At 37°C, the level of RV strains remained constant regardless of the method used. At 68°C, 72°C, and 80°C, the genomic titer of the RV strains was found to be constant by using the RT-qPCR method regardless of the RT-qPCR assay tested. The S_i,res confidence intervals indicate that the fraction of virus that can survive thermal treatment differs depending on the titration method used.

For the Wa RV strain, with EMA-RT-qPCR and RT-qPCR assay A, the S_2,res value was approximately −1.3 log₁₀ which means 1 virus out of 20 was quantifiable after 20 min of treatment regardless of the temperature. With EMA-RT-qPCR and RT-qPCR assays B or C, between 1 virus out of 10⁴ and 1 virus out of 10⁵, was still quantifiable after treatment at 68°C, 72°C or 80°C (i.e. S_2,res ranged between −4 log₁₀ and −5 log₁₀). The S_3,res values obtained with the infectious titration method were similar to the S_2,res values of RT-qPCR assays B and C.

For the SA11 RV strain, with PMA-RT-qPCR and RT-qPCR assay A, S_2,res value is approximately −1.2 log₁₀. With RT-qPCR assays B and C, S_2,res ranged from −2.4 log₁₀ and −3.9 log₁₀. The value of S_2,res with these RT-qPCR assays decreased significantly when the temperature of treatment increases. S_3,res values cannot be estimated as inactivation after 1 minute of treatment for 68°C, 72°C or 80°C was higher than the LOQ.

For SA11, the k_max value increased with the temperature. Using a Bigelow-type model, this increase corresponds to z-values ranging from 15°C to 19°C according to the RT-qPCR assays for the PMA-RT-qPCR method and of 28°C for the infectious titration method. For the Wa strain and EMA-RT-qPCR, the large confidence interval observed for k_max did not make it possible to detect a temperature effect. Very fast inactivation of Wa strain, (after 1 minute of treatment, infectious titers were below the limit of detection (LOD)) only allows to argue that k_max values were higher than 8.

In conclusion, assays conducted to examine the efficiency of pre-treatment RT-qPCR in minimizing detection signals from thermally-inactivated viruses were dependent on virus species, on the temperature of inactivation and on the RT-qPCR assays.

Viruses and cells

HAV strain HM175/18f, clone B (VR-1402) was obtained from the American Type Culture Collection (ATCC). This clone replicates rapidly and has cytopathic effects in cell culture [35]. HAV stock was produced by propagation in foetal rhesus monkey kidney (FRhK-4) cells (ATCC, CRL-1688) [36] and titrated by plaque assay [37]. Results were expressed in plaque-forming units/mL (PFU/mL) and HAV stock contained 10⁷ PFU/mL.

Rotavirus strains SA11 (simian rotavirus A) and Wa (human rotavirus) were obtained from the Pasteur Institute (Paris, France) and were propagated in MA-104 rhesus monkey epithelial cell line (ATCC CRL-2378). MA-104 cells were grown in Minimum Essential Medium - Glutamax™ (MEM), 1% non-essential amino acids, 10% foetal bovine serum and 0.5% penicillin-streptomycin (Life Technologies, France). Cells were incubated at 37°C in an atmosphere containing 5% CO₂ and grown to sub-confluence. Rotavirus viral stock solutions consisted of an infected cell culture supernatant. Infected cells were frozen and thawed once and then clarified using low-speed centrifugation (6000 × g) at 4°C to remove residual debris. The supernatant of SA11 contained 10⁷ TCID₅₀ / mL. The supernatant containing Wa was then ultracentrifugated at 151,000 ×g for 1 h at 4°C to obtain a higher viral titer. The pellet was resuspended in PBS to obtain a Wa stock containing 10⁵ TCID₅₀ / mL. Both virus stocks were divided into aliquots and stored at −80°C. For the infectivity assay, sub-confluent MA-104 cells seeded in 96-well plates were washed twice with MEM. Samples were trypsin-activated for 30 min at 37°C, and then added to MA-104 cells. Plates were incubated 3 days at 37°C. Infectious titers of RV were expressed as TCID₅₀/mL, according to the Kärber method.

RNA purification of Rotaviruses and HAV

HAV and RV RNA stocks were produced from infected cell culture supernatants. They were centrifugated at 4,000 g for 30 minutes at 4°C and then the supernatants were ultracentrifugated at 25,000 g for 25 min at 4°C. Finally, supernatants were ultracentrifugated at 151,000 g for 50 min at 4°C and the pellets were suspended in aliquots of 0.7 mL of 1× PBS and incubated overnight at 4°C before virus titration. The viral stocks were then vortexed for about 10 s before RNA extraction. Volumes of 350 μL were supplemented with NucliSens® easyMAG™ lysis buffer (BioMérieux) up to 3 mL and subjected to the NucliSens® easyMAG™ platform for RNA extraction by the “off-board Specific A protocol” according to the manufacturer’s instructions. Lastly, nucleic acids were eluted in 70 μL of elution buffer and pooled to obtain a homogenized RNA stock. To avoid contamination of cellular DNA from the HAV and RV RNA stocks, the samples were treated with the Turbo DNase free-kit (Life Technologies) according to the manufacturer’s instructions. The purified RNA stocks were quantified by measuring absorbance at 260 / 280 nm with a Nanodrop ND-100 (Thermoscientific, France) and the free software available on the “http://endmemo.com/bio/dnacopynum.php” website. All viral RNA stocks (from HAV, SA11 and Wa) containing 10⁹ copies / μL were aliquoted and stored at - 80°C.

Propidium monoazide (PMA), ethidium monoazide (EMA)

PMA (phenanthridium, 3-amino-8-azido-5-[3-(diethylmethylammonio)propyl]-6-phenyl dichloride) was purchased from VWR (Fontenay sous Bois, France) at 20 mM and diluted in ultra pure RNAse-free water to obtain the solutions used in this study. EMA (phenanthridium, 3-amino-8-azido-5-ethyl-6-phenyl bromide) (Life Technologies) was dissolved in absolute ethanol to create the stock concentration of 5 mg / mL and then dissolved in ultra pure RNAse-free water to obtain the solutions used in this study. The EMA and PMA solutions were stored at −20°C in the dark. All the experiments with dyes were performed in light-transparent 1.5 mL microcentrifuge tubes (VWR).

Binding of dyes to purified viral RNA

The effect of several EMA and PMA treatment processes on 10⁸ copies genome of viral RNA (RV, HAV) in 100 μL of phosphate-buffered saline (PBS) 1 ×, pH 7.0, were evaluated by testing several final dye concentrations (10, 20, 50, 100, 200 μM), with incubation of 2 h at 4°C in the dark and sample exposure to light for 15 min using the LED-Active® Blue system (IB - Applied Science, Barcelona, Spain). To determine whether PMA / EMA interfere with the ability of RT-qPCR to detect viruses, controls consisting of viral RNA that was treated with PMA / EMA without photoactivation were included with each dye concentration used. To attempt to remove the inhibitory effects of residual EMA / PMA on RT-qPCR, viral RNA treated with each dye concentration without photoactivation was purified using the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. Finally, to determine the efficiency of each concentration of PMA / EMA tested, treated viral RNA samples were subjected to photoactivation before the purification step using the QIAquick PCR purification kit. The negative control was a non-treated 1× PBS sample. The positive control was a non-treated viral RNA sample in 1× PBS. A non-treated viral RNA control sample was subjected to the photoactivation step to check the effect of the lamp. Finally, all these samples were subjected to RNA detection by RT-qPCR assays A. The experiments were performed three times for all viral RNA.

Determination of the optimal dye concentration for viruses

The best dye (PMA / EMA) and its optimised concentration were determined for each viral target by testing five dye concentrations (5 μM, 20 μM, 50 μM, 75 μM, 100 μM). Briefly, in 100 μL of 1× PBS samples of 10⁵ TCID₅₀ of RV (SA11), 10³ TCID₅₀ of RV (Wa) and 6 × 10⁴ PFU of HAV were conserved at 4°C or inactivated at 80°C for 10 minutes. Next, samples were incubated with different dye concentrations for 2 h at 4°C in the dark and then exposed to light for 15 min using the LED-Active® Blue system. The negative control was an untreated 1× PBS sample. The positive controls were the non-dye-treated viral samples kept at 4°C or inactivated at 80°C for 10 minutes, used to calculate the reduction rates of the viral load. To check the effect of the lamp, the non-dye-treated viral samples kept at 4°C or inactivated at 80°C for 10 minutes and subjected to the photoactivation step were used as the controls. To check the effect of the dyes, the viral samples at 4°C or inactivated at 80°C for 10 minutes treated with 50 μM of dye without the photoactivation step were used as the controls.

Finally, all these samples were subjected to RNA extraction and detection by RT-qPCR assays A. The experiments were performed three times for each virus.

Evaluation of the combined effect of dyes and surfactants

Tween 20 and IGEPAL CA-630 were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and Triton X-100 from Fisher Bioblock Scientific (Illkirch, France). These surfactants were dissolved in ultra pure RNAse-free water to obtain solutions at 1% and 10%. In 100 μL of 1× PBS, samples of 10⁵ TCID₅₀ of RV (SA11), 10³ TCID₅₀ of RV (Wa) and 6 × 10⁴ PFU of HAV were stored at 4°C or inactivated at 80°C for 10 minutes. The HAV and RV (Wa, SA11) samples were further treated with EMA 20 μM to which different final concentrations (0.1%, 0.5% and 1%) of the surfactants were added. The HAV and RV (SA11) samples were treated with PMA 50 μM to which different concentrations (0.1%, 0.5% and 1%) of the surfactants were added. The RV (Wa) samples were treated with PMA 75 μM to which different concentrations (0.1%, 0.5% and 1%) of the surfactants were added. Next, the samples were incubated for 2 h at 4°C in the dark and then exposed to light for 15 min using the LED-Active® Blue system.

The negative control was a non-inactivated and untreated 1× PBS sample. For the experiments at 4°C, the positive control was a non-inactivated and untreated virus sample incubated for 2 h at 4°C. For the experiments at 80°C, the positive control was an inactivated (10 min at 80°C) and untreated virus sample incubated for 2 h at 4°C. All non-inactivated samples and positive controls were subjected to infectious titration to check the effect of the surfactants on the infectious viruses. Finally, all these samples were subjected to RNA extraction and detection by RT-qPCR assays A. The experiments were performed three times for each virus. Concentrations of the surfactant (Tween 20, Triton ×100 and IGEPAL CA-630) added to the treated samples were applied to MA-104 cells in order to check their cytotoxicity (negative control). The experiments were performed three times for each virus.

Evaluation of the incubation time with dyes and surfactants

The influence of the incubation time with dyes and surfactant were determined for HAV treated with EMA 20 μM + IGEPAL CA-630 0.5%, SA11 treated with PMA 50 μM and Wa treated with EMA 20 μM. Briefly, samples of 10⁵ TCID₅₀ of RV (SA11), 10³ TCID₅₀ of RV (Wa) and 6 × 10⁴ PFU of HAV were stored in 100 μL of 1× PBS at 4°C or inactivated at 80°C for 10 minutes and were further incubated with the corresponding selected dyes and surfactants for 30 min, 2 h and overnight in the dark and then exposed to light for 15 min using the LED-Active® Blue system.

The negative control was a non-inactivated and untreated 1× PBS sample incubated for 2 h at 4°C. For the experiments at 4°C, the positive control was a non-inactivated and untreated virus sample incubated for 2 h at 4°C. For the experiments at 80°C, the positive control was an inactivated (10 min at 80°C) and untreated virus sample incubated for 2 h at 4°C. Additional controls were performed to check the effect of the IGEPAL CA-630 0.5% alone on HAV regardless of the thermal inactivation and photoactivation. Finally, all these samples were subjected to RNA extraction and detection by RT-qPCR assays A. The experiments were performed three times for each virus.

Thermal inactivation of viruses

Three series of HAV and RV strain (Wa, SA11) samples were inactivated thermally in 1× PBS by using a water bath set at 37°C and dry baths at 68°C, 72°C and 80°C. Aliquots of 50 μL of each virus were incubated for each temperature for 0, 1, 5, 10 and 20 min. Then, 150 μL of 1× PBS at 4°C were added to the samples and placed on ice. The negative control was a non-inactivated and untreated 1× PBS sample. The positive control was a non-inactivated and untreated virus sample stored at 4°C. Three 100 μL series of aliquots corresponding to 10⁵ TCID₅₀ of RV (SA11), 10³ TCID₅₀ of RV (Wa) and 6 × 10⁴ PFU of HAV were performed. The first series was kept to monitor loss of infectivity by performing virus titration on cells. The second series was subjected to direct RNA extraction. Finally, the third series was treated with selected dyes and surfactant. Typically, a final dye concentration of 20 μM of EMA and IGEPAL CA-630 0.5% were added to HAV aliquots, a final dye concentration of 20 μM EMA was added to RV (Wa) aliquots, and a final dye concentration of 50 μM of PMA was added to RV (SA11) aliquots. Then, all samples were incubated for 2 h at 4°C in the dark and then exposed to light for 15 min using the LED-Active® Blue system. After photo-activation, the virus samples were also subjected to nucleic acid extraction. Finally, RNA extracts obtained from the second and third series were quantified by testing the three RT-qPCR assays designed for each viral target. The experiments were performed three times for each virus.

Viral RNA extraction

Nucleic acid extraction was performed in untreated virus samples and samples treated with dyes and surfactants. A hundred μL of the virus sample were supplemented with NucliSens® easyMAG™ lysis buffer (BioMérieux) up to 3 mL and subjected to the NucliSens® easyMAG™ platform for total nucleic acid extraction by the “off-board Specific A protocol” according to the manufacturer’s instructions. Lastly, nucleic acids were eluted in 70 μL of elution buffer and stored at −80°C.

Primers and probes

Three RT-qPCR assays targeting the non-coding region at the 5’ end (5’-NCR) of HAV which have been described by Costafreda et al. [38], and adapted from Costafreda et al. [38] and Di Pasquale et al. [39, 40] were used. The sequences of the primer pairs and the TaqMan probes used were as follows:

The HAV RT-qPCR assay A generates amplification products of 174 bp [38] and was recommended in the CEN/ISO/TS 15216 (qualitative / quantitative methods) for detection of HAV in foodstuffs. The sense primer (HAV68) was 5′-TCACCGCCGTTTGCCTAG-3′, the antisense primer (HAV241) was 5′-GGAGAGCCCTGGAAGAAAG-3′ and the TaqMan probe (HAV150 -) was 5′-FAM-CCTGAACCTGCAGGAATTAA–MGB-3′.

HAV RT-qPCR assay B generates amplification products of 353 bp. It exhibits the same sense primer and probe as HAV RT-qPCR model A associated with another antisense primer named HAV-399R: 5′ -GCCTAAGAGGTTTCACCCGTAG -3′ designed with Beacon Designer software.

Finally, the HAV RT-qPCR assay C adapted from Di Pasquale et al. [39, 40] generates amplification products of 77 bp. The sense primer (HAVf ISS (459–478)) was 5′- GCGGCGGATATTGGTGAGTT-3′, the antisense primer (HAVr ISS (535–515)) was 5′- CAATGCATCCACTGGATGAGA-3′ and the TaqMan probe (HAVp ISS (484–511)) was 5′ ROX- Δ GACAAAAACCATTCAACGCCGGAGGACT-BHQ2-3′. When comparing to the model published by Di Pasquale et al. [39, 40], “Δ” corresponds to a deletion of 4 nucleotides and the nucleotides in bold corresponds to insertions.

Three RT-qPCR assays targeting the rotaviruses were used. The RT-qPCR assay which has been described by Pang et al. [41] in the NSP3 region was used with a sense primer slightly modified with degenerated bases for matching with both human and simian strains.

Thus, RV RT-qPCR assay A generates amplification products of 87 bp. The sense primer (Rota NVP3-F) (positions: 963–982) was 5′-RYCATCTAYRCATRACCCTC-3′, the antisense primer (Rota NVP3-R) (positions 1034–1049) was 5′-GGTCACATAACGCCCC-3′ and the TaqMan probe (positions 984–1016) was 5′- FAM- ATGAGCACAATAGTTAAAAGCTAACACTGTCAA-BHQ1-3′.

RV RT-qPCR assay B generates amplification products of 313 bp. It exhibits the same antisense primer and probe as RV RT-qPCR assay A associated with another sense primer named Rota NSP3-736 F : 5′-GARTGGTATYTAAGATCWATGGAAT-3′ designed with Beacon Designer software.

RV RT-qPCR assay C designed in the NSP4 region with Beacon Designer software generates amplification products of 352 bp. The sense primer (rotaNSP4_166-188 F) was: 5′-ATTGCRYTGAAAACRTCAAAATG-3′, the antisense primer (rotaNSP4_517-493R) was: 5′-GCAGTCACTTCTYTTGGTTCATAAG-3′ and the TaqMan probe (rotaNSP4_486-462P) was 5′-ROX-YCCACTTTCCCAYTCTTCTAGCGTT-BHQ2-3′. Primers and probes were purchased from Eurofins (Les Ulis, France) and Applied Biosystems (Courtaboeuf, France).

Real-time RT-qPCR conditions

One-step RT-qPCR amplifications were performed in duplicate on a CFX96™ real-time PCR detection system from Bio-Rad (Marnes-la-Coquette, France). Reactions were performed in a 25 μL reaction mixture containing 1× of thermoscript reaction mix, and 0.5 μL of Thermoscript Plus / Platinum Taq enzyme mix, which are components of the Platinum® Quantitative RT-PCR ThermoScript™ One-Step System (Fisher Bioblock Scientific, Illkirch, France), as well as 2 U RNAse inhibitor (Applied Biosystems), 5 μg of BSA (Ambion), 500 nM of forward primer, 900 nM of reverse primer, 250 nM of probe and 5 μL of RNA extract. The one-step RT-qPCR program was as follows: 60 min reverse transcription of RNA at 55°C, followed by a 15 min denaturation step at 95°C, and finally 45 cycles of 15 s at 95°C, 1 min at 60°C and 1 min at 65°C. The fluorescence was recorded at the end of the elongation steps (1 minute at 65°C) by the apparatus for each amplification cycle. Ct was defined as the PCR cycle at which the fluorescence intensity exceeded the threshold value. All samples were characterised by a corresponding Ct value. Negative samples gave no Ct value. A standard curve for each system was generated using 10-fold dilution of purified RNA. The slopes (S) of the regression lines were used to calculate the amplification efficiency (E) of the real-time qRT-PCR reactions, according to the formula: E = 10^|-1/s| -1 [42].

Data analysis

The viral titers were obtained with cell culture assay and RT-qPCR according to the pre-treatment. Virus inactivation was determined by calculating the log₁₀ (N_t/N₀), where N₀ is the titre of the virus recovered on the positive control and N_t is the titre of the virus recovered on the tested sample.

where N_i(t) is the virus concentration measured with method i at time t and N₀ is the virus concentration obtained by the RT-qPCR method.

GInaFiT, a freeware Add-in for Microsoft® Excel developed by Geeraerd et al. [43] was used to model inactivation kinetics. GInaFiT makes it possible to choose from different types of microbial survival models (nine) according to different statistical criteria (i.e., sum of squared errors, mean sum of squared errors and its root, R², and adjusted R²). According to these criteria, the “log-linear + tail” inactivation model was found to be the most appropriate for describing inactivation curves regardless of the virus and the temperature of inactivation.

where k_max (min⁻¹), S_i,res and S_i,0 are the model parameters.

k_max is the first order inactivation constant, i.e. it characterizes the slope of the linear decrease of concentration expressed as a logarithm. k_max is directly linked to the D value, the decimal reduction time, k_max = ln(10)/D. S_i,res characterizes the fraction of the population remaining constant in time, or, otherwise stated, not undergoing any significant subsequent inactivation regardless of the duration of the inactivation treatment. S_i,0 is the initial survival ratio.

This ratio was expected to be equal to zero if the RT-qPCR method (i = 1) was used to quantify virus titer. S_i,0 can also help to quantify the difference between RT-qPCR and pretreatment-RTqPCR (i = 2) or the cultural titration method (i = 3).

GInaFiT also returns the standard error values of the estimated parameter. These standard errors were used to construct asymptotic parameter confidence intervals. When no inactivation was observed, k_max and S_i,res were presented as zero with no confidence intervals, and the considered experiments were simply represented with S_i,0. When no quantification was possible after 1 minute of treatment, corresponding to very fast inactivation, the limit of quantification (LOQ) value was used to set a value for k_max and S_i,res. k_max was set at its minimum possible value, ln(10)·LOQ and S_i,res were set to their maximum possible value, i.e. LOQ. No confidence intervals were given for either parameter.