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 (Nt/N0), where N0 is the titre of the virus recovered on the positive control and Nt is the titre of the virus recovered on the tested sample.
Thermal inactivation kinetics were expressed as the virus survival ratio
where Ni(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.
The log-linear + tail model can be expressed as followed:
(2)
where kmax (min−1), Si,res and Si,0 are the model parameters.
kmax is the first order inactivation constant, i.e. it characterizes the slope of the linear decrease of concentration expressed as a logarithm. kmax is directly linked to the D value, the decimal reduction time, kmax = ln(10)/D. Si,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. Si,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. Si,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, kmax and Si,res were presented as zero with no confidence intervals, and the considered experiments were simply represented with Si,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 kmax and Si,res. kmax was set at its minimum possible value, ln(10)·LOQ and Si,res were set to their maximum possible value, i.e. LOQ. No confidence intervals were given for either parameter.
