2. Materials and Methods
2.1. Virus Strain and Titration
Experiments were performed in BSL3 facilities using a clinical SARS-CoV-2 strain (Ref-SKU: 026V-03883) isolated at Charite University (Berlin, Germany) and obtained from the European Virus Archive catalog (EVA-GLOBAL H2020 project) (https://www.european-virus-archive.com
). The strain was inoculated at a 0.001 MOI in 90% confluent Vero-E6 cells (ATCC number CRL-1586) and incubated at 37 °C for 24–48 h, after which the medium was changed and incubation continued for 24 h; then, the supernatant was collected, clarified by spinning at 1500× g
for 10 min, supplemented with 25mM HEPES (Sigma-Aldrich, Lyon, France), and aliquots were stored at −80 °C. One aliquot was thawed and used for titration using 50% tissue culture infectivity dose (TCID50
); briefly, when cells were at 90% confluence, six replicates were infected with 150μL of ten-fold serial dilutions of the virus sample, and incubated for 4 days at 37 °C under 5% CO2
. Cytopathic effect (CPE) was read using an inverted microscope, and infectivity was expressed as TCID50
/mL based on the Karber formula [7
]. All samples were quantified by end-point titration on Vero E6 cells with a limit of detection of about 100.5
TCID50/mL (3.16 TCID50/mL).
2.2. Samples Used for Heat Inactivation
Three types of sample were used for assessing the efficacy of heat inactivation protocols: (i) SARS-CoV-2 infected Vero-E6 cell supernatants (with or without supplementation with 3g/L bovine serum albumine [BSA]), (ii) nasopharyngeal samples (NPS) collected in patients, (iii) and sera from blood donors (BD); the two latter were collected before the COVID-19 pandemic period, and were negative for SARS-CoV-2 RNA and for SARS-CoV-2 antibodies, respectively.
NPS were collected into 1 mL of viral transport media (Virocult®, Sigma). They were pooled in order to constitute a homogeneous material that was spiked with infectious SARS-CoV-2 to a final titer ranging from 105 to 106 TCID50/mL depending on the sample type. Spiked material was then distributed in 300 µL aliquots before performing the different heating protocols. The same approach was applied to BD sera.
2.3. Heat Inactivation of SARS-CoV-2 Samples
The virucidal activity of different heat protocols was determined according to the European Standards NF EN 14476-A2 (https://www.analytice.com/en/nf-en-14476-laboratory-biocide-efficacy-test/
). Briefly, a 300-µL sample containing 105
/mL was incubated in a pre-warmed dry heat block using either of the three following protocols: 56 °C-30 min, 60 °C-60 min and 92 °C-15 min, after which the treated sample was immediately titrated (TCID50
) and tested for RNA copies (Table 1
). Virus titration and RT-qPCR were performed before and after heating to measure the viral load reduction factor and variation in RNA copies. Samples were tested in duplicates (cell supernatants) or in six replicates (NPS and BD sera). For NPS and BD sera, the 92 °C-15 min protocol was not performed because of its poor suitability for practical applications in clinical microbiology laboratories [8
2.4. Integrity of SARS-CoV-2 RNA before and after Heat Inactivation
Heat inactivated samples and control samples were extracted using the Qiacube HT and the Cador pathogen extraction kit (both from Qiagen, Venlo, The Netherlands). Viral RNA was quantified by RT-qPCR (qRT-PCR EXPRESS One-Step Superscript™, ThermoFisher Scientific, Waltham, Massachusetts) (10 min-50 °C, 2 min-95 °C, and 40 times 95 °C-3 s/60 °C-30 s) using serial dilutions of a T7-generated synthetic RNA standard. Primers and probe target the N gene (Fw: GGCCGCAAATTGCACAAT; Rev: CCAATGCGCGACATTCC; Probe: FAM-CCCCCAGCGCTTCAGCGTTCT-BHQ1. The calculated limit of detection was 10 RNA copies per reaction.
2.5. Impact of 56 °C-30 min Heating on Results of Serological Assays
To address whether heating sera at 56 °C for 30 min may affect the results observed with two serological assays, a total of 38 SARS-CoV-2 positive human sera were selected, processed and reanalyzed comparatively as detailed hereunder.
2.5.1. Detection of SARS-CoV-2 IgG by ELISA
The semi-quantitative anti-SARS-CoV-2 ELISA for immunoglobulin class G (EI 2606-9601 G, Euroimmun AG, Lübeck, Germany) was used as recommended by the manufacturer. The optical density (OD) was detected at 450 nm, and a ratio of the reading of each sample to the reading of the calibrator was calculated for each sample (OD ratio). Samples were considered positive when OD ratio >1.1.
2.5.2. Detection of SARS-CoV-2 Neutralizing Antibodies
A virus neutralization test (VNT) was performed as previously described [9
]. Briefly, VNT was performed in a 96-well format, using Vero-E6 cells and virus strain described in 2.1. Two-fold serial dilutions of sera were mixed with 100 TCID50
, resulting in final serum dilutions ranging from 1/20 to 1/160, and incubated for 1 h at 37 °C. Serum+virus was transferred onto the confluent cell monolayer, and incubated at 37 °C in a 5% CO2
atmosphere. Positive and negative control wells, containing virus+cells and cells only, respectively, were included in each series. After 4 days, the plates were examined for the presence (no neutralization) or absence (neutralization) of CPE using an inverted microscope.
Understanding the potential effect of heat inactivation on the novel coronavirus SARS-CoV-2 is important in environmental and laboratory conditions in order to elaborate adapted biosafety protocols. Owing to the contagiousness of SARS-CoV-2, heat inactivation can be considered (i) for abolishing virus infectivity or (ii) for reducing the infectivity by viral load reduction. In this study, we used three different types of sample in order to mimic situations encountered in laboratory and non-laboratory environments. SARS-CoV-2-infected cell culture supernatant is commonly manipulated in BSL3 laboratories where the virus is grown for research or diagnostic activities. Nasopharyngeal samples (NPS) are clinical specimens that are manipulated by medical and laboratory personnel in routine care and diagnostic activities. Lastly, serum specimens are commonly manipulated in various types of laboratory activities in and outside of the field of virology. Therefore, addressing the effects of three heating protocols onto these different types of samples is important to better understand their possible use to reduce or suppress the infectivity of the SARS-CoV-2 and to define biosafety measures. The possible influence of heating protocols on the results of virological techniques is also important to address to avoid a loss of sensitivity and false negative results in diagnostic procedures. As 60 °C-60 min can be detrimental for serology and 92 °C 15 min results in a clear drop in RNA quantity detection, these protocols were excluded from certain modules of our study.
According to the European norm NF EN 14476-A2, the protocols tested in this study can be considered as virucidal, i.e., achieving a 4 Log10
reduction in infectivity for all tested samples (cell supernatant, virus-spiked serum or nasopharyngeal samples). However, in our experimental conditions, samples containing viral loads > 6 Log10
remain infectious after 56 °C-30 min and 60 °C-60 min, although the risk of infection at the individual level with samples containing viral loads below 10 TCID50
is not established. In any case, the postulate is that if a given viral load can infect the cell monolayer, the risk of human infection exists. Interestingly, the efficacy of the 56 °C-30 min and 60 °C-60 min protocols are in line with results observed using canine coronavirus and mouse hepatitis coronavirus (3.88 to 4.51 Log10
reduction factor) with 60 °C for 30 min [11
]. However, 56 °C-30 min appears much less efficient on SARS-CoV-2 than on transmissible swine gastroenteritis virus, an alpha coronavirus, showing a reduction in infectivity of >7.5 Log10
]. This suggests that the inactivation of clinical samples ahead of molecular diagnosis should also consider chemical inactivation as an alternative to heat inactivation for SARS-CoV-2 diagnostics [13
In this study, the lack of impact of 56 °C-30 min and 60 °C-60 min protocols on RNA copies detection confirms recently reported results [14
]; however, in contrast, the latter reported a lower decrease in copy number detectable after 95 °C-3 min (ΔCt = 2.2) compared to the ΔCt > 5 after 92 °C-15 min we observed. This is not totally unexpected because of differences in the two protocols. This also raises questions about the possibility to inactivate high viral loads within respiratory samples by short heating at high temperature, an option that is increasingly debated with protocol replacing nucleic acid extraction by heat denaturation [15
]. It is clear that additional studies are needed to elaborate heating protocols achieving infectivity loss together with unchanged detectable copy number.
Complement inactivation through 56 °C-30 min heating is common before ELISA serology. Additionally, 60 °C-60 min and 56 °C-30 min are used for reducing the potential infectivity of the samples processed for serology [18
]; however, either of these treatments may have a deleterious impact on the results qualitatively or quantitatively [19
]. The aim was to address whether 56 °C-30 min could influence the results of two serological assays or not. In contrast with Hu et al. [19
], our results suggest that 56 °C-30 min does not affect, either qualitatively nor quantitatively, the detection of SARS-CoV-2 specific IgG using a commercially available ELISA test. However, 56 °C-30 min had a noticeable effect on the detection of neutralizing antibodies. To the best of our knowledge, this has not been described previously, either for coronaviruses or for other viruses. One recent study reported that combining 56 °C-30 min with phosphate buffered saline-Tween20 (0.3%) supplementation (vol/vol) with (0.15% final concentration) influenced neutralization titers, specifically when they ranged from 20 to 80 (up to 30% of sera showed measurable impact on the result) [20
]. Although we have no evidence-based information to explain this phenomenon, it could be due to (i) the partial heat denaturation of antibodies, changing their ability to neutralize the virus but not the antigen binding or (ii) the presence in the serum of components, other than antibodies, that are heat-sensitive, such as the complement as described for Junín virus and human cytomegalovirus [21
]. Further studies are needed to assess whether this phenomenon is virus-dependent or more widely observed.
In conclusion, the results observed in this study should be taken into consideration for sorting serum samples according to the subsequent serological assays to be performed. Upon reception, aliquoting of serum samples is necessary to allow different processes downstream. Since ELISA is commonly used as a screening test before ELISA-positive sera are confirmed by neutralization assay, performing VNT using heat-inactivated sera can result in reduced titers and in false negative results. Therefore, we advocate that heat inactivation is performed on aliquots before ELISA and that confirmation VNT is performed with unheated aliquots.
Finally, this study should help to choose the best-suited protocol for inactivation in order to prevent the exposure of laboratory personnel in charge of direct and indirect detection of SARS-CoV-2 for diagnostic or research purposes.