β-Propiolactone (BPL)-inactivation of SARS-Co-V-2: In vitro validation with focus on saliva from COVID-19 patients for scent dog training

⁎ Correspondence to: Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany.

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Associated Data

Written agreement was obtained prior to sample collection. The study was conducted according to the ethical requirements established by the Declaration of Helsinki. The local Ethics Committee of Hannover Medical School (MHH) and Hamburg Medical Association at the University Medical-Center Hamburg-Eppendorf (UKE) approved the study (ethic consent number 9042_BO_K_2020 and PV7298, respectively).

Abstract

β-Propiolactone (BPL) is an organic compound widely used as an inactivating agent in vaccine development and production, for example for SARS-CoV, SARS-CoV-2 and Influenza viruses. Inactivation of pathogens by BPL is based on an irreversible alkylation of nucleic acids but also on acetylation and cross-linking between proteins, DNA or RNA. However, the protocols for BPL inactivation of viruses vary widely. Handling of infectious, enriched SARS-CoV-2 specimens and diagnostic samples from COVID-19 patients is recommended in biosafety level (BSL)− 3 or BSL-2 laboratories, respectively. We validated BPL inactivation of SARS-CoV-2 in saliva samples with the objective to use saliva from COVID-19 patients for training of scent dogs for the detection of SARS-CoV-2 positive individuals. Therefore, saliva samples and cell culture medium buffered with NaHCO₃ (pH 8.3) were comparatively spiked with SARS-CoV-2 and inactivated with 0.1 % BPL for 1 h (h) or 71 h ( ± 1 h) at 2–8 °C, followed by hydrolysis of BPL at 37 °C for 1 or 2 h, converting BPL into non-toxic beta-hydroxy-propionic acid. SARS-CoV-2 inactivation was demonstrated by a titre reduction of up to 10^4 TCID₅₀/ml in the spiked samples for both inactivation periods using virus titration and virus isolation, respectively. The validated method was confirmed by successful inactivation of pathogens in saliva samples from COVID-19 patients. Furthermore, we reviewed the currently available literature on SARS-CoV-2 inactivation by BPL. Accordingly, BPL-inactivated, hydrolysed samples can be handled in a non-laboratory setting. Furthermore, our BPL inactivation protocols can be adapted to validation experiments with other pathogens.

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current COVID-19 pandemic that still impacts the world´s health care systems and economics. Therefore, biosafety level-2 laboratory conditions (BSL-2) are currently recommended for diagnostic analysis of COVID-19 patient samples and BSL-3 for the propagation and handling of enriched infectious SARS-CoV-2 samples in Germany (ABAS, 2021). Recently, the detection of COVID-19 and SARS-CoV-2 infection in humans by scent dogs was demonstrated to be a rapid, highly specific and sensitive detection method (Else, 2020, Jendrny et al., 2020, Jendrny et al., 2021b, ten Hagen et al., 2022). The susceptibility of dogs to SARS-CoV-2 infection as spillover hosts and their role in spillback transmission is considered relatively low (Hobbs and Reid, 2021, WOAH, 2020). However, for biosafety reasons, to prevent virus transmission to scent dogs and especially their handlers during training, inactivation of SARS-CoV-2 without affecting the scent of the sample is highly recommended.

β-Propiolactone (BPL) is an organic compound widely used for virus inactivation in vaccine development and production (Wiesner et al., 2021, Zhugunissov et al., 2018), including vaccines against SARS-CoV (Roberts et al., 2010, Tang et al., 2004) and SARS-CoV-2 (Abdoli et al., 2021, Chen et al., 2021, Gao et al., 2020, Kozlovskaya et al., 2021, Wang et al., 2020, Xia et al., 2020). Furthermore, BPL was confirmed not only to inactivate a wide range of viruses, but also other pathogens including bacteria and fungi (LoGrippo, 1957).

The biochemical mechanism of BPL inactivation is based on irreversible alkylation of nucleic acids but also on acetylation and cross-linking between proteins, DNA or RNA (Brusick, 1977, Perrin and Morgeaux, 1995). As a result of those reactions, BPL forms adducts with nucleobases, mainly with guanine (Španinger and Bren, 2020) and induces extensive modifications on nucleobase analogues, nucleosides and synthetic peptides (Uittenbogaard et al., 2011). Importantly, the successful pathogen-inactivation, and at the same time conservation of the BPL-inactivated samples, depends on a combination of various influencing factors. Namely, BPL concentration, pH, temperature, inactivation time and the stability of the pathogen itself (Gupta et al., 2021; Li and Breaker, 1999; LoGrippo, 1960; Sasaki et al., 2016; Tai et al., 2021; Taubman and Atassi, 1968; Weismiller et al., 1990) ( Appendix A , Appendix A). Safe handling of BPL-inactivated samples requires a hydrolysis step that converts toxic and carcinogenic BPL to hazard-free, non-toxic beta-hydroxy-propionic acid after the inactivation of a pathogen (Brusick, 1977).

Scent dogs trained with (inactivated) COVID-19 patient samples are able to distinguish between SARS-CoV-2 positive and negative individuals when they are exposed to different body fluids including saliva, bronchoalveolar lavage fluids (BALFs) (Jendrny et al., 2020), urine (Essler et al., 2021, Jendrny et al., 2021b) and sweat (Grandjean et al., 2022, Hag-Ali et al., 2021, ten Hagen et al., 2021). Besides the need for the elimination of infectivity (Meller et al., 2022), the key feature for an adequate inactivation procedure of SARS-CoV-2 samples prepared for scent dog training is the maintenance of integrity in order to ensure that the dogs still recognise the original volatile organic compound (VOC) pattern characteristic for SARS-CoV-2-infected or diseased individuals (Jendrny et al., 2021a). VOCs are chemical compounds including gases, acids and alcohols released in body fluids for instance as a result of an infection in humans (Sethi et al., 2013). Their composition is disease specific and was proven to be specific for SARS-CoV-2 (Lichtenstein et al., 2021, Wang et al., 2022).

According to current literature, samples prepared for scent dog training can be inactivated with BPL (Jendrny et al., 2020, Jendrny et al., 2021b), heat, ultraviolet (UV) light or protein detergent NP-40 (Essler et al., 2021, Mendel et al., 2021). However, the outcome of the study using NP-40 and heat for inactivation of SARS-CoV-2 were inconclusive. While volatilisation of VOCs due to the calefaction step might impact the sample integrity and odour, BPL treatment does not seem to alter disease-specific VOCs and/or odour imprint required for dogs to detect and distinguish between SARS-CoV-2 infected or diseased and healthy individuals (Jendrny et al., 2021b, Jendrny et al., 2020, ten Hagen et al., 2022, ten Hagen et al., 2021). Dogs trained with BPL-inactivated samples generally revealed a high diagnostic sensitivity (DSe) and specificity (DSp) for the detection of SARS-CoV-2 in different body fluids (82–95 % DSe, 94–99.9 % DSp) and in comparison to other respiratory pathogens (74 % DSe, 96 % DSp) (Jendrny et al., 2021b, Jendrny et al., 2020, ten Hagen et al., 2022, ten Hagen et al., 2021).

The available BPL-inactivation protocols for SARS-CoV and SARS-CoV-2 differ widely. Therefore, the objective of our study was to perform a literature review summarising suitable SARS-CoV and SARS-CoV-2 inactivation protocols using BPL. Furthermore, we focused on the in vitro validation of a BPL-inactivation protocol of SARS-CoV-2 in human saliva samples prepared for scent dog training under field conditions without biosafety level requirements.

2. Materials and methods

2.1. Virus

SARS-CoV-2 human isolate (Human 2019-nCov ex China, BavPat1/2020, Ref-SKU 026 V-03883, kindly provided by Christian Drosten, Charité, Berlin) was passaged once on Vero E6 cells (kindly provided by Bart Haagmans, Erasmus MC, Netherlands) using Opti-MEM™ I Reduced Serum Medium, GlutaMAX™ Supplement (Opti-MEM; ThermoFisher, Karlsruhe, Germany) with 1 % penicillin/streptomycin (Pen/Strep; Sigma, MO, USA). All experiments with infectious SARS-CoV-2 were performed under biosafety level (BSL)− 3 conditions.

2.2. Experimental setup for SARS-CoV-2 spiking experiment

Saliva from SARS-CoV-2 negative healthy volunteers (see section Data availability statement) and, for comparison, Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco™, Thermo Fisher Scientific Inc., MA, USA) with 1 % Pen/Strep were used as spiking matrices or negative controls (NC) in three independent experiments. A detailed graphical description of the workflow is shown in Fig. 1 , Fig. 2 . Briefly, to each sample consisting of 1 ml of saliva or DMEM, 20 µL of 7.5 % sodium bicarbonate (NaHCO₃; Thermo Fisher Scientific) with pH 8.3 was added, resulting in a final concentration of 0.15 % NaHCO₃. After incubation on ice for 10 min, 10 µL of 10 % β-propiolactone (BPL; ACROS Organics™, 1.146 g/ml) pre-diluted in DMEM medium was added to 1 ml of matrix achieving a final concentration of 0.1 % BPL. The vials were subsequently transported on dry ice from biosafety level (BSL)− 2 to BSL-3 laboratory. In the BSL-3 lab, samples were kept on cooling racks (Corning, Arizona, USA) at 2–8 °C for up to 1 h (handling time). For spiking of samples (3 saliva and 3 DMEM samples in each experiment) and the positive control PC 1 (one DMEM or saliva sample used in one of three experiments), 100 µL of SARS-CoV-2 of 10^7.14 (E#1, virus stock M4), 10^7.17 (E#2, virus stock M3) and 10^5.51 (E#3, virus stock M1) TCID₅₀/ml was added to 900 µL of DMEM/saliva and mixed. The spiking procedure took up to one hour and was performed on cooling racks at 2–8 °C. To convert cytotoxic BPL to non-toxic derivative, from each vial (spiked samples and negative controls) aliquots of 100 µL were hydrolysed for 1 h (E#1) or 2 h (E#2, E#3) at 37 °C and stored at − 80 °C. The remaining spiked, PC 1 and NC samples (1 ml each), and the virus stock used for spiking (positive control PC 2) were incubated for 70–72 h at 2–8 °C. Samples were hydrolysed at 37 °C for 1 or 2 h before they were stored at − 80 °C until further analysis to avoid any possible remaining reactivity of BPL at − 80 °C, (Jones et al., 1967). A third freshly thawed positive control PC 3 was used directly as control for the end-point-dilution assay (see section virus quantification and isolation) ( Fig. 2 , Table 1 ).

Schematic workflow of the study concept for validation of SARS-CoV-2 inactivation by β-propiolactone (BPL) in saliva samples. 1) Saliva collection from healthy volunteers. 2) Spiking of saliva and Dulbecco’s Modified Eagle’s Medium (DMEM) with SARS-CoV-2 (see also Fig. 2 ). 3) Inactivation of spiked samples with BPL with subsequent hydrolysis. 4) Verification of SARS-CoV-2 inactivation using virus titration and/or isolation on Vero E6 cells. 5) Validation of the inactivation method on field samples from COVID-19 positive patients. 6) Scent dog training with BPL-inactivated saliva samples for recognition of samples from SARS-CoV-2 positive patients. The Figure was created with BioRender.com (Agreement number GF2570P7K5).

Schematic workflow of the validation of SARS-CoV-2 inactivation with β-Propiolactone (BPL). The light blue, dark blue and white backgrounds represent work conducted under biosafety level (BSL) 2, 3 and 0 conditions, respectively. Negative controls (NC) (A, B) and spiked samples (SPK) (C, D) were treated with sodium bicarbonate (NaHCO₃) and BPL. In the BSL-3 facility, samples C and D were spiked with SARS-CoV-2 (same handling time as NC A and B) and subsequently incubated at 2–8 °C, hydrolysed at 37 °C, stored at − 80 °C and later analysed. Positive controls (PC) Dulbecco’s Modified Eagle’s Medium (DMEM, E) and saliva samples (F) were spiked with SARS-CoV-2 without any chemical pre-treatment and were used in one of three experiments (n = 1). The SARS-CoV-2 stocks used for spiking (G) were incubated as positive controls together with the samples under the same conditions before storage. A freshly thawed SARS-CoV-2 virus stock (H) (corresponding with the virus stock used for spiking in each experiment) was directly used for virus quantification. This protocol was validated with clinical field samples. Inactivated samples were used for scent dog training without any biosafety measures. For more detailed information see Table 1 . The Figure was created with BioRender.com (Agreement number KT25711ESU).

Table 1

Study design and virological results of saliva and medium samples spiked with SARS-CoV-2 and inactivated with beta-Propiolactone (BPL) including the respective controls. Within each individual experiment the same virus stock was used for spiking and the positive control. The schematic workflow is presented in Fig. 2 .

NC: negative control; PC: positive control; CPE: cytopathic effect; DMEM: Dulbecco’s Modified Eagle’s Medium; Opti-MEM: Improved Minimal Essential Medium; TCID50: tissue culture infectious dose; ND: not done; 1 h: sample inactivated with BPL for < 1 h at 2–8 °C at day 0 and hydrolysed for 1 or 2 h at 37 °C, 72 h: sample inactivated with BPL for 71 ± 1 h at 2–8 °C and hydrolysed for 1 or 2 h at 37 °C; M: internal virus stock identification number.

2.3. Confirmation of the performance characteristics of BPL inactivation of SARS-CoV-2 in saliva samples from COVID-19 patients

A total of 8 saliva samples (PS1 to PS8) were obtained from hospitalised COVID-19 patients with clinical symptoms that were proven SARS-CoV-2-RNA positive with PCR by the respective authorised laboratory ( Table 2 ). All samples were subjected to SARS-CoV-2 specific real-time quantitative reverse transcription-PCR (RT-qPCR) according to Jendrny et al. 2020 and Ciurkiewicz et al., 2021 (see supplemental materials), titrated and isolated on Vero E6 cells (see Section 2.4. Virus quantification and isolation). All obtained clinical field samples were subjected to the validated BPL inactivation assay (see Section 2.2. Experimental setup of SARS-CoV-2 spiking experiment) independent from titration and isolation results. All 8 BPL-inactivated samples were subsequently successfully used for training of scent dogs to detect COVID-19 patients (Jendrny et al., 2020).

Table 2

Virological results including real-time reverse transcription-PCR (Cq value from 1:10 diluted sample), virus titration and virus isolation before and after β-Propiolactone (BPL) inactivation of clinical saliva samples from COVID-19 patients.

PS: patient sample, MHH: Hannover Medical School, Hanover, Germany UKE: University Medical-Center Hamburg-Eppendorf, Hamburg, Germany, Cq: quantification cycle value, CPE: cytopathic effect, pos.: positive, TCID50: tissue culture infectious dose 50; neg.: negative, ND: not done since the virus isolation revealed no CPE. # cytotoxic bacterial and/or fungal contamination; § SARS-CoV-2-like CPE not confirmed with subsequent RT-qPCR and end-point dilution assay. * internal RNA extraction/amplification controls were positive (data not shown), Jendrny et al. 2020 (RNA extraction and PCR amplification protocol described in supplemental material).

2.4. Virus quantification and isolation

All samples of the three BPL validation experiments ( Table 1 ) as well as clinical samples ( Table 2 ) were subjected to virus infectivity quantification with end-point dilution assay. Therefore, Vero E6 cells were seeded into 96-well plates (Sarstedt, Germany) in DMEM with 2 % fetal bovine serum (FBS; SUPERIOR stabil®, Bio&SELL GmbH, Germany), 1 % Pen/Strep and 1 % L -Glutamin (GlutaMAX™-I, Gibco™, Thermo Fisher Scientific Inc., MA, USA). For titration of clinical samples, medium was additionally supplemented with 0.2 % gentamycin/amphotericin (Gent/Amph; Gibco™, Thermo Fisher Scientific Inc., MA, USA). All samples were 1:10 pre-diluted with PBS (Thermo Fisher Scientific Inc.) and then ten-fold serially diluted in triplicates on 96-well plates. Cell morphology was checked for cytopathic effects (CPE) for 5–7 days post infection (dpi). Virus titres are given as 50 % tissue culture infective dose per millilitre (TCID₅₀/ml) and were calculated according to Spearman and Kärber (Mayr et al., 1974). The limit of detection of the end-point dilution assay was 10^2.65 TCID₅₀/ml (Mayr et al., 1974, Binder, 2017).

The list of samples subjected to virus isolation is given in Table 1 , Table 2 . Therefore, a sample volume of 20 µL was pre-diluted in 500 µL of DMEM medium and added to 70–80 % confluent Vero E6 cells in a T25 flask with ventilated cap (Sarstedt, Gemany). The medium was supplemented with 2 % FBS, 1 % Pen/Strep, 1 % Glutamax and, in case of clinical samples, additionally with 0.2 % of Gent/Amph. Flasks with infected cells were incubated for 1 h at 37 °C, 5 % CO₂ and rocked every 15 min. The inoculum was discarded and cells were washed once with pre-warmed (37 °C) phosphate buffered saline (PBS). After adding 3 ml of medium, samples were incubated at 37 °C and 5 % CO₂ and checked daily for cytopathic effect (CPE) from 2 dpi to 6 or 7 dpi (Pyke et al., 2020, Wölfel et al., 2020).

2.5. Criteria of literature review

Literature research was conducted in PubMed (https://pubmed.ncbi.nlm.nih.gov/) and VetSearch databanks (https://www.tiho-hannover.de/universitaet/bibliothek/bibliothek, a local platform combining several databases, e.g. Web of Science, CABI, PubMed, Science Direkt and others) using “SARS-CoV”, “inactivat* ” and “propiolactone” or “SARS-CoV-2″, “inactivat* ” and “propiolactone” as keywords. Only peer-reviewed original research articles published between 2003 and 2022 were included. Pre-prints (apart from those clearly marked) and reviews were excluded. Publications that did not contain any description of the inactivation process/conditions or only referred to other articles were not included. We considered all studies that provided at least one of the three major parameters - BPL concentration, incubation time and temperature ( Appendix A , Appendix A).

3. Results

3.1. BPL-inactivation of SARS-CoV-2 in spiked and field samples

All samples incubated with BPL for 1 h or 70–72 h at 2–8 °C were successfully inactivated (no CPE observed) as determined by end-point dilution and virus isolation assays ( Table 1 ). Due to the 1:10 dilution of the inocula in the spiked samples with an initial titre 10^5.52–10^7.17 TCID₅₀/ml, the use of 20 µL for virus isolation (inoculation of cells in cell culture flask) and the lack of CPE, we demonstrated a complete inactivation with a respective titre reduction of up to 10^4 TCID₅₀/ml. Saliva samples used as negative controls or as spiking matrix were SARS-CoV-2 negative with RT-qPCR (data not shown).

Of the 8 specimen PS1 to PS8 from COVID-19 patients, four (PS1, PS2, PS4 and PS5) were confirmed positive with RT-qPCR quantification cycle (Cq) values between 26.81 and Cq 33.34 ( Table 2 ). A 1:10 pre-dilution of the samples was conducted due to the thick and sticky consistency of the saliva. Therefore, we expect 1 log-step lower SARS-CoV-2 RNA values (approximately 3.33 Cq values lower) in the undiluted samples. Virus titration results of saliva samples PS4, PS6, PS7 and PS8 conducted before inactivation revealed cytotoxic effects, indicating bacterial and/or fungal contaminations in the three dilution rows. The sample PS5 that showed the second highest SARS-CoV-2-RNA load (=second lowest Cq value, Cq 28.48) with RT-qPCR revealed a SARS-CoV-2-like CPE using end-point dilution (10^2.65 TCID₅₀/ml) and virus isolation assays. However, RT-qPCR analysis and end-point dilution assay from the supernatant of the virus isolation did not confirm SARS-CoV-2 isolation (no Cq and no CPE). After BPL treatment, successful inactivation of the respective pathogens was proven with end-point dilution and “pathogen isolation” assays from all samples by the absence of cytotoxic effects (PS4, PS5 and PS7) ( Table 2 ).

3.2. Summary of the literature review of SARS-CoV-2 and SARS-CoV inactivation with BPL

Our literature research (supplemental Table 1 ) was conducted in two parts, in 2020 before our experiments and in 2022, since no studies on BPL inactivation were available for SARS-CoV-2 in 2020. We focused on three major BPL-inactivation parameters (BPL concentration, incubation time and temperature) and found in total 37 publications, 27 focusing on an inactivation of SARS-CoV-2 and 10 on SARS-CoV. Selected virus strain and specimen was provided in 30 and 31 out of the 37 studies, respectively, including mainly (20 of 31) virus cell culture or similar matrices. One publication (Jendrny et al. 2020) associated with the present study used saliva. About half (56.8 %, 21 of 37) of the publications used BPL concentrations between 0.05 % and 1 %, 11 studies used 0.025 %, 4 described concentrations below 0.025 % (0.0125–0.001 %) and no concentrations were given in 3 of the studies. Two studies combined BPL (0.025 % and 0.05 %) with formaldehyde (0.01 % and 0.025 %, respectively). An incubation temperature of samples with BPL at 2–8 °C was used in more than two thirds of the studies (73 %, 27 of 37), two and one studies used room temperature or 37 °C, respectively. In contrast, no temperature was given for 8 studies. The BPL-incubation time at 2–8 °C varied between 16 h and 72 h, while at 37 °C 2 h period and at room temperature 3 × 3 h or 24 h period was used. Information about the pH (pH 6–7) or its adjustment with a buffer was given in 4 studies. Hydrolysis step for 1–4 h at 37 °C was described in 13 of 37 studies. Verification of the inactivation method by virus titration, isolation or plaque assay was shown in 21 of 37 publications. However, the initial virus titre was only given in a quarter of all studies (9 of 37) and the calculation of the virus titer reduction after inactivation was not available in any of the selected articles.

4. Discussion

We validated the chemical inactivation of SARS-CoV-2 with BPL in saliva samples with the objective to use saliva from COVID-19 patients for training of scent dogs without biosafety requirements. BPL is an organic compound widely used as an inactivant in vaccine development and production, including for SARS-CoV (Tang et al., 2004) and SARS-CoV-2 (Xia et al., 2020) candidate vaccines. Handling of infectious, enriched SARS-CoV-2 specimen and diagnostic samples from COVID-19 patients is recommended in biosafety level (BSL)− 3 or BSL-2 laboratories, respectively.

Therefore, the successful inactivation of SARS-CoV-2 in human saliva samples by BPL was demonstrated in SARS-CoV-2-spiked and in field samples from COVID-19 patients using a final concentration of 0.1 % BPL buffered with NaHCO₃ (final concentration of 0.15 %) at a temperature of 2–8 °C for 1 h or 71 h (+/- 1 h). The inactivation was stopped after hydrolysis of BPL by incubation at 37 °C for 1 or 2 h ( Fig. 2 , Table 1 , Table 2 ). In our literature review focusing on BPL inactivation protocols for SARS-CoV and SARS-CoV-2, we considered all studies that provided at least one of the three major parameters BPL concentration, incubation time and temperature for the validation of the inactivation ( Appendix A , Appendix A). The hitherto published inactivation protocols of SARS-CoV and SARS-CoV-2 with BPL vary widely. Successful virus inactivation demonstrated by absence of infectious virus in cell culture including the initial virus titre (10^7–10^6 PFU/ml) was only proven in six studies (16 % of 37 studies) (de Castro Barbosa et al., 2022, Gao et al., 2020, Jureka et al., 2020, Kongsomros et al., 2022, Petráš et al., 2020, Saleh et al., 2021) ( Appendix A , Appendix A). Similarly, we demonstrated BPL-inactivation of SARS-CoV-2 by a titre reduction of up to 10^4 TCID₅₀/ml verified by virus titration and isolation, respectively ( Table 1 , Table 2 ). Using the later three studies as an example, the conditions given for the parameters BPL concentration (0.0125–0.5 % or twice 0.05–01 %), incubation time (16–66 h), temperature (2–8 °C or room temperature), pH (6–6.5 or not given) and hydrolysis step (2 h for 37 °C or not given) varied considerably or were not provided by the authors (de Castro Barbosa et al., 2022, Jureka et al., 2020, Petráš et al., 2020) ( Appendix A , Appendix A).

The sample matrix in all reviewed studies was virus cell culture supernatant, except for one study (saliva) that is associated with our study (Jendrny et al. 2020). One third (11 of 37) of the studies described the same (0.1 %) or higher concentrations of BPL for SARS-CoV-2 or SARS-CoV-2 inactivation compared to our study. Two thirds (27 of 37) used the same incubation temperature of 2–8 °C.

BPL was reported to cause chemical modifications on nucleotides and nucleobase analogues depending on the concentration (Gupta et al., 2021, Sasaki et al., 2016). Concentrations 0.2–0.4 % lead to loss of protein content, antigenic integrity and cause aggregation of the virions and therefore, a maximum concentration of 0.05 % was recommended (Gupta et al., 2021, Sasaki et al., 2016). However, for scent dog training VOCs (Meller et al., 2022), but not the antigenic integrity of the sample play a major role (Jendrny et al., 2021a).

We assume that in saliva the protein content and pH is variable and in general higher compared to cell culture associated matrices. In particular field samples may vary in consistency and content such as protein load, pH or inhibiting contaminations including other pathogens. Physiologically non-homogeneous sample matrices of various protein contents infected with unknown viral loads are a particular challenge for the validation of protocols (Elveborg et al., 2022; Pilchová et al. in revision). Another important factor influencing BPL reactivity and hydrolysis is the pH: acidic conditions may impact the reactivity of BPL Taubman and Atassi, 1968 and Lei et al. (2018). The physiological pH of saliva ranges between 6.2 and 7.6 and can turn acidic if the sample is taken after food consumption (Bibby et al., 1986). Therefore, NaHCO₃ for pH correction/buffering of the sample prior to BPL was added. Furthermore, we found that despite of the supplementation with antibiotics and antifungals (combination of Pen/Strep and Gent/Amph), four out of eight clinical samples were contaminated with bacteria and/or fungi ( Table 2 ). However, after BPL treatment, repeated virus titration and isolation analyses revealed inactivation of the contaminating pathogens, at least using cell culture for isolation. No characterisation of the contaminating pathogens or isolation with pathogen-specific assays were conducted to further confirm the inactivation of the unknown pathogens. Hence, we confirm that 0.1 % BPL in saliva also inactivates other pathogens, but the extent has to be further characterised/determined in more in depth studies in the future. LoGrippo (1957) confirmed that BPL inactivates a wide range of viruses and other pathogens including bacteria and fungi (LoGrippo, 1957).

For virus isolation of SARS-CoV-2 on Vero E6 cells from samples of COVID-19 patients, a sample volume of 20 µL diluted in 500 µL medium was chosen to avoid detrimental effects of clinical samples on cell cultures. An MOI of 0.0001, 0.001 and 0.01 with an incubation time of 60 min, 15 min and 1 min were sufficient to successfully infect Vero E6 cells with SARS-CoV-2 according to (Zupin et al., 2021). Therefore, we assumed a successful isolation of SARS-CoV-2 in case of the presence of infectious SARS-CoV-2 virus. Nevertheless, re-isolation did not confirm successful virus isolation from the field samples.

We chose a comparatively high concentration of BPL (0.1 %) to safely inactivate SARS-CoV-2 and potentially other unknown pathogens in inhomogeneous patients´ specimen matrices. Scent dogs may discriminate non-infected individuals from COVID-19 patients in different sample materials such as BALF (Jendrny et al., 2020), urine (Essler et al., 2021, Jendrny et al., 2021b) and sweat (Grandjean et al., 2022, Hag-Ali et al., 2021, ten Hagen et al., 2021). Our BPL-inactivation protocol was also used successfully in follow up studies with other sample matrices such as urine and sweat (sweat was obtained by squeezing sweat containing cotton pads after soaking the pads in PBS) (Jendrny et al., 2021, ten Hagen et al. 2021) with protein contents considered generally lower compared to saliva.

We showed BPL-inactivation of SARS-CoV-2 in medium and saliva samples after incubation at a temperature of 2–8 °C and time of 1 h or 70 h (+/−1 h) followed by hydrolysis for 1 or 2 h at 37 °C. A rapid successful inactivation of SARS-CoV-2 after 1 h at 4 °C is in accordance with other studies (Cerutti et al., 2021, Kusters et al., 2009). In 1960, LoGrippo et al. (1960) newly presented BPL as a virus inactivant. The study showed that at 37 °C successful inactivation of Eastern equine encephalitis virus (EEEV) can be achieved within fifteen minutes - before the half-life of BPL – using a BPL concentration of 4000 mg/L. In contrast, at 2–8 °C the inactivation with BPL required 8–12 h (same concentration) and the half-life of BPL was approximately 20 h (LoGrippo, 1960). Hence, an increase of the incubation temperature to room temperature and higher boosts the reactivity of BPL, but simultaneously leads to a hydrolytic reaction that decreases and finally deactivates the BPL reactivity. The half-life of BPL at 37 °C and 4 °C was described 24–32 min and 16–20 h, respectively (LoGrippo et al., 1960. We used a BPL concentration of 1146 mg/l (28.65 % of 4000 mg/l) at 2–8 °C to inactivate SARS-CoV-2 for 1 h or 72 h and hydrolysed BPL at 37 °C for 1 h or 2 h. A hydrolysis time of 2–3 h at 37 °C was recommended for an initial concentration of 4000 mg/L (LoGrippo et al., 1960. Of the 37 reviewed studies 13 authors provided the hydrolysed BPL at 37 °C. Ten used a hydrolysis time of 2 h at 37 °C, while three used 4, 6 or 24 h ( Appendix A , Appendix A). BPL is reported to be highly toxic, cytotoxic, carcinogenic and tumorogenic in experimental animals, causing severe skin irritation or permanent eye damage (Hearn and Dawson, 1961, Španinger and Bren, 2020). Accordingly, the hydrolysis of BPL in our study was possibly not complete. Although the remaining reactivity of BPL is considered negligible, we recommend 2 h of hydrolysis at 37 °C for samples subjected to scent dog training. Futhermore, no cytotoxic effect of BPL in BPL-treated negative control samples in spiking experiments was observed and therefore, the conversion of BPL to non-toxic compounds was complete ( Table 1 , Table 2 ).

One key factor for the successful training of scent dogs with inactivated samples is the containment of the odour of disease-specific VOCs and/or odour imprint during the sample inactivation procedure, which is required for dogs to detect and distinguish between SARS-CoV-2 infected or diseased and healthy individuals (Jendrny et al., 2021b, Jendrny et al., 2020, ten Hagen et al., 2022, ten Hagen et al., 2021).

An alternative inactivation method of SARS-CoV-2 for scent dog training could be UV-irradiation. The research group of Mendel et al. (2021) found no significant impact of UV light on VOCs using headspace solid phase microextraction gas chromatography mass spectrometry. Respiratory masks, previously worn by patients diagnosed with COVID-19, were subjected to UV-C irradiation for 10 min (each side) and used for a scent dog training. (Mendel et al., 2021). However, adaptability of this inactivation method on liquid samples including the preservation of VOCs required for scent dog training is unknown (Meller et al. 2022). In fact, the effectivity of UV inactivation in fluid samples decreases with the required depth of penetration, as shown for Escherichia coli (Ngadi et al., 2003). Additionally, saliva as a specimen is not homogenous and may include sputum or phlegm if taken from respiratory-ill patients complicating successful inactivation. The protein detergent NP-40 and heat were also used for inactivation of SARS-CoV-2 in COVID-19 patient urine and saliva samples used for training of dogs to detect SARS-CoV-2 infected individuals (Essler et al., 2021), but the study results were inconclusive. Accordingly, for the proposed alternative inactivation methods the containment of VOCs and the integrity of field samples required for reliable scent dog training requires further investigation.

In conclusion, 0.1 % BPL is a suitable inactivating agent for SARS-CoV-2 in saliva samples and is effective already after 1 h at 2–8 °C combined with hydrolysis at 37 °C for 2 h. This way, a transfer of BPL inactivated SARS-CoV-2 containing samples from BSL-3/BSL-2 facility to no biosafety level was allowed. We proved in previous studies that this inactivation protocol does not disrupt the odour imprint of VOCs essential for the scent dog training (Jendrny et al., 2021b, Jendrny et al., 2020, ten Hagen et al., 2021) and samples can be used in high-through put screening events without biosafety requirements (ten Hagen et al., 2022). Furthermore, our protocol can be used for future spiking and validation experiments with other pathogens required for permission of the responsible authority.

Funding sources

The project was funded as a special research project of the German Armed Forces Medical Service (02Z9-S-852121) and supported by the COVID-19 Research Network of the State of Lower Saxony (COFONI) (5FT21) through funding from the Ministry of Science and Culture of Lower Saxony in Germany (14-76403-184). DZIF-Fasttrack 1.921 provided means for biosampling.

CRediT authorship contribution statement

Veronika Pilchová: Investigation, Laboratory and Formal analysis, Validation, Writing – original draft. Prajeeth Chittappen: Conceptualization, Methodology, Writing – review & editing. Paula Jendrny: Writing – review & editing. Friederike Twele: Sample acquisition, Data curation, Writing – review & editing. Sebastian Meller: Writing – review & editing. Isabell Pink: Organisation of the ethical requirements and consents, COVID-19 sample acquisition, review and editing. Anahita Fathi: Organisation of the ethical requirements and consents, COVID-19 sample acquisition, review and editing. Marylyn Martina Addo: Organisation of the ethical requirements and consents, COVID-19 sample acquisition, review and editing. Holger Volk: Writing – review & editing, Resources, Funding acquisition. Albert Osterhaus: Conceptualization, Writing – review & editing, Resources. Maren von Köckritz-Blickwede: Conceptualization, Writing – review & editing, Supervision, Resources, Funding acquisition, Project administration. Claudia Schulz: Conceptualization, Sample acquisition, Data curation, Methodology, Investigation, Laboratory and Formal analysis, Validation, Writing – review & editing, Supervision, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We would like to thank all study participants that provided the saliva samples and Rouwen Stucke, Katharina Meyer and the laboratory management team of the BSL3 facility for their excellent technical support.

Footnotes

Appendix A Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jviromet.2023.114733.

Appendix A. Supplementary material

Data availability

Written agreement was obtained prior to sample collection. The study was conducted according to the ethical requirements established by the Declaration of Helsinki. The local Ethics Committee of Hannover Medical School (MHH) and Hamburg Medical Association at the University Medical-Center Hamburg-Eppendorf (UKE) approved the study (ethic consent number 9042_BO_K_2020 and PV7298, respectively).

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