key: cord-0838860-ba2nli51 authors: Hindawi, Salwa I; El-Kafrawy, Sherif A; Hassan, Ahmed M; Badawi, Maha A.; Bayoumi, Maiman M.; Almalki, Ahmad A.; Zowawi, Hosam M.; Tolah, Ahmed M; Alandijany, Thamir A; Abunada, Qossay; Picard-Maureau, Marcus; Damanhouri, Ghazi A; Azhar, Esam I title: Efficient inactivation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in human apheresis platelet concentrates with amotosalen and ultraviolet A light date: 2021-08-16 journal: Transfus Clin Biol DOI: 10.1016/j.tracli.2021.08.005 sha: 5fa351fb4c7b82f4bad248f0417e0ecc377687fb doc_id: 838860 cord_uid: ba2nli51 Objectives: the detection of SARS-CoV-2 RNA in blood and platelet concentrates from asymptomatic donors, and the detection of viral particles on the surface and inside platelets during in vitro experiments, raised concerns over the potential risk for transfusion-transmitted-infection (TTI). The objective of this study was to assess the efficacy of the amotosalen/UVA pathogen reduction technology for SARS-CoV-2 in human platelet concentrates to mitigate such potential risk. Material and Methods: five apheresis platelet units in 100% plasma were spiked with a clinical SARS-CoV-2 isolate followed by treatment with amotosalen/UVA (INTERCEPT Blood System), pre- and post-treatment samples were collected as well as untreated positive and negative controls. The infectious viral titer was assessed by plaque assay and the genomic titer by quantitative RT-PCR. To exclude the presence of infectious particles post-pathogen reduction treatment below the limit of detection, three consecutive rounds of passaging on permissive cell lines were conducted. Results: SARS-CoV-2 in platelet concentrates was inactivated with amotosalen/UVA below the limit of detection with a mean log reduction of >3.31 ± 0.23. During three consecutive rounds of passaging, no viral replication was detected. Pathogen reduction treatment also inhibited nucleic acid detection with a log reduction of >4.46 ± 0.51 PFU equivalents. Conclusion: SARS-CoV-2 was efficiently inactivated in platelet concentrates by amotosalen/UVA treatment. These results are in line with previous inactivation data for SARS-CoV-2 in plasma as well as MERS-CoV and SARS-CoV-1 in platelets and plasma, demonstrating efficient inactivation of human coronaviruses. Résultats : le SARS-CoV-2 dans les concentrés plaquettaires a été inactivé en dessous de la limite de détection avec une réduction logarithmique moyenne > 3,31 ± 0,23. Aucune réplication virale n'a été détectée au cours de trois passages consécutifs. Le traitement de réduction des agents pathogènes a également inhibé la détection des acides nucléiques avec une réduction logarithmique > 4,46 ± 0,51. Conclusion : le SARS-CoV-2 a été efficacement inactivé dans les concentrés plaquettaires par traitement amotosalen/UVA. Ces résultats sont conformes aux données d'inactivation. réduction des agents pathogènes, sécurité transfusionnelle, amotosalen, SARS-CoV-2 J o u r n a l P r e -p r o o f 5 On 11 th of March 2020 the WHO declared a pandemic of the respiratory COVID-19 disease caused by SARS-CoV-2, a betacoronavirus spreading rapidly by respiratory transmission throughout the globe. To date, more than 195 million infections have been confirmed and more than 4.1 million disease-related deaths were reported globally according to the WHO. The detection of SARS-CoV-2 genomic RNA at low viral loads in serum and blood samples of symptomatic patients was reported, but only in a minority of approximately 10% of the samples (linked to disease severity) in a quantity often close to the limit of detection (CT values above 35) (1) . Low viral load SARS-CoV-2 genomic RNA was also detected in rare occasions in screening samples, post donation information (PDI) samples and blood products from asymptomatic donors, as well as in platelet units, but infectious virus could not be isolated (2) (3) (4) . Interestingly, platelets of symptomatic patients were shown to be associated with viral RNA (5) . In vitro studies showed the attachment of viral particles to the surface of platelets, as well as the presence of viral particles inside platelets, but the meaning of this findings is still unclear (6) . The transmission of infectious SARS-CoV-2 through blood transfusion has not been reported yet, but even unlikely it cannot be excluded and is perceived as a theoretical risk (2) (3) (4) . The amotosalen/UVA pathogen reduction (PR) process uses a photochemical reaction to crosslink nucleic acids, resulting in the inhibition of cell and pathogen replication and transcription (7, 8) . Efficient inactivation was shown for a broad variety of viruses and parasites (9) . A recent study showed an effective inactivation of many bacterial species with a breakpoint of >7 log cfu/mL (10) in human platelet concentrates, which is in line with former findings (11) . The treatment of platelet units also efficiently inactivates white blood cells, eliminating the need for gamma-irradiation (12, 13) . The treated platelets have been shown clinically comparable to untreated platelets with respect to hemostasis (14) and component utilization (15) . To address potential concerns regarding the theoretical transmission of human coronaviruses by blood transfusion, and after the demonstration of efficient inactivation of J o u r n a l P r e -p r o o f 6 MERS-CoV-in plasma and platelets with amotosalen/UVA (16, 17) , we recently demonstrated the efficient inactivation of a local clinical SARS-CoV-2 isolate in human plasma with amotosalen/UVA (18) . In the current study we expanded that work assessing the pathogen inactivation efficacy of the amotosalen/UVA PR treatment for SARS-CoV-2 in human apheresis platelet concentrates in 100% plasma. We used a clinical SARS-CoV-2 isolate (SARS-CoV-2/human/SAU/85791C/2020, gene bank accession number: MT630432) maintained in Vero E6 cells (ATCC# CRL-1586) in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). Vero E6 cells SARS-CoV-2/human/SAU/85791C/2020 were used in all experiments as described previously (18) . The SARS-CoV-2 stock was prepared as described previously (18) . Briefly, 90-95% confluent Vero E6 cells were inoculated with a multiplicity of infection (MOI) of one and incubated at 37°C with 5% CO2 in a tissue culture incubator until 80%-90% of cells showed a cytopathic effect (CPE). Supernatant was then collected; cellular debris removed, and aliquots of the viral stock were subsequently stored at -80°C. The infectious titer was determined by plaque assay. Subsequently the illumination container was exposed to 3J/cm 2 FBS and re-transferred to non-infected Vero E6 cells for two more passages. Supernatants were collected at day 3 of incubation of each passage for viral load determination. Plaque assays were conducted as previously described (18) with CT values in the exponential phase. Each run included a positive viral template control and no-template negative control. Each sample was tested in duplicate, and the mean is reported as PEq/mL. The study was approved by the Unit of Biomedical Ethics of the King Abdulaziz University Hospital (approval #285-20). The platelet units used for this experiment were tested negative for the presence of SARS-CoV-2 neutralizing antibodies using the MN assay. Five human apheresis platelet units (A-E) in 100% donor plasma were collected and spiked with SARS-CoV-2. The units were subsequently treated with amotosalen/UVA. The mean infectious titer in the pre-PR treatment samples was 3.31 ± 0.23 log10 PFU/mL (3.68-3.11 log10 PFU/mL) (table 1). The PR treatment resulted in a reduction of >3.31 ± 0.23 log10 PFU/mL, since no infectious virus was detected in the PR samples in the plaque assay (table 1). Figure 1 (20) . However, the implementation of PR takes a certain time, to be prepared against newly emerging pathogens it should be already in place (20) . We showed complete inactivation of >3.31 ± 0.23 log PFU/mL and >4.64 ± 0.5 log PEq/mL SARS-CoV-2 in human apheresis platelets in 100% donor plasma. In the present study, the genomic titer was 10-fold higher compared to the infectious titer (table 1, table 2 (21) . The maximum inactivation capacity which can be shown is dependent on the input titer in a given experiment and is usually several magnitudes lower in clinical isolates compared to tissue-culture attenuated viral stock preparations. Therefore, it is needed to ensure experimentally that the entire virus inoculum is inactivated, underlining the importance of passaging experiments or, as is the case for bacteria, the inclusion of enrichment culture analyses. A study evaluating the inactivation of SARS-CoV-2 using the Riboflavin/UVB PR technology reported a reduction capacity of ≥4.79 ± 0.15 log in human plasma (22) , ≥4.35 log in human apheresis platelets in 100% plasma (23) and ≥3.3 ± 0.26 log in whole blood (22) . This difference in results between the two studies could be attributed to the different SARS-CoV-2 isolates translating into different maximum input titers reported in the studies. The inactivation efficacy of a photochemical PR system largely depends on the pathogen structure and genomic organization (24) . For nucleic acid targeting PR technologies using photoactive compounds and UV light illumination, the photoactive substance must penetrate the viral particle and reach the pathogen's genome, as well as the UV-light during illumination. That points towards a comparable sensitivity of taxonomically closely related pathogens to a PR-system due to their closely related morphology and genome structure. Considering previous data showing the efficient inactivation of MERS-CoV (16, 17) and SARS-CoV-1 (21, 25) , coronavirus sensitivity to amotosalen/UVA PR is high likely. Amotosalen/UVA-treatment of platelet concentrates and plasma units could present additional benefits for coronaviruses TTI risk mitigation. Post-treatment sample ND ND ND * Data are shown as log10 PEq/mL. † Samples in Table 1 were used in this experiment. Samples were used at 1:10 dilution and titer was determined on day 3-post inoculation. ND indicates not detected. 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