key: cord-0742017-5d8hi2pe
authors: Eickmann, Markus; Gravemann, Ute; Handke, Wiebke; Tolksdorf, Frank; Reichenberg, Stefan; Müller, Thomas H.; Seltsam, Axel
title: Inactivation of three emerging viruses – severe acute respiratory syndrome coronavirus, Crimean–Congo haemorrhagic fever virus and Nipah virus – in platelet concentrates by ultraviolet C light and in plasma by methylene blue plus visible light
date: 2020-01-12
journal: Vox Sang
DOI: 10.1111/vox.12888
sha: ef48b8004f3b7732acc9c56b4d1ccb45760a1445
doc_id: 742017
cord_uid: 5d8hi2pe

BACKGROUND: Emerging viruses like severe acute respiratory syndrome coronavirus (SARS‐CoV), Crimean–Congo haemorrhagic fever virus (CCHFV) and Nipah virus (NiV) have been identified to pose a potential threat to transfusion safety. In this study, the ability of the THERAFLEX UV‐Platelets and THERAFLEX MB‐Plasma pathogen inactivation systems to inactivate these viruses in platelet concentrates and plasma, respectively, was investigated. MATERIALS AND METHODS: Blood products were spiked with SARS‐CoV, CCHFV or NiV, and then treated with increasing doses of UVC light (THERAFLEX UV‐Platelets) or with methylene blue (MB) plus increasing doses of visible light (MB/light; THERAFLEX MB‐Plasma). Samples were taken before and after treatment with each illumination dose and tested for residual infectivity. RESULTS: Treatment with half to three‐fourths of the full UVC dose (0·2 J/cm(2)) reduced the infectivity of SARS‐CoV (≥3·4 log), CCHFV (≥2·2 log) and NiV (≥4·3 log) to the limit of detection (LOD) in platelet concentrates, and treatment with MB and a fourth of the full light dose (120 J/cm(2)) decreased that of SARS‐CoV (≥3·1 log), CCHFV (≥3·2 log) and NiV (≥2·7 log) to the LOD in plasma. CONCLUSION: Our study demonstrates that both THERAFLEX UV‐Platelets (UVC) and THERAFLEX MB‐Plasma (MB/light) effectively reduce the infectivity of SARS‐CoV, CCHFV and NiV in platelet concentrates and plasma, respectively.

There is a large group of emerging viruses known to be occasionally transmitted by blood or to have properties suggesting their transmissibility by this route. These pathogens include severe acute respiratory syndrome coronavirus (SARS-CoV), Crimean-Congo haemorrhagic fever virus (CCHFV) and Nipah virus (NiV), which have been identified by the World Health Organization (WHO) as major infectious threats with the potential to cause a global pandemic [1] [2] [3] .

There are different pathogen inactivation techniques that have been developed to reduce or eliminate the threat of infectivity from known and emerging transfusion-transmissible agents [4] . THERAFLEX UV-Platelets (Macopharma, Tourcoing, France) is a novel method for pathogen inactivation treatment of platelet concentrates (PCs) [5] [6] [7] . This purely physical system is based on shortwave UVC light, which penetrates the fluid of PCs and inactivates micro-organisms and leucocytes by damaging nucleic acids. THERAFLEX MB-Plasma (Macopharma) is a photodynamic pathogen inactivation procedure for treatment of plasma [8, 9] . Plasma units derived from single blood donations are illuminated with visible light in the presence of the phenothiazine dye methylene blue (MB). When plasma is MB/light-treated, singlet oxygen is generated, which leads to the destruction of viral nucleic acids. The MB/light-based method is in routine use in Europe for about 17 years [10].

Both pathogen inactivation systems have been tested in vitro to be effective against many different types of viruses, including emerging viruses such as West Nile virus and yellow fever virus [6, 8, 9, [11] [12] [13] [14] [15] [16] [17] [18] [19] . In this study, we investigated the capacity of THERAFLEX UV-Platelets and THERAFLEX MB-Plasma systems to inactivate the emerging viruses SARS-CoV, CCHFV and NiV in PCs and plasma, respectively.

Selection of volunteer donors was based on local standard practices. Only regular blood donors that fulfilled the requirements for blood donation and had given their informed consent approved by the local ethics committee were included in the study.

Plasma-reduced PCs in platelet additive solution SSP + (Macopharma) were prepared from pools of five buffy coats as previously described and were stored under agitation at 22 -2°C [14] . The target specifications of the PCs were a platelet concentration of approximately 1x10 9 /mL and a plasma content of approximately 35% in accordance with Macopharma's specifications for UVC treatment of PCs. Air was removed from all plasma units and PCs.

Pathogen inactivation of PCs was performed using the THERAFLEX UV-Platelets system (Macopharma) according to the manufacturer's instructions as described previously [14] . All PCs were irradiated with UVC light to a total dose of 0Á2 joules per square centimetre (J/cm 2 ) with constant vigorous agitation to ensure uniform treatment [6] .

Pathogen inactivation of plasma units was performed using the THERAFLEX MB-Plasma system (Macopharma) as described previously [14] . Plasma for pathogen inactivation was processed by filtration for leucodepletion, addition of MB and subsequent illumination with visible light to a total dose of 120 J/cm 2 according to the instructions of the manufacturer for this system [20] . The MB removal step, an integral processing step in routine use of the THERAFLEX MB-Plasma system, was omitted for exclusive analysis of the virus inactivation effects of illumination.

Virus titres were determined by assessing for virus-induced changes in morphology (cytopathic effects) of indicator cells and calculated according to the Spearman-K€ arber method and expressed as the log of the 50% tissue culture infectious dose (log TCID50) [14, 21, 22] . Titration was performed at the initial sample dilutions at which no cytotoxicity of indicator cells was observed. The effectiveness of virus inactivation was calculated as the log reduction factor (RF) using the formula RF = log 10 A 0log 10 A n , (R, reduction factor; A 0 , spiked total virus load before treatment; and A n , total virus load after treatment). The overall reduction factor was expressed as the sum of RFs for all steps. The limit of detection (LOD) of the assay was defined as the lowest TCID 50 achievable at non-cytotoxic sample concentrations.

SARS-CoV, strain Frankfurt 1 [23] , was grown and assessed in Vero E6 cells (ATCC CCL-22), CCHFV, strain Afg09-2990 [24] , was propagated and assessed in Huh7 cells (JCRB 0403), and NiV, strain Malaysia [25] , was grown and assayed in Vero 76 cells (ATCC CRL-1587). For preparation of the virus stocks, viral supernatants were collected on days 2-4 of cell culture, when a cell confluence of approximately 80% was achieved, centrifuged, aliquoted and frozen at -80°C until further use in spiking experiments.

PCs (volume: 375 ml; n = 2 per virus) and plasma units (volume: 315 ml; n = 2 per virus) were spiked 1:10 with supernatant of each virus and treated with UVC and MB/light, respectively. After spiking, PCs and plasma units were still within the specifications of the respective pathogen inactivation method. The light doses were applied incrementally until the full light doses of each treatment were achieved. After each process step, samples were collected and serially diluted for virus titration. In order to test for intrinsic virus inactivation of the blood product, reference samples were collected from each bag before pathogen inactivation treatment, stored at room temperature and tested at the end of the experiments to account for any intrinsic virus inactivation by the blood product.

The results of the infectivity assays demonstrated that UVC irradiation and MB/light dose-dependently inactivated SARS-CoV, CCHFV and NiV in plasma-reduced PCs and plasma units, respectively. In PCs, at half of the full UVC dose (0Á1 J/cm 2 ) SARS-CoV and CCHFV infectivity levels were below the LOD, while at three-fourth of the full UVC dose (0Á15 J/cm 2 ) also NIV infectivity levels were below the LOD (Table 1 ). Thus, virus reduction factors ≥3Á4 for SARS-CoV, ≥2Á2 for CCHFV and ≥4Á3 for NiV were achieved with the UVC-based pathogen inactivation system in PCs.

In plasma, already at one-fourth of the full light dose (30 J/cm 2 ) SARS-CoV, CCHFV and NiV were inactivated to levels below the LOD (Table 2) . These results correspond to virus log reduction factors of ≥3Á1, ≥3Á2 and ≥2Á7 that were achieved by MB/Light treatment for SARS-CoV, CCHFV and NiV, respectively, in plasma.

For SARS-CoV, we observed a loss of infectivity of about 1 log lower after spiking in some cases. This significant loss of infectivity was probably caused by nonspecific innate immune factors that neutralize viruses in plasma [23, 24] . However, virus titres did not further decrease in controls during the course of the experiments. In particular, there were no significant differences between load and reference samples, indicating that the observed virus inactivation was solely caused by the treatment with UVC and MB/light.

A major argument for using pathogen inactivation technologies to treat blood components is that they support a proactive approach providing more generalized protection against new and emerging infectious agents which continuously challenge the safety of the blood supply. The conventional reactive approach to wait until the threat from an emerging transfusion-transmitted agent has been identified before responding by modifying donor screening programmes takes time and, ultimately, the response may not be quick enough to prevent the transfusion of contaminated blood products. Because there are hundreds of known emerging or re-emerging human pathogens [26] , the manufacturers of pathogen inactivation methods are required to continuously test the inactivation capacity of their systems for new infectious agents.

In this study, the inactivation efficacy of UVC and MB/ light was for the first time tested against CCHFV and NiV Bag 2 log 10 TCID 50 /ml log 10 RF log 10 TCID 50 /ml log 10 RF log 10 TCID 50 /ml log 10 RF log 10 TCID 50 /ml log 10 RF log 10 TCID 50 /ml log 10 RF log 10 TCID 50 /ml log 10 RF or other members of the Nairoviridae and Paramyxoviridae families. SARS-CoV was also included in the study to confirm the efficacy of the two pathogen inactivation systems for coronaviruses, as has previously been demonstrated for MERS-CoV [14] . The results of this study show that both pathogen inactivation systems effectively inactivated all three viruses spiked into the PC and plasma samples, even at light dose increments below the full doses recommended by the manufacturers. One limitation of this study is that the number of replicates was small due to safety constraintslaboratory studies with these zoonotic viruses must be performed in accordance with the highest biosafety requirements. In addition, large-volume plating which would have allowed increasing the sensitivity of the assay and consequently improving the log reduction value could not be performed. SARS-CoV is an enveloped, positive-sense singlestranded RNA coronavirus. It emerged in 2002 in China and spread to 29 additional countries and is thought to be an animal virus that spread to humans from civets most likely infected by bats [27] . Similar to Middle East respiratory syndrome coronavirus (MERS-CoV), the main route of human-to-human transmission of SARS-CoV is nosocomial transmission. However, transmission between family members has also been observed, suggesting that SARS-CoV might continue to spread via transmission by infected persons returning from affected areas. Transmission by blood transfusion has not been described yet. Nevertheless, the high mortality of the disease and the not yet fully understood transmission mechanisms of SARS-CoV pose a potential threat to the safety of the blood supply [27] . Interestingly, the detection of low-level viremia in asymptomatic patients during an SARS-CoV outbreak suggests a theoretical risk of transmission via blood products. As a precautionary measure, the World Health Organization introduced a recommendation for the deferral of blood donations from donors potentially exposed to SARS-CoV, and the Australian Red Cross Blood Service amended its donor screening questionnaire to include questions to identify persons with SARS-CoVrelated symptoms [28] .

Crimean-Congo haemorrhagic fever virus is an enveloped, negative-sense single-stranded RNA virus of the Nairoviridae family. CCHFV often results in a mild, nonspecific febrile illness but may occasionally cause severe haemorrhagic disease. This disease was first identified in the Crimean region of the former Soviet Union in 1944 and is a significant public health concern. CCHFV occurs across a wide geographic region, including Europe, Asia and Africa, and may expand into new regions [29] . The virus is usually transmitted to humans through contact with infected ticks and animal blood, but it is also transmissible from human to human via exposure to infected 

a TCID 50 , 50% tissue culture infectious dose; RF, reduction factor; RefÁ sample, pretreatment reference sample.

blood and other body fluids. Although no cases of CCHFV transmission by blood transfusion have been reported to date, incidences of hospital-acquired CCHFV infection due to contaminated medical instruments have been documented [30] . These cases are strongly reminiscent of the transmission routes of many other transfusion-relevant viruses.

NiV is an enveloped, single-stranded negative-sense virus that belongs to the Paramyxoviridae family. It was reported for the first time in the Malaysian population in 1998 and reappeared on different occasions in Asia. The NiV disease spectrum ranges from asymptomatic infection to acute respiratory illness and fatal encephalitis [31] . NiV is a zoonotic virus transmitted to humans from animals such as bats or pigs, but it can also be transmitted through contaminated foods or directly person-to-person through close contact with virus-containing body fluids and excretions [32] . The available data, particularly the findings on viral load in different body fluids, are too limited to provide a full understanding of its transmission routes [33] . Transfusion has not been implicated as a potential transmission pathway to date. The incubation period of up to 14 days and the occurrence of latent infections with subsequent reactivation of NiV months and even years after exposure suggest that infected persons may be overlooked by donor screening programmes. However, the potential transfusion risk may be limited by the fact that asymptomatic and mild NiV infections are rare.

Future studies are needed to determine whether SARS-CoV, CCHFV and/or NiV can be transmitted through transfusion. If one or more of these viruses is transfusion transmissible, its threshold concentration to elicit disease must be examined to determine whether the capacity of these pathogen inactivation technologies to inactivate the respective virus in plasma and PCs is sufficient to prevent transfusion transmission. Interpreting pathogen load in relationship to infectivity and inactivation efficacy is generally a very complex task [34] . When attempting to do so, it is important to consider that because quantitative polymerase chain reaction (qPCR), the most commonly used approach, measures viral load by detecting a small fragment of the viral genome, the results may not reflect infectivity and that qPCR usually overestimates the titre of circulating infectious agents [34] . In contrast, infectivity assays, which were used in this and previous studies [5, [12] [13] [14] , determine the inactivation capacity of a pathogen inactivation method based on intact, functional viral units. Nevertheless, the log reduction factors observed in this study and the safety margins calculated from the inactivation levels achieved using only a fraction of the standard light dose suggest that the THERA-FLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation technologies may effectively reduce the potential risk of SARS-CoV, CCHFV and NiV and related viruses for platelet or plasma transfusion.

Bacterial inactivation of platelet concentrates with the THERAFLEX UV-Platelets pathogen inactivation system

Main properties of the THERAFLEX MB-plasma system for pathogen reduction

The method of "right and wrong cases" ("constant stimuli") without Gauss's formulae

Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche

Complete sequence and phylogenetic characterisation of Crimean-Congo hemorrhagic fever virus from Afghanistan

Henipaviruses: a new family of emerging Paramyxoviruses

Emerging pathogens: the epidemiology and evolution of species jumps

SARS and MERS: recent insights into emerging coronaviruses

Emerging viral threats to the Australian blood supply

Crimean-Congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity

Health care response to CCHF in US soldier and nosocomial transmission to health care providers, Germany

Emerging trends of Nipah virus: A review

Nipah virus infection

Transmission of nipah virus -14 years of investigations in Bangladesh

Interpretation of pathogen load in relationship to infectivity and pathogen reduction efficacy

We would like to thank Katharina Kowalski for excellent technical assistance and the staff of the blood collection and blood preparation departments for their support.

FT and SR are employees of Macopharma, manufacturer and distributor of the THERAFLEX pathogen inactivation (PI) system. UG, WH, THM and AS received project grants from the German Red Cross Blood Services and Macopharma for the development of the UVC-based PI technology for platelets. ME has no conflicts of interest to disclose.

M. Eickmann designed the study, interpreted the data and co-wrote the manuscript. U. Gravemann designed the study, performed the in vitro experiments and analysed the data. W. Handke performed the in vitro experiments and analysed the data. F. Tolksdorf interpreted the data and edited the manuscript. S. Reichenberg interpreted the data and edited the manuscript. T. H. M€ uller interpreted the data and edited the manuscript. A. Seltsam designed the study, interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.

This work was supported by the German Red Cross Blood Services (Deutsche Forschungsgemeinschaft der Blutspendedienste des Deutschen Roten Kreuzes) and Macopharma S.A.S.