Vox Sanguinis (2015) 109, 417–419 © 2015 International Society of Blood Transfusion DOI: 10.1111/vox.12305

SHORT REPORT

Efficacy of the Mirasol pathogen reduction technology system against severe fever with thrombocytopenia syndrome virus (SFTSV) N. Shinohara,1 C. Matsumoto,1 M. Chatani,2 S. Uchida,1 T. Yoshikawa,3 M. Shimojima,3 M. Satake1 & K. Tadokoro1 1

Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Tokyo, Japan Kanto-Koshinetsu Block Blood Center, Japanese Red Cross Society, Tokyo, Japan 3 Special Pathogens Laboratory, Department of Virology I, National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo, Japan 2

Received: 12 March 2015, revised 30 April 2015, accepted 30 April 2015, published online 1 June 2015

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne virus in the Bunyaviridae family. This virus has recently been found in China, Japan and Korea. The risk of transfusion-transmitted SFTSV infection (TTI-SFTSV) is a concern because person-to-person transmission resulting from contact with SFTSV-contaminated blood has been reported. Therefore, we investigated the efficacy of the Mirasol pathogen reduction technology (PRT) system for inactivating SFTSV in vitro. The Mirasol PRT system achieved a > 4
11 log10 reduction value (LRV) for SFTSV. In conclusion, we showed that the Mirasol PRT system could potentially be used to reduce the risk of TTI-SFTSV. Key words: Mirasol PRT System, pathogen reduction technology, platelet concentrate, severe fever with thrombocytopenia.

Introduction Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne virus that is classified as a member of Bunyaviridae family. In 2009, an outbreak of an unknown infectious disease in China that caused high fever and thrombocytopenia was subsequently confirmed as having been caused by SFTSV in 2011 [1]. The virus was characterized as an enveloped, single-stranded, negativesense RNA virus comprising three segments. A variety of domestic animals (e.g. sheep, cattle, dog, pig and chicken) are infected by SFTSV through bites by ectoparasitic ticks that occur in areas of East Asia where SFTSV is endemic [2]. Humans can also develop severe fever with thrombocytopenia syndrome (SFTS) if they are bitten by SFTSVinfected ticks, which are distributed throughout Japan. The first case of SFTS in the country was confirmed in 2012 [3], and as of 4 February 2015, 110 Japanese patients with SFTS have been reported [4]. In an initial report, fatality

Correspondence: Naoya Shinohara, Infectious Disease Research Department, Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, 2-1-67 Tatsumi, Koto-ku, Tokyo 135-8521, Japan E-mail: [email protected]

due to SFTS was reported to be as high as 30%. Human SFTS cases were also reported in Korea in 2013 [5]. The maximum viremia level of SFTSV observed in Japanese fatalities was approximately 108 copies ml-1 serum [6]. In an SFTSV survey of Chinese blood donors in regions where SFTSV is endemic, seroprevalence rates in Xinyang were 0
54% (80/14,752); Mianyang, 0
27% (3/1130); and Luoyang, 0
28% (3/1326), and the RNA prevalence rate in Xinyang was 0
02% (2/9960) [7]. Moreover, since personto-person transmission resulting from contact with SFTSVcontaminated blood has been reported previously [8], the risk of transfusion-transmitted SFTSV infection (TTISFTSV) is a concern. Given the current situation in which no screening measures have been implemented for the virus, this study investigated the efficacy of the Mirasol pathogen reduction technology (PRT) system (Terumo, Tokyo, Japan) to inactivate SFTSV in the contaminated blood component of platelet concentrate (PC). The Mirasol system utilizes 35 ml of riboflavin solution as a light sensitizer (500 lM) and UV-B light irradiation dose of 6
24 J/ml to inactivate the virus.

Materials and methods The ability of the Mirasol PRT system to reduce the infectivity titre was examined using PC derived from apheresis

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418 N. Shinohara et al.

Table 1 Inactivation of severe fever with thrombocytopenia syndrome virus in PCs using the Mirasol PRT system

Table 2 Calculated severe fever with thrombocytopenia syndrome virus copy number required for one unit of TCID50

Platelet concentrate sample

Pretreatment (log 10 TCID50/ml)

Post-treatment (log 10 TCID50/ml)

Log10 reduction value (LRV)

Platelet concentrate sample

Spiked viral load (copies/component)

Pre-treatment (copies/TCID50)

Post-treatment (copies/TCID50)

A B C

5
47 6
3 6
63

4
11 4
77 4
76

A B C

109
45 109
45 109
45

101
67 100
83 100
49

>105
78 105
23 105
19

donation, as described previously [9]. The infectivity titre of SFTSV was evaluated using Vero cells and Japanese SFTSV strain SPL010 [3]. PC in approximately 200 ml of whole plasma was inoculated with SFTSV and then treated using the PRT system according to the manufacturer’s instructions. Aliquots were removed from the PC samples before and after PRT treatment, serially diluted and then added to wells containing cultured Vero cells. The Vero cells that were infected with the virus were detected by indirect immunofluorescence assay using a rabbit polyclonal antibody to SFTSV nucleoprotein and fluorescenceconjugated Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Life Technologies, Carlsbad, CA) for the second antibody [3]. The 50% tissue culture infective dose (TCID50) was measured by counting the number of wells containing infected fluorescence-positive cells; a total of six wells were prepared for each virus dilution. The experiment was repeated three times using PCs obtained from different donors. Viral inactivation by the PRT system was assessed by using the log10 reduction value (LRV) [10]. To evaluate the relationship between viral infectious ability and viral load, the SFTSV copy number required for one unit of TCID50 in the PRT-untreated and PRTtreated PC samples was determined by extracting the viral RNA from PC samples using a QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) and then subjecting the RNA to quantitative one-step reverse transcription polymerase chain reaction (qRT-PCR) using a slight modification of a previously reported method [6]. Briefly, we used a Qantitect Probe RT-PCR Kit (Qiagen) and an ABI Step One Plus system (Applied Biosystems, Foster City, CA), and the primer and probe sequences targeting the viral nucleoprotein were as follows: forward primer, 50 - TGT CAG AGT GGT CCA GGA TT -30 , reverse primer, 50 - ACC TGT CTC CTT CAG CTT CT -30 , and the minor groove-binding probe, FAM 50 -TTT GGT GAG CAG CAG C -30 . The SFTSV copy number spiked into the three PCs was determined to be 109
45 copies/PC.

The SFTSV RNA copy numbers required for one unit of TCID50 pre- and post-treatment are shown in Table 2, which shows that at least 105 copies of SFTSV are required to infect Vero cells in terms of TCID50 post-treatment.

Discussion There is a risk that blood donors who are either infected with SFTSV but have not yet presented with symptoms, or who have a subclinical course of SFTSV infection, may transmit SFTSV through transfusion of their blood products. Although the viremia levels in such individuals have not yet been clarified, viremia levels have been inferred to be less than approximately 105
2–106
4 copies ml-1, which is the average level among individuals presenting with SFTSV symptoms [6]. Also, it has been reported that the incubation period of SFTSV was 6-13 days after coming into contact with, or being exposed to, blood from SFTSV-contaminated blood [11]. We demonstrated that the Mirasol PRT system is capable of reducing the infectivity of SFTSV spiked into PC with an LRV >4
11 log10, in terms of TCID50 against Vero cells, implying that there was a marked decrease in the infectivity of PCs contaminated with SFTSV. However, no information is currently available on the minimal infectious dose of SFTSV in blood products, or the prevalence of asymptomatic blood donors with levels of viremia that exceed the levels that the Mirasol PRT system can effectively cope with (i.e., >105
19 copies/ml). In addition, the extent to which the in vitro infection system employed in this study reflects real infection in humans through transfusion of contaminated blood components is not known. In either case, it is considered that the Mirasol PRT system has the potential to decrease the possible risk of TTI-SFTSV infection as well as other emerging pathogens for which blood-screening methodologies have not yet been established.

Results The LRVs obtained with the Mirasol PRT system against SFTSV in PCs were >4 (range: >4
11 to 4
77) (Table 1).

Conflict of interest The authors declare no conflict of interests. © 2015 International Society of Blood Transfusion Vox Sanguinis (2015) 109, 417–419

SFTSV inactivation level of Mirasol PRT System 419

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© 2015 International Society of Blood Transfusion Vox Sanguinis (2015) 109, 417–419

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