Development of a reverse transcription polymerase chain reaction for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals

Background Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble to those of SFTS patients and SFTS-contracted cats shows high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. Methodology/Principle findings Four primer sets were newly designed from consensus sequences constructed by 108 strains of SFTSV. A reverse transcription polymerase chain reaction (RT-PCR) with these four primer sets were successfully and specifically detected several clades of SFTSV. Their limits of detection are 1-10 copies/reaction. By this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients. Conclusion/Significance This newly developed RT-PCR could detect SFTSV RNA of several clades from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study. Author summary This study developed RT-PCR to detect SFTS animal cases. This assay could detect SFTSV RNA belonging to different clades. Cats diagnosed as SFTS had IgM or IgG, but not neutralizing antibodies. SFTS cat cases were distributed in the area where SFTS patients have been reported highly, indicating the establishment of the circulation of SFTSV in the environment. These diagnostic assays could be helpful tools to detect and not to miss SFTS animal cases.

SFTSV. A reverse transcription polymerase chain reaction (RT-PCR) with these four primer sets 23 were successfully and specifically detected several clades of SFTSV. Their limits of detection are 24 1-10 copies/reaction. By this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were 25 diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked 26 immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization 27 titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients.

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This newly developed RT-PCR could detect SFTSV RNA of several clades from SFTS-suspected 30 animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-31 suspected animals has been made in this study.

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Author summary 33 This study developed RT-PCR to detect SFTS animal cases. This assay could detect SFTSV RNA 34 belonging to different clades. Cats diagnosed as SFTS had IgM or IgG, but not neutralizing Introduction 40 Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic 41 fever that was first identified in China (1), with cases since reported in Japan, South Korea,42 Vietnam and Taiwan (2, 3). SFTS virus (SFTSV)  in preparation). Thus, it is important to diagnose SFTS-suspected animals. Seven to eight clades 52 of SFTSV are reportedly spread throughout Japan, China and South Korea [9].

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In this study, a reverse transcription polymerase chain reaction (RT-PCR) was developed 54 to establish a laboratory diagnosis system for detecting all the clades of SFTSV in the specimens 55 of SFTS-suspected animals.  The nucleotide sequences of 100 strains of SFTSV S segment and M segment were selected 67 randomly from Genbank to cover all the clades and aligned and phylogenetically analyzed ( Figure   68 1). In brief, phylogenic trees were constructed using with the maximum likelihood method with 69 the Tamura-Nei model using the MEGA 7 software program [10]. The robustness of the resulting 70 branching patterns was tested using the bootstrap method with 1,000 replicates. From this analysis, 71 it was confirmed that the seven to eight clades of SFTSV correlate with their geographical location, 72 as has been reported previously [9]. The nucleotide identity, determined using the Bioedit sequence  reported study (Park ES). RT-PCR was performed using the Superscript III one-step RT-PCR RT-PCR-positive RNAs were used to determine the viral genome of the S segment for a 99 phylogenetic analysis. RT-PCR was performed using a Superscript III one-step RT-PCR system 100 with platinum Taq DNA polymerase (Invitrogen) with primers covering the entire S segment 101 region according to a previously reported study [9]. The PCR products were determined by 6 102 electrophoresis on 1% agarose gels with GR Red Loading Buffer (GRR-1000, Bio-Craft, Tokyo, 103 Japan). The PCR products were then extracted and purified using an illustra TM GFX TM PCR DNA 104 and Gel Band Purification kit (GE Healthcare, Buckinghamshire, UK). The samples were 105 sequenced using the general Sanger sequencing technique.

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The nucleotide sequences determined in this study were deposited in the DDBJ GenBank 107 databases. For the phylogenetic analysis, three nucleotide data points per cluster were selected. The 50% plaque reduction neutralization titer (PRNT 50 ) 136 The PRNT test was performed to determine the neutralizing antibodies against SFTSV using Vero

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Four primer pairs (2 for the S segment and 2 for the M segment) successfully detected the RNAs 153 of the three SFTSV strains belonging to different clusters with a detection limit of 1-10 154 copies/reaction (Figures 3 and 4 and Table 1). For the detection of SFTSV RNA from samples, 155 these four primer sets were used.  (Table 2). These nucleotide 160 sequences of the S segment from five cases were phylogenetically analyzed with the corresponding 161 segment of the Heartland virus as an outgroup ( Figure 6). As a result, four strains were clustered 162 into genotype J1, and one strain was clustered into genotype J3 of the Japanese clade, according 163 to previous studies.

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In addition to SFTSV RNA, antibodies against SFTSV were detected in the sera of the five 165 cats that were positive on RT-PCR (Table 3). Three samples had IgM and IgG against SFTSV, 166 and two had IgM or IgG, respectively (Table 3). Serum samples were collected at a one-week 167 interval from one case. IgM was detected in these two interval sera, and IgG was detected in the 168 serum collected one week later. This seroconversion pattern was similar to that of our previous 169 study. Antibodies were not detected in the RT-PCR-negative animals.

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The neutralizing antibodies against SFTSV were then measured with the PRNT 50 according 171 to our previous study. The titer of neutralizing antibodies was below the limit of detection, 172 indicating that the antibodies detected by the ELISA were not functional, similar to those of fatal 173 human cases.

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In this study, four primer sets were able to detect SFTSV RNA belonging to different 176 genotypes with a low detection limit. Two pairs were specific for the S segment, and two pairs 177 were specific for the M segment. SFTSV RNAs were detected from five cases using these primers.