Sensitive and specific PCR systems for detection of both Chinese and Japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.

FIG 1

Establishment of the cvPCR method and the limits of detection. (A) Authentic SFTSV strain HB29 viral RNA (HB29), the plasmid pCR-SFTSV-posicon (P.C.), which contains the artificial sequence, and the nontemplate control (H₂O) were amplified by cvPCR using primer set 1 or 2. The PCR products were digested by EcoRI (+EcoRI). The sizes of the products were estimated by using a 2-log DNA ladder marker (NEB). (B and C) Isolated strains HB29, YG1, and SPL005, expanded in Vero cells, were diluted with serum from a healthy donor. The purified viral RNAs from the dilutions of virus-spiked serum were amplified. The resulting HB29, YG1, or SPL005 was amplified by primer set 1 (B) or 2 (C). The listed values are the dilutions of the viral strains. NTC, nontemplate control.

FIG 2

qPCR standard curves and amplification curves. The curves for NP (A and B), GPC (C and D), and RdRp (E and F) were derived from a dilution series of reference RNA. Values of the slope, correlation coefficient (r²), and PCR efficacy (E_PCR) were calculated.

FIG 3

Limits of detection of the singleplex or multiplex qPCR to detect either the Chinese or the Japanese strain of SFTSV. Isolated strains HB29, YG1, and SPL005 were diluted with serum as described in Fig. 1, and purified RNA was amplified. The singleplex (A to C) or multiplex (D to F) qPCR copy numbers at each dilution of HB29 (A and D), YG1 (B and E), or SPL005 (C and F) that were detected by NP (square), GPC (triangle), or RdRp (diamond) are shown as dots and lines. The theoretical copy number of each strain estimated by undiluted viruses whose viral titers (TCID₅₀/ml) were known are shown in orange.

FIG 4

Agreement of cvPCR and qPCR results validated by clinical specimens from patients with suspected SFTS. The results (SFTSV positive or negative) determined by cvPCR (x axis) and the viral RNA copy number determined by qPCR (y axis) from each specimen are plotted as dots.

FIG 5

Relationship between the viral RNA level in the SFTS patient blood samples and the patient survival outcomes. (A) Viral RNA copy numbers in the blood samples from SFTS patients are plotted as dots and arranged based on the patients' survival outcomes. The mean of each group is indicated by a horizontal bar and the value. One-way ANOVA with Bonferroni's multiple-comparison test was used to determine the level of statistical significance. The calculated P values are shown above the groups that were compared. (B) Kinetics of the viral NP RNA copy number in the blood specimens after the onset of symptoms. The copy numbers in the blood specimens from SFTS patients who died (red) or survived (black) are plotted as dots for each of the collection days (days after the onset of symptoms). Horizontal solid lines for each day indicate the mean RNA copy number in the blood specimens collected from patients who died (red) or survived (black). The dots connected by dashed lines indicate specimens collected from the same patients.