Abstract
Severe fever with thrombocytopenia syndrome virus (SFTSV) causes lethal hemorrhagic diseases in human, cats, and dogs. Several human cases involving direct transmission of SFTSV from diseased animals have been reported. Therefore, rapid diagnosis in veterinary clinics is important for preventing animal-to-human transmission. Previously, we developed a simplified reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for human that does not require RNA extraction for detecting the SFTSV genome. In this study, we improved the simplified RT-LAMP assay for cats by introducing a dried reaction reagent and investigated the applicability of this method for diagnosing SFTS in cats. SFTSV RNA was detected in 11 of 12 cats naturally infected with SFTSV by RT-LAMP assay using both liquid and dried reagents. The RT-LAMP assay using liquid and dried reagents was also applicable to the detection of SFTSV genes 3–4 days after challenge in cats experimentally infected with SFTSV. The minimum copy number of SFTSV genes for 100% detection using the RT-LAMP assay with liquid and dried reagents was 4.3 × 104 and 9.6 × 104 copies/mL, respectively. Although the RT-LAMP assay using the dried reagent was less sensitive than that using the liquid reagent, it was sufficiently sensitive to detect SFTSV genes in cats with acute-phase SFTS. As the simplified RT-LAMP assay using a dried reagent enables detection of SFTSV genes more readily than the assay using a liquid reagent, it is applicable for use in veterinary clinics.
Keywords: cat, reverse transcription-loop-mediated isothermal amplification, reverse transcription-polymerase chain reaction, severe fever with thrombocytopenia syndrome virus, zoonosis
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV) that has spread throughout East Asia [4, 6, 15, 17, 20]. Since the first case of SFTS was reported in Japan in December 2012 [14], numerous SFTSV infections accompanied by lethal hemorrhagic fever have been reported in humans, cats, and dogs [2, 7]. In our unpublished data, we identified 449 SFTS cats and 24 SFTS dogs in Japan by the end of March 2022. In addition, as several cases of direct animal-to-human transmission of SFTSV have been reported [3, 5, 8, 10, 12, 16], pet owners and veterinarians are considered at high risk of infection with SFTSV from cats and dogs. SFTSV is currently one of the most important zoonotic pathogens in Japan.
Conventional RT-PCR has been officially used to diagnose SFTS in companion animals in Japan [2, 7], while qRT-PCR has been used in the research laboratories [18]. Although qRT-PCR has the advantages of less nonspecific reaction and rapid diagnosis, the both RT-PCR assays can be difficult to perform in animal hospitals because they require special equipment and complicated manipulations. To prevent the transmission of SFTSV from diseased animals to pet owners, veterinarians, and veterinary associates, a simple and rapid diagnostic method that can be performed at animal hospitals is needed.
The loop-mediated isothermal amplification (LAMP) method was originally developed by Eiken Chemical Co. [9]. The LAMP method can be performed without a thermal cycler and requires less reaction time than PCR methods. When combined with reverse transcription (RT) reactions, RNA can be also detected using LAMP (RT-LAMP) [1]. An RT-LAMP method involving a simple RNA extraction step (simplified RT-LAMP) was developed by Sano et al. to detect SFTSV RNA for human [13].
In this study, the simplified RT-LAMP assay was further improved to facilitate its routine use in veterinary clinics for the diagnosis of SFTS in cats.
At first, serum or ethylenediaminetetraacetic acid (EDTA)-treated plasma was collected from 28 cats at 22 animal hospitals between May and November 2021. All cats exhibited SFTS-like symptoms that led veterinarians to consider possible SFTSV infection, such as fever, lethargy, anorexia, leukopenia, and thrombocytopenia. The viral genome was analyzed according to a previously reported method [2]. Viral RNA was extracted from serum or plasma using a Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Oral and anal swab samples were collected from some cats using cotton swabs and RNA was extracted from these samples as described previously [2]. Conventional RT-PCR was performed using a QIAGEN OneStep RT-PCR Kit (QIAGEN) with two primer sets, SFTSV-S2-200s and SFTSV-S2-360a, and SFTSV-S7F and SFTSV S7R. Conventional RT-PCR using SFTSV-S2-200s and SFTSV-S2-360a primers was performed as follows: RT reaction at 50°C for 30 min and at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min, with final extension at 72°C for 7 min. RT-PCR using the SFTSV-S7F and SFTSV-S7R primers was conducted as follows: RT reaction at 50°C for 30 min and at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, and extension at 72°C for 30 sec, with final extension at 72°C for 5 min. The PCR products were confirmed by electrophoresis on a 2% agarose gel. The expected sizes of the fragments amplified using the SFTSV-S2-200s and SFTSV-S2-360a and SFTSV-S7F and SFTSV-S7R primer pairs were 201 bp and 125 bp, respectively. The results of RT-PCR indicated that 12 of the 28 serum or plasma samples were positive for the SFTSV genome. These 12 cats were therefore diagnosed as having natural SFTSV infection (Table 1). In one case, #617, the SFTSV genome was only detected when using the primer set SFTSV-S7F and SFTSV-S7R. As the SFTSV genome was also detected from an oral swab of cat #617 using RT-PCR with both primer sets, we finally diagnosed the cat as having SFTSV infection.
Table 1. Results of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay and reverse transcription polymerase chain reaction (RT-PCR) in cats naturally infected with severe fever with thrombocytopenia syndrome virus (SFTSV).
| Cat ID | Sample | SFTSV RNA (copies/mL) |
RT-LAMP |
RT-PCR |
||
|---|---|---|---|---|---|---|
| Liquid reagent | Dried reagent | S2 primers | S7 primers | |||
| #520 | Plasma | 7.4 × 106 | + | + | + | + |
| #525 | Serum | 9.8 × 105 | + | + | + | + |
| #528 | Serum | 5.0 × 106 | + | + | + | + |
| #533 | Plasma | 2.3 × 105 | + | + | + | + |
| #548 | Serum | 1.6 × 107 | + | + | + | + |
| #551 | Plasma | 3.6 × 106 | + | + | + | + |
| #552 | Serum | 2.0 × 108 | + | + | + | + |
| #556 | Plasma | 4.3 × 104 | + | - | + | + |
| #560 | Serum | 5.1 × 105 | + | + | + | + |
| #567 | Serum | 8.0 × 106 | + | + | + | + |
| #596 | Serum | 3.7 × 105 | + | + | + | + |
| #617 | Serum | <5.0 × 102 | - | + | -* | +* |
| #594 | Serum | <5.0 × 102 | - | - ** | - | - |
| #595 | Serum | <5.0 × 102 | - | - | - | - |
| #597 | Serum | <5.0 × 102 | - | - | - | - |
| #598 | Serum | <5.0 × 102 | - | - | - | - |
| #600 | Serum | <5.0 × 102 | - | - | - | - |
| #601 | Serum | <5.0 × 102 | - | - | - | - |
| #602 | Serum | <5.0 × 102 | - | - | - | - |
| #604 | Serum | <5.0 × 102 | - | - | - | - |
| #606 | Plasma | <5.0 × 102 | - | - | - | - |
| #607 | Serum | <5.0 × 102 | - | - | - | - |
| #609 | Serum | <5.0 × 102 | - | - | - | - |
| #610 | Serum | <5.0 × 102 | - | - | - | - |
| #611 | Serum | <5.0 × 102 | - | - | - | - |
| #612 | Serum | <5.0 × 102 | - | - | - | - |
| #614 | Plasma | <5.0 × 102 | - | - | - | - |
| #615 | Serum | <5.0 × 102 | - | - | - | - |
*SFTSV genome was detected in the oral swab using RT-PCR. **The sample was automatically judged to be positive.
To quantify the SFTSV RNA in serum and plasma samples, quantitative one-step RT-PCR (qRT-PCR) was performed using a previously described method for the determination of SFTSV RNA copy number [11]. A total of 100 µL of RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan) from 100 µL of serum from cats experimentally infected with SFTSV, and 60 µL of RNA was extracted from 140 µL of clinical samples using a Viral RNA Mini Kit (QIAGEN). qRT-PCR was performed using a QIAGEN QuantiTect Probe RT-PCR Kit (QIAGEN) with primers and probe targeting the nucleoprotein gene reported by Yoshikawa et al. [18]. qRT-PCR was performed as follows using a LightCycler480II system (Roche, Basel, Swiss Confederation): RT reaction at 50°C for 30 min and at 95°C for 15 min, followed by 45 cycles of denaturation at 94°C for 15 sec and extension at 60°C for 1 min. The result indicated that all SFTSV-positive clinical samples except for cat #617 contained from 4.3 × 104 to 2.0 × 108 copies/mL of SFTSV RNA (Table 1).
Next, serum or plasma samples were examined by RT-LAMP assay using the liquid reagent as described previously [13]. In brief, a mixture of 5 µL of serum or plasma and 40 µL of Loopamp viral RNA extraction solution (Eiken Chemical, Tokyo, Japan) was incubated for 1 min at 90°C. The primer/probe sets used in this study and their final concentrations were as follows: 5 pmol of SFTS_L_F3, 2.5 pmol of SFTS_L_B3-1, 2.5 pmol of SFTS_L_B3-2, 60 pmol of SFTS_L_FIP-1, 40 pmol of SFTS_L_FIP-2, 60 pmol of SFTS_L_BIP-1, 40 pmol of SFTS_L_BIP-2, 20 pmol of SFTS_L_LF, 10 pmol of SFTS_L_LB-1, 10 pmol of SFTS_L_LB-2, and 1 pmol of SFTS_L_QProbe. For primer/probe sets, 1.4 mM dNTPs, 0.5% Tween 20, 8 mM MgSO4, 30 mM KCl, 20 mM tricine (pH 8.6), 25 U of Bst DNA polymerase (New England Biolabs, Ipswich, MA, USA), 1 U of avian myeloblastosis virus reverse transcriptase (Roche), and 5 µL of extracted RNA were mixed and adjusted to a final volume of 25 µL. The mixture was incubated for 30 min at 63°C using an ESEQuant TS2 tube scanner (QIAGEN), which was set to collect fluorescence signals at 30 sec intervals to construct the amplification curves. Fluorescence data were automatically analyzed using ESEQuant TS2 studio software, version 2.0.2., which was pre-installed on the ESEQuant TS2 tube scanner. Finally, the amplification curves were directly checked to confirm the results. The result of RT-LAMP assay using the liquid reagent showed that SFTSV RNA was detected in samples from 11 of 12 SFTSV-positive cats (Table 1). The only sample with no detectable RNA by RT-LAMP assay using the liquid reagent was cat #617. All SFTSV-negative samples were negative by RT-LAMP assays using the liquid reagent. The comparison of the qRT-PCR and RT-LAMP assay indicated that the minimum SFTSV RNA copy number in cat serum or plasma positive by the RT-LAMP assay using the liquid reagent was 4.3 × 104 copies/mL (equivalent to 104.6 copies/mL). These results suggested that RT-LAMP assay using the liquid reagent must be available for diagnosis of SFTSV infection in cats as well as in humans. The median number of RNA copies in samples positive by RT-LAMP assay using the liquid reagent was 106.6 copies/mL, and the median number of RNA copies in samples negative by the RT-LAMP assay was 103.0 copies/mL (Fig. 1). Sano et al. [13] reported a median value of SFTSV RNA copies in human serum positive by RT-LAMP assay using the liquid reagent of 105.6 copies/mL, with a median number of RNA copies in samples that tested negative of 103.5 copies/mL. These results indicate that the RT-LAMP assay using the liquid reagent is suitable for analyzing samples from cats as well as humans. Matsuu et al. [7] reported a median RNA copy number of 106.1 copies/mL in the first examination of cats with SFTS. Alternatively, the number of samples in this study was limited. It is necessary to study with more samples for discussion of sensitivity of the RT-LAMP assay.
Fig. 1.
Comparison of severe fever with thrombocytopenia syndrome virus (SFTSV) RNA copy number in serum and plasma of cats. Cats were divided into two groups based on the result (i.e., positive and negative) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay using liquid or dried reagent. The bars indicate the median value.
To further simplify the RT-LAMP assay, we developed a dried reagent. The liquid reagent described above, including primer and probe solutions, dNTPs, DNA polymerase, and reverse transcriptase, was lyophilized by a freeze dryer FD-550P (EYELA, Tokyo, Japan) and used as a dried reagent. The dried reagent can be stored at room temperature over 6 months. For assays, the dried reagent, 12.5 µL of DEPC-treated water (Nippon Gene), and 12.5 µL of serum treated according to the simplified method were mixed, reacted for 30 min at 63°C, and used as the liquid reagent. This RT-LAMP assay using the dried reagent could detect approximately 100 focus-forming units of SFTSV SPL010 strain per mL as well as both standard RT-LAMP assay using purified RNA and simplified RT-LAMP assay using the liquid reagent. In addition, since the RT-LAMP assay using the dried reagent and 12.5 µL of the treated sera showed similar sensitivity to the standard RT-LAMP assay using the purified RNA, 12.5 µL of the treated sera was added to RT-LAMP assay using dried reagent in further experiments (data not shown). The result of the RT-LAMP assay using the dried reagent showed that SFTSV RNA was also detected in samples from 11 of 12 cats. No SFTSV RNA was detected in the sample from cat #556, which included 4.3 × 104 copies/mL of RNA in the result of qRT-PCR. All SFTSV-negative samples were negative. Only one serum sample, from cat #594, was positive by analysis of RT-LAMP assay with the dried reagent using ESEQuant TS2 studio software. As the shape of the absorbance curve was not sigmoid, this sample was finally judged negative for SFTSV RNA by RT-LAMP assay.
The serum of cat #617 was positive by the RT-LAMP assay only when the dried reagent was used. The SFTSV RNA copy number was under the qRT-PCR limit of detection, and the results of RT-PCR using two sets of primers were positive only when using one primer set. The amount of RNA in the serum sample was close to the detection limit, which could have caused a false-negative result in the qRT-PCR assay. In addition, a mismatch in the primer binding regions could have occurred in the RT-PCR assay using the S2 primer set and in the qRT-PCR assay. As this sample was positive by RT-PCR using the S7 primer set, the possibility of contamination in the RT-LAMP assay using the dried reagent can be excluded. The reason the sample from cat #617 was positive by the RT-LAMP assay with the dried reagent but not by the assay using the liquid reagent might simply be attributed to the volume of sample added to the reaction tube, indicating that the RT-LAMP assay using the dried reagent has an advantage in this regard over the assay using the liquid reagent.
The RT-LAMP assay using the dried reagent was applied for experimentally SFTSV-infected cats and the results were compared with those by qRT-PCR and RT-LAMP assay using liquid reagent. SFTSV strain SPL010 [19] was used for challenge experiments. Four cats (#1–6, #2–8, #2–9, #2–10) were inoculated intravenously with 107 TCID50/mL of SFTSV strain SPL010A. Blood samples were collected from cat #1–6 at 3, 6, and 9 days post-infection (dpi), cat #2–8 at 1, 3, and 4 dpi, cat #2–9 at 1, 3, 4, and 7 dpi, and cat #2–10 at 3, 4, 7, 9, and 10 dpi. Serum samples were prepared by centrifugation at 1,750 × g for 5 min. All experiments involving animals were performed according to the Animal Experimentation Guidelines of the National Institute of Infectious Diseases (NIID). The protocol was approved under permission number 117103, 118071, and 119130 by the Institutional Animal Care and Use Committee of the NIID. These cats were used for infection control in our unpublished study (manuscript in preparation). Serum samples were analyzed by qRT-PCR and RT-LAMP assay using the liquid and dried reagents (Table 2). The RNA copy number reached a maximum of 9.6 × 104 copies/mL at 6 dpi in cat #1–6, 2.1 × 108 copies/mL at 4 dpi in cat #2–8, 2.6 × 109 copies/mL at 7 dpi in cat #2–9, and 1.1 × 107 copies/mL at 9 dpi in cat #2–10. The results of RT-LAMP assays of samples from cats #1–6 and #2–8 were the same with the liquid and dried reagents. For cat #1–6, RNA was detected by the RT-LAMP assay only at 6 dpi, with the highest RNA copy number, 9.6 × 104 copies/mL. For cat #2–8, no RNA was detected by the RT-LAMP assay at 1 dpi, and this sample had the lowest RNA copy number, 2.3 × 104 copies/mL. At 3 and 4 dpi, RNA was detected by RT-LAMP assay using both reagents. For cat #2–9, the serum collected 1 dpi was negative by the RT-LAMP assays using both reagents, and samples collected 4 and 7 dpi were positive in the RT-LAMP assays using both reagents. However, at 3 dpi, SFTSV RNA was detected only in the RT-LAMP assay using the liquid reagent, with 3.1 × 105 copies/mL detected using qRT-PCR. For cat #2–10, all serum samples collected after 4 dpi were positive by RT-LAMP assays using both reagents. At 3 dpi, SFTSV RNA was detected only in the RT-LAMP assay using the liquid reagent, with 1.1 × 105 copies/mL detected.
Table 2. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay in cats experimentally infected with severe fever with thrombocytopenia syndrome virus (SFTSV).
| Cat ID | Days post SFTSV infection | Sample | SFTSV RNA (copies/mL) |
RT-LAMP |
|
|---|---|---|---|---|---|
| Liquid reagent | Dried reagent | ||||
| #1–6 | 3 | Serum | 1.1 × 103 | - | - |
| #1–6 | 6 | Serum | 9.6 × 104 | + | + |
| #1–6 | 9 | Serum | 1.2 × 104 | - | - |
| #2–8 | 1 | Serum | 2.3 × 104 | - | - |
| #2–8 | 3 | Serum | 4.3 × 106 | + | + |
| #2–8 | 4 | Serum | 2.1 × 108 | + | + |
| #2–9 | 1 | Serum | <5.0 × 102 | - | - |
| #2–9 | 3 | Serum | 3.1 × 105 | + | - |
| #2–9 | 4 | Serum | 6.8 × 106 | + | + |
| #2–9 | 7 | Serum | 2.6 × 109 | + | + |
| #2–10 | 3 | Serum | 1.1 × 105 | + | - |
| #2–10 | 4 | Serum | 3.4 × 105 | + | + |
| #2–10 | 7 | Serum | 7.2 × 106 | + | + |
| #2–10 | 9 | Serum | 1.1 × 107 | + | + |
| #2–10 | 10 | Serum | 1.5 × 106 | + | + |
The sensitivities of RT-LAMP assays using the liquid and dried reagents were compared by analyzing all cat data in this study. The minimum SFTSV RNA copy numbers in cat serum or plasma positive by the RT-LAMP assay using the liquid and dried reagents were 4.3 × 104 copies/mL (equivalent to 104.6 copies/mL) and 9.6 × 104 copies/mL (equivalent to 105.0 copies/mL), respectively. Furthermore, the median number of RNA copies in samples positive by RT-LAMP assay using the liquid and dried reagents was 106.6 and 106.7 copies/mL, respectively. The median numbers of RNA copies in samples negative by the RT-LAMP assay using the liquid and dried reagents was 103.0 and 104.4copies/mL, respectively (Fig. 1). In the results, both dry and liquid assays performed similarly. Although, it was noted that the RNA copy number of negative samples for RT-LAMP assay using the dried reagent was slightly higher than that for the assay using the liquid reagent.
In conclusion, as cases of cat-to-human and dog-to-human transmission of SFTSV have been reported [3, 5, 8, 10, 12, 16], a simple and rapid diagnostic method that can be performed in animal hospitals is needed to help prevent transmission from diseased animals to veterinary workers and pet owners. The RT-LAMP assay using the liquid reagent can be used to diagnose animals with SFTS. The RT-LAMP assay using the dried reagent was found to be sensitive as well as the assay using the liquid reagent, furthermore use of the premixed dried reagent is simpler and can reduce the risk of contamination. The RT-LAMP assay using the dried reagent is thus suitable for use in veterinary clinics.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Acknowledgments
This research was funded by AMED under grant numbers JP22fk0108634, JP21fk0108615, 21fk0108081, JP20wm0225009, JP22fk0108625, JP21fk0108613, 20fk0108069, and JP19fk0108081; Environment Research and Technology Development Fund grant number 4-2005, MEXT/JSPS KAKENHI grant number 19K06395 and 20H00652; and Health Labour Sciences Research grant number 21HA2006. We thank all the participating veterinarians and staff in the animal hospitals.
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