Clinical and epidemiological characteristics of severe fever with thrombocytopenia syndrome in dogs: an observational study in the Republic of Korea

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic disease with a high mortality rate for humans and cats. The clinical course and prognosis of SFTS in dogs remains unclear. In the present study, we investigated the clinical and epidemiological characteristics of SFTS virus (SFTSV) infection in dogs. All evaluated dogs exhibited an acute course and symptoms such as fever (57.1%), anorexia (57.1%), depression (42.9%), and vomiting (35.7%). Thrombocytopenia was observed in 45.5% dogs, while jaundice was not observed. C-reactive protein, alanine transaminase, and alkaline phosphatase were elevated in some cases. Viral clearance occurred within 6 to 26 days. Phylogenetic analysis revealed that the SFTSV sequences were consistent with viruses in the regions of the Republic of Korea. Given that dogs often live in close contact with humans, awareness of the clinical and epidemiological features of SFTS in dogs is crucial. Further large-scale studies are necessary to investigate SFTSV infection in dogs.

Introduction

Severe fever with thrombocytopenia (SFTS) is a zoonotic disease, with reported cases of direct transmission from dogs to humans (Saijo et al. 2018) and from cats to humans, including veterinary personnel (Skida et al. 2019; Yamanaka et al. 2020). Moreover, antibodies against the SFTS virus (SFTSV) have been identified in veterinary hospital workers in Japan (Kirino et al. 2021). In the Republic of Korea (ROK), there have been cases where SFTS was presumed to have been transmitted from domestic dogs to humans, while an in vivo study in dogs confirmed that intraspecies SFTSV transmission could occur by contact; these findings suggest that infection can occur in animals as well as humans (Chung et al. 2020; Park et al. 2021).

Many studies have assessed the presence of SFTSV in samples from various dogs in China, Japan, and the ROK. For canine SFTS, the antigen positive rate is 0.23–0.88%, while the antibody seroprevalence is 7.4–68.18% (Ding et al. 2014a; Huang et al. 2019; Kang et al. 2019; Kimura et al. 2018; Lee et al. 2017; Lee et al. 2018; Li et al. 2014; Tian et al. 2017). Reported SFTSV prevalence rates in dogs are 5.3% (19/359) (Niu et al. 2013) in China, 0.9% (1/114) (Matsuu et al. 2021) in Japan, and 0.2% (1/426) in shelter dogs (Lee et al. 2017) and 2.9% (3/103) in military dogs (Kang et al. 2019) in the ROK. Meanwhile, reported SFTSV seroprevalence rates are 37.9% (136/359) (Niu et al. 2013) and 7.4% (23/311) (Li et al. 2014) in China, 9.1% (3/33) in outdoor dogs and 14.3% (2/14) in impounded dogs in Japan (Kimura et al. 2018), and 13.9% (59/426) in shelter dogs (Lee et al. 2018) and 21.4% (22/103) in military dogs (Kang et al. 2019) in the ROK.

To our knowledge, clinical infection has been reported only in two dogs with natural infection and 16 dogs with experimental infection (Han et al. 2020; Nam et al. 2020; Park et al. 2021). Dogs typically exhibit fever, anorexia, depression, vomiting, and pink eyes. Hematological features include thrombocytopenia; anemia; leukopenia; leukocytosis; and increased alanine transaminase (ALT), alkaline phosphatase (ALP), C-reactive protein (CRP) levels. Fever and thrombocytopenia are major clinical features. Fever is the most common symptom and is associated with an increase in tumor necrosis factor alpha, which acts on the endothelium to increase vasodilation and vascular permeability (Seynhaeve et al. 2006), while thrombocytopenia is caused by macrophage phagocytosis in the spleen, when the virus attaches to platelets (Jin et al. 2012). Although fever is not a prognostic factor for humans (Liu et al. 2020; Xu et al. 2018), the severity of thrombocytopenia is an important prognostic factor (Li et al. 2020). There is no treatment protocol for SFTS infection in humans and animals, and treatment is symptomatic. Because natural clinical infection in dogs with SFTS has been reported in a limited number of cases till date, the clinical characteristics and survival rate are not well known.

The aim of this study was to investigate clinical (symptoms and results of blood examinations) and epidemiological characteristics, including the findings of phylogenetic analysis, of SFTSV infection in dogs in the Republic of Korea.

Material And Methods

Sample collection

From April 2019 to December 2020, blood was collected from 448 dogs exposed to hard ticks or exhibiting clinical symptoms similar to those of SFTS at 166 animal hospitals in the ROK. Collected blood samples were centrifuged to harvest the sera, and all sera samples were stored at −80°C until analysis. When the SFTSV antigen was confirmed, blood samples, urine samples, and swab samples (rectal, nose, eye, oral) were requested for follow-up, and complete blood count (CBC) and serum chemistry results were requested. 

Detection of SFTSV antigen and sequencing

RNA was extracted from 200-µL aliquots of serum using a Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Republic of Korea) according to the manufacturer’s instructions. RNA was also extracted from urine samples and swab samples (rectal, nose, eye, oral) obtained for follow-up. The viral RNA was stored at −80°C until use. Each RNA sample was tested using nested reverse transcription-polymerase chain reaction (RT-PCR) assays to detect the small (S) segment of SFTSV. Primary PCR was performed using one-step RT-PCR premix (Solgent, Daejeon, Republic of Korea) with previously designed primers: NP-2F (5’-CAT CAT TGT CTT TGC CCT GA-3’) and NP-2R (5’-AGA AGA CAG AGT TCA CAG CA-3’) (Yoshikawa et al. 2014). The primary reaction was performed with an initial step of 30 min at 50°C and 15 min at 95°C for denaturation, followed by 40 cycles of 20 s at 95°C, 40 s at 52°C, and 30 s at 72°C, with a final extension step of 5 min at 72°C. Nested PCR was conducted using 1 µL of the primary PCR product as a template and PCR premix (BIOFACT, Daejeon, Korea) with previously described primers: N2-F (5’-AAY AAG ATC GTC AAG GCA TCA-3’) and N2-R (5’TAG TCT TGG TGA AGG CAT CTT-3’) (Oh et al. 2016). The reaction for the nested PCR was 25 cycles of 20 s at 94°C, 40 s at 55°C, and 30 s at 72°C. Secondary PCR products were separated by electrophoresis on 1.2% agarose gels, visualized after staining with EcoDye ™ Nucleic Acid Staining Solution (BIOFACT, Daejeon, Korea), and purified using a DNA Gel Extraction Kit (Bionics, Seoul, Korea). In addition to nested RT-PCR, viral RNA was quantified using a PowerChek SFTSV Real-time PCR kit (Kogenebiotech, Seoul, ROK), according to the manufacturer’s instructions, in the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). 

Sequencing and phylogenetic analysis

To confirm the SFTSV PCR products, positive PCR amplicons were directly sequenced using an Applied Biosystems 3730 DNA Analyzer (PE Applied Biosystems, Foster City, CA, USA). Sequence homology with other deposited sequences were identified by searches using the Basic Local Alignment Search Tool network service (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). The obtained sequences were aligned and analyzed using Molecular Evolutionary Genetics Analysis Version 7.0 (MEGA7) software. Phylogenetic trees were constructed using the maximum likelihood method. Sequences of the SFTSV S segment previously identified in the ROK, China, and Japan were obtained from GenBank and used for a comparative analysis.  

Indirect immunofluorescence assay (IFA)

IFA slides were prepared using SFTSV-infected Vero E6 cells. Vero cells were resuspended at 1×10³ cells/well in medium (2% fetal bovine serum in Dulbecco’s modified Eagle medium), added to each well of a 24-well slide, and incubated in 5% CO₂ for 16 h. The slides were fixed with 100% acetone for 10 min at −20°C. After blocking with 5% goat serum for 2 h, the diluted serum was applied to IFA antigen slides and incubated in the 5% CO₂ incubator for 90 min. The serum samples were diluted to 1:50, 1:100, 1:200, 1:400, and 1:800 with phosphate-buffered saline (PBS) for attaching the primary antibody, and the diluted serum was spotted onto the wells of IFA antigen slides and incubated in 5% CO₂ for 1 h. After washing with PBS, fluorescein isothiocyanate -conjugated anti-dog immunoglobulin G (IgG) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well of the antigen slide and incubated in 5% CO₂ for 1 h. The IFA slides were visualized using the EVOS™ M7000 Imaging System (Invitrogen, Frederick, MD, USA). 

Enzyme-linked immunosorbent assay (ELISA) 

For the detection of SFTSV-specific antibodies in the horse sera, 96-well plates (Thermo Scientific, Waltham, MA, USA) were coated with 100 ng/well of purified recombinant nucleoprotein (NP) at 4°C overnight. For preparation of the recombinant protein, the NP-encoding gene from a human SFTSV isolate was cloned into the pET28a(+) vector (Novagen, Gibbstown, NJ, USA) and the plasmid transfected into the Escherichia coli strain BL21. After induction with 0.1-mM isopropyl β-D-thiogalactoside, the recombinant protein was purified using HisTrap HP histidine-tagged protein columns (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions. After coating with the purified recombinant NP, the 96-well plates were blocked with 5% skim milk in PBS containing 0.05% Tween 20 for 2 h at room temperature (RT); they were subsequently incubated for 1 h at RT with 1 µL/well of horse serum in 5% skim milk. Binding antibody in the serum samples was detected using horseradish peroxidase-conjugated goat anti-horse IgG (Abcam, Cambridge, UK). Substrate solution containing 3,3′,5,5′-tetramethylbenzidine was added to allow color development. The reaction was performed for 10 min at RT, and 1M H₃PO₄ solution was used to stop the reaction. The optical density was measured at 450 nm for each well using a microplate reader (BioTek Instruments, Winooski, VT, USA). 

Virus isolation

Collected canine serum samples were inoculated onto monolayers of Vero E6 cells for virus isolation, as previously described (Han et al. 2020). After adaptation and proliferation of SFTSV in Vero E6 cells, the virus was confirmed by RT-PCR and indirect IFA using supernatants of infected cells and infected cells, respectively. In the ROK, SFTSV is a biosafety level 3 (BSL 3) pathogen; therefore, all experiments were conducted in a BSL 3 laboratory.  

Full genome sequencing

For complete genetic sequencing of SFTSV, nucleotide sequence analyses of the full segments of S, M, and L were completed through rapid amplification of cDNA ends. All generated nucleotide sequences of Dog 1 and Dog 12 have been deposited in GenBank under accession numbers OL773687 and OK423754 for the S segment, OM179920 and OM179921 for the M segment, and OM179922 and OM179923 for the L segment. 

Statistical analysis

Statistical analyses were performed using a statistical software (Prism 9.3.1, Graphpad). The Shapiro–Wilk test was used to test normality. Because age, CBC, and serum chemistry values were not normally distributed, median values were calculated. P < 0.05 was considered statistically significant.

Results

The infected dogs showed fever (57.1%, 8/14; reference, 38.0℃–39.2℃), anorexia (57.1%, 8/14), depression (42.9%, 6/14), vomiting (35.7%, 5/14), diarrhea (21.4%, 3/14), hematochezia (21.4%, 3/14), hematuria (7.1%, 1/14), tachypnea (7.1%, 1/14), eyelid swelling (7.1%, 1/14), and oral mucosal erosion (7.1%, 1/14; Fig. 2). The median body temperature was 39.7°C (range, 38.4°C–40.2°C) and exceeded 40°C in only two dogs.

Laboratory results

Complete blood count was performed for 11 dogs (Fig. 3, Table 1). The platelet count decreased below the reference range in 45.5% (5/11) dogs, while it decreased but remained within the reference range in 27.3% (3/11) dogs. Leukogram analysis showed leukocytosis (27.3%, 3/11), leukopenia (18.2%, 2/11), lymphocytosis (18.2%, 2/11), and lymphopenia (27.3%, 3/11), while erythrogram analysis showed decreased hematocrit and a red blood cell (RBC) count lower than the reference range in 27.3% (3/11) dogs and a decrease in the RBC count and hemoglobin level within the reference range in 7.1% (1/11) dogs. C-reactive protein was increased in all six tested dogs. Eight dogs were tested for ALT and ALP; two (25%) and six (75%) exhibited increased levels of ALT and ALP, respectively.

Other tick-borne diseases

Only one animal showed antibody positivity for Lyme disease. All other tick-born disease antigen and antibody tests showed negative results.

Detection of the SFTSV antigen by RT-PCR

Among 448 serum samples, 14 were positive for SFTSV in PCR; the positive rate was 3.1% (Fig. 4A). Specifically, virus clearance was confirmed through follow-up in 11 of the 14 dogs. Although daily blood samples were not collected, the duration of virus clearance was found to be 6 to 26 days (Table 2). In addition, SFTSV was detected in the urine sample of Dog 12 (Table 3). Accordingly, a serum sample of the dog cohabiting with Dog 12 was obtained and tested for the SFTSV antigen, and the result was negative. In addition, all other cohabiting dogs showed negativity for the SFTSV antigen (Fig. 4B).

Detection of SFTSV antibodies by IFA and ELISA

IFA was used to detect anti-SFTSV antibodies in the samples of 10 of the 14 dogs, and four showed positivity (Table 2; Fig. 4C). In the case of Dog 1, it was confirmed that the IgG titer was higher on day 12 (≤ 800) than on day 5 (≤ 400). Dog 3 was negative on day 0 (≤ 100) and positive on day 15. Although blood tests were not conducted every day, the results suggested that it took 5 (Dog 1) to 15 days (Dog 3) for the formation of antibodies. ELISA was used to detect anti-SFTSV antibodies in three dogs, and seropositivity was confirmed for Dog 11 and Dog 12. In addition, continuous follow-up of Dog 1 confirmed that antibodies were formed for approximately 1 year.

Sequencing and phylogenetic analysis

The nucleotide sequences obtained from our study have been deposited in GenBank under accession numbers MN398158, MW004841-MW004855, and MZ171135-MZ171136. The partial sequence (MW004854) of SFTSV detected in the urine of Dog 12 was confirmed to be the same as the sequence detected in the serum (MW004855). The thirteen sequences of sera obtained in this study showed 94.2–100.0% identity with each other. Phylogenetic analysis identified four, two, and two subgenotypes of B-3, B-2, and B-1, respectively, and five were identified as genotype D. It was confirmed that most of the samples belonged to genotype B (Fig. 5). The sequence of Dog 5 could not be obtained because of the lack of samples.

Treatment and prognosis

The dogs received symptomatic and antibiotic treatment. Antibiotics such as doxycycline, metronidazole, ciprofloxacin, cefaclor, and amoxicillin were used differently depending on each case, and famotidine, metoclopramide, maropitant, and sucralfate were used for symptomatic treatment. Two dogs were hospitalized for 1 day each, and both showed severe anemia and received a single blood transfusion. All dogs survived with complete resolution of symptoms.

Discussion

Till date, natural clinical infection with SFTSV has been reported only in two dogs (Han et al. 2020; Nam et al. 2020). In the present observational study, we investigated the clinical features of SFTS infection in 14 dogs in the ROK. The results suggested that dogs show a clinical course different from that observed in humans and cat. Moreover, all dogs recovered and survived.

According to the new classification method for the SFTSV genome, eight (8/13, 61.5%) of the sequences identified in this study were confirmed to be genotype B, which is the most prevalent in the ROK (Yun et al. 2020). The remaining five (38.5%) were identified as genotype D, which has not been recently identified in the ROK and is a genotype mainly found in China. All genotypes of Dog 1 and Dog 12, after virus isolation and full genome sequencing, were genotype B, and they were confirmed to correspond to subgenotypes B-3 and B-2, respectively. The subgenotype B-3, to which the sequences of Dogs 1, 2, 3, and 4 in the present study belonged, is not only a human isolate (MK301482, ROK); horse (MT989463, ROK), wild boar (MT502543, ROK), goat, chicken, and cat (MW004853, ROK) have been confirmed to be in the same cluster. This means that subgenotype B-3 is dominant in various animals in the ROK. In our follow-up study, Dog 12, which showed the SFTSV antigen in its urine sample, was identified as genotype B-2, with confirmation of 96.4% similarity to a human isolate (KP663742, ROK). Dogs 6, 7, 8, 9, and 10, identified as genotype D, showed 99.4–100.0% similarity, and a human isolate sequence (KP663733, ROK) and a wild boar sequence (MT502563, ROK) were confirmed in the same cluster. When the sequence of a Chinese domestic dog (JQ693003, China) was compared with the sequences of Dogs 6, 7, 8, 9, and 10 in this study, the similarity was found to be quite consistent at 99.8–99.1%.

Human and feline SFTS patients present with symptoms such as fever, thrombocytopenia, encephalitis, multiple organ failure, and death (Kim et al. 2013; Takahashi et al. 2014; Yu et al. 2011). The dogs in the present study showed some similar symptoms, although there was no case of encephalitis, multiple organ failure, or death. Clinical symptoms occurred as early as 1 day after exposure to ticks. Fever was observed in 57.1% dogs, with severe fever (over 40°C) only in two dogs. In a previous study in which dogs were experimentally infected with SFTSV, immunosuppressed dogs showed high fever (above 40°C) while immunocompetent dogs showed slightly high fever (above 39.5°C) (Park et al. 2021). Only one immunocompetent dog had high fever. It can be assumed that the host’s immune status influences the severity of fever. In our study of dogs with natural infection, 42.9% did not have fever; however, SFTS was considered because they were exposed to ticks. Thrombocytopenia was present in 45.5% dogs, with 27.3% showing a decrease within the reference range. Only one dog showed hematuria and hematochezia. In feline SFTS, thrombocytopenia was seen in 91.7% (22/24) (Matsuu et al. 2019). Similar to the results observed in the previous study of canine SFTS, in our study, even when thrombocytopenia occurred, severe anemia occurred in only one dog, and most of the dogs did not show anemia (Han et al. 2020; Nam et al. 2020; Park et al. 2021). This result is similar to that reported for feline SFTS (Matsuu et al. 2019). In human SFTS, a link between the platelet count and clinical outcome has been suggested; this may be related to activation of the cytokine network, the vascular endothelium, and the coagulation/fibrinolysis system, rather than virus attachment to platelets (Li et al. 2020).

In all studies of canine SFTS, including the present study, there was no case of death from SFTS infection (Han et al. 2020; Nam et al. 2020). In the present study, only symptomatic treatment with drugs such as doxycycline and metronidazole was administered, and only one dog with severe anemia required blood transfusion while two dogs were hospitalized for 1 day. All other dogs were managed as outpatients, and most clinical symptoms resolved within 2 weeks. Cats are highly sensitive to SFTSV, with a mortality rate of 62.5% (Matsuno et al. 2018; Matsuu et al. 2019; Park et al. 2019; Sakai et al. 2021; Seto et al. 2020). Moreover, the mortality rate for humans is approximately 20% (Seo et al. 2021). Reported factors associated with poor survival in feline SFTS infection included fever (> 39.5°C; P = 0.020) and elevated creatine phosphokinase (CPK) levels (P = 0.002), SFTSV copy numbers (P = 0.027), and IgG levels (P = 0.004) (Matsuu et al. 2019). All cats also developed jaundice, although it was not related to survival (P = 0.179). In our study, severe fever was observed in only two dogs, and quantitative IgG using ELISA and the CPK level were not measured; therefore, comparisons cannot be made with cats. IgG was confirmed using IFA, and two dogs showed positivity. Because all dogs survived, it was difficult to determine risk factors for mortality. The severity of fever and disease in viral infections, including SFTS, are cytokine-related, and cytokine storm is associated with patient prognosis (Deng et al. 2012; Ding et al. 2014b; Hu et al. 2018; Sun et al. 2014; Sun et al. 2012). In human SFTS, an increase in several cytokines such as interferon (IFN)-α (IFN-α), IFN-γ, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein-1α, interleukin-6, and IFN-inducible protein-10 was associated with disease severity. In animals, such studies are limited. It is necessary to study a larger number of dogs and determine if they exhibit disease characteristics consistent with our findings.

There were no instances of dogs transmitting SFTSV to other dogs or owners in this study. In Korea and Japan, cases of SFTSV transmission from companion animals such as dogs (Chung et al. 2020; Kim et al. 2021; Park et al. 2021) and cats (Kida et al. 2019; Tsuru et al. 2021; Yamanaka et al. 2020) to other animals or humans in close contact, such as owners or veterinarians, have been reported. The seropositivity rate for veterinarians and nurses working in veterinary hospitals has been reported as 2.2–4.2%, with positivity despite the absence of clinical symptoms, and it was significantly higher than that reported for healthy blood donors (Ando et al. 2021; Kirino et al. 2021). Human-to-human transmission occurs through close contact with infected body fluids (Kim et al. 2015; Liu et al. 2012; Yoo et al. 2016; Yoo et al. 2018), and it can be assumed that transmission between animals occurs in a similar manner (Park et al. 2021). In our study, viral antigen testing was performed for various samples, namely serum and urine samples and oral, eye, nose, and rectal swab samples. For one dog, the antigen was detected in the urine. In a previous study of dogs, SFTS viral shedding in the urine and rectal swabs persisted until 16 days after infection (Park et al. 2021). SFTSV invades multiple organs (Huang et al. 2019; Park et al. 2019; Park et al. 2020). Dogs can function as a reservoir host for SFTSV (Wang et al. 2021), and even if animals and humans are not directly bitten by ticks, infection can occur through various indirect routes. Despite these risks, no treatment strategy has been established for humans and animals. Depending on the patient’s symptoms, symptomatic treatments such as fluids, antibiotics, transfusion, antipyretics, G-CSF, intravenous immunoglobulin, plasma exchange, and monoclonal antibodies are available. In humans, antiviral agents such as ribavirin and favipiravir have been tested, but their effectiveness was insufficient (Seo et al. 2021). Because cytokine storm is an important pathological process in SFTS, steroids have also been tested; however, there is no well-established treatment protocol, and steroids should be used with caution as the incidence of complications may increase (Jung et al. 2021; Kim et al. 2016; Nakamura et al. 2018). It is necessary to establish a treatment protocol for SFTS and develop effective antiviral agents and vaccines.

In our study, all dogs infected with SFTSV were exposed to ticks in urban parks and trails. This was confirmed by checking the aerial photos obtained by tracking their locations. Therefore, even if it is an urban area, an area with bushes is likely to be inhabited by ticks because of the entry of abandoned animals and wild animals. Accordingly, even an urban area is not safe in terms of SFTS infection, and parental education, such as instructions to avoid places with bushes or avoid the use of tick repellents during outdoor activities, is necessary to reduce exposure to ticks. When handling tick-exposed animals, personal protective equipment and infection control should be given prime importance, and awareness among veterinary personnel should be increased.

This study has some limitations. First, the sample size was small. The clinical characteristics of SFTS may change if a larger scale study is performed. Second, this was not a controlled study in terms of time of diagnosis, examination items, and treatment methods; it was an observational study of patient samples and data from several different veterinary hospitals. It is necessary to study the clinical characteristics and risk factors for survival while testing and treating a larger number of patients in the same environment.

Conclusions

In the present study of clinical infection with SFTSV in dogs, all dogs received only symptomatic treatment and survived, and they showed a clinical course different from that observed in humans and cats. Further studies should identify the reasons for differences in the clinical course among species. Even if the severity of SFTSV infection in dogs is mild to moderate, interspecies transmission is possible, so guidelines for prevention and diagnosis focusing on SFTS in dogs are warranted.

Declarations

Acknowledgements

This study was supported by the BK21 FOUR Future Veterinary Medicine Leading Education and Research Center. 

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. 

Funding

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Animal Disease Management Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) [grant number 119053-02]. 

Ethics approval

This study was approved by the Seoul National University Institutional Animal Care and Use Committee (IACUC No. SNU-190617-6), and conducted in strict accordance with the recommendations in the National Guideline. The owners of the dogs provided informed consent. 

Consent to participate

Not applicable 

Consent for publication

Not applicable 

Conflict of Interest 

The authors have no conflict of interest. 

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Han, S.-W, and Oh, Y.-I, Conceptualization: Han SW, Chae JS; Methodology and Data curation: Han SW; Formal analysis: Han SW, Oh, YI; Funding acquisition: Chae JS; Investigation: Han SW; Project administration: Chae JS; Resources: Rim JM, Kim DH, Kang JG; Supervision: Choi KS, Chae JS; Validation: Han SW, Oh YI, Chae JS; Writing - original draft: Han SW, Oh YI; Writing - review & editing: Han SW, Oh YI.

References

1. Ando T, Nabeshima T, Inoue S, Tun MMN, Obata M, Hu W, Shimoda H, Kurihara S, Izumikawa K, Morita K (2021) Severe fever with thrombocytopenia syndrome in cats and Its prevalence among veterinarian staff members in Nagasaki, Japan. Viruses 13:1142. http://doi.org/10.3390/v13061142
2. Chung JK, Kim CM, Kim DM, Yun NR, Park JW, Seo J, Kim YS (2020) Severe fever with thrombocytopenia syndrome associated with manual de-ticking of domestic dogs. Vector Borne Zoonotic Dis 20:285–294. http://doi.org/10.1089/vbz.2019.2463
3. Deng B, Zhang S, Geng Y, Zhang Y, Wang Y, Yao W, Wen Y, Cui W, Zhou Y, Gu Q (2012) Cytokine and chemokine levels in patients with severe fever with thrombocytopenia syndrome virus. PLoS ONE 7:e41365. http://doi.org/ 10.1371/journal.pone.0041365
4. Ding S, Yin H, Xu X, Liu G, Jiang S, Wang W, Han X, Liu J, Niu G, Zhang X (2014) A cross-sectional survey of severe fever with thrombocytopenia syndrome virus infection of domestic animals in Laizhou City, Shandong Province, China. JpnJ Infect Dis 67:1–4. http://doi.org/10.7883/yoken.67.1
5. Ding YP, Liang MF, Ye JB, Liu QH, Xiong CH, Long B, Lin WB, Cui N, Zou ZQ, Song YL (2014) Prognostic value of clinical and immunological markers in acute phase of SFTS virus infection. Clin Microbiol Infect 20:O870–O878. http://doi.org/10.1111/1469-0691.12636
6. Han SW, Kang JG, Byeon AR, Cho YK, Choi KS, Chae JS (2020) Severe fever with thrombocytopenia syndrome in canines from the Republic of Korea. Ticks Tick Borne Dis 11(4):101454. http://doi.org/10.1016/j.ttbdis.2020.101454
7. Hu LF, Wu T, Wang B, Wei YY, Kong QX, Ye Y, Yin HF, Li JB (2018) The regulation of seventeen inflammatory mediators are associated with patient outcomes in severe fever with thrombocytopenia syndrome. Sci Rep 8(1):159. http://doi.org/ 10.1038/s41598-017-18616-z
8. Huang XY, Du YH, Wang HF, You AG, Li Y, Su J, Nie YF, Ma HX, Xu BL (2019) Prevalence of severe fever with thrombocytopenia syndrome virus in animals in Henan Province, China. Infect Dis Poverty 8(1):56. http://doi.org/ 10.1186/s40249-019-0569-x
9. Jin C, Liang M, Ning J, Gu W, Jiang H, Wu W, Zhang F, Li C, Zhang Q, Zhu H (2012) Pathogenesis of emerging severe fever with thrombocytopenia syndrome virus in C57/BL6 mouse model. Proc Natl Acad Sci U S A 109(25):10053–10053. http://doi.org/10.1073/pnas.1120246109
10. Jung SI, Kim YE, Yun NR, Kim CM, Kim DM, Han MA, Kim UJ, Kim SE, Kim J, Ryu SY (2021) Effects of steroid therapy in patients with severe fever with Thrombocytopenia syndrome: A multicenter clinical cohort study. PLoS Negl Trop Dis 15(2):e009128. http://doi.org/ 10.1371/journal.pntd.0009128
11. Kang JG, Cho YK, Jo YS, Chae JB, Joo YH, Park KW, Chae JS (2019) Severe fever with thrombocytopenia syndrome virus in dogs, South Korea. Emerg Infect Dis 25(2):376–378. http://doi.org/10.3201/eid2502.180859
12. Kida K, Matsuoka Y, Shimoda T, Matsuoka H, Yamada H, Saito T, Imataki O, Kadowaki N, Noguchi K, Maeda K (2019) A case of cat-to-human transmission of severe fever with thrombocytopenia syndrome virus. Jpn J Infect Dis 72(5):356–358. http://doi.org/10.7883/yoken.JJID.2018.526
13. Kim JH, Choi YJ, Lee KS, Kim JE, Oh JW, Moon JH (2021) Severe fever with thrombocytopenia syndrome with Q fever coinfection in an 8-year-old girl. Pediatr Infect Dis J 40(1):e31–e34. http://doi.org/10.1097/INF.0000000000002948
14. Kim KH, Yi J, Kim G, Choi SJ, Jun KI, Kim NH, Choe PG, Kim NJ, Lee JK, Oh M (2013) Severe fever with thrombocytopenia syndrome, South Korea, 2012. Emerg Infect Dis 19(11):1892–1894. http://doi.org/10.3201/ eid1911.130792
15. Kim UJ, Kim DM, Ahn JH, Kang SJ, Jang HC, Park KH, Jung SI (2016) Case report Successful treatment of rapidly progressing severe fever with thrombocytopenia syndrome with neurological complications using intravenous immunoglobulin and corticosteroid. Antivir Ther 21(7):637–640. http://doi.org/10.3851/IMP3036
16. Kim WY, Choi W, Park SW, Wang EB, Lee WJ, Jee Y, Lim KS, Lee HJ, Kim SM, Lee SO (2015) Nosocomial transmission of severe fever with thrombocytopenia syndrome in Korea. Clin Infect Dis 60(11):1681–1683. http://doi.org/ 10.1093/cid/civ128
17. Kimura T, Fukuma A, Shimojima M, Yamashita Y, Mizota F, Yamashita M, Otsuka Y, Kan M, Fukushi S, Tani H (2018) Seroprevalence of severe fever with thrombocytopenia syndrome (SFTS) virus antibodies in humans and animals in Ehime prefecture, Japan, an endemic region of SFTS. J Infect Chemother 24(10):802–806. http://doi.org/10.1016/j.jiac.2018.06.007
18. Kirino Y, Ishijima K, Miura M, Nomachi T, Mazimpaka E, Sudaryatma PE, Yamanaka A, Maeda K, Sugimoto T, Saito A (2021) Seroprevalence of severe fever with thrombocytopenia syndrome virus in small-animal veterinarians and nurses in the Japanese prefecture with the highest case load. Viruses 13(2):229. http://doi.org/ 10.3390/v13020229
19. Lee SH, Kim HJ, Byun JW, Lee MJ, Kim NH, Kim DH, Kang HE, Nam HM (2017) Molecular detection and phylogenetic analysis of severe fever with thrombocytopenia syndrome virus in shelter dogs and cats in the Republic of Korea. Ticks Tick Borne Dis 8(4):626–630. http://doi.org/10.1016/ j.ttbdis.2017. 04.008
20. Lee SH, Kim HJ, Lee MJ, Byun JW, Kim DY, Kim NH, Kim DH, Kwak D, Kang HE, Nam HM (2018) Prevalence of antibodies against severe fever with thrombocytopenia syndrome virus in shelter dogs in the Republic of Korea. Ticks Tick Borne Dis 9(2):183–187. http://doi.org/10.1016/j.ttbdis.2017.09.002
21. Li XK, Dai K, Yang ZD, Yuan C, Cui N, Zhang SF, Hu YY, Wang ZB, Miao D, Zhang PH (2020) Correlation between thrombocytopenia and host response in severe fever with thrombocytopenia syndrome. PLoS Negl Trop Dis 14(10):e0008801. http://doi.org/10.1371/journal.pntd.0008801
22. Li Z, Hu J, Bao C, Li P, Qi X, Qin Y, Wang S, Tan Z, Zhu Y, Tang F (2014) Seroprevalence of antibodies against SFTS virus infection in farmers and animals, Jiangsu, China. J Clin Virol 60(3):185–189. http://doi.org/10.1016/j. jcv.2014.03.020
23. Liu J, Fu H, Sun D, Wu S, Wang L, Yao M, Yuan G (2020) Analysis of the laboratory indexes and risk factors in 189 cases of severe fever with thrombocytopenia syndrome. Medicine 99(2):e18727. http://doi.org/10.1097/MD.0000000000018727
24. Liu Y, Li Q, Hu W, Wu J, Wang Y, Mei L, Walker DH, Ren J, Wang Y, Yu XJ (2012) Person-to-person transmission of severe fever with thrombocytopenia syndrome virus. Vector Borne Zoonotic Dis 12(2):56–160. http://doi.org/10.1089/vbz.2011.0758
25. Matsuno K, Nonoue N, Noda A, Kasajima N, Noguchi K, Takano A, Shimoda H, Orba Y, Muramatsu M, Sakoda Y (2018) Fatal tickborne phlebovirus infection in captive cheetahs, Japan. Emerg Infect Dis 24(9):1726–1729. http://doi.org/ 10.3201/eid2409.171667
26. Matsuu A, Hamakubo E, Yabuki M (2021) Seroprevalence of severe fever with thrombocytopenia syndrome virus in animals in Kagoshima Prefecture, Japan, and development of Gaussia luciferase immunoprecipitation system to detect specific IgG antibodies. Ticks Tick Borne Dis 12(5):101771. http://doi.org/ 10.1016/j.ttbdis.2021.101771
27. Matsuu A, Momoi Y, Nishiguchi A, Noguchi K, Yabuki M, Hamakubo E, Take M, Maeda K (2019) Natural severe fever with thrombocytopenia syndrome virus infection in domestic cats in Japan. Vet Microbiol 236:108346. http://doi.org/ 10.1016/j.vetmic.2019.06.019
28. Nakamura S, Azuma M, Maruhashi T, Sogabe K, Sumitani R, Uemura M, Iwasa M, Fujii S, Miki H, Kagawa K (2018) Steroid pulse therapy in patients with encephalopathy associated with severe fever with thrombocytopenia syndrome. J Infect Chemother 24(5):389–392. http://doi.org/ 10.1016/j.jiac.2017.11.004
29. Nam SJ, Oh YI, Kim HJ, Cheon DS, Noh SJ, Hong YJ (2020) Unusual case of severe fever with thrombocytopenia syndrome showing clinical manifestations in a companion dog. Vet Med Sci 6(3):353–358. http://doi.org/10.1002/vms3.261
30. Niu G, Li J, Liang M, Jiang X, Jiang M, Yin H, Wang Z, Li C, Zhang Q, Jin C (2013) Severe fever with thrombocytopenia syndrome virus among domesticated animals, China. Emerg Iinfect Dis 19(5):756–763. http://doi.org/ 10.3201/eid1905.120245
31. Oh SS, Chae JB, Kang JG, Kim HC, Chong ST, Shin JH, Hur MS, Suh JH, Oh MD, Jeong SM (2016) Detection of severe fever with thrombocytopenia syndrome virus from wild animals and Ixodidae ticks in the Republic of Korea. Vector Borne Zoonotic Dis 16(6):408–414. http://doi.org/10.1089/vbz.2015.1848
32. Park E, Shimojima M, Nagata N, Ami Y, Yoshikawa T, Iwata-Yoshikawa N, Fukushi S, Watanabe S, Kurosu T, Kataoka M (2019) Severe fever with thrombocytopenia syndrome phlebovirus causes lethal viral hemorrhagic fever in cats. Sci Rep 9(1):11990. http://doi.org/10.1038/s41598-019-48317-8
33. Park SC, Park JY, Choi JY, Lee SG, Eo SK, Oem JK, Tark DS, You M, Yu DH, Chae JS (2020) Pathogenicity of severe fever with thrombocytopenia syndrome virus in mice regulated in type I interferon signaling. Lab Anim Res 36:38. http://doi.org/10.1186/s42826-020-00070-0
34. Park SC, Park JY, Choi JY, Oh B, Yang MS, Lee SY, Kim JW, Eo SK, Chae JS, Lim CW (2021) Experimental infection of dogs with severe fever with thrombocytopenia syndrome virus: Pathogenicity and potential for intraspecies transmission. http://doi.org/10.1111/tbed.14372. Transboundary Emerg Dis Online ahead of print
35. Saijo M, Shimojima M (2018) A human case report of severe fever with thrombocytopenia syndrome infected from a domestic dog. 92th Conference on Japanese Association for Infectious Diseases; 2018 May 31–June 2; Okayama, Japan. Kansenshogaku zasshi. 2018; 92: extra issue, 228. Japanese
36. Sakai Y, Kuwabara Y, Ishijima K, Kagimoto S, Mura S, Tatemoto K, Kuwata R, Yonemitsu K, Minami S, Kuroda Y (2021) Histopathological Characterization of Cases of Spontaneous Fatal Feline Severe Fever with Thrombocytopenia Syndrome, Japan. Emerg Infect Dis 27(4):1068–1076. http://doi.org/10.3201/ eid2704.204148
37. Seo JW, Kim D, Yun N, Kim DM (2021) Clinical update of severe fever with thrombocytopenia syndrome. Viruses 13(7):1213. http://doi.org/10.3390/v13071213
38. Seto J, Tanaka S, Kawabata H, Ito Y, Ikeda T, Mizuta K (2020) Detection of tick-borne pathogens in ticks from dogs and cats in Yamagata Prefecture of Japan in 2018. Jpn J Infect Dis 74(2):122–128. http://doi.org/10.7883/ yoken.JJID.2020.462
39. Seynhaeve A, Vermeulen C, Eggermont A, ten Hagen T (2006) Cytokines and vascular permeability: an in vitro study on human endothelial cells in relation to tumor necrosis factor-alpha-primed peripheral blood mononuclear cells. Cell Biochem Biophys 44(1):157–169. http://doi.org/10.1385/CBB:44:1:157
40. Sun L, Hu Y, Niyonsaba A, Tong Q, Lu L, Li H, Jie S (2014) Detection and evaluation of immunofunction of patients with severe fever with thrombocytopenia syndrome. Clin Exp Med 14(4):389–395. http://doi.org/10.1007/s10238-013-0259-0
41. Sun Y, Jin C, Zhan F, Wang X, Liang M, Zhang Q, Ding S, Guan X, Huo X, Li C (2012) Host cytokine storm is associated with disease severity of severe fever with thrombocytopenia syndrome. J Infect Dis 206(7):1085–1094. http://doi.org/ 10.1093/infdis/jis452
42. Takahashi T, Maeda K, Suzuki T, Ishido A, Shigeoka T, Tominaga T, Kamei T, Honda M, Ninomiya D, Sakai T (2014) The first identification and retrospective study of severe fever with thrombocytopenia syndrome in Japan. J Infect Dis 209(6):816–827. http://doi.org/10.1093/infdis/jit603
43. Tian H, Yu P, Chowell G, Li S, Wei J, Tian H, Lv W, Han Z, Yang J, Huang S 2017 Severe fever with thrombocytopenia syndrome virus in humans, domesticated animals, ticks, and mosquitoes, Shaanxi Province, China.Am J Trop Med Hyg96(6):1346–1349. http://doi.org/10.4269/ajtmh.16-0333
44. Tsuru M, Suzuki T, Murakami T, Matsui K, Maeda Y, Yoshikawa T, Kurosu T, Shimojima M, Shimada T, Hasegawa H (2021) Pathological characteristics of a patient with severe fever with thrombocytopenia syndrome (SFTS) infected with SFTS virus through a sick cat’s bite. Viruses 13(2):204. http://doi.org/10.3390/v13020204
45. Wang JN, Li TQ, Liu QM, Wu YY, Luo MY, Gong ZY (2021) Vectors, Hosts, and the Possible Risk Factors Associated with Severe Fever with Thrombocytopenia Syndrome. Can J Infect Dis Med Microbiol. 2021: 8518189. http://doi.org/ 10.1155/2021/8518189
46. Xu X, Sun Z, Liu J, Zhang J, Liu T, Mu X, Jiang M (2018) Analysis of clinical features and early warning indicators of death from severe fever with thrombocytopenia syndrome. Int J Infect Dis 73:43–48. http://doi.org/10.1016/j.ijid.2018.05.013
47. Yamanaka A, Kirino Y, Fujimoto S, Ueda N, Himeji D, Miura M, Sudaryatma PE, Sato Y, Tanaka H, Mekata H (2020) Direct transmission of severe fever with thrombocytopenia syndrome virus from domestic cat to veterinary personnel. Emerg Infect Dis 26(12):2994–2998. http://doi.org/10.3201/eid2612.191513
48. Yoo JR, Heo ST, Park D, Kim H, Fukuma A, Fukushi S, Shimojima M, Lee KH (2016) Family cluster analysis of severe fever with thrombocytopenia syndrome virus infection in Korea. Am J Trop Med Hyg 95:1351–1357. http://doi.org/10.4269/ajtmh.16-0527
49. Yoo JR, Lee KH, Heo ST (2018) Surveillance results for family members of patients with severe fever with thrombocytopenia syndrome. Zoonoses Public Health 65(8):903–907. http://doi.org/10.1111/zph.12481
50. Yu XJ, Liang MF, Zhang SY, Liu Y, Li JD, Sun YL, Zhang L, Zhang QF, Popov VL, Li C (2011) Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med 364(16):1523–1532. http://doi.org/ 10.1056/NEJMoa1010095
51. Yun SM, Park SJ, Kim YI, Park SW, Yu MA, Kwon HI, Kim EH, Yu KM, Jeong HW, Ryou J (2020) Genetic and pathogenic diversity of severe fever with thrombocytopenia syndrome virus (SFTSV) in South Korea. JCI Insight 5(2):e129531. http://doi.org/10.1172/jci.insight.129531