Unusual case of severe fever with thrombocytopenia syndrome showing clinical manifestations in a companion dog

Abstract Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging zoonotic virus in East Asia. However, SFTS virus (SFTSV) has not been reported to cause clinical infection in companion dogs to date. We report the case of a 4‐year‐old companion dog that presented with fever, vomiting, leukocytopenia and thrombocytopenia at a veterinary hospital in the Republic of Korea. It was diagnosed with SFTS, which was confirmed using real‐time reverse transcription PCR, sequencing and an indirect immunofluorescence assay, and recovered after supportive care. Further studies are required to investigate SFTSV infection in companion animals, living in close contact with humans, as well as animal‐to‐human transmission.


| INTRODUC TI ON
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonotic disease in East Asia, including the Republic of Korea, China, Japan and Vietnam (Chen et al., 2019;Tran et al., 2019), and has a high mortality rate of 16.2%-55% in humans (Im et al., 2019;Li et al., 2018;Takahashi et al., 2014;Yu et al., 2011;Zhang et al., 2012) and of 62.5% in cats (Matsuu et al., 2019). The tick Haemaphysalis longicornis is the main vector of the SFTS virus (SFTSV; Huaiyangshan banyangvirus: Phenuiviridae), previously described as Phlebovirus (Maes et al., 2019), and is the most dominant tick species (88.9%) in the Republic of Korea (Kim et al., 2015). Although antibodies to SFTSV have been detected in various animal species, with a seroprevalence ranging from 0.2% to 52.1% in dogs, most animals are asymptomatic (Chen et al., 2019;Ding et al., 2014;Kang et al., 2019;Lee et al., 2017Lee et al., , 2018Oh et al., 2016). However, in stray and pet cats, there have been reports of apparent SFTSV infections manifesting with clinical symptoms (Kida et al., 2019;Matsuu et al., 2019), and there was a report of transmission of SFTSV from these cats to the attending veterinarian (Kida et al., 2019).

O R I G I N A L A R T I C L E
Unusual case of severe fever with thrombocytopenia syndrome showing clinical manifestations in a companion dog 2 | MATERIAL S AND ME THODS

| Clinical history of the canine patient
The dog, a 4-year-old, castrated male, Bichon Frise, presented with a 2-day history of fever, anorexia, lethargy and vomiting in October 2018 (day 0: first day of admission). The owner had noticed multiple tick bites and applied a tick-preventative medicine 19 days prior to admission. Physical examination and a complete blood count revealed fever (40.6°C; reference, 38.0°C-39.2°C), leukopenia and thrombocytopenia without bleeding (Table 1). On day 2, diagnostic imaging revealed slight enlargement of the spleen and pancreas and a thickened gall bladder wall. Real-time polymerase chain reaction (PCR) for common infectious diseases in dogs (Anaplasma spp., Ehrlichia spp., Babesia spp., Leptospira spp., Bartonella spp., haemotropic mycoplasma and Rickettsia spp.) showed negative results. We suspected SFTSV infection on the basis of the history, fever and bicytopenia without anaemia.
To confirm SFTSV infection, we acquired a whole blood sample and performed real-time reverse transcription (RT)-PCR for specific identification of the S-segment genome, a Vero E6 cell culture for virus isolation, and an indirect fluorescence assay (IFA) (day 2). To determine the genetic identity of the virus, we amplified the full length of the S-segment of SFTSV.

| Detection of SFTSV RNA in canine serum samples
SFTSV RNA was extracted from the patient's serum samples using an RNAeasy extraction kit (QIAgen) with an automated extraction system (QIAcube, QIAgen). The eluted RNA was added to a reaction mixture provided in the RT-PCR kit (Bioneer). SFTSV-specific TaqMan primers and probe were used to detect viral RNA reliably.

| Quantification of RNA of SFTSV
To measure viral loads of SFTSV in the dog's serum samples, qRT-PCR was performed using TaqMan probes. Based on a standard curve produced using the Ct values of a plasmid containing the S segment, the SFTSV RNA copy number (per ml) was determined.

| Virus isolation and identification of SFTSV by IFA
Canine serum samples collected from the patient were inoculated onto monolayers of Vero E6 cells for virus isolation, as previously described (Lee et al., 2016;Yu et al., 2011). After adaptation and proliferation of viruses in Vero E6 cells, we confirmed SFTSV replication by IFA using anti-SFTSV rabbit polyclonal antibody, as described

| Phylogenetic analysis of SFTSV
The S segment of SFTSV RNA was amplified using one-step RT-PCR, as previously described (Hwang et al., 2017;Li et al., 2018). Positive samples of RT-PCR were sequenced using the Sanger sequencing technique. Sequence alignment was computed using the software program (DNAstar). The phylogenetic trees were analysed using the Clustal W method in the DNAstar package. RT-PCR were negative, and the viral load was below the detection limit; therefore, the patient was discharged.

| RE SULTS
Real-time PCR facilitated the detection of viral RNA up to day 13; the viral RNA load on day 2 was 8.5 × 10 8 copies/ml (Figure 1). The Vero E6

| D ISCUSS I ON
Although SFTSV is primarily transmitted through tick bites, human- ver, rearing animals is a risk factor for human SFTS (Song, Lee, & Ju, 2017). Consequently, SFTS is attracting attention as an emerging zoonotic disease. In particular, companion animals, such as dogs and cats, which live in close contact with humans, serve as important mediators for transmitting infectious diseases to humans. Therefore, it is important to investigate infectious diseases in companion animals to gain information related to preventing infection in humans.
In the present case, the dog's owner showed no clinical signs of SFTS and did not undergo the required examinations; hence, we could not identify any association. However, SFTSV isolated from the dog strongly resembled that previously isolated from humans in the Republic of Korea (Liu et al., 2012); therefore, we cannot negate the possibility of dog-to-human transmission. Further studies are necessary, given that cat-to-human transmission has been proven previously (Kida et al., 2019).
Human SFTS presents various clinical manifestations, including high fever, gastrointestinal signs, neurological signs, thrombocytopenia, leukocytopenia, lymphadenopathy and multiple organ failure (Sun et al., 2012). The symptoms observed in our case also included fever, vomiting, leukocytopenia and thrombocytopenia. Although a high viral load is a strong risk factor for death in humans (Sun et al., In conclusion, SFTS exhibiting clinical manifestations in a companion dog has not been reported to date, to the best of our knowledge. SFTS is an emerging zoonotic disease with an annual increase in incidence (Im et al., 2019); thus, thorough tick prevention measures for companion dogs and measures for preventing animal-to-human transmission are necessary.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests. Validation; Writing-review & editing.

E TH I C A L S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as this is a description of the diagnosis and treatment of one case and no experimentation was conducted on the treated dog.