Pathological characteristics of severe fever with thrombocytopenia syndrome (SFTS) virus in a patient infected with SFTS virus from a sick cat’s bite Running title: Pathology in a cat-associated SFTS patient

1 Department of Hematology, Kanmon Medical Center, Choufu Sotoura-chou, Shimonoseki City, Yamaguchi 752-8510, Japan 2 Department of Pathology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan 3 Department of Pathology, Kanmon Medical Center, Choufu Sotoura-chou, Shimonoseki City, Yamaguchi 752-8510, Japan 4 Maeda Animal Hospital, 698 Takura, Shimonoseki city, Yamaguchi 751-0883, Japan 5 Department of Virology 1, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama City, Tokyo 208-0011, Japan 6 Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 January 2021 doi:10.20944/preprints202101.0207.v1


Introduction
Severe fever with thrombocytopenia syndrome (SFTS) (formerly SFTS virus, SFTSV) is caused by the Dabie bandavirus, which belongs to the Bandavirus genus (formerly Phlebovirus genus) of the Phenuiviridae family (formerly Bunyaviridae family). SFTS was first discovered in China [1,2] and was then reported to be endemic to Japan [3], South Korea [4], Taiwan [5], and Vietnam [6]. Patients with SFTS are usually infected with SFTSV through a tick bite such as that from Haemaphysalis longicornis or Amblyomma testudinarium [7].
The case fatality rate of patients with SFTS in Japan is reported to be approximately 30% [8,9]. Notably, there is always a risk for people living in SFTS-endemic regions to suffer from SFTS, a fatal disease with a high fatality rate.
Recently, it has been reported that cats including cheetahs were infected with and sickened by SFTSV [10,11]. Furthermore, patients with SFTS who were infected by sick cats have also been reported [12].
A woman who died from multiorgan failure of unknown cause was retrospectively diagnosed as having SFTS following a pathological examination. A sick cat bit her on her left hand, and symptoms appeared 2 days later. The cat also died of multiorgan failure. In this study, the clinical and pathological characteristics of the patient with SFTS who was infected by a cat positive for SFTSV infection were examined.

Patient
A woman in her 50s became ill after she was bitten by a sick cat. She was retrospectively diagnosed as having SFTS as described below. The clinical course comprising her symptoms, laboratory findings including total blood cell (TBC) counts, serum chemistry, computed tomography imaging, and postmortem examination were retrospectively retrieved from her medical records.

Cat
The clinical course and laboratory findings of the cat that bit the reported patient were retrospectively retrieved from its medical records.

Measurement of SFTSV genome load with real-time RT-PCR in blood
To measure copy numbers of the SFTSV S segment in sera, quantitative one-step reverse-transcription polymerase chain reaction (RT-PCR) was performed as described previously [13].

Antibody detection with indirect immunofluorescence assay
Immunofluorescence assay using SFTSV-infected cells was performed to evaluate the presence of IgM and IgG to SFTSV as 6 described previously [14].

Measurement of SFTSV genome load with real-time RT-PCR in tissues
The SFTSV copy number was determined by a quantitative real-time RT-PCR on RNA samples extracted from paraffin-embedded sections (10 µm; × 3) as described previously with some modifications [3,16]. Briefly, RNA was extracted using a Pure Link FFPE RNA isolation kit (Invitrogen, Carlsbad, CA), and RT-PCR was performed using a QuantiTect Multiplex RT-PCR Kit (Qiagen, Hilden, Germany) and Agilent Mx3000P system (Agilent, Santa Clara, CA) according to the manufacturer's protocol. Quantitative real-time RT-PCR amplified the N region within the S segment of the SFTSV genome. The amount of human β-actin mRNA in the RNA extracted from each section was also determined and used as an internal reference for normalization.

Pathological and immunohistochemical analyses 7
Histopathological studies of formalin-fixed and paraffin-embedded specimens were performed using hematoxylineosin staining. Immunohistochemical detection of the SFTSV nucleoprotein antigen (SFTSV-NP) was performed on paraffin-embedded sections, as previously described [3,15].

Patient presentation
A previously healthy woman in her 50s living in Yamaguchi prefecture in western Japan was bitten by an ill cat, which she

Cat presentation
The patient had found a cat near her house on Day -2 and noticed that it was sick. The cat seemed to have lost its appetite, was frequently vomiting, and its breathing did not seem to be

Dynamics of SFTSV loads and immunological responses in sera
The SFTSV copy number on Day 5 was 2.0 × 10 4 copies/mL and reached a maximum of 2.7 × 10 9 copies/mL on Day 11. No IgM and IgG antibodies reactive with SFTSV were detected in any sera collected during the course of the patient's hospitalization.

Gross pathology
Gross examination on autopsy revealed mild bilateral pleural effusion (L 200 mL; R 100 mL), retention of ascites (600 mL), liver congestion, splenic congestion, and many enlarged lymph nodes (short-axis diameter of ~7 mm) throughout the mesentery, omentum, and para-aortic regions. The stomach contained 300 mL of blood clots, and gastric mucosa showed severe congestion in the absence of ulceration.

Histopathology and immunohistochemistry
Histopathological analyses showed infiltration of numerous atypical large lymphocytes in red pulp and periarteriolar sheaths, white pulp depletion with massive nuclear debris, necrotic debris, hemophagocytosis, and congestion in the spleen Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 January 2021 doi:10.20944/preprints202101.0207.v1 (Fig. 1A). Spleen, liver, multiple lymph nodes, bone marrow, kidney, thyroid, adrenal gland, and lung were positive for SFTSV antigen in the immunohistochemistry (IHC) analysis. Numerous atypical lymphocytes positive for SFTSV antigens were present in the spleen (Fig. 1B). In liver, viral antigens were detected in the sinusoids amid a background of hemophagocytosis, centrilobular necrosis, hemorrhage, and mild lymphocytic inflammation around the portal tracts (Fig. 1C, D). The lymph nodes in the left subclavian regions showed focal necrotizing lymphadenitis with viral antigens positive within necrotic regions (Fig. 1E, F). IHC assays for Bartonella henselae were negative in the lymph nodes. Atypical large lymphoid cells with viral antigens were present in follicles of the mesentery and subcarinal lymph nodes (Fig. 1G, H). Prominent hemophagocytosis was also evident in these lymph nodes and bone marrow (Fig. 1I, J).

SFTSV RNA measurement in organs
SFTSV-RNA was detected in all organs and tissues tested, and the numbers of SFTSV-RNA copies/cell calculated using the β-actin mRNA copy number are shown in Table 3. The level of SFTSV genome copies was consistent with the IHC results. *(Copies/cell): copy/cell = SFTSV/β-actin × 1500.

Discussion
In the present patient, findings associated with hemophagocytosis were present in bone marrow and lymph nodes.
Necrotizing lymphadenitis in lymph nodes and hemophagocytosis in bone marrow are the relatively specific and common findings in the pathology of SFTS patients [16]. The presence of necrotizing lymphadenitis and hemophagocytosis in bone marrow are reported to be positive in fatal patients with SFTS [3,[15][16][17][18][19][20]. Because the presence of these factors was observed in the patient, her pathological materials were further examined for SFTSV infection.
The data in Table 3 indicate that SFTSV replicated predominantly in the spleen in this patient. SFTSV antigen was detected with IHC analyses in regional lymph nodes such as the subclavian and subcarinal lymph nodes, indicating that SFTSV replicates dominantly in the regional lymph nodes (Fig. 1E, H).
It was unclear whether SFTSV replicated in the non-regional lymph nodes of the patient because these lymph nodes were not tested for SFTSV antigens in the IHC analyses. Similar pathological findings have already been reported in another SFTS patient, in whom regional lymph nodes were enlarged and SFTSV was detected in these nodes but not in the non-regional lymph nodes [3]. SFTSV antigen was detected in all organs tested in the present patient. Other reports also noted that SFTSV antigens were positive in most of the organs tested as shown in the present patient [17,18]. There were no pathological characteristics specific to our patient. All of her pathological features were similar to those reported so far [3,[15][16][17][18][19][20].
However, the incubation time in the present patient was only The clinical signs and laboratory findings of SFTSV-infected cats were characterized in 24 cats with confirmed SFTSV infection in western Japan [10]. All cats showed clinical signs of anorexia and lethargy, with fever and vomiting in 68% and 42%, respectively, and the case fatality rate was 63%.
Laboratory characteristics included a TBC count indicating thrombocytopenia and leukocytopenia and elevation of serum total bilirubin, amyloid A, and CPK. Another study summarizing the cases of 3 sick cats infected with SFTSV, showed that fever and loss of appetite were the typical signs, and the TBC count revealed the presence of thrombocytopenia and leukocytopenia [12].
The clinical signs and laboratory findings of the cat who bit the present patient were similar to those previously reported. Therefore, it is highly possible that this patient was infected with SFTSV from the sick cat, although the cat was not specifically diagnosed as having SFTSV infection virologically.
A male veterinarian in western Japan was also infected with SFTSV by a sick cat [12].