Patient 1
Patient 1 was a 41-year-old male with a medical history of hyperuricemia and hyperlipidemia, who lived in Sapporo, Hokkaido. In mid-May 2019, he visited a forest area near Sapporo for approximately 4 h. The next day, he noticed and removed an arthropod attached to his right abdomen. Four days after visiting the forest, he had a fever over 39 °C, followed by gait disturbance and leg pain. After the fever continued for 4 days, he was admitted to our hospital with a temperature of 38.9 °C (Table 1). On admission, a review of systems was negative except for a fever, appetite loss, and bilateral lower extremity pain. Physical examination was unremarkable except for a small papule on the abdomen, which was suspected to be the site of a tick bite. Laboratory tests showed leukopenia and lymphocytopenia with a white-blood cell (WBC) count of 1,600/µl and a lymphocyte ratio of 24.0% on differential count (Fig. 1a), thrombocytopenia with a platelet count of 87,000/µl (Fig. 1b), and significantly increasing levels of aspartate aminotransferase (AST, 3703 units/l), alanine aminotransferase (ALT, 1,783 unit/l), creatine kinase (CK; 5,847 unit/l), lactose dehydrogenase (LDH, 4,069 unit/l) (Fig. 1c), and ferritin (55,200 ng/ml) (Supplementary Table 1). Computed tomography scans of the chest, abdomen, and pelvis with contrast showed findings consistent with fatty liver disease without organomegaly.
Table 1 Summary of two patients.Fig. 1: Laboratory test values of two patients infected with Yezo virus (YEZV).
a White-blood cell count shown as the total of the differential counts. Cell types not indicated in the key were undetectable. *Neutrophils were further differentiated into band cells and segmented neutrophils except for the sample from patient 1 on day 3. Gray shading behind the graphs indicates the approximate normal ranges of values. b Platelet counts for patient 1 (orange) and patient 2 (light green). c Aspartate aminotransferase (AST, circle), alanine aminotransferase (ALT, square), lactose dehydrogenase (LDH, triangle), and creatinine kinase (CK, cross) values for patients 1 (orange) and 2 (green). d Activated partial thromboplastin time (APTT, circle), fibrinogen degradation products (FDP, square), and D-dimer (rhombus) plotted for patients 1 (orange) and 2 (green). e Viral loads determined for serum (dark color with circle) and urine (regular color with square) for patients 1 (orange) and 2 (green) using RT-qPCR. Limit of detection (LOD) was approximately 100 copies/µl. f, g Serum antibodies reacting with YEZV N protein were detected using enzyme-linked immunosorbent assay (ELISA) for patients 1 (f) and 2 (g). The reciprocal of the highest serum dilution in which the difference in OD value between YEZV N and the negative control was >0.3, was determined as the serum antibody titer. Anti-human IgM (light blue) or IgG (dark blue) were used to detect specific antibodies binding to the antigen.
During hospitalization, thrombocytopenia progressed until day 6 (6 days after the onset of fever, the second day in hospital) when the lowest platelet count reached 75,000/µl. This was followed by thrombocytosis and a peak platelet count of 638,000/µl. Atypical lymphocytosis was observed throughout the hospitalization period with the peak level of atypical lymphocytes reaching 76% in a WBC count of 8,200/µl. Significant elevation of AST, ALT, LDH, and CK levels lasted until day 10. Activated partial thromboplastin time (APTT) was extended on days 5–9 (up to 52 s on day 8), and elevations of fibrinogen degradation products (FDP; maximum of 14.1 µg/ml) and D-dimer (maximum of 6.6 µg/ml) were recorded on day 6 (Fig. 1d).
On the basis of the history of a suspected tick bite, the patient was treated empirically with 8 days of ceftriaxone for suspected Lyme disease or Borrelia miyamotoi infection, 14 days of doxycycline for suspected rickettsioses, and 6 days of gentamicin for suspected tularemia. His fever and other symptoms gradually resolved, and the patient was discharged without complications after day 19 (15 days of hospitalization).
The patient’s sera, collected on days 5 (the day of hospitalization) and 19, were negative on both days for IgM and IgG antibodies for Lyme disease (the Borrelia spp.) and relapsing fever (i.e., B. miyamotoi), neutralizing antibodies for tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus, and specific antibodies for Francisella tularensis and Rickettsia spp. Polymerase chain reaction (PCR) assays were performed on whole blood taken on day 5; these assays also were negative for Borrelia spp. and severe fever with thrombocytopenia syndrome virus (SFTSV).
A skin biopsy around the papule of the suspected tick bite was performed on the second day of hospitalization, and a fragment of the gltA gene of R. helvetica was detected in this specimen.
Patient 2
Patient 2 was a 59-year-old previously healthy male with no remarkable medical history living in Sapporo, Hokkaido. In mid-July 2020, he hiked on a mountain near Sapporo. During the hike, he received a bite on his lower leg from an unidentified arthropod that remained attached for at least 30 min. He remained in his usual state of health until 9 days after the hike when he lost his appetite and then, developed a fever of 37.4 °C on 17 days after the hike. Following two visits to different hospitals on days 3 and 4 after the onset of fever (Table 1), where he was found to have a fever (38.5 °C on day 3) with leukopenia (WBC count 1,170/µl and lymphocytes at 38.0% on differential count, Fig. 1a) and thrombocytopenia (platelet count 65,000/µl, Fig. 1b), he visited our hospital on day 5 post-onset of fever. Our laboratory tests on day 5 confirmed progressing thrombocytopenia (52,000/µl), continuing leukopenia (WBC count 1700/µl and lymphocytes at 38.0% on differential count), and elevated AST, ALT, LDH, ferritin, and CK levels (Fig. 1c and Supplementary Table 1). On day 6, pruritic urticarial rash developed on his extremities (Supplementary Fig. 1), but the fever resolved.
During his visits to the hospitals, thrombocytopenia progressed to a minimum of 42,000/µl on day 6. Lymphocytopenia began to resolve on day 5 and atypical lymphocytosis appeared with the atypical lymphocyte population increasing to 58% on day 8. Elevation of AST, ALT, LDH, and CK levels continued to resolve after day 8. An extended APTT (up to 41 s on day 5) and elevations of FDP (maximum of 19.5 µg/ml on day 5) and D-dimer (maximum of 10.7 µg/ml on day 7) were observed by day 7 (Fig. 1d).
The patient was administered doxycycline after the second hospital visit on day 4. The patient’s blood was collected on day 5 and tested positive for B. miyamotoi and negative for B. burgdorferi by PCR, while serum collected on day 7 was positive for B. miyamotoi IgG antibody and Lyme disease Borrelia IgM and IgG antibodies. RNAs of TBEV and SFTSV were not detected in serum collected on day 5.
Detection of a novel orthonairovirus in patients
A serum sample from patient 1 on day 4 was initially used for virus isolation using Vero E6 cells. After 14 days without any cytopathic effect (CPE), RNA fragments of a possible orthonairovirus, Yezo virus (YEZV) tentatively named after the historical name of Hokkaido, were detected from the supernatant using high-throughput sequencing. Then, primers for reverse-transcription PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR) were designed to detect the YEZV RNA fragments.
Serum and urine samples from the two patients were subjected to RT-qPCR to reveal the viral load during febrile illness (Fig. 1e). The highest viral load observed was 7.3 × 107 copies/µl in the serum of patient 1 at day 5. Serum samples collected from patient 2 during the acute phase of infection were also positive for YEZV RNA, with the highest value being 5.2 × 105 copies/µl. The serum viral load dropped steeply at days 7–8 in patient 1 and at days 5–6 in patient 2. YEZV RNA was detectable in the serum of patient 1 until day 19. Urine viral loads were lower than serum viral loads during the acute phase of infection but were maintained even after the steep drop in the serum viral load in patients 1 and 2. Serum and/or urine were negative for YEZV by RT-qPCR after day 33 for patient 1 and day 56 for patient 2.
Antibody levels in patient sera were measured using enzyme-linked immunosorbent assay (ELISA), with YEZV nucleocapsid (N) protein-expressing cells as the antigen. IgM antibodies were detected at day 6 in patient 1 (Fig. 1f) and day 5 in patient 2 (Fig. 1g) followed by a peak IgM antibody titer of 25,600 and disappearance approximately 6 months later (day 168 for patient 1 and day 184 for patient 2). IgG antibody titer increased on day 11 in patient 1 and day 7 in patient 2 and was still high at the end of the study period.
Retrospective identification of patients infected with YEZV
We retrospectively screened serum samples of 248 patients suspected as having a tick-borne disease since 2014 using RT-PCR and RT-qPCR assays. Five positive samples were identified and were tested by ELISA, along with two available convalescent serum samples in conjunction with the positive samples. In total, seven patients, including patients 1 and 2, were confirmed to be infected with YEZV in the acute phase of febrile illness with thrombocytopenia after tick bite, four of whom were found to have seroconverted from undetectable levels of IgG at their acute phase to more than 6,400 ELISA titers at their convalescent phase (Table 2). Four out of the seven patients tested positive for Borrelia spp. infections.
Table 2 Summary of clinical findings and pathogen detection in patients infected with YEZV.Isolation and genetic identification of YEZV
After the identification of YEZV RNA fragments in Vero E6 cells, no further growth of the virus was confirmed during consecutive passages of the samples. Then, sequential passage of the virus from patient 2 through AG129 mice and Vero E6 cells was performed. While two AG129 mice intraperitonially inoculated with a plasma sample did not show clinical signs until 14 days, seroconversion with an ELISA IgG titer of 409,600 was identified. Swelling of the spleen was found in another two mice 5 days after inoculation when they were sacrificed for serum collection. The serum samples were passaged onto Vero E6 cells and infectious YEZV was recovered without causing any CPE in Vero E6 cells. Spherical and oval virions of approximately 100–200 nm in diameter at the widest point were observed in the supernatant of the infected Vero E6 cells by transmission electron microscopy (Fig. 2a, b, and Supplementary Fig. 2). Ixodes scapularis embryo-derived ISE6 cells5 supported the replication of YEZV to titers similar to those in Vero E6 cells, while no viral RNA replication was detected in Rhipicephalus microplus embryo-derived BME/CTVM23 cells6 over 14 days (Fig. 2c). Immunofluorescent staining with convalescent sera from patients 1 and 2 revealed that viral antigens were detected in the cytoplasm of Vero E6 cells infected with YEZV (Fig. 2d, e).
Fig. 2: Isolation of a novel orthonairovirus, YEZV.
a Transmission electron microscopy of YEZV particles negatively stained with 2% phosphotungstic acid (pH 7.0). Scale bar = 100 nm. Similar particle images were obtained in two independent experiments. b Particle size distribution shown by the long diameters of spherical and oval virions in the microscopy images. Diameters of 98 particles were manually measured, and the number of particles in each 20-nm range are shown. c Growth of YEZV in different cell lines. YEZV was inoculated at a multiplicity of infection of 0.1 (high, dark color) or of 0.001 (low, regular color) into mammalian-derived Vero E6 cells (orange), tick ISE6 (green) and BME/CTVM23 (BME, gray) cells, and incubated for 14 days. Viral RNA copies at each time point were measured using RT-qPCR. d, e Detection of YEZV antigens in Vero E6 cells. Vero E6 cells were infected with YEZV and fixed after seven days. Convalescent serum collected from patient 1 on day 168 (d) and from patient 2 on day 184 (e) after the onset of fever, were used in an immunofluorescent assay. Antigens reacting with human IgG are shown in green. Cell nuclei are shown in blue, scale bar = 100 µm. Similar results were obtained in two independent experiments.
Sequence analysis of RNA extracted from culture supernatant identified entire YEZV RNA sequences constituting three genome segments: large (L), medium (M), and small (S) (Fig. 3a). Each genome segment encoded a single open reading frame whose deduced amino acid sequence was similar to each corresponding orthonairovirus protein, i.e., RNA-dependent RNA polymerase (L protein), glycoprotein precursor (GPC), and N protein. Sequences obtained from patients are shown in Supplementary Table 2. The sequences of the YEZV strains were nearly identical to each other (>97% at the nucleotide level and >99% at the protein level). All three RNA segments of YEZV were phylogenetically grouped with Sulina virus, which was discovered in I. ricinus ticks collected in Romania7 (Fig. 3b–d). Sequence identity between YEZV and Sulina virus was 70.1% (L segment), 58.4% (M segment), and 54.1% (S segment), and 82.2% (L protein), 56.7% (GPC), and 60.2% (N protein). Based on the high sequence identity of the N protein sequence between YEZV and Sulina virus, YEZV will be assigned to the genogroup Sulina8.
Fig. 3: Genetic characterization of YEZV.
a Schematic diagram of YEZV genomic RNA segments. The YEZV genome comprises three RNA segments, L, M, and S. Each segment encodes a single open reading frame, as indicated by an arrow: L protein by L segment, GPC by M segment, and N protein by S segment. Phylogenetic relationships of (b) the N protein, (c) the GPC, and (d) the L protein coding sequences of orthonairoviruses including YEZVs, as shown surrounded by a broken line. Nucleotide sequences of orthonairovirus coding sequences available in public databases were aligned together with those of YEZV strains. The trees were constructed using the maximum-likelihood method with 1000 bootstraps. Branch support (%) higher than 70% is indicated beside each branch. Orthonairovirus genogroups are indicated to the right of the trees.
Detection of antibodies to YEZV in wild animals and YEZV RNA in ticks
To investigate the possible natural reservoir of YEZV in Hokkaido, serological screening was performed with serum samples collected from wild animals in Hokkaido between 2010 and 2020 (Table 3). Among Hokkaido shika deer (Cervus nippon yesoensis) and raccoons (Procyon lotor) in Hokkaido, 6/785 (0.8%) and 3/182 (1.6%) animals were found to be positive for YEZV antibodies, respectively, while no Hokkaido raccoon dogs (Nyctereutes procyonoides albus) or rodents (Myodes rufocanus bedfordiae and Apodemus speciosus) tested positive. The three major tick species (i.e. Haemaphysalis megaspinosa, Ixodes ovatus, and Ixodes persulcatus), collected from vegetation in Hokkaido in 2016–2020, were screened for YEZV RNA using RT-qPCR (Table 4). The highest detection rate of YEZV among these three species was 3.8% in H. megaspinosa.
Table 3 Detection of YEZV antibodies in serum samples from wild animals.Table 4 Detection of YEZV RNA in adult ticks.
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