A novel nairovirus associated with acute febrile illness in Hokkaido, Japan

Abstract

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014-2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan.

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.

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.

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.