The genus Orthoflavivirus within the family Flaviviridae includes mosquito-borne flaviviruses (MBFs) and dual-host affiliated insect-specific flaviviruses (dISFs). MBFs infect both vertebrates and mosquitoes, while dISFs are thought to infect only mosquitoes, despite their phylogenetic proximity to MBFs. Some dISFs have been shown to be capable of infecting mammalian cells in the setting of a suppressed immune response and subphysiological temperatures. Therefore, temperature sensitivity is considered one of the key factors restricting viral host tropism. To investigate the effect of temperature on dISFs propagation, we evaluated the growth of two dISFs, Psorophora flavivirus (PSFV) and Barkedji virus (BJV), in mosquito-derived C6/36 cells at 28 and 37 °C. While MBFs, including dengue virus (DENV) and Japanese encephalitis virus (JEV) could propagate efficiently at both temperatures, both PSFV and BJV failed to propagate at 37 °C. Serial passaging of PSFV with a gradual increase in temperature resulted in PSFV adaptation to 37 °C, and whole-genome sequencing revealed that the 37 °C-adapted PSFV acquired non-synonymous amino acid substitutions in the non-structural proteins, NS4B and NS5. We demonstrated that both NS4B and NS5 amino acid mutations in PFSV confer the ability to propagate at 37 °C, suggesting that the replication machinery contributes to the thermal restriction on dISFs. This study provides new insights into the temperature sensitivity of dISFs and their relationship to host tropism.

BACKGROUND:Mpox virus (MPXV) is the causative agent of mpox, a disease characterized by skin lesions, and remains a global threat since the recent 2022 outbreak. Tecovirimat is an antiviral agent that inhibits MPXV replication in vitro and in vivo by targeting the viral phospholipase VP37 protein and blocking viral envelope wrapping. Although tecovirimat has been administered to many mpox patients, the emergence of tecovirimat-resistant MPXVs carrying mutations in the VP37 protein has been reported, particularly in immunocompromised individuals. However, the impact of these respective mutations on drug susceptibility and viral fitness have not been fully characterized. METHODS:Tecovirimat was applied as a selective pressure in vitro to isolate MPXV variants carrying clinically observed VP37 substitutions. Drug susceptibility among the major VP37 mutations was compared, and the cellular fitness of the isolated viruses was evaluated. RESULTS:The VP37-N267D and -Y258C mutants showed markedly reduced susceptibility to tecovirimat compared with the VP37-I372N and -T245I (+I7L-A388S) mutants, while retaining susceptibility to an alternative anti-MPXV agent, brincidofovir. The VP37-I372N, T245I (+I7L-A388S), Y258C, and N267D mutants exhibited viral fitness comparable to that of the parental virus in A549 and Vero cells, as well as in MRC-5 and HaCaT cells. CONCLUSIONS:These findings provide insights into the phenotype of each VP37 mutation and emphasize the importance of developing antivirals with distinct mechanisms of action to effectively combat tecovirimat-resistant MPXV.

Pteropine orthoreovirus (PRV) is an emerging zoonotic virus that causes pneumonia in humans. We previously isolated the PRV strain Nachunsulwe-57 (N57) from a Zambian fruit bat and demonstrated its low virulence in laboratory mice. Here, we have attempted to identify factors responsible for differences in the virulence between strain N57 and the human-derived clinical strain Miyazaki-Bali/2007 (MB). Characterization of the virulence of recombinant monoreassortant PRVs derived from highly virulent MB and low virulent N57 strains in mice revealed that compared with wild-type (WT) MB, MB-based monoreassortants carrying the L1, S1, or S2 segment from N57 exhibited attenuated virulence. Among these, the monoreassortants carrying the S1 or S2 segment exhibited reduced viral loads and reduced cytokine gene expression levels in the lungs. Genetic mapping of virulence determinants using the reciprocal monoreassortant viruses with increased virulence demonstrated that N57-based monoreassortants carrying the S1 or S2 segment of MB exhibited enhanced virulence, resulting in lower survival rate compared with WT N57. Unlike the S1 segment, the functions of the S2 segment in pathogenesis are unclear. Thus, we further investigated the functional region of the inner-capsid σA protein encoded by the S2 segment. Notably, Ser-46 of σA was identified as a key amino acid determinant of PRV virulence and is present in strains derived from humans, monkeys, and bat flies, but not those identified from bats (their natural host). Collectively, these findings demonstrate that PRV σA is one factor that regulates virulence, and that σA Ser-46 may be related to potential interspecies transmission events from bats.

Oz virus (OZV), a member of the genus Thogotovirus in the family Orthomyxoviridae , is an emerging tick-borne virus reported in Japan. A fatal human case and seroepidemiological evidence of widespread exposure among wild animals and humans suggest its potential public health significance. However, no animal models suitable for pathogenic studies or evaluation of countermeasures are available for OZV. Here, we have established a lethal mouse model of OZV infection using cell-adapted virus and mice lacking type I interferon signaling (B6 Ifnar1 KO mice). OZV infection resulted in 100% mortality and was characterized by robust viral replication in the liver and spleen, severe hepatitis, and acute liver injury. Using this model, we also demonstrated that oral administration of T-705, an antiviral drug widely used against RNA viruses, as well as immunization with an inactivated whole virus particle vaccine, protected B6 Ifnar1 KO mice from lethal OZV infection by mitigating the acute hepatitis. The present study provides critical insights into OZV pathogenesis and establishes a practical in vivo platform for the development of countermeasures against OZV infection. ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, 25K18810, 25K09431 Japan Agency for Medical Research and Development, JP223fa627005, JP24wm0225044

Abstract West Nile virus (WNV) is the causative agent of fatal West Nile encephalitis. To date, no human vaccine against WNV has been approved. Adjuvants are important for developing effective and affordable vaccines that enhance the immunogenicity and decrease the required antigen doses. In this study, we assessed the efficacy of AddaS03, a synthetic adjuvant analogous to AS03, in a WNV subunit vaccine composed of soluble recombinant envelope protein (sEnv). Using a passive immunization mouse model, we defined the neutralizing antibody titer threshold required for protection against lethal WNV infections and applied this threshold as a surrogate marker to evaluate adjuvant efficacy. AddaS03-adjuvanted formulations elicited markedly higher neutralizing antibody titers compared to Alhydrogel adjuvant 2% (Alhydrogel), even at suboptimal antigen doses, and consistently exceeded the defined protective threshold titer. Moreover, in a sequential challenge mouse model, AddaS03-adjuvanted vaccines completely protected mice from symptomatic WNV infections, whereas Alhydrogel-adjuvanted vaccines failed to confer full protection. Collectively, these findings demonstrate that AddaS03 is a promising adjuvant for WNV subunit vaccine development and highlights the utility of a passive immunization model for defining protective antibody thresholds as a surrogate marker for vaccine evaluation.

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for Escherichia coli transformation and cloning of RABV full-genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.IMPORTANCEReverse genetics systems allow researchers to generate recombinant viruses with precise genetic modifications, advancing studies of viral replication, pathogenicity, and vaccine development. However, constructing a full-length viral genome expressing plasmids is often time-consuming and technically demanding. To bypass the cloning process, a simple, cloning-free reverse genetics platform based on the circular polymerase extension reaction (CPER) has been applied for positive-sense RNA viruses. In this study, we applied the CPER-based reverse genetics system for rabies virus (RABV), a mononegavirus, enabling rapid and flexible generation of recombinant RABVs, including mutant, reporter-expressing, and chimeric clones. Our approach greatly facilitates genetic engineering of RABV and provides a versatile framework that can be extended to other mononegaviruses, thereby accelerating both basic and applied virology research.

Alphaviruses in the family Togaviridae include zoonotic arthropod-borne viruses, including Sindbis virus (SINV), chikungunya virus, as well as insect-specific viruses such as Eilat virus (EILV). Previous investigations of alphaviruses in Zambia have identified a novel insect-specific alphavirus, Mwinilunga alphavirus in mosquitoes. Further ongoing surveillance resulted in the isolation of EILV and SINV for the first time in Zambia. Here, these alphaviruses were characterized in terms of growth kinetics in cells, and molecular phylogenetic relatedness to other alphaviruses. Zambian EILV (strain zmq19_M44) exhibited a close phylogenetic relationship with other insect-specific alphaviruses and shared a close nucleotide identity to those of EILV isolate (90.4 %) and Mwinilunga alphavirus (75.5 %). EILV zmq19_M44 attained a saturating titer in C6/36 cells at 6-8-days post infection but was unable to replicate in mammalian cells. Phylogenetic analysis revealed the Zambian SINV (strain zmq17_M115) belongs in Clade D of SINV Genotype 1 along with the Kenyan isolate BONI 584 from Central Africa. The growth of the SINV zmq17_M115 was comparable to that of the prototype SINV strain AR339 in mammalian cells but was statistically different in insect cells. Our findings will contribute to public health measures for the control of alphaviral diseases in Zambia.

The spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which dictates the viral entry pathway. SARS-CoV-2 utilizes two different pathways for cellular entry mediated by both a host type II transmembrane serine protease (TMPRSS2) and cathepsin proteases. These host proteases cleave the viral S protein and initiate membrane fusion allowing viral infection. We previously isolated a SARS-CoV-2 mutant with deletion in the furin cleavage site of the S gene (del2) and revealed differences in cell tropism between wild-type (WT) and del2 viruses. Here, we evaluated the antiviral activities of cellular protease inhibitors against SARS-CoV-2 WT and del2 viruses using several different cell lines. The TMPRSS2 inhibitor, camostat, exhibited strong antiviral activity against WT virus but not del2, while the cathepsin B/L inhibitor, K11777, exhibited potent antiviral activity against the del2 virus. We isolated K11777-escape mutants of SARS-CoV-2 and SARS-CoV and demonstrated that these mutations facilitated S protein cleavage at the S2' site mediated by cathepsin L. Finally, we demonstrated that combination treatment of K11777 and camostat potently inhibited SARS-CoV-2 WT infection in vitro and in vivo, suggesting the usefulness of combination therapeutics targeting host TMPRSS2 and cathepsin proteases against coronavirus infection. In summary, our study characterized K11777 as an inhibitor of S2' cleavage by cathepsins, highlighting the critical role of the S2' site in SARS-CoV-2 cellular entry. This research sheds light on the infection process and has implications for potential therapeutic interventions for SARS-CoV-2 infection.

Viral infections produce double-stranded RNA (dsRNA) during replication, which trigger host innate immune responses. Immunoassays using anti-dsRNA antibodies have been widely employed to detect viral dsRNA. In this study, we used a luciferase-based dsRNA biosensor which consists of protein kinase R (PKR)-derived dsRNA binding domains fused to split luciferase subunits. Here, we demonstrate the use of the dsRNA biosensor to measure viral dsRNA in RNA specimens extracted from cells infected with Japanese encephalitis virus (JEV). Moreover, the biosensor reacts to a broad-spectrum of dsRNAs from infection with representatives of various viral families including positive- and negative-sense ssRNA viruses, dsRNA viruses, and DNA viruses. We validated the specific interaction between the dsRNA biosensor and viral RNA through RNA immunoprecipitation. Additionally, we observed luminescence signals directly from lysates of JEV-infected cells after cell lysis and phase separation with Triton X-114. Finally, we used the biosensor to assess the activity of antiviral compounds. Collectively, our results demonstrate that the luciferase-based dsRNA biosensor provides a simple, homogeneous, and high-throughput platform for the quantification of viral replication, offering a promising alternative to antibody-based dsRNA detection methods. ### Competing Interest Statement The authors K.K. and A.S. are employees and shareholders of Shionogi & Co., Ltd. The remaining authors declare no competing interests.

La Crosse virus (LACV) infection, the causative agent of La Crosse encephalitis, can lead to severe neurological symptoms and sequelae, particularly in children. Despite annual reports of neurologically symptomatic cases, no effective treatment has yet been established. Bunyaviruses, including LACV, utilize a cap-snatching mechanism for transcription, with a cap-dependent endonuclease (CEN) serving as a promising target for antiviral treatment. Specifically, we now demonstrate that a CEN inhibitor, carbamoyl pyridone carboxylic acid (CAPCA)-1, exhibits potent anti-LACV activity in vitro and in vivo. CAPCA-1 exhibited 50% effective concentration values below 1 µM in neuronal and non-neuronal cells, demonstrating a higher in vitro activity than the nucleoside analogs, ribavirin and favipiravir. Multiple passages of LACV in the presence of CAPCA-1 produced numerous amino acid mutations in the CEN active site. Notably, using a lethal infection model in mice, CAPCA-1 treatment reduced viral loads in the brain and extended the survival rate of LACV-infected mice. These findings highlight the potential of CEN inhibitors as treatment options for La Crosse encephalitis.

BACKGROUND:Infections with various RNA viruses and certain DNA viruses may produce double-stranded RNA (dsRNA) during replication, which trigger host innate immune responses. Immunoassays using anti-dsRNA antibodies have been widely employed to detect viral dsRNA. In this study, we used a luciferase-based dsRNA biosensor for viral dsRNA detection, which consists of protein kinase R (PKR)-derived dsRNA binding domains fused to split luciferase subunits and is available as part of a commercial system. RESULTS:We demonstrate the use of the dsRNA biosensor to measure viral dsRNA in RNA specimens extracted from cells infected with Japanese encephalitis virus (JEV). Moreover, the biosensor reacts to a broad-spectrum of dsRNAs from infection with representatives of various viral families including positive- and negative-sense single-stranded RNA (ssRNA) viruses, dsRNA viruses, and DNA viruses. We validated the specific interaction between the dsRNA biosensor and viral RNA including subgenomic flavivirus RNA (sfRNA) through RNA immunoprecipitation. Additionally, we observed luminescence signals directly from lysates of JEV-infected cells after cell lysis and phase separation with Triton X-114. Finally, we used the biosensor to assess the activity of antiviral compounds. CONCLUSIONS:Our results demonstrate that the luciferase-based dsRNA biosensor offers a simple, homogeneous, and high-throughput platform for quantifying viral replication, presenting a promising alternative to antibody-based dsRNA detection methods.

Rotavirus C (RVC) causes acute gastroenteritis in neonatal piglets. Despite the clinical importance of RVC infection, the distribution and prevalence in pig populations in most African countries remains unknown. In this study, we identified RVC in Zambian pigs by metagenomic analysis. The full genome sequence of the RVC revealed two different VP4 sequences, implying that two different RVC strains (ZP18-77-c1 and ZP18-77-c2) were present in the same sample. Genetic analyses demonstrated that all segments of ZP18-77-c1 and ZP1877-c2 showed high nucleotide sequence identities (87.7-94.5%) to known porcine RVC strains, and ZP18-77c1 and ZP18-77-c2 strains were assigned to genotype constellations, G1-P[4]/P[14]-I13-R5-C5-M1-A7-N9-T10E5-H1. We further screened RVC genomes among pig feces collected in Zambia (n = 147) by RT-qPCR, and 78 samples (53.1%) were positive. This study demonstrated the first full genome sequence of African RVC strains with a relatively high prevalence of RVC infection in the pig populations in Zambia.

Antibody-dependent enhancement (ADE) is one of the mechanisms associated with severe clinical outcomes in infections caused by certain viruses, including dengue virus (DENV). Several ADE assay systems have been established, including flow cytometric assays using live viruses, enzyme-linked immuno-sorbent assay (ELISA) for the detection of viral NS1, and luciferase reporter gene assays. Among these, the flow cytometric assay is the most commonly used to evaluate ADE activity; however, it has limitations such as high operational costs due to fixation and immunostaining procedures, as well as a long analysis time. Fluorescent protein-expressing single-round infectious particles (SRIPs) enables label-free detection of ADE activity, but the flow cytometric procedure still requires a long analysis time. In this study, to simplify and expedite the ADE assay using enhanced green fluorescent protein (EGFP)-expressing SRIPs, we developed a plate reader-based ADE assay as an alternative to the conventional flow cytometry-based method. To evaluate effectiveness of this assay, we measured ADE activities in K562 cells induced by pan-orthoflavivirus 4G2 and pan-dengue 4E11 monoclonal antibodies (mAbs) using both flow cytometric assays using live viruses and plate-reader-based EGFP-expressing SRIPs assays. The results showed strong correlations between the two different ADE assays with R² values of 0.92 for 4G2 mAb and 0.94 for 4E11 mAb (Pearson correlation coefficients). In summary, this newly established assay offers a high-throughput and cost-effective method for comprehensive characterization of the relationship between vaccine- or infection-induced antibodies and ADE in orthoflavivirus infections.

Japanese encephalitis virus (JEV) infection causes encephalitis in humans and animals. Following intradermal infection, JEV crosses the blood-brain barrier (BBB) and reaches target cells in the brain parenchyma. However, the cellular dynamics and pathological niches involved in JEV neuroinvasion remain poorly understood. In this study, we investigated the early stages of JEV infection in the mouse brain employing a highly multiplexed spatial transcriptomics platform to map viral RNA and host gene expressions in intact brain sections at a single-cell resolution. Although JEV RNA was undetectable in brain sections at 1-day postinfection (dpi), innate immune responses were transiently activated across the brain. At 4 dpi, we detected limited viral RNA and mapped its spatial distribution, identifying glial cells surrounding microvessels as early targets of brain infection. We further characterized transcriptional changes in infected and surrounding bystander cells, revealing cell-type-specific antiviral responses. Notably, JEV neuroinvasion led to the downregulation of endothelial tight junction genes, indicative of an early event that precedes BBB impairment during subsequent disease progression. Our spatial transcriptomic analysis provides insights into cell-type- and region-specific responses to JEV infection, and highlights the early role of glial cells in shaping the immune response landscape of the brain. These findings greatly improve our understanding of JEV pathogenesis before the onset of clinical encephalitis.

Rabies is a fatal neurological disorder caused by rabies virus (RABV) infection. Approximately 60,000 patients die from rabies annually, and there are no effective treatments for this disease. Nucleoside analogs are employed as antiviral drugs based on their broad antiviral spectrum, and certain nucleoside analogs have been reported to exhibit anti-RABV activity. The nucleoside analog β-d-N4-hydroxycytidine (NHC) has antiviral effects against a range of RNA viruses. Molnupiravir (MPV), a prodrug of NHC, is clinically used as an oral antiviral drug for coronavirus infections. Despite its broad-spectrum activity, the antiviral activity of NHC against RABV remains unclear. In this study, we reveal that NHC exhibits comparable in vitro anti-RABV activity as ribavirin and favipiravir (also known as T-705) with a 90% effective concentration of 6 μM in mouse neuroblastoma cells. NHC reduced viral loads in neuronal and nonneuronal cells in a dose-dependent manner. Both laboratory and field RABVs (fixed and street strains, respectively) were susceptible to NHC. However, no increase in survival or reduction in viral titers in the brain was observed in RABV-infected mice treated prophylactically with MPV. These findings highlight the potential and challenges of NHC in the treatment of RABV infection.

Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains.

Emerging and reemerging tick-borne virus infections caused by orthonairoviruses (family Nairoviridae), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129 mice) both for a better understanding of the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel orthonairovirus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment.

Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, the treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. Here, we show that the oral antiviral ensitrelvir combined with the anti-inflammatory corticosteroid methylprednisolone has higher therapeutic effects and better outcomes in a delayed dosing model of SARS-CoV-2 infected hamsters compared to the monotherapy with ensitrelvir or methylprednisolone alone. Combination therapy with these drugs improved respiratory conditions and the development of pneumonia in hamsters even when the treatment was started after 2 days post infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulated expressions of genes involved in inflammatory response. Furthermore, we found that the combination treatment is effective in infection with both highly pathogenic delta and circulating omicron variants. Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment. Since both drugs are available as oral medications, this combination therapy could provide a clinical and potent therapeutic option for COVID-19.

Rotavirus B (RVB) causes diarrhea in humans and pigs. Although various RVB strains were identified in humans and various animals globally, little is known about the epidemiology RVB infection in Africa. In this study, we attempted to examine the prevalence of RVB infection in pig populations in Zambia. Metagenomic analyses were conducted on pig feces collected in Zambia to detect double stranded RNA viruses, including RVB. To clarify the prevalence of RVB infection in pig populations in Zambia, 147 fecal samples were screened for the RVB detection by RT-qPCR. Full genome sequence of a detected RVB was determined by Sanger sequencing and genetically analyzed. The metagenomic analyses revealed that RVB sequence reads and contigs of RVB were detected from one fecal sample collected from pigs in Zambia. RT-qPCR screening detected RVB genomes in 36.7

Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.