Detection and evolutionary characterization of arboviruses in mosquitoes and biting midges on Hainan Island, China, 2019-2023.

We conducted a large-scale survey of arboviruses in mosquitoes and biting midges to assess the presence and spread of mosquito-borne pathogens currently circulating on Hainan Island, China. RT-PCR assays were used to detect the arbovirus species, distribution, and infection rates in mosquitoes and biting midges. Cell inoculation and high throughput sequencing were performed to isolate the viruses and assemble full viral genomes. Phylogenetic analysis was conducted to identify the viral genotypes and evolutionary relationships with known viruses. During 2019-2023, 32,632 mosquitoes and 21,000 biting midges were collected from 14 of 18 cities/counties on Hainan Island. Japanese encephalitis virus (JEV) was detected in Culex mosquitoes from five cities/counties, where the minimum infection rate (MIR) was 1.6 (0.6-2.6) per 1,000 females tested. Tembusu virus (TMUV) was detected in Culex mosquitoes from three cities/counties with MIR1.0 (0-2.2) per 1,000. Getah virus (GETV) was detected in Armigeres mosquitoes from Qionghai city with MIR 7.1 (0-15.2) per 1,000. Oya virus (OYAV) and Bluetongue virus (BTV) were detected in biting midges from Wanning city with MIRs of 0.4 (0-1.2) and 0.1 (0-10.2) per 1,000, respectively. Three JEV strains were isolated and clustered within the genotype I group, which is presently the dominant genotype in China. Three TMUV strains were isolated for the first time on Hainan Island that belonged to Cluster 3. Three isolated GETVs were identified as Group 3. BTV was reported for the first time on Hainan Island, and the complete genome for one BTV strain was successfully assembled, which was classified as serotype 1 based on the sequences of segment 2. These results stress the need to develop adequate surveillance plan measures to better control the public health threat of arboviruses carried by mosquitoes and biting midges in local regions.

Rational design of self-amplifying virus-like vesicles with Ebola virus glycoprotein as vaccines.

As emerging and re-emerging pathogens, filoviruses, especially Ebola virus (EBOV), pose a great threat to public health and require sustained attention and ongoing surveillance. More vaccines and antiviral drugs are imperative to be developed and stockpiled to respond to unpredictable outbreaks. Virus-like vesicles, generated by alphavirus replicons expressing homogeneous or heterogeneous glycoproteins (GPs), have demonstrated the capacity of self-propagation and shown great potential in vaccine development. Here, we describe a novel class of EBOV-like vesicles (eVLVs) incorporating both EBOV GP and VP40. The eVLVs exhibited similar antigenicity as EBOV. In murine models, eVLVs were highly attenuated and elicited robust GP-specific antibodies with neutralizing activities. Importantly, a single dose of eVLVs conferred complete protection in a surrogate EBOV lethal mouse model. Furthermore, our VLVs strategy was also successfully applied to Marburg virus (MARV), the representative member of the genus Marburgvirus. Taken together, our findings indicate the feasibility of an alphavirus-derived VLVs strategy in combating infection of filoviruses represented by EBOV and MARV, which provides further evidence of the potential of this platform for universal live-attenuated vaccine development.

A highly effective ferritin-based divalent nanoparticle vaccine shields Syrian hamsters against lethal Nipah virus.

The Nipah virus (NiV), a highly deadly bat-borne paramyxovirus, poses a substantial threat due to recurrent outbreaks in specific regions, causing severe respiratory and neurological diseases with high morbidity. Two distinct strains, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), contribute to outbreaks in different geographical areas. Currently, there are no commercially licensed vaccines or drugs available for prevention or treatment. In response to this urgent need for protection against NiV and related henipaviruses infections, we developed a novel homotypic virus-like nanoparticle (VLP) vaccine co-displaying NiV attachment glycoproteins (G) from both strains, utilizing the self-assembling properties of ferritin protein. In comparison to the NiV G subunit vaccine, our nanoparticle vaccine elicited significantly higher levels of neutralizing antibodies and provided complete protection against a lethal challenge with NiV infection in Syrian hamsters. Remarkably, the nanoparticle vaccine stimulated the production of antibodies that exhibited superior cross-reactivity to homologous or heterologous henipavirus. These findings underscore the potential utility of ferritin-based nanoparticle vaccines in providing both broad-spectrum and long-term protection against NiV and emerging zoonotic henipaviruses challenges.

A cell atlas of the adult female Aedes aegypti midgut revealed by single-cell RNA sequencing.

Aedes aegypti is a primary vector for transmitting various arboviruses, including Yellow fever, dengue and Zika virus. The mosquito midgut is the principal organ for blood meal digestion, nutrient absorption and the initial site of arbovirus infection. Although a previous study delineated midgut's transcriptome of Ae. aegypti at the single-nucleus resolution, there still lacks an established protocol for isolating and RNA sequencing of single cells of Ae. aegypti midgut, which is required for investigating arbovirus-midgut interaction at the single-cell level. Here, we established an atlas of the midgut cells for Ae. aegypti by single-cell RNA sequencing. We annotated the cell clusters including intestinal stem cells/enteroblasts (ISC/EB), cardia cells (Cardia), enterocytes (EC, EC-like), enteroendocrine cells (EE), visceral muscle (VM), fat body cells (FBC) and hemocyte cells (HC). This study will provide a foundation for further studies of arbovirus infection in mosquito midgut at the single-cell level.

Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake virus.

BACKGROUND: Mosquito-borne viruses cause various infectious diseases in humans and animals. Oya virus (OYAV) and Ebinur Lake virus (EBIV), belonging to the genus Orthobunyavirus within the family Peribunyaviridae, are recognized as neglected viruses with the potential to pose threats to animal or public health. The evaluation of vector competence is essential for predicting the arbovirus transmission risk. METHODS: To investigate the range of mosquito vectors for OYAV (strain SZC50) and EBIV (strain Cu20-XJ), the susceptibility of four mosquito species (Culex pipiens pallens, Cx. quinquefasciatus, Aedes albopictus, and Ae. aegypti) was measured through artificial oral infection. Then, mosquito species with a high infection rate (IR) were chosen to further evaluate the dissemination rate (DR), transmission rate (TR), and transmission efficiency. The viral RNA in each mosquito sample was determined by RT-qPCR. RESULTS: The results revealed that for OYAV, Cx. pipiens pallens had the highest IR (up to 40.0%) among the four species, but the DR and TR were 4.8% and 0.0%, respectively. For EBIV, Cx. pipiens pallens and Cx. quinquefasciatus had higher IR compared to Ae. albopictus (1.7%). However, the EBIV RNA and infectious virus were detected in Cx. pipiens pallens, with a TR of up to 15.4% and a transmission efficiency of 3.3%. CONCLUSIONS: The findings indicate that Cx. pipiens pallens was susceptible to OYAV but had an extremely low risk of transmitting the virus. Culex pipiens pallens and Cx. quinquefasciatus were susceptible to EBIV, and Cx. pipiens pallens had a higher transmission risk to EBIV than Cx. quinquefasciatus.

Characterization of vB_SalS_PSa2, a T5-like Demerecviridae bacteriophage, and its potential use in food matrices.

This study characterizes a newly isolated Demerecviridae phage, named vB_SalS_PSa2, belonging to the phage T5 group. The main variations between vB_SalS_PSa2 and T5 concern structural proteins related to morphology and host recognition. vB_SalS_PSa2 is infective to 19 out of the 25 tested Salmonella enterica (including the rare "Sendai" and "Equine" serotypes) and Escherichia coli isolates, most of them being multidrug resistant. vB_SalS_PSa2 displayed good thermal stability (4-60 °C) and broad pH stability (4.0-12.0). It also exhibited antibacterial activity against S. enterica sv. Paratyphi A Enb50 at 4 °C in milk during the whole tested period (5 d), and for 3-6 h at both 25 and 37 °C. Furthermore, vB_SalS_PSa2 was able to inhibit biofilm formation and to show degradation activity on mature biofilms of E. coli K12 and S. enterica sv. Paratyphi Enb50 in both LB and milk. Altogether, these results indicate that phage vB_SalS_PSa2 is a valuable candidate for controlling foodborne S. enterica and E. coli pathogens.

Favipiravir Treatment Prolongs Survival in a Lethal BALB/c Mouse Model of Ebinur Lake Virus Infection.

Orthobunyavirus is the largest and most diverse genus in the family Peribunyaviridae. Orthobunyaviruses are widely distributed globally and pose threats to human and animal health. Ebinur Lake virus (EBIV) is a newly classified Orthobunyavirus detected in China, Russia, and Kenya. This study explored the antiviral effects of two broad-spectrum antiviral drugs, favipiravir and ribavirin, in a BALB/c mouse model. Favipiravir significantly improved the clinical symptoms of infected mice, reduced viral titer and RNA copies in serum, and extended overall survival. The median survival times of mice in the vehicle- and favipiravir-treated groups were 5 and 7 days, respectively. Favipiravir significantly reduced virus titers 10- to 100-fold in sera at all three time points compared to vehicle-treated mice. And favipiravir treatment effectively reduced the virus copies by approximately 10-fold across the three time points, relative to vehicle-treated mice. The findings expand the antiviral spectrum of favipiravir for orthobunyaviruses in vivo.

Evaluation of vector susceptibility in Aedes aegypti and Culex pipiens pallens to Tibet orbivirus.

Mosquito-borne viruses cause various infectious diseases in humans and animals. Tibet orbivirus (TIBOV), a newly identified arbovirus, efficiently replicates in different types of vertebrate and mosquito cells, with its neutralizing antibodies detected in cattle and goats. However, despite being isolated from Culicoides midges, Anopheles, and Culex mosquitoes, there has been a notable absence of systematic studies on its vector competence. Thus, in this study, Aedes aegypti and Culex pipiens pallens were reared in the laboratory to measure vector susceptibility through blood-feeding infection. Furthermore, RNA sequencing was used to examine the overall alterations in the Ae. aegypti transcriptome following TIBOV infection. The results revealed that Ae. aegypti exhibited a high susceptibility to TIBOV compared to Cx. p. pallens. Effective replication of the virus in Ae. aegypti midguts occurred when the blood-feeding titer of TIBOV exceeded 105 plaque-forming units mL-1. Nevertheless, only a few TIBOV RNA-positive samples were detected in the saliva of Ae. aegypti and Cx. p. pallens, suggesting that these mosquito species may not be the primary vectors for TIBOV. Moreover, at 2 dpi of TIBOV, numerous antimicrobial peptides downstream of the Toll and Imd signaling pathways were significantly downregulated in Ae. aegypti, indicating that TIBOV suppressed mosquitos' defense to survive in the vector at an early stage. Subsequently, the stress-activated protein kinase JNK, a crucial component of the MAPK signaling pathway, exhibited significant upregulation. Certain genes were also enriched in the MAPK signaling pathway in TIBOV-infected Ae. aegypti at 7 dpi.IMPORTANCETibet orbivirus (TIBOV) is an understudied arbovirus of the genus Orbivirus. Our study is the first-ever attempt to assess the vector susceptibility of this virus in two important mosquito vectors, Aedes aegypti and Culex pipiens pallens. Additionally, we present transcriptome data detailing the interaction between TIBOV and the immune system of Ae. aegypti, which expands the knowledge about orbivirus infection and its interaction with mosquitoes.

Cryo-EM structures of Banna virus in multiple states reveal stepwise detachment of viral spikes.

Banna virus (BAV) is the prototype Seadornavirus, a class of reoviruses for which there has been little structural study. Here, we report atomic cryo-EM structures of three states of BAV virions-surrounded by 120 spikes (full virions), 60 spikes (partial virions), or no spikes (cores). BAV cores are double-layered particles similar to the cores of other non-turreted reoviruses, except for an additional protein component in the outer capsid shell, VP10. VP10 was identified to be a cementing protein that plays a pivotal role in the assembly of BAV virions by directly interacting with VP2 (inner capsid), VP8 (outer capsid), and VP4 (spike). Viral spikes (VP4/VP9 heterohexamers) are situated on top of VP10 molecules in full or partial virions. Asymmetrical electrostatic interactions between VP10 monomers and VP4 trimers are disrupted by high pH treatment, which is thus a simple way to produce BAV cores. Low pH treatment of BAV virions removes only the flexible receptor binding protein VP9 and triggers significant conformational changes in the membrane penetration protein VP4. BAV virions adopt distinct spatial organization of their surface proteins compared with other well-studied reoviruses, suggesting that BAV may have a unique mechanism of penetration of cellular endomembranes.

A Chimeric Classical Insect-Specific Flavivirus Provides Complete Protection Against West Nile Virus Lethal Challenge in Mice.

West Nile virus (WNV), an arthropod-borne flavivirus, can cause severe symptoms, including encephalitis, and death, posing a threat to public health and the economy. However, there is still no approved treatment or vaccine available for humans. Here, we developed a novel vaccine platform based on a classical insect-specific flavivirus (cISF) YN15-283-02, which was derived from Culicoides. The cISF-WNV chimera was constructed by replacing prME structural genes of the infectious YN15-283-02 cDNA clone with those of WNV and successfully rescued in Aedes albopictus cells. cISF-WNV was nonreplicable in vertebrate cells and nonpathogenic in type I interferon receptor (IFNAR)-deficient mice. A single-dose immunization of cISF-WNV elicited considerable Th1-biased antibody responses in C57BL/6 mice, which was sufficient to offer complete protection against lethal WNV challenge with no symptoms. Our studies demonstrated the potential of the insect-specific cISF-WNV as a prophylactic vaccine candidate to prevent infection with WNV.

Development and evaluation of RT-qPCR assays for two neglected orthobunyaviruses: Oya virus and Ebinur Lake virus.

OBJECTIVES: Oya virus (OYAV) and Ebinur lake virus (EBIV) belong to the genus Orthobunyavirus within the Peribunyaviridae family, and both are recognized as the novel virus with potential threat to the animal or public health. Given their potential to cause outbreaks and their detection in diverse samples across different regions, the need for a reliable and efficient molecular detection method for OYAV and EBIV becomes imperative. METHODS: The S-segment of OYAV and EBIV was used for designing specific primer and probe sets, which were employed in a real-time reverse transcription quantitative PCR (RT-qPCR) assay. The analytical performance of these assays, encompassing specificity, sensitivity, reproducibility, and fitness for purpose, was thoroughly evaluated across various sample matrices. RESULTS: The developed RT-qPCR assays were very specific to their respective targets. Both assays were highly reproducible (%CV<3) and sensitive with the 95% limit of detection (LOD) of 0.80 PFU/mL for OYAV primer probe set and 0.37 PFU/mL for EBIV primer probe set. Furthermore, the assays fitness for purpose was good as it could detect the specific viruses in virus-spiked serum samples, virus-inoculated mosquito samples, field caught mosquitoes and biting midge samples. CONCLUSIONS: Our study has successfully developed specific, sensitive, and reliable RT-qPCR assays for the detection of OYAV and EBIV. These assays hold great promise for their potential application in clinical and field samples in the future.

HostNet: improved sequence representation in deep neural networks for virus-host prediction.

BACKGROUND: The escalation of viruses over the past decade has highlighted the need to determine their respective hosts, particularly for emerging ones that pose a potential menace to the welfare of both human and animal life. Yet, the traditional means of ascertaining the host range of viruses, which involves field surveillance and laboratory experiments, is a laborious and demanding undertaking. A computational tool with the capability to reliably predict host ranges for novel viruses can provide timely responses in the prevention and control of emerging infectious diseases. The intricate nature of viral-host prediction involves issues such as data imbalance and deficiency. Therefore, developing highly accurate computational tools capable of predicting virus-host associations is a challenging and pressing demand. RESULTS: To overcome the challenges of virus-host prediction, we present HostNet, a deep learning framework that utilizes a Transformer-CNN-BiGRU architecture and two enhanced sequence representation modules. The first module, k-mer to vector, pre-trains a background vector representation of k-mers from a broad range of virus sequences to address the issue of data deficiency. The second module, an adaptive sliding window, truncates virus sequences of various lengths to create a uniform number of informative and distinct samples for each sequence to address the issue of data imbalance. We assess HostNet's performance on a benchmark dataset of "Rabies lyssavirus" and an in-house dataset of "Flavivirus". Our results show that HostNet surpasses the state-of-the-art deep learning-based method in host-prediction accuracies and F1 score. The enhanced sequence representation modules, significantly improve HostNet's training generalization, performance in challenging classes, and stability. CONCLUSION: HostNet is a promising framework for predicting virus hosts from genomic sequences, addressing challenges posed by sparse and varying-length virus sequence data. Our results demonstrate its potential as a valuable tool for virus-host prediction in various biological contexts. Virus-host prediction based on genomic sequences using deep neural networks is a promising approach to identifying their potential hosts accurately and efficiently, with significant impacts on public health, disease prevention, and vaccine development.

The Potential Role of Nitric Oxide as a Therapeutic Agent against SARS-CoV-2 Infection.

The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the greatest worldwide public health threat of this century, which may predispose multi-organ failure (especially the lung) and death despite numerous mild and moderate symptoms. Recent studies have unraveled the molecular and clinical characteristics of the infectivity, pathogenicity, and immune evasion of SARS-CoV-2 and thus improved the development of many different therapeutic strategies to combat COVID-19, including treatment and prevention. Previous studies have indicated that nitric oxide (NO) is an antimicrobial and anti-inflammatory molecule with key roles in pulmonary vascular function in the context of viral infections and other pulmonary disease states. This review summarized the recent advances of the pathogenesis of SARS-CoV-2, and accordingly elaborated on the potential application of NO in the management of patients with COVID-19 through antiviral activities and anti-inflammatory properties, which mitigate the propagation of this disease. Although there are some limits of NO in the treatment of COVID-19, it might be a worthy candidate in the multiple stages of COVID-19 prevention or therapy.

Both chimpanzee adenovirus-vectored and DNA vaccines induced long-term immunity against Nipah virus infection.

Nipah virus (NiV) is a highly lethal zoonotic paramyxovirus that poses a severe threat to humans due to its high morbidity and the lack of viable countermeasures. Vaccines are the most crucial defense against NiV infections. Here, a recombinant chimpanzee adenovirus-based vaccine (AdC68-G) and a DNA vaccine (DNA-G) were developed by expressing the codon-optimized full-length glycoprotein (G) of NiV. Strong and sustained neutralizing antibody production, accompanied by an effective T-cell response, was induced in BALB/c mice by intranasal or intramuscular administration of one or two doses of AdC68-G, as well as by priming with DNA-G and boosting with intramuscularly administered AdC68-G. Importantly, the neutralizing antibody titers were maintained for up to 68 weeks in the mice that received intramuscularly administered AdC68-G and the prime DNA-G/boost AdC68-G regimen, without a significant decline. Additionally, Syrian golden hamsters immunized with AdC68-G and DNA-G via homologous or heterologous prime/boost immunization were completely protected against a lethal NiV virus challenge, without any apparent weight loss, clinical signs, or pathological tissue damage. There was a significant reduction in but not a complete absence of the viral load and number of infectious particles in the lungs and spleen tissue following NiV challenge. These findings suggest that the AdC68-G and DNA-G vaccines against NiV infection are promising candidates for further development.

Vaccines based on the fusion protein consensus sequence protect Syrian hamsters from Nipah virus infection.

Nipah virus (NiV), a bat-borne paramyxovirus, results in neurological and respiratory diseases with high mortality in humans and animals. Developing vaccines is crucial for fighting these diseases. Previously, only a few studies focused on the fusion (F) protein alone as the immunogen. Numerous NiV strains have been identified, including 2 representative strains from Malaysia (NiV-M) and Bangladesh (NiV-B), which differ significantly from each other. In this study, an F protein sequence with the potential to prevent different NiV strain infections was designed by bioinformatics analysis after an in-depth study of NiV sequences in GenBank. Then, a chimpanzee adenoviral vector vaccine and a DNA vaccine were developed. High levels of immune responses were detected after AdC68-F, pVAX1-F, and a prime-boost strategy (pVAX1-F/AdC68-F) in mice. After high titers of humoral responses were induced, the hamsters were challenged by the lethal NiV-M and NiV-B strains separately. The vaccinated hamsters did not show any clinical signs and survived 21 days after infection with either strain of NiV, and no virus was detected in different tissues. These results indicate that the vaccines provided complete protection against representative strains of NiV infection and have the potential to be developed as a broad-spectrum vaccine for human use.

Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants.

The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike (S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the wild-type, B.1.1.7, B.1.351, B.1.617.2, and the newly emerging Omicron variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. Furthermore, our safety assessment of RQ3013 in NHP showed no observable adverse effects. These results provide strong support for the evaluation of RQ3013 in clinical trials and suggest that it may be a promising candidate for broad protection against COVID-19 and its variants.

Protection of the receptor binding domain (RBD) dimer against SARS-CoV-2 and its variants.

IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants achieved immune escape and became less virulent and easily transmissible through rapid mutation in the spike protein, thus the efficacy of vaccines on the market or in development continues to be challenged. Updating the vaccine, exploring compromise vaccination strategies, and evaluating the efficacy of candidate vaccines for the emerging variants in a timely manner are important to combat complex and volatile SARS-CoV-2. This study reports that vaccines prepared from the dimeric receptor-binding domain (RBD) recombinant protein, which can be quickly produced using a mature and stable process platform, had both good immunogenicity and protection in vivo and could completely protect rodents from lethal challenge by SARS-CoV-2 and its variants, including the emerging Omicron XBB.1.16, highlighting the value of dimeric recombinant vaccines in the post-COVID-19 era.

Colorectal Signet Ring Cell Carcinoma Presenting as Ulcerating Rectosigmoid Stricture.

Colorectal signet ring cell carcinoma is a rare type of colon cancer. Early diagnosis remains challenging because of nonspecific colonoscopy findings, such as diffuse circumferential thickening, stricture, and ulcerations, and the potential absence of typical pathological features in the initial biopsy sample. In this article, we report a 41-year-old man with ulcerating rectosigmoid stricture in the rectosigmoid colon with inconclusive histology. Subsequently, the patient developed small bowel obstruction and was diagnosed with stage 4 colorectal signet ring cell carcinoma with peritoneal carcinomatosis.

In vivo and in vitro characterization of a new Oya virus isolate from Culicoides spp. and its seroprevalence in domestic animals in Yunnan, China.

Biting midges are one of the most common hematophagous insects. They are capable of transmitting a wide range of arboviruses and have a significant impact on public health and veterinary medicine. Herein, from midge samples collected in 2013 in Yunnan, China, one sample induced a cell cytopathic effect (CPE) in BHK-21, MA104, and PK15 cell lines. Next-generation sequencing data, RACE and PCR determined the genome sequence of the sample and designated as an Oya virus (OYAV) isolate SZC50. Phylogenetic analysis of the sample revealed that it was cluster into viruses from species Orthobunyavirus catqueense. The open reading frames of S, M, and L segment of OYAV SZC50 were closest to those of OYAV SC0806. Moreover, 831 serum samples (736 pigs, 45 cattle, and 50 sheep) were gathered from 13 cities in Yunnan Province to detect neutralizing antibody of OYAV SZC50. A significant proportion of OYAV SZC50 antibody (more than 30%) was found in Yunnan pig populations, with the positive rate of OYAV SZC50 antibody in pigs from Malipo reaching 95%. To determine the pathogenicity of OYAV SZC50, we chose three animal models: specific pathogen-free Kunming mice, C57BL/6 mice lacking the interferon α/ß receptor, and chicken embryos. At 5, 6, and 7 days post-infection, all adult and suckling C57BL/6 mice, and specific pathogen-free suckling Kunming mice were dead. Our finding was expanding the knowledge about the infection and pathogenic risk of the neglected virus in the Orthobunyavirus.