Zhiming Yuan

Nipah virus (NiV) comprises two major genotypes, the Malaysia (NiV-M) and Bangladesh (NiV-B) strains. It is a zoonotic pathogen with high virulence in humans and pigs. The attachment glycoprotein (G) is considered a target for NiV vaccine development. Here, we report an Fc-G fusion protein, generated by fusing the ectodomain of NiV-B G to an immunoglobulin G1 (IgG1) fragment crystallizable (Fc), and evaluated its immunogenicity in three animal species. In mice, Fc-G was evaluated together with an unfused G comparator and induced potent G-specific antibody levels and strong neutralizing activity against NiV pseudovirus. Robust antibody responses were elicited in Fc-G-immunized Syrian hamsters and pigs; high-level neutralizing antibodies against both NiV-M and NiV-B were also detected. Neutralizing antibody titers remained high at 91 days post-immunization in hamsters. These findings demonstrate that Fc-G elicits robust, durable binding and cross-reactive neutralizing antibody responses.

Orthobunyaviruses, comprising more than 130 species, constitute a significant threat to human and animal health, yet no effective broad-spectrum treatments or vaccines are currently available. Here, we initially identified an evolutionarily constrained lysine residue, highly conserved across all 22 Orthobunyavirus serogroups, within the α30 helix of the RNA-dependent RNA polymerase (RdRp). Functional interrogation of this site by charge-reversal substitution (K-to-E) substantially impaired polymerase activity and led to marked attenuation of Ebinur Lake virus (EBIV), as evidenced by impaired replication, attenuated cytopathogenicity, and reduced virulence in mice. The similar attenuation observed in Bunyamwera virus (BUNV), the prototype virus of the genus, and Oya virus (OYAV), a member of the Simbu serogroup, demonstrates that this residue fulfills a conserved functional role. Biochemical and cellular analyses further revealed a graded, charge-dependent mutational effect, indicating that attenuation arises predominantly from disruption of the essential interaction between RdRp and the 3' terminus of viral genomic RNA (3' vRNA). Structural modeling and mutagenesis further suggest that the K-to-E substitution creates a new hydrogen bond that may also contribute to viral attenuation. Together, these findings establish the conserved lysine as a critical regulator of RdRp function and offer a rational strategy for broad-spectrum attenuation of orthobunyaviruses.

Orthobunyaviruses are segmented negative-sense RNA viruses that encompass several life-threatening human and animal pathogens. However, no licensed antivirals are currently available. The viral RNA-dependent RNA polymerase (RdRp) is a multi-domain enzyme critical for genome replication and transcription, representing a promising target for antiviral drug development. Here, we establish robust in vitro enzymatic activity assays for the Ebinur Lake virus (EBIV) polymerase and identify suramin, a century-old drug, as an inhibitor of EBIV polymerase. We further determine cryo-EM structures of the EBIV polymerase in apo, elongation, and suramin-bound states. These structures reveal conformational rearrangements of the polymerase during RNA synthesis, including conformational transitions of the prime-and-realign (PR) loop and a unique β-hairpin that bridges the zinc-binding domain (ZBD) to the RdRp core. The structural observations are correlated with in vitro enzymatic activity and cell-based minireplicon assays. The suramin-bound structure reveals two distinct inhibitory binding sites. One site sterically clashes with the vRNA promoter. The other site directly blocks RNA template strand binding, thereby inhibiting polymerase activity. These findings advance our understanding of orthobunyavirus RNA synthesis mechanisms and offer a structural framework for the rational design and optimization of antiviral drugs.

The type I interferon (IFN-I) system serves as a frontline defense against viral infection, yet how orthobunyaviruses counteract this pathway remains poorly defined. Here, we identify cytochrome P450 1A1 (CYP1A1) as a crucial host factor promoting infection by two emerging orthobunyaviruses-Oya virus (OYAV) and Ebinur Lake virus (EBIV). Transcriptomic and functional analyses demonstrate that CYP1A1 overexpression enhances viral RNA synthesis, whereas its CRISPR-Cas9-mediated knockout attenuates infection. Mechanistically, OYAV and EBIV activate the aryl hydrocarbon receptor (AhR), driving its nuclear translocation and subsequent upregulation of CYP1A1. Deficiency of CYP1A1 potentiates IFN-β production and interferon-stimulated gene (ISG) expression, while its overexpression suppressed antiviral signaling, revealing an immunomodulatory role that is distinct from its canonical metabolic function. Collectively, this work defines the AhR-CYP1A1 axis as a conserved immune-evasion module exploited by emerging orthobunyaviruses and highlights the innate immune pathway as a potential therapeutic target against these emerging threats.

Mosquitoes harbor diverse microbial communities that influence their potential to transmit pathogens. However, the ecological drivers shaping these microbiomes, particularly in under-sampled regions like Africa, remain poorly resolved. We conducted a large-scale metatranscriptomic survey of 3,940 Aedes and Culex mosquitoes from diverse ecological zones across Kenya. Our analyses revealed that viruses dominated the overall transcriptome, while bacteria exhibited the greatest taxonomic richness. Geographic location emerged as the primary driver of microbial community structure, whereas host genus identity shaped virome diversity at local or city-level scales. Culex mosquitoes harbored higher viral richness, particularly in coastal regions, while Aedes supported more diverse bacterial assemblages. Microbial co-occurrence networks exhibited distinct topologies across hosts: Culex networks featured cross-domain interactions and viral keystone taxa, whereas Aedes networks were more cohesive and robust, centered on bacterial hubs. We identified 102 distinct viruses from 24 families, including 31 putative novel RNA viruses. Segment-resolved phylogenies revealed cryptic clades within Bunyavirales, Picornavirales, and other lineages. Collectively, our findings highlight the scale-dependent influences of geography and host identity on mosquito microbiomes in East Africa and demonstrate the utility of metatranscriptomics in uncovering hidden microbial diversity and ecological interactions. These insights provide a foundation for ecologically informed arthropod vector surveillance and microbiome-based intervention strategies. IMPORTANCE: Mosquitoes are more than just flying syringes; they are complex ecosystems hosting a variety of microbes. Understanding what shapes this microbial world inside mosquitoes is key to developing new control strategies. Our study of nearly 4,000 mosquitoes from Kenya reveals that where a mosquito lives matters most for its overall microbial makeup, but its genus dictates which viruses it carries. We discovered that different mosquito types have distinct microbial social networks: one type has a fragile network centered on viruses, while the other has a resilient network built around bacteria. This means that strategies to disrupt disease transmission by targeting mosquito microbes may need to be tailored to a specific mosquito genus. Our work provides a map of these microbial ecosystems, highlighting potential new viruses and offering insights for future public health surveillance and interventions.

Effective disinfectant validation is essential for ensuring biosafety in high-containment laboratories when lethal pathogens are being handled. Micro-Chem Plus™ (MCP) is widely used in high-containment facilities for pathogen disinfection and routine decontamination. However, it induces severe cytotoxicity in cell culture, which may lead to an overestimation of its virucidal efficacy during disinfectant validation assays. To resolve this problem, we systematically evaluated the effects of three neutralisation methods (dilution, chemical neutralisation, and chromatographic separation) on MCP. The results showed that a 400-fold dilution with assay medium completely neutralised MCP, but reliable detection required high viral titers (≥6 log₁₀ TCID₅₀/mL). Chemical neutralisation using Dey–Engley broth showed inherent cytotoxicity, while chromatographic separation (MicroSpin S-400 HR/DetergentOUT™ columns) was the most effective but necessitated an additional 8-fold dilution. Validation in a BSL-4 facility with the risk group 4 (RG-4) agent Ebola virus confirmed MCP’s concentration- and time-dependent virucidal activity, achieving a ≥6 log₁₀ TCID₅₀ reduction within 1–5 min. This study establishes an optimised framework for disinfectant validation in high-containment laboratories, addressing critical gaps in current protocols.

Dengue virus (DENV) is a mosquito-borne flavivirus with the capability of replicating and spreading between vertebrate and invertebrate hosts, which poses a huge threat to global public health. Understanding the molecular mechanism of its replication and transmission in mosquitoes is crucial for controlling the dengue epidemic. The 3′ untranslated region (UTR) has been identified as a mediator for viral replication, pathogenicity, and transmission, but its effect on DENV in the mosquito host remains unknown. This study investigated the role of the dumbbell (DB) structure within the 3′UTR of DENV-2 during mosquito infection. The results showed that the absence of the DB structure reduced DENV-2 viral replication efficiency in both mosquito cell lines and Aedes mosquito adults. The DB structure is essential for DENV-2 to escape from the midgut infection and scape barriers, but not for the salivary gland infection and escape barriers. The transcriptome analysis also revealed that the absence of the DB structure increased the expression of Defensin A and Defensin C, which can inhibit viral replication in midgut and spread to hemolymph at 7–10 days post-infection. Through siRNA-mediated knockdown and overexpression experiments in C6/36 cells, we further established that Defensin C exhibits particularly inhibitory effects on the absence of DB structure DENV-2. Our findings highlight the critical role of the DB structure in DENV-2 adaptation to the mosquito host and provide insights into the molecular mechanisms underlying flavivirus transmission, contributing to the understanding of flavivirus-host interactions and assisting the development of strategies for controlling dengue fever. IMPORTANCE: Dengue virus (DENV) transmitted by Aedes mosquitoes threatens global health. This study focuses on the dumbbell (DB) structure in DENV-2’s 3′ untranslated region, which is crucial for viral replication and transmission in mosquitoes. We discovered that the absence of the DB structure reduces DENV-2’s replication efficiency and impairs its ability to evade the mosquito midgut barrier. Transcriptome data suggest that the DB structure may facilitate viral escape by modulating the expression of Defensin A and Defensin C. Functional validation in C6/36 cells demonstrates that Defensin C acts as an inhibitor, suppressing the replication of DENV-2 with the absence of DB structure and revealing a novel RNA structure-mediated immune evasion strategy. The findings of this study highlight the critical role of the DB structure in DENV-2 adaptation to mosquito hosts and provide valuable insights into the mechanisms of flavivirus transmission and potential strategies for dengue control.

Mosquitoes are the most important arthropod vectors of human diseases globally, with their midgut serving as both a digestive organ and the primary site of viral infection. Single-cell RNA sequencing (scRNA-seq) studies in insects, including mosquitoes, remain limited due to the absence of established protocols. Here, we fill this gap by developing a compatible microwell-based scRNA-seq workflow, and generated midgut cell atlas and virome for four important mosquito species: Aedes aegypti, Aedes albopictus, Culex pipiens pallens and Culex tritaeniorhynchus. Eight distinct cell types were identified, and the cell composition was inter-generic conserved in types but divergent in proportions. Culex mosquitoes exhibited higher proportions of intestinal stem cells/enteroblasts (ISC/EBs) than Aedes mosquitoes. In response to dengue virus 2 (DENV-2) infection, stress-associated genes were broadly downregulated, whereas endoenterocytes (EEs) and ISC/EBs showed marked upregulation of diverse immune related genes. Notably, we identified a novel midgut cell cluster (termed SV2/UNC-93) characterized by high expression of synaptic vesicle protein-2 and UNC-93, along with broad upregulation of apical-basal polarity-related genes linked to viral infection. The midguts of Aedes and Culex mosquitoes harbored diverse virome belonging to 5 viral families and one unclassified group. Hanko iflavirus 1 (HKIFV1) was the most common virus, distributed in all four mosquito species, while Tiger mosquito bi-segmented tombus-like virus (TMBSTLV) was the most abundant virus in Ae. albopictus and exhibited strong tropism in hemocytes. DENV-2 only detected in experimental infected Ae. aegypti, with no obvious midgut cell tropism but highest viral reads occurred in enterocytes (ECs) and EEs.Our study provides fundamental insights into the cellular composition of these four medically important mosquitoes and facilitate future research on virus-midgut interactions.

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.