The step-by-step assembly mechanism of secreted flavivirus NS1 tetramer and hexamer captured at atomic resolution.

Flaviviruses encode a conserved, membrane-associated nonstructural protein 1 (NS1) with replication and immune evasion functions. The current knowledge of secreted NS1 (sNS1) oligomers is based on several low-resolution structures, thus hindering the development of drugs and vaccines against flaviviruses. Here, we revealed that recombinant sNS1 from flaviviruses exists in a dynamic equilibrium of dimer-tetramer-hexamer states. Two DENV4 hexameric NS1 structures and several tetrameric NS1 structures from multiple flaviviruses were solved at atomic resolution by cryo-EM. The stacking of the tetrameric NS1 and hexameric NS1 is facilitated by the hydrophobic ß-roll and connector domains. Additionally, a triacylglycerol molecule located within the central cavity may play a role in stabilizing the hexamer. Based on differentiated interactions between the dimeric NS1, two distinct hexamer models (head-to-head and side-to-side hexamer) and the step-by-step assembly mechanisms of NS1 dimer into hexamer were proposed. We believe that our study sheds light on the understanding of the NS1 oligomerization and contributes to NS1-based therapies.

Characterization of Two Highly Specific Monoclonal Antibodies Targeting the Glycan Loop of the Zika Virus Envelope Protein.

Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.

Circulation of West Nile Virus and Usutu Virus in Europe: Overview and Challenges.

West Nile Virus (WNV) and Usutu Virus (USUV) are both neurotropic mosquito-borne viruses belonging to the Flaviviridae family. These closely related viruses mainly follow an enzootic cycle involving mosquitoes as vectors and birds as amplifying hosts, but humans and other mammals can also be infected through mosquito bites. WNV was first identified in Uganda in 1937 and has since spread globally, notably in Europe, causing periodic outbreaks associated with severe cases of neuroinvasive diseases such as meningitis and encephalitis. USUV was initially isolated in 1959 in Swaziland and has also spread to Europe, primarily affecting birds and having a limited impact on human health. There has been a recent expansion of these viruses' geographic range in Europe, facilitated by factors such as climate change, leading to increased human exposure. While sharing similar biological traits, ecology, and epidemiology, there are significant distinctions in their pathogenicity and their impact on both human and animal health. While WNV has been more extensively studied and is a significant public health concern in many regions, USUV has recently been gaining attention due to its emergence in Europe and the diversity of its circulating lineages. Understanding the pathophysiology, ecology, and transmission dynamics of these viruses is important to the implementation of effective surveillance and control measures. This perspective provides a brief overview of the current situation of these two viruses in Europe and outlines the significant challenges that need to be addressed in the coming years.

Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses.

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

Dissemination of the Flavivirus Subgenomic Replicon Genome and Viral Proteins by Extracellular Vesicles.

Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis.

CDK5-mediated rearrangement of vimentin during Duck Tembusu virus infection inhibits viral replication.

Duck Tembusu virus (DTMUV) is a newly emerging pathogen that causes massive economic losses to the poultry industry in China and neighbouring countries. Vimentin, an intermediate filament protein, has been demonstrated to be involved in viral replication during infection. However, the specific role of vimentin in DTMUV replication has not been determined. In this study, we found that overexpression of vimentin in BHK-21 cells can inhibit DTMUV replication. Moreover, DTMUV replication was enhanced after vimentin expression was reduced in BHK-21 cells via small interfering RNA (siRNA). Further research indicated that DTMUV infection had no effect on the transcription or expression of vimentin. However, we found that DTMUV infection induced vimentin rearrangement, and the rearrangement of vimentin was subsequently confirmed to negatively modulate viral replication through the use of a vimentin network disrupting agent. Vimentin rearrangement is closely associated with its phosphorylation. Our experiments revealed that the phosphorylation of vimentin at Ser56 was promoted in the early stage of DTMUV infection. In addition, by inhibiting the phosphorylation of vimentin at Ser56 with a CDK5 inhibitor, vimentin rearrangement was suppressed, and DTMUV replication was significantly enhanced. These results indicated that DTMUV infection induced vimentin phosphorylation and rearrangement through CDK5, resulting in the inhibition of DTMUV replication. In summary, our study reveals a role for vimentin as a negative factor in the process of DTMUV replication, which helps to elucidate the function of cellular proteins in regulating DTMUV replication.

Single-Dose Immunogenic DNA Vaccines Coding for Live-Attenuated Alpha- and Flaviviruses.

Single-dose, immunogenic DNA (iDNA) vaccines coding for whole live-attenuated viruses are reviewed. This platform, sometimes called immunization DNA, has been used for vaccine development for flavi- and alphaviruses. An iDNA vaccine uses plasmid DNA to launch live-attenuated virus vaccines in vitro or in vivo. When iDNA is injected into mammalian cells in vitro or in vivo, the RNA genome of an attenuated virus is transcribed, which starts replication of a defined, live-attenuated vaccine virus in cell culture or the cells of a vaccine recipient. In the latter case, an immune response to the live virus vaccine is elicited, which protects against the pathogenic virus. Unlike other nucleic acid vaccines, such as mRNA and standard DNA vaccines, iDNA vaccines elicit protection with a single dose, thus providing major improvement to epidemic preparedness. Still, iDNA vaccines retain the advantages of other nucleic acid vaccines. In summary, the iDNA platform combines the advantages of reverse genetics and DNA immunization with the high immunogenicity of live-attenuated vaccines, resulting in enhanced safety and immunogenicity. This vaccine platform has expanded the field of genetic DNA and RNA vaccines with a novel type of immunogenic DNA vaccines that encode entire live-attenuated viruses.

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a survey of DENV susceptibility using a panel of seven field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain.

Role of long non-coding RNA DLY6E in regulating TMUV infection.

Long non-coding RNA (lncRNA) is a type of RNA with a length greater than 200 nt and lacking coding ability. In recent years, a considerable number of lncRNAs have been found to have important functions. The lncRNA plays an important role in growth and development, body metabolism, immune function, and regulation of viral replication. A lncRNA, MSTRG8505.2, was screened and named lncRNA DLY6E, which was a new duck-derived lncRNA. The lncRNADLY6E in this study has a complex secondary structure, specifically distributed in the heart, liver and other organs. The expression of lncRNA DLY6E was significantly up-regulated after TMUV infection, which was time-dependent and non-dose-dependent. Overexpression of three structural proteins and seven non-structural proteins of TMUV in DEF cells showed no significant difference in the expression of lncRNADLY6E. Meanwhile, using lipopolysaccharides (LPS) and poly (I:C) to stimulate DEF cells, the results showed that the induced expression of lncRNA DLY6E was associated with the dsRNA-related TLR3/RIG-I/MDA5 pathway rather than the LPS activated signaling pathway. To further explore the function of lncRNA DLY6E, an eukaryotic expression vector was constructed. Overexpression of lncRNA DLY6E in DEF cells can increase the replication of TMUV. After overexpression of lncRNADLY6E, the transcriptional level of its target gene LY6E was detected, and the results showed that lncRNADLY6E did not act through its target gene. Overexpression of lncRNA DLY6E significantly inhibited the mRNA levels of OAS, Mx and PKR, suggesting that lncRNA DLY6E may promote the virus by inhibiting the transcription of antiviral proteins in innate immunity. This phenomenon provides new ideas for the prevention and control of TMUV, which is worth further thinking and exploration.

A Review on The Pathogenesis of Cardiovascular Disease of Flaviviridea Viruses Infection.

Members of the Flaviviridae family, encompassing the Flavivirus and Hepacivirus genera, are implicated in a spectrum of severe human pathologies. These diseases span a diverse spectrum, including hepatitis, vascular shock syndrome, encephalitis, acute flaccid paralysis, and adverse fetal outcomes, such as congenital heart defects and increased mortality rates. Notably, infections by Flaviviridae viruses have been associated with substantial cardiovascular compromise, yet the exploration into the attendant cardiovascular sequelae and underlying mechanisms remains relatively underexplored. This review aims to explore the epidemiology of Flaviviridae virus infections and synthesize their cardiovascular morbidities. Leveraging current research trajectories and our investigative contributions, we aspire to construct a cogent theoretical framework elucidating the pathogenesis of Flaviviridae-induced cardiovascular injury and illuminate prospective therapeutic avenues.

Peptide-Based Flavivirus Biosensors: From Cell Structure to Virological and Serological Detection Methods.

In tropical and developing countries, mosquito-borne diseases by flaviviruses pose a serious threat to public health. Early detection is critical for preventing their spread, but conventional methods are time-consuming and require skilled technicians. Biosensors have been developed to address this issue, but cross-reactivity with other flaviviruses remains a challenge. Peptides are essentially biomaterials used in diagnostics that allow virological and serological techniques to identify flavivirus selectively. This biomaterial originated as a small protein consisting of two to 50 amino acid chains. They offer flexibility in chemical modification and can be easily synthesized and applied to living cells in the engineering process. Peptides could potentially be developed as robust, low-cost, sensitive, and selective receptors for detecting flaviviruses. However, modification and selection of the receptor agents are crucial to determine the effectiveness of binding between the targets and the receptors. This paper addresses two potential peptide nucleic acids (PNAs) and affinity peptides that can detect flavivirus from another target-based biosensor as well as the potential peptide behaviors of flaviviruses. The PNAs detect flaviviruses based on the nucleotide base sequence of the target's virological profile via Watson-Crick base pairing, while the affinity peptides sense the epitope or immunological profile of the targets. Recent developments in the functionalization of peptides for flavivirus biosensors are explored in this Review by division into electrochemical, optical, and other detection methods.

The infection kinetics and transmission potential of two Guaico Culex viruses in Culex quinquefasciatus mosquitoes.

Guaico Culex virus (GCXV) is a newly identified segmented Jingmenvirus from Culex spp. mosquitoes in Central and South America. The genome of GCXV is composed of four or five single-stranded positive RNA segments. However, the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown. In this study, we used reverse genetics to rescue two GCXVs (4S and 5S) that contained four and five RNA segments, respectively, in C6/36 â€‹cells. Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics, protein expression and viral titers. Importantly, GCXV RNAs were detected in the bodies, salivary glands, midguts and ovaries of Culex quinquefasciatus at 4-10 days after oral infection. In addition, two GCXVs can colonize Cx. quinquefasciatus eggs, resulting in positive rates of 15%-35% for the second gonotrophic cycle. In conclusion, our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx. quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.

NS1: a promising novel target antigen with strong immunogenicity and protective efficacy for avian flavivirus vaccine development.

Tembusu virus (TMUV), an avian pathogenic flavivirus, has emerged as a significant threat to the duck industry in Southeast Asia, causing substantial economic losses. Due to the antibody-dependent enhancement (ADE) effect of TMUV subneutralizing antibodies, there is a pressing need to further develop new TMUV vaccine target antigens that ensure both safety and efficacy. Here, the TMUV non-structural protein 1 (NS1) as a target for development of effective anti-TMUV vaccines was unveiled. The amino acid sequences of TMUV NS1 exhibit a high degree of conservation across different strains (92.63-100%). To investigate the potential of TMUV NS1 as a vaccine target, the TMUV NS1-based plasmids were constructed and identified the C-terminal 30 amino acids residues of TMUV E (EC30) as an effective signal peptide for promoting NS1 expression and secretion. Subsequently, the plasmid pVAX1-EC30-NS1 was employed to immunize ducks, resulting in specific anti-NS1 IgG responses being stimulated, while without inducing anti-TMUV neutralizing antibodies. Furthermore, the cellular immune responses triggered by the TMUV NS1 were evaluated, observing a notable increase in lymphocyte proliferation at 4 wk and 6 wk postinjection with the pVAX1-EC30-NS1. Additionally, there was a significant up-regulation of NS1-specific Il-4 and Ifnγ levels at these time points. Following this, ducks from different groups were challenged with TMUV, and remarkably, those immunized with the NS1 vaccine displayed significantly lower viral copies both at 3 d postinfection (dpi) and 7 dpi (P < 0.05) compared to ducks immunized with the control vector. Notably, the NS1 demonstrated remarkable protection against TMUV challenge without causing severe gross lesions. Collectively, these findings highlighted the impressive immunogenicity and protectivity of the TMUV NS1. Consequently, NS1 holds great promise as a novel antigen target for the development of efficient and safe TMUV vaccines.

Recruitment of the 40S ribosomal subunit by the West Nile virus 3' UTR promotes the cross-talk between the viral genomic ends for translation regulation.

Flaviviral RNA genomes are composed of discrete RNA structural units arranged in an ordered fashion and grouped into complex folded domains that regulate essential viral functions, e.g. replication and translation. This is achieved by adjusting the overall structure of the RNA genome via the establishment of inter- and intramolecular interactions. Translation regulation is likely the main process controlling flaviviral gene expression. Although the genomic 3' UTR is a key player in this regulation, little is known about the molecular mechanisms underlying this role. The present work provides evidence for the specific recruitment of the 40S ribosomal subunit by the 3' UTR of the West Nile virus RNA genome, showing that the joint action of both genomic ends contributes the positioning of the 40S subunit at the 5' end. The combination of structural mapping techniques revealed specific conformational requirements at the 3' UTR for 40S binding, involving the highly conserved SL-III, 5'DB, 3'DB and 3'SL elements, all involved in the translation regulation. These results point to the 40S subunit as a bridge to ensure cross-talk between both genomic ends during viral translation and support a link between 40S recruitment by the 3' UTR and translation control.

Temperature affects viral kinetics and vectorial capacity of Aedes aegypti mosquitoes co-infected with Mayaro and Dengue viruses.

BACKGROUND: Increasing global temperatures and unpredictable climatic extremes have contributed to the spread of vector-borne diseases. The mosquito Aedes aegypti is the main vector of multiple arboviruses that negatively impact human health, mostly in low socioeconomic areas of the world. Co-circulation and co-infection of these viruses in humans have been increasingly reported; however, how vectors contribute to this alarming trend remains unclear. METHODS: Here, we examine single and co-infection of Mayaro virus (D strain, Alphavirus) and dengue virus (serotype 2, Flavivirus) in Ae. aegypti adults and cell lines at two constant temperatures, moderate (27 °C) and hot (32 °C), to quantify vector competence and the effect of temperature on infection, dissemination and transmission, including on the degree of interaction between the two viruses. RESULTS: Both viruses were primarily affected by temperature but there was a partial interaction with co-infection. Dengue virus quickly replicates in adult mosquitoes with a tendency for higher titers in co-infected mosquitoes at both temperatures, and mosquito mortality was more severe at higher temperatures in all conditions. For dengue, and to a lesser extent Mayaro, vector competence and vectorial capacity were higher at hotter temperature in co- vs. single infections and was more evident at earlier time points (7 vs. 14 days post infection) for Mayaro. The temperature-dependent phenotype was confirmed in vitro by faster cellular infection and initial replication at higher temperatures for dengue but not for Mayaro virus. CONCLUSIONS: Our study suggests that contrasting kinetics of the two viruses could be related to their intrinsic thermal requirements, where alphaviruses thrive better at lower temperatures compared to flaviviruses. However, more studies are necessary to clarify the role of co-infection at different temperature regimes, including under more natural temperature settings.

Development of a live-attenuated chimeric vaccine against the emerging Usutu virus.

Usutu virus (USUV) is an emerging arthropod-borne flavivirus that has expanded into multiple European countries during the past several decades. USUV infection in human has been linked to severe neurological complications, and no vaccine is now available against USUV. In this work, we develop a live-attenuated chimeric USUV vaccine (termed ChinUSUV) based on the full-length infectious cDNA clone of the licensed Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. In vitro studies demonstrate that ChinUSUV replicates efficiently and maintains its genetic stability. Remarkably, ChinUSUV exhibits a significant attenuation phenotype in multiple mouse models even compared with the licensed JEV vaccine. A single immunization with ChinUSUV elicits potent IgG and neutralizing antibody responses as well as T cell response. Passive transfer of sera from ChinUSUV-immunized mice confers significant protection against lethal homologous challenge in suckling mice. Taken together, our results suggest that ChinUSUV represents a potential USUV vaccine candidate that merits further development.

The Role of Noncoding RNA in the Transmission and Pathogenicity of Flaviviruses.

Noncoding RNAs (ncRNAs) constitute a class of RNA molecules that lack protein-coding capacity. ncRNAs frequently modulate gene expression through specific interactions with target proteins or messenger RNAs, thereby playing integral roles in a wide array of cellular processes. The Flavivirus genus comprises several significant members, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV), which have caused global outbreaks, resulting in high morbidity and mortality in human populations. The life cycle of arthropod-borne flaviviruses encompasses their transmission between hematophagous insect vectors and mammalian hosts. During this process, a complex three-way interplay occurs among the pathogen, vector, and host, with ncRNAs exerting a critical regulatory influence. ncRNAs not only constitute a crucial regulatory mechanism that has emerged from the coevolution of viruses and their hosts but also hold potential as antiviral targets for controlling flavivirus epidemics. This review introduces the biogenesis of flavivirus-derived ncRNAs and summarizes the regulatory roles of ncRNAs in viral replication, vector-mediated viral transmission, antiviral innate immunity, and viral pathogenicity. A profound comprehension of the interplay between ncRNAs and flaviviruses will help formulate efficacious prophylactic and therapeutic strategies against flavivirus-related diseases.