A ~40-kb flavi-like virus does not encode a known error-correcting mechanism.

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

The pathogenicity and potential risk evaluation of Zika virus to cause mysterious "Disease X": A brief report.

Background and Aims: Disease X is included to represent an unknown pathogen that has the potential to spread globally and trigger significant epidemics. The World Health Organization (WHO) and the Global Alliance for Vaccine and Immunization (GAVI) recommend 10 potential disease organisms responsible for disease X. Among them, the Zika virus (ZIKV) is one of the potential organisms. The present work aimed to evaluate the current pathogenicity and potential risk of the ZIKV for public health emergencies. Methods: We performed a brief review of existing literature from available sources using Zika fever, Zika virus, and Flaviviridae as keywords and extracted relevant information for this review. Results: The primary way that the ZIKV transmits to humans is by mosquito bites (Aedes aegypti and Aedes albopictus). Typically, Aedes mosquitoes bite during the day. As an RNA virus, the ZIKV is well-known for its rapid mutation rate. ZIKV strains or variations can arise due to genomic mutations, and they occur through various processes, including replication errors, recombination, and selection pressure. Nowadays, the ZIKV has also evolved into a global pandemic because the four-amino acid sequence fits the envelope protein's 154 glycosylation motif, which may be involved in virulence. Conclusion: There is no specific medicine or vaccine available for the prevention or treatment of ZIKV infection. Therefore, personal protective measures should be the way to prevent ZIKV disease. Therefore, avoiding exposure is the best way to prevent problems from ZIKV to mosquitoes.

Spatiotemporal Evolution of the Yellow Fever Epidemic in Southeast Brazil from 2016 to 2019.

Background: Yellow fever (YF) is a zoonotic disease transmitted by mosquitoes among humans and nonhuman primates. Although urban YF is eradicated, the sylvatic YF has reemerged in some areas of Brazil in the twenty-first century. From 2016 to 2019, a sylvatic YF epidemic occurred in Southeast Brazil, where it had been eradicated in the 1940s. Methods: This study's objective was to describe the epidemic in the states of the Southeast region, based on descriptive, cluster, and mobility analyses. Results: Both the descriptive and cluster analyses showed that the YF cases spread from the state of Minas Gerais southward, causing peaks in cases during the summer months. None of the state capitals was included in the clusters, but the connectivity between the municipalities in Greater Metropolitan São Paulo highlighted potential paths of spread. Despite differences in sociodemographic profiles between the Southeast and North of Brazil (the latter region considered endemic), the epidemiological profile was similar, except for patients' occupation, which was not related to rural work in the Southeast. Conclusion: The results contributed to our understanding of the paths by which YF spread across Southeast Brazil and the epidemiological profile in an area that had gone decades without autochthonous cases.

Porcine low-density lipoprotein receptor plays an important role in classical swine fever virus infection.

Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

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.

Untranslated Regions of a Segmented Kindia Tick Virus Genome Are Highly Conserved and Contain Multiple Regulatory Elements for Viral Replication.

Novel segmented tick-borne RNA viruses belonging to the group of Jingmenviruses (JMVs) are widespread across Africa, Asia, Europe, and America. In this work, we obtained whole-genome sequences of two Kindia tick virus (KITV) isolates and performed modeling and the functional annotation of the secondary structure of 5' and 3' UTRs from JMV and KITV viruses. UTRs of various KITV segments are characterized by the following points: (1) the polyadenylated 3' UTR; (2) 5' DAR and 3' DAR motifs; (3) a highly conserved 5'-CACAG-3' pentanucleotide; (4) a binding site of the La protein; (5) multiple UAG sites providing interactions with the MSI1 protein; (6) three homologous sequences in the 5' UTR and 3' UTR of segment 2; (7) the segment 2 3' UTR of a KITV/2017/1 isolate, which comprises two consecutive 40 nucleotide repeats forming a Y-3 structure; (8) a 35-nucleotide deletion in the second repeat of the segment 2 3' UTR of KITV/2018/1 and KITV/2018/2 isolates, leading to a modification of the Y-3 structure; (9) two pseudoknots in the segment 2 3' UTR; (10) the 5' UTR and 3' UTR being represented by patterns of conserved motifs; (11) the 5'-CAAGUG-3' sequence occurring in early UTR hairpins. Thus, we identified regulatory elements in the UTRs of KITV, which are characteristic of orthoflaviviruses. This suggests that they hold functional significance for the replication of JMVs and the evolutionary similarity between orthoflaviviruses and segmented flavi-like viruses.

Extracellular Vesicles in Flaviviridae Pathogenesis: Their Roles in Viral Transmission, Immune Evasion, and Inflammation.

The members of the Flaviviridae family are becoming an emerging threat for public health, causing an increasing number of infections each year and requiring effective treatment. The consequences of these infections can be severe and include liver inflammation with subsequent carcinogenesis, endothelial damage with hemorrhage, neuroinflammation, and, in some cases, death. The mechanisms of Flaviviridae pathogenesis are being actively investigated, but there are still many gaps in their understanding. Extracellular vesicles may play important roles in these mechanisms, and, therefore, this topic deserves detailed research. Recent data have revealed the involvement of extracellular vesicles in steps of Flaviviridae pathogenesis such as transmission, immune evasion, and inflammation, which is critical for disease establishment. This review covers recent papers on the roles of extracellular vesicles in the pathogenesis of Flaviviridae and includes examples of clinical applications of the accumulated data.

Characterizing the West Nile Virus's polyprotein from nucleotide sequence to protein structure - Computational tools.

Objectives: West Nile virus (WNV) belongs to the Flaviviridae family and causes West Nile fever. The mechanism of transmission involves the culex mosquito species. Infected individuals are primarily asymptomatic, and few exhibit common symptoms. Moreover, 10 % of neuronal infection caused by this virus cause death. The proteins encoded by these genes had been uncharacterized, although understanding their function and structure is important for formulating antiviral drugs. Methods: Herein, we used in silico approaches, including various bioinformatic tools and databases, to analyse the proteins from the WNV polyprotein individually. The characterization included GC content, physicochemical properties, conserved domains, soluble and transmembrane regions, signal localization, protein disorder, and secondary structure features and their respective 3D protein structures. Results: Among 11 proteins, eight had >50 % GC content, eight proteins had basic pI values, three proteins were unstable under in vitro conditions, four were thermostable according to >100 AI values and some had negative GRAVY values in physicochemical analyses. All protein-conserved domains were shared among Flaviviridae family members. Five proteins were soluble and lacked transmembrane regions. Two proteins had signals for localization in the host endoplasmic reticulum. Non-structural (NS) 2A showed low protein disorder. The secondary structural features and tertiary structure models provide a valuable biochemical resource for designing selective substrates and synthetic inhibitors. Conclusions: WNV proteins NS2A, NS2B, PM, NS3 and NS5 can be used as drug targets for the pharmacological design of lead antiviral compounds.

Outcome and Sequelae of Infectious Encephalitis.

Acute infectious encephalitis is a widely studied clinical syndrome. Although identified almost 100 years ago, its immediate and delayed consequences are still neglected despite their high frequency and possible severity. We reviewed the available data on sequelae and persisting symptoms following infectious encephalitis with the aim of characterizing the clinical picture of these patients at months to years after hospitalization. We searched PubMed for case series involving sequelae after infectious encephalitis. We carried out a narrative review of the literature on encephalitis caused by members of the Herpesviridae family (herpes simplex virus, varicella zoster virus, and human herpesvirus-6), members of the Flaviviridae family (West Nile virus, tick-borne encephalitis virus, and Japanese encephalitis virus), alphaviruses, and Nipah virus. We retrieved 41 studies that yielded original data involving 3,072 adult patients evaluated after infectious encephalitis. At least one of the five domains of cognitive outcome, psychiatric disorders, neurological deficits, global functioning, and quality of life was investigated in the reviewed studies. Various tests were used in the 41 studies and the investigation took place at different times after hospital discharge. The results showed that most patients are discharged with impairments, with frequent deficits in cognitive function such as memory loss or attention disorders. Sequelae tend to improve within several years following flavivirus or Nipah virus infection, but long-term data are scarce for other pathogens. Further research is needed to better understand the extent of sequelae after infectious encephalitis, and to propose a standardized assessment method and assess the rehabilitation efficacy in these patients.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

Identification of a Novel Hepacivirus in Southeast Asian Shrew (Crocidura fuliginosa) from Yunnan Province, China.

The genus Hepacivirus contains single-stranded positive-sense RNA viruses belonging to the family Flaviviridae, which comprises 14 species. These 14 hepaciviruses have been found in different mammals, such as primates, dogs, bats, and rodents. To date, Hepacivirus has not been reported in the shrew genus of Crocidura. To study the prevalence and genetic evolution of Hepacivirus in small mammals in Yunnan Province, China, molecular detection of Hepacivirus in small mammals from Yunnan Province during 2016 and 2017 was performed using reverse-transcription polymerase chain reaction (RT-PCR). Our results showed that the overall infection rate of Hepacivirus in small mammals was 0.12% (2/1602), and the host animal was the Southeast Asian shrew (Crocidura fuliginosa) (12.5%, 2/16). Quantitative real-time PCR showed that Hepacivirus had the highest viral RNA copy number in the liver. Phylogenetic analysis revealed that the hepaciviruses obtained in this study does not belong to any designated species of hepaciviruses and forms an independent clade. To conclude, a novel hepacivirus was identified for the first time in C. fuliginosa specimens from Yunnan Province, China. This study expands the host range and viral diversity of hepaciviruses.

Viromes of Tabanids from Russia.

Advances in sequencing technologies and bioinformatics have greatly enhanced our knowledge of virus biodiversity. Currently, the viromes of hematophagous invertebrates, such as mosquitoes and ixodid ticks, are being actively studied. Tabanidae (Diptera) are a widespread family, with members mostly known for their persistent hematophagous behavior. They transmit viral, bacterial, and other pathogens, both biologically and mechanically. However, tabanid viromes remain severely understudied. In this study, we used high-throughput sequencing to describe the viromes of several species in the Hybomitra, Tabanus, Chrysops, and Haematopota genera, which were collected in two distant parts of Russia: the Primorye Territory and Ryazan Region. We assembled fourteen full coding genomes of novel viruses, four partial coding genomes, as well as several fragmented viral sequences, which presumably belong to another twelve new viruses. All the discovered viruses were tested for their ability to replicate in mammalian porcine embryo kidney (PEK), tick HAE/CTVM8, and mosquito C6/36 cell lines. In total, 16 viruses were detected in at least one cell culture after three passages (for PEK and C6/36) or 3 weeks of persistence in HAE/CTVM8. However, in the majority of cases, qPCR showed a decline in virus load over time.

CRISPR-Cas system to discover host-virus interactions in Flaviviridae.

The Flaviviridae virus family members cause severe human diseases and are responsible for considerable mortality and morbidity worldwide. Therefore, researchers have conducted genetic screens to enhance insight into viral dependency and develop potential anti-viral strategies to treat and prevent these infections. The host factors identified by the clustered regularly interspaced short palindromic repeats (CRISPR) system can be potential targets for drug development. Meanwhile, CRISPR technology can be efficiently used to treat viral diseases as it targets both DNA and RNA. This paper discusses the host factors related to the life cycle of viruses of this family that were recently discovered using the CRISPR system. It also explores the role of immune factors and recent advances in gene editing in treating flavivirus-related diseases. The ever-increasing advancements of this technology may promise new therapeutic approaches with unique capabilities, surpassing the traditional methods of drug production and treatment.

Dangerous Risk Group-4 (RG-4) emergent viruses.

World Health Organization (WHO) Risk Group-4 (RG-4) pathogens are among the most dangerous of the emergent and re-emergent viruses. International health agencies, working in concert, bridge the gaps in health care for populations at risk for RG-4 viral pathogen exposure. RG-4 virus research incorporates Biodefense Program and Biosafety Laboratory (BSL)-4 technologies. RG-4 viruses include Arena-viridae, Filo-viridae, Flavi-viridae, Herpes-viridae, Nairo-viridae, Paramyxo-viridae, and Pox-viridae.

Tick-Borne Encephalitis-Review of the Current Status.

The tick-borne encephalitis virus (TBEV) is the arboviral etiological agent of tick-borne encephalitis (TBE), considered to be one of the most important tick-borne viral diseases in Europe and Asia. In recent years, an increase in the incidence of TBE as well as an increasing geographical range of the disease have been noted. Despite the COVID-19 pandemic and the imposition of restrictions that it necessitated, the incidence of TBE is rising in more than half of the European countries analyzed in recent studies. The virus is transmitted between ticks, animals, and humans. It seems that ticks and small mammals play a role in maintaining TBEV in nature. The disease can also affect dogs, horses, cattle, and small ruminants. Humans are incidental hosts, infected through the bite of an infected tick or by the alimentary route, through the consumption of unpasteurized milk or milk products from TBEV-infected animals. TBEV infections in humans may be asymptomatic, but the symptoms can range from mild flu-like to severe neurological. In Europe, cases of TBE are reported every year. While there is currently no effective treatment for TBE, immunization and protection against tick bites are critical in preventing this disease.

Jingmen tick virus: an emerging arbovirus with a global threat.

Jingmen tick virus (JMTV), belonging to the Flaviviridae family, is a novel segmented RNA virus identified in 2014 in the Jingmen region of Hubei Province, China. Up to now, JMTV has been detected in a variety of countries or regions in Asia, Europe, Africa, and the Americas, involving a wide range of arthropods and mammals, and even humans. The JMTV genome is composed of four linear RNA segments, two of which are derived from flaviviruses, while the other two segments are unique to JMTV and has no matching virus. Currently, JMTV has been shown to have a pathogenic effect on humans. Humans who had been infected would develop viremia and variable degrees of clinical symptoms. However, the pathogenic mechanism of JMTV has not been elucidated yet. Therefore, it is crucial to strengthen the epidemiological surveillance and laboratory studies of JMTV.

Packaging defects in pestiviral NS4A can be compensated by mutations in NS2 and NS3.

The non-structural (NS) proteins of the Flaviviridae members play a dual role in genome replication and virion morphogenesis. For pestiviruses, like bovine viral diarrhea virus, the NS2-3 region and its processing by the NS2 autoprotease is of particular importance. While uncleaved NS2-3 in complex with NS4A is essential for virion assembly, it cannot replace free NS3/4A in the viral replicase. Furthermore, surface interactions between NS3 and the C-terminal cytosolic domain of NS4A were shown to serve as a molecular switch between RNA replication and virion morphogenesis. To further characterize the functionality of NS4A, we performed an alanine-scanning mutagenesis of two NS4A regions, a short highly conserved cytoplasmic linker downstream of the transmembrane domain and the C-terminal domain. NS4A residues critical for polyprotein processing, RNA replication, and/or virion morphogenesis were identified. Three double-alanine mutants, two in the linker region and one close to the C-terminus of NS4A, showed a selective effect on virion assembly. All three packaging defective mutants could be rescued by a selected set of two second-site mutations, located in NS2 and NS3, respectively. This phenotype was additionally confirmed by complementation studies providing the NS2-3/4A packaging molecules containing the rescue mutations in trans. This indicates that the linker region and the cytosolic C-terminal part of NS4A are critical for the formation of protein complexes required for virion morphogenesis. The ability of the identified sets of second-site mutations in NS2-3 to compensate for diverse NS4A defects highlights a surprising functional flexibility for pestiviral NS proteins. IMPORTANCE Positive-strand RNA viruses have a limited coding capacity due to their rather small genome size. To overcome this constraint, viral proteins often exhibit multiple functions that come into play at different stages during the viral replication cycle. The molecular basis for this multifunctionality is often unknown. For the bovine viral diarrhea virus, the non-structural protein (NS) 4A functions as an NS3 protease cofactor, a replicase building block, and a component in virion morphogenesis. Here, we identified the critical amino acids of its C-terminal cytosolic region involved in those processes and show that second-site mutations in NS2 and NS3 can compensate for diverse NS4A defects in virion morphogenesis. The ability to evolve alternative functional solutions by gain-of-function mutations highlights the astounding plasticity of the pestiviral system.

Dengue virus 3 genotype I (GI) lineage 1 (L1) isolates elicit differential cytopathic effect with syncytium formation in human glioblastoma cells (U251).

BACKGROUND: Dengue virus (DENV) is a Flaviviridae member classified into four antigenically distinct serotypes (DENV 1, 2, 3, and 4) and further subdivided genotypes. DENV3 is subdivided into four or five genotypes, depending on the classification adopted. Despite their high genetic proximity, as revealed by phylogenetic complete polyprotein analysis, DENV3 MG-20 and DENV3 PV_BR showed different neurovirulence in mice models. Our group identified six amino acid mutations in protein E, including the E62K and E123Q, which may affect interactions of hydrophobic clusters on domain II, thus leading to the observed differences in the studied viruses. METHODS: Human glioblastoma cells (U251) derived from a malignant glioblastoma tumor by explant technique were infected by the DENV3 GIL1 isolates DENV3 MG-20 and DENV3 PV_BR and analyzed by plaque assays and titration, optical, immunofluorescence, and transmission electronic microscopy. RESULTS: The two isolates showed different cytopathic effects (CPE) and fusogenic patterns, further confirmed by indirect immunofluorescence. Transmission electron microscopy revealed intense cytopathic effects in DENV3 MG-20 infected U251 cells, displaying endoplasmic reticulum hypertrophy and turgid vesicles with proteins and multiple viruses, distinct from DENV3 PV_BR infected cells. It is hypothesized that the different amino acids in the DENV3 MG-20 isolate are related to an increased membrane fusion ability in viral infection, thus facilitating immune system evasion and increased chances of central nervous system cell infection. CONCLUSION: These results emphasize the biological differences between the isolates, which could be a critical factor in host-virus interaction and severe dengue development. Our study presents comparative results of highly similar isolates with the potential to generate more subsidies for a deeper understanding of the DENV pathogenesis. The neurotropism of the isolate DENV3 MG-20 (belonging to the DENV3 GI L1 genotype) showing infection of nervous system cells (U251) could contribute to understanding neurological dengue disease.

[Hepatitis C Virus Nonstructural Protein 3 Increases Secretion of Interleukin-lbeta in HEK293T Cells with a Reconstructed NLRP3 Inflammasome].

The pathology of diseases arising from infections by viruses of Flaviviridae is largely determined by the development of systemic inflammation. The cytokines interleukin-1beta and interleukin-18 play a key role in triggering inflammation. Their secretion from cells, in its turn, is induced upon activation of inflammasomes. Activation of NLRP3 (NLR pyrin domain-containing family 3) inflammasomes was detected in cells infected with Flaviviridae. Some nonstructural proteins of these viruses have been shown to be able to activate or to inhibit the NLRP3 inflammasome, in particular, through interaction with its components. In this study, a functional NLRP3 inflammasome was reconstructed in human HEK293T cells and the effect of some nonstructural proteins of individual Flaviviridae viruses on it was studied. This model did not reveal any impact of nonstructural NS1 proteins of the West Nile virus, NS3 of hepatitis C virus, or NS5 of tick-borne encephalitis virus on the inflammasome components content. At the same time, in the presence of the NS1 of the West Nile virus and NS5 of the tick-borne encephalitis virus, the level of secretion of interleukin-1beta did not change, whereas in the presence of the NS3 protein of the hepatitis C virus, it increased by 1.5 times. Thus, NS3 can be considered as one of the factors of NLRP3 inflammasome activation and inflammatory pathogenesis in chronic hepatitis C virus infection.

Structures of dengue virus RNA replicase complexes.

Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.