The Role of the Stem-Loop A RNA Promoter in Flavivirus Replication

An essential challenge in the lifecycle of RNA viruses is identifying and replicating the viral genome amongst all the RNAs present in the host cell cytoplasm. Yet, how the viral polymerase selectively recognizes and copies the viral RNA genome is poorly understood. In flaviviruses, the 5′-end of the viral RNA genome contains a 70 nucleotide-long stem-loop, called stem-loop A (SLA), which functions as a promoter for genome replication. During replication, flaviviral polymerase NS5 specifically recognizes SLA to both initiate viral RNA synthesis and to methylate the 5′ guanine cap of the nascent RNA. While the sequences of this region vary between different flaviviruses, the three-way junction arrangement of secondary structures is conserved in SLA, suggesting that viruses recognize a common structural feature to replicate the viral genome rather than a particular sequence. To better understand the molecular basis of genome recognition by flaviviruses, we recently determined the crystal structures of flavivirus SLAs from dengue virus (DENV) and Zika virus (ZIKV). In this review, I will provide an overview of (1) flaviviral genome replication; (2) structures of viral SLA promoters and NS5 polymerases; and (3) and describe our current model of how NS5 polymerases specifically recognize the SLA at the 5′ terminus of the viral genome to initiate RNA synthesis at the 3′ terminus.     

Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission

The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention.     

Usutu Virus: An Emerging Flavivirus in Europe

Usutu virus (USUV) is an African mosquito-borne flavivirus belonging to the Japanese encephalitis virus serocomplex. USUV is closely related to Murray Valley encephalitis virus, Japanese encephalitis virus, and West Nile virus. USUV was discovered in South Africa in 1959. In Europe, the first true demonstration of circulation of USUV was reported in Austria in 2001 with a significant die-off of Eurasian blackbirds. In the subsequent years, USUV expanded to neighboring countries, including Italy, Germany, Spain, Hungary, Switzerland, Poland, England, Czech Republic, Greece, and Belgium, where it caused unusual mortality in birds. In 2009, the first two human cases of USUV infection in Europe have been reported in Italy, causing meningoencephalitis in immunocompromised patients. This review describes USUV in terms of its life cycle, USUV surveillance from Africa to Europe, human cases, its cellular tropism and pathogenesis, its genetic relationship with other flaviviruses, genetic diversity among USUV strains, its diagnosis, and a discussion of the potential future threat to Asian countries.     

West Nile and Usutu Viruses’ Surveillance in Birds of the Province of Ferrara,Italy, from 2015 to 2019

West Nile (WNV) and Usutu (USUV) viruses are mosquito-borne flaviviruses. Thanks to their importance as zoonotic diseases, a regional plan for surveillance of Arboviruses was implemented in Emilia-Romagna in 2009. The province of Ferrara belongs to the Emilia-Romagna region, and it is an endemic territory for these viruses, with favorable ecological conditions for abundance of mosquitoes and wild birds. From 2015 to 2019, we collected 1842 dead-found birds at a wildlife rehabilitation center, which were analysed by three different PCRs for the detection of WNV and USUV genomes. August was characterized by the highest infection rate for both viruses. Columbiformes scored the highest USUV prevalence (8%), while Galliformes and Strigiformes reported the highest prevalence for WNV (13%). Among Passeriformes (the most populated Order), Turdus merula was the most abundant species and scored the highest prevalence for both viruses. To optimize passive surveillance plans, monitoring should be focused on the summer and towards the avian species more prone to infection by both viruses.     

Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.     

First serological evidence of West Nile virus activity in horses in Serbia

West Nile virus (WNV), the most widely distributed flavivirus worldwide, has lately reemerged in Europe, causing worrisome outbreaks in humans and horses. Serological analysis by enzyme-linked immunoassay and plaque reduction neutralization test showed for the first time in Serbia that 12% of 349 horses presented specific neutralizing WNV antibodies, which in one case also cross-neutralized Usutu virus (USUV). This is the first time that anti-USUV high neutralizing antibody titers are reported in horses. All these data indicate that WNV and USUV are circulating in the region and advise on the convenience of implementing surveillance programs.     

Culex flavivirus and West Nile virus mosquito coinfection and positive ecological association in Chicago, United States

Culex flavivirus (CxFV) is an insect-specific flavivirus globally distributed in mosquitoes of the genus Culex. CxFV was positively associated with West Nile virus (WNV) infection in a case-control study of 268 mosquito pools from an endemic focus of WNV transmission in Chicago, United States. Specifically, WNV-positive Culex mosquito pools were four times more likely also to be infected with CxFV than were spatiotemporally matched WNV-negative pools. In addition, mosquito pools from residential sites characterized by dense housing and impermeable surfaces were more likely to be infected with CxFV than were pools from nearby urban green spaces. Further, 6/15 (40%) WNV-positive individual mosquitoes were also CxFV positive, demonstrating that both viruses can coinfect mosquitoes in nature. Phylogenetic analysis of CxFV from Chicago demonstrated a pattern similar to WNV, consisting of low global viral diversity and lack of geographic clustering. These results illustrate a positive ecological association between CxFV and WNV, and that coinfection of individual mosquitoes can occur naturally in areas of high flaviviral transmission. These conclusions represent a challenge to the hypothesis of super-infection exclusion in the CxFV/WNV system, whereby an established infection with one virus may interfere with secondary viral infection with a similar virus. This study suggests that infection with insect-specific flaviviruses such as CxFV may not exclude secondary infection with genetically distinct flaviviruses such as WNV, and that both viruses can naturally coinfect mosquitoes that are epidemic bridge vectors of WNV to humans.     

Make Yourself at Home: Viral Hijacking of the PI3K/Akt Signaling Pathway

As viruses do not possess genes encoding for proteins required for translation, energy metabolism or membrane biosynthesis, they are classified as obligatory intracellular parasites that depend on a host cell to replicate. This genome limitation forces them to gain control over cellular processes to ensure their successful propagation. A diverse spectrum of virally encoded proteins tackling a broad spectrum of cellular pathways during most steps of the viral life cycle ranging from the host cell entry to viral protein translation has evolved. Since the host cell PI3K/Akt signaling pathway plays a critical regulatory role in many cellular processes including RNA processing, translation, autophagy and apoptosis, many viruses, in widely varying ways, target it. This review focuses on a number of remarkable examples of viral strategies, which exploit the PI3K/Akt signaling pathway for effective viral replication.     

Influenza A virus NS1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling

Influenza A virus NS1 is a multifunctional protein, and in virus-infected cells NS1 modulates a number of host-cell processes by interacting with cellular factors. Here, we report that NS1 binds directly to p85beta, a regulatory subunit of phosphatidylinositol-3-kinase (PI3K), but not to the related p85alpha subunit. Activation of PI3K in influenza virus-infected cells depended on genome replication, and showed kinetics that correlated with NS1 expression. Additionally, it was found that expression of NS1 alone was sufficient to constitutively activate PI3K, causing the phosphorylation of a downstream mediator of PI3K signal transduction, Akt. Mutational analysis of a potential SH2-binding motif within NS1 indicated that the highly conserved tyrosine at residue 89 is important for both the interaction with p85beta, and the activation of PI3K. A mutant influenza virus (A/Udorn/72) expressing NS1 with the Y89F amino acid substitution exhibited a small-plaque phenotype, and grew more slowly in tissue culture than WT virus. These data suggest that activation of PI3K signaling in influenza A virus-infected cells is important for efficient virus replication.     

Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

Usutu virus (USUV), the etiological agent of an emerging infectious disease across Europe, is of significance because of its commonly asymptomatic prevalence, an increasing incidence of neuroinvasive human disease and the potential for rapid, geographical spread to Asia and the Americas (1–3). USUV circulates across Africa through enzootic cycles involving birds and Culex spp. mosquitoes, and avian migration facilitated multiple introductions in Europe (4, 5). USUV belongs to clade XIV in the Japanese encephalitis antigenic serocomplex of flaviviruses and is related to Murray Valley encephalitis virus (MVEV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) (nucleotide identity of 73, 71, and 68%, respectively) (6, 7). African isolates (lineage Africa 1 to 3, including the prototype SAAR-1776 and CAR_1969) tend to cause mild illness, but severe infections, such as encephalitis or acute meningoencephalitis, are associated with the European strains (Europe 1 to 5, including Italy_2009) (8–10). Yet USUV strains share 97% nucleotide and 96 to 99% amino acid sequence identity of the complete genome and polyprotein, respectively (6, 7, 11, 12). USUV can also possibly induce Guillain Barré syndrome (13). Expansion of USUV in countries with simultaneous WNV infections, cocirculation with WNV in the mosquito vector and cross-reactivity in serological testing, has undermined the timely recognition of the scale and threat posed by USUV to public health (1, 14, 15). No preventative measures or treatment avenues currently exist for treating USUV disease.Significant breakthroughs in the mechanistic understanding of pathobiology of flaviviruses come from structures representing prefusion and postfusion states of the flavivirus (Dengue virus [DENV], JEV, WNV, Zika virus [ZIKV], MVEV, yellow fever virus, and tick-borne encephalitis virus [TBEV]) envelope (E) glycoprotein (16–21). Investigations of the structures of virus/glycoprotein complexes with antibodies or Fabs complemented by serological data have uncovered the structural basis for mechanisms of virus neutralization (22, 23). However, structural studies on USUV are currently limited to the crystal structure of E (24). Here, we report two structures of the mature USUV strain SAAR-1776, solved with single-particle cryogenic electron microscopy (cryo-EM) to a resolution of 2.4 Å. We describe the E architecture, its association with the M protein, and detailed interactions between E, M, and the viral membrane. The structure reveals the location of the second N-linked glycosylation site in USUV that has implications for binding to lectins. Detailed views of three potential receptor-binding regions provide clues for the molecular basis of viral tropism. With unprecedented resolution of the structures, we identify residues that anchor the structural proteins into the viral membrane and interactions that may contribute to virion stability. Because of the high resolution of the reconstructions, asymmetry in disulfide bonds is observed in the fusion loops (FLs), suggesting the presence of more than a single population.     

Zika virus employs the host antiviral RNase L protein to support replication factory assembly

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

The flavivirus genus contains arthropod-transmitted viruses with a positive-sense single-stranded RNA (ssRNA) genome, including Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV). These viruses are transmitted by mosquitos and globally distributed, with high associated morbidity and mortality in humans (1–3). More recently, ZIKV sexual and vertical transmission has been recognized, the latter involving transplacental migration of the virus, potentially resulting in fetal microcephaly (4–9). Due to diversity in ZIKV tissue tropism, disease, and route of transmission as compared with other flaviviruses, it is possible that variances in ZIKV infection at the molecular level confer the observed shifts in clinical outcome at the organismal level. For this reason, we are interested in host machinery that supports the ZIKV replication cycle but not that of other flaviviruses, as this may improve our understanding of the molecular determinants of ZIKV pathogenesis.After entry into the host cell, the flavivirus genome, which also serves as the messenger RNA (mRNA), is directly translated at the endoplasmic reticulum (ER). Proximal to sites of translation, flaviviruses create replication factories (RFs) through extensive cytoskeletal rearrangements that generate invaginations in the folds of the ER membrane, within which new genome synthesis occurs (10–15). These RFs contain ZIKV replication complex proteins, including the NS3 protease, the replication intermediate double-stranded RNA (dsRNA), as well as template genomic ssRNA. New genome is packaged into compartments at opposite ER folds, and new virions traffic through the transgolgi network and eventually bud from the plasma membrane (16). RFs therefore enable efficient throughput of key viral processes as centers of new genome synthesis linked with viral protein translation as well as new virus assembly. In addition, RFs serve as a protective barrier to impede cytosolic innate immune sensing, as flavivirus RNA predominantly resides within RFs during the bulk of the intracellular replication cycle.Innate immune sensors within the cytoplasm of the infected cell detect viral RNA to activate antiviral responses, including the type I interferon (IFN) and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) pathways. Extensive research has demonstrated that flaviviruses, including ZIKV, have evolved strategies for counteracting the type I IFN response (17–22). Since OAS genes are IFN-stimulated genes and therefore up-regulated by type I IFN signaling, the OAS/RNase L pathway can also be potentiated by type I IFN production. However, activation of RNase L can occur in the absence of type I IFN responses when basal OAS expression is sufficient (23, 24). In either event, OAS sensors detect viral dsRNA and generate 2''-5''-oligoadenylates (2-5A). RNase L, which is constitutively expressed in an inactive form, homodimerizes upon 2-5A binding to become catalytically active (25). Active RNase L cleaves both host and viral ssRNA within the cell (Fig. 1). While there are three OAS isoforms, we have shown that the OAS3 isoform is the predominant activator of RNase L during infection with a variety of viruses including ZIKV (23, 26). Activated RNase L cleavage of host ribosomal RNA (rRNA) and mRNA as well as viral ssRNA ultimately inhibits virus infection (26–31).Open in a separate windowFig. 1.Noncanonical RNase L function promotes infectious ZIKV production. (Left) Canonical RNase L antiviral activity. Viral dsRNA is detected by OAS3, which produces the small molecule 2-5A that binds inactive RNase L, inducing its homodimerization and catalytic activation, resulting in cleavage of host and viral ssRNA, leading to inhibition of viral infection. (Right) RNase L activity during ZIKV infection. ZIKV dsRNA is recognized by OAS3, which activates RNase L resulting in ssRNA cleavage; however, ZIKV production is improved with RNase L expression.Once activated, RNase L can restrict infection of a diverse range of DNA and RNA viruses, including flaviviruses DENV and WNV (30, 32–34). Many viruses have subsequently developed mechanisms for evading RNase L antiviral effects, most of which target this pathway upstream of RNase L activation through sequestration of dsRNA, which prevents OAS activation, or by degradation of 2-5A (32, 35–40). We recently showed that ZIKV avoids antiviral effects of activated RNase L and that this evasion strategy requires assembly of RFs to protect genome from RNase L cleavage (23). Despite substantial RNase L–mediated cleavage of intracellular ZIKV genome, a portion of uncleaved genome was shielded from activated RNase L within RFs. This genome was sufficient to produce high levels of infectious virus particles, as infectious ZIKV released from wild-type (WT) cells was significantly higher than from RNase L knockout (KO) cells. Unlike ZIKV, infectious DENV production was decreased by canonical RNase L antiviral activity (23, 33). These results indicated that RNase L expression was ultimately proviral during ZIKV infection (Fig. 1). As this was the initial report of viral resistance to catalytically active RNase L during infection, we sought to isolate the differences between ZIKV RFs and those constructed by other flaviviruses, to identify factors that enable this ZIKV evasion mechanism.In this study, we focused on elucidating how RNase L increases ZIKV production. An earlier study reported that an inactive form of RNase L interacts with the actin cytoskeleton to reorganize cellular framework during viral infection (41). Since flaviviruses reorganize the cellular cytoskeletal and organellar network during infection (11), we investigated the possibility that RNase L was exploited by ZIKV to assemble protective RFs that dually serve as a barrier against host sensors in addition to providing sites of replication.     

West Nile Virus Drug Discovery

The outbreak of West Nile virus (WNV) in 1999 in the USA, and its continued spread throughout the Americas, parts of Europe, the Middle East and Africa, underscored the need for WNV antiviral development. Here, we review the current status of WNV drug discovery. A number of approaches have been used to search for inhibitors of WNV, including viral infection-based screening, enzyme-based screening, structure-based virtual screening, structure-based rationale design, and antibody-based therapy. These efforts have yielded inhibitors of viral or cellular factors that are critical for viral replication. For small molecule inhibitors, no promising preclinical candidate has been developed; most of the inhibitors could not even be advanced to the stage of hit-to-lead optimization due to their poor drug-like properties. However, several inhibitors developed for related members of the family Flaviviridae, such as dengue virus and hepatitis C virus, exhibited cross-inhibition of WNV, suggesting the possibility to re-purpose these antivirals for WNV treatment. Most promisingly, therapeutic antibodies have shown excellent efficacy in mouse model; one of such antibodies has been advanced into clinical trial. The knowledge accumulated during the past fifteen years has provided better rationale for the ongoing WNV and other flavivirus antiviral development.     

The Role of the Host Ubiquitin System in Promoting Replication of Emergent Viruses

Ubiquitination of proteins is a post-translational modification process with many different cellular functions, including protein stability, immune signaling, antiviral functions and virus replication. While ubiquitination of viral proteins can be used by the host as a defense mechanism by destroying the incoming pathogen, viruses have adapted to take advantage of this cellular process. The ubiquitin system can be hijacked by viruses to enhance various steps of the replication cycle and increase pathogenesis. Emerging viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses like Zika and dengue, as well as highly pathogenic viruses like Ebola and Nipah, have the ability to directly use the ubiquitination process to enhance their viral-replication cycle, and evade immune responses. Some of these mechanisms are conserved among different virus families, especially early during virus entry, providing an opportunity to develop broad-spectrum antivirals. Here, we discuss the mechanisms used by emergent viruses to exploit the host ubiquitin system, with the main focus on the role of ubiquitin in enhancing virus replication.     

Antibodies targeting linear determinants of the envelope protein protect mice against West Nile virus

The flavivirus envelope (E) protein mediates cellular attachment and fusion with host cell membranes and is recognized by virus-neutralizing antibodies. We raised antibodies against a broad range of epitopes by immunizing a horse with recombinant West Nile virus (WNV) E protein. To define epitopes recognized by protective antibodies, we selected, by affinity chromatography, immunoglobulins against immobilized linear peptides derived from parts of the E protein. Immunoglobulins binding 9 different peptides from domains I, II, and III of the E protein neutralized WNV in vitro. This indicates that multiple protective epitopes can be found in the E protein. Immunoglobulins recognizing 3 peptides derived from domains I and II of E protein protected mice against a lethal challenge with WNV. These immunoglobulins recognized the E proteins of related flaviviruses, demonstrating that antibodies targeting specific E protein epitopes could be developed for prevention and treatment of multiple flavivirus infections.     

Overview of flavivirus molecular biology and future vaccine development via recombinant DNA

Studies in many laboratories over the last several years have elucidated the structures of several different flavivirus genomes. Conserved features include the production of at least 10 different virus encoded proteins from a single long open reading frame by a combination of host and virus-encoded proteases. The established gene order is 5'-C- prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS 5-3' and these proteins exhibit varying degrees of homology in comparisons among flaviviruses. Conserved RNA sequences and structures have also been identified for the mosquito-borne flaviviruses but are absent in sequenced tick-bone viruses. Relevant to the development of efficacious flavivirus vaccines, studies aimed at defining the antigenic determinants necessary for eliciting protective immunity have focused primarily on the structural proteins, in particular the E protein, as well as the nonstructural secreted glycoprotein, NS1. Other work, which has led to the derivation of live-attenuated flavivirus strains, should eventually allow the genetic determinants of flavivirus attenuation and pathogenesis to be understood at the molecular level. The successful recovery infectious flaviviruses from cloned cDNA raises the possibility of manipulating these viral genomes as cDNA to construct or propagate candidate live-attenuated vaccine strains. Several applications of this technology are discussed.     

Novel flaviviruses detected in different species of mosquitoes in Spain

We report the characterization of three novel flaviviruses isolated in Spain. Marisma Mosquito virus, a novel mosquito borne virus, was isolated from Ochlerotatus caspius mosquitoes; Spanish Ochlerotatus flavivirus and Spanish Culex flavivirus, two novel insect flaviviruses, were isolated from Oc. caspius and Culex pipiens, respectively. During this investigation, we designed a sensitive RT-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus NS5 gene and could be directly used in clinical or environmental samples for flavivirus characterization and surveillance. Analysis of the sequence generated from that amplicon contains enough phylogenetic information for proper taxonomic studies. Moreover, the use of this tool allowed the detection of additional flavivirus DNA forms in Culex, Culiseta, and Ochlerotatus mosquitoes.