Orthoflavivirus
Orthoflavivirus is a genus of enveloped, positive-sense single-stranded RNA viruses in the family Flaviviridae, characterized by a genome of approximately 9.2–11.0 kb that encodes a single long open reading frame flanked by 5' and 3' non-coding regions.[1] These viruses exhibit spherical virions about 50 nm in diameter with icosahedral-like symmetry, featuring envelope proteins E and M on the surface.[1] Primarily arthropod-borne, Orthoflavivirus species cycle between hematophagous arthropod vectors—such as mosquitoes (about 50% of species) and ticks (about 28%)—and vertebrate hosts, including mammals, birds, and reptiles, with some capable of direct transmission between vertebrates without vectors.[1] Notable members include mosquito-borne pathogens like dengue virus (DENV), Zika virus (ZIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV), many of which cause significant human diseases ranging from mild febrile illness to severe neurological disorders and hemorrhagic fever.[1] Formerly classified as the genus Flavivirus, the taxon was renamed Orthoflavivirus in 2023 by the International Committee on Taxonomy of Viruses (ICTV) to distinguish it from newly established genera for insect-specific and no-known-vector flaviviruses, reflecting phylogenetic analyses that highlight distinct evolutionary branches within the family.[2] This renaming also involved adopting binomial species nomenclature, such as Orthoflavivirus denguei for the species including DENV, to improve clarity in viral taxonomy.[3] Replication occurs in the cytoplasm, where the viral RNA is translated into a polyprotein cleaved into structural (C, prM/M, E) and non-structural (NS1–NS5) proteins; the viruses enter host cells via receptor-mediated endocytosis and assemble at the endoplasmic reticulum before maturing in the Golgi apparatus.[4] With 53 recognized species, Orthoflavivirus pose major public health challenges globally, particularly in tropical and subtropical regions, due to their vector competence, human encroachment on wildlife habitats, and potential for emergence via mutation or recombination.[5]History and nomenclature
Early discoveries
The discovery of the yellow fever virus (YFV) marked a pivotal moment in understanding orthoflaviviruses, when in 1901, the U.S. Army Yellow Fever Commission, led by Walter Reed, demonstrated through human volunteer experiments that the disease was caused by a filterable agent transmitted by Aedes aegypti mosquitoes.[6] This work built on earlier hypotheses by Carlos Finlay and confirmed mosquito-borne transmission, shifting focus from contaminated water to arthropod vectors as the primary mode of spread.[7] Although actual laboratory isolation of YFV occurred later in 1927, Reed's findings established YFV as the prototype orthoflavivirus and highlighted the role of urban mosquito cycles in epidemics.[8] Subsequent isolations expanded recognition of the genus. The West Nile virus (WNV) was first isolated in 1937 from the blood of a febrile woman in the West Nile district of Uganda, during surveys of undiagnosed fevers, using intracerebral inoculation in mice; it was later recognized as a mosquito-borne pathogen causing mild febrile illness in humans and birds.[9] The Japanese encephalitis virus (JEV) was first isolated in 1935 from the brain of a fatal human case in Japan, during investigations into encephalitis outbreaks linked to rice farming seasons and Culex mosquitoes.[10] This isolation, achieved via intracerebral inoculation in mice, confirmed JEV as a neurotropic orthoflavivirus distinct from poliomyelitis.[11] Similarly, the tick-borne encephalitis virus (TBEV) was isolated in 1939 by Soviet scientists from patients and ticks in the Far Eastern taiga during an outbreak investigation, establishing it as the prototype tick-borne orthoflavivirus transmitted by Ixodes ticks.[12] Dengue virus (DENV) followed with its initial isolation in 1943 by Albert Sabin from serum of U.S. soldiers in New Guinea, using similar mouse inoculation techniques during World War II outbreaks.[13] These efforts identified DENV as another mosquito-transmitted agent, primarily by Aedes species, causing febrile illness rather than severe encephalitis.[14] Early serological studies in the 1930s and 1940s played a crucial role in linking these viruses to arthropod vectors. Complement-fixation and neutralization assays revealed cross-reactivity among YFV, JEV, DENV, WNV, and TBEV, suggesting a shared antigenic group transmitted by hematophagous insects.[15] Experimental transmissions, such as infecting monkeys with mosquito-fed blood or tick bites, further corroborated vector competence, distinguishing orthoflaviviruses from non-arthropod-borne pathogens.[16] Initial classification faced challenges due to limited morphological data, relying on serological profiles and clinical syndromes until the 1950s. Orthoflaviviruses were tentatively grouped as "Group B arboviruses" based on hemagglutination-inhibition tests showing antigenic relatedness among mosquito-borne agents like YFV, DENV, WNV, and JEV, as well as tick-borne ones like TBEV.[17] The advent of transmission electron microscopy in the mid-1950s resolved these ambiguities by visualizing the enveloped, spherical virions approximately 40-50 nm in diameter, confirming their distinct ultrastructure and separating them from non-enveloped enteroviruses.[18]Taxonomic evolution and name change
The genus Flavivirus was formally established in 1985 by the International Committee on Taxonomy of Viruses (ICTV) as part of the newly created family Flaviviridae, encompassing small enveloped viruses with positive-sense single-stranded RNA genomes primarily transmitted by arthropods and infecting vertebrates.[19] This taxonomic placement separated these agents from related groups like togaviruses, based on shared morphological, biochemical, and serological properties, with the genus initially including viruses such as yellow fever virus and dengue virus as prototypes.[20] In 2004, the Flaviviridae Study Group proposed refined species demarcation criteria for the genus Flavivirus, emphasizing genetic relatedness alongside ecological and antigenic factors; specifically, viruses sharing more than 84% amino acid identity in the envelope (E) protein were considered members of the same species, while those below this threshold, combined with differences in vector or host associations, warranted separate classification. These criteria facilitated the organization of over 50 recognized species by integrating phylogenetic data from complete genome sequences, addressing ambiguities in earlier serological-based groupings. To resolve ongoing nomenclatural ambiguity—where terms like "flavivirus" could refer interchangeably to the genus, family, or vernacular usage—the ICTV approved the renaming of the genus Flavivirus to Orthoflavivirus in April 2023, following a 2022 proposal by the Flaviviridae Study Group.[2] The prefix "ortho-" denotes "true" flaviviruses sensu stricto, distinguishing the arthropod-borne members from other Flaviviridae genera like Hepacivirus (e.g., hepatitis C virus); concurrently, the ICTV extended binomial nomenclature to all species across the family, adopting formats such as Orthoflavivirus dengue for dengue virus species.[21] Recent updates in 2024 and 2025 have incorporated protein structure predictions alongside sequence data to reclassify unassigned flaviviruses, enabling the integration of structural phylogenies for glycoprotein and polymerase domains to refine genus boundaries and assign novel isolates.[22] For instance, analyses of envelope glycoprotein structures across Flaviviridae have revealed evolutionary divergences that support the reallocation of certain insect-specific viruses previously unclassified within Orthoflavivirus. These advancements, ratified in the ICTV's 2024 taxonomy release, enhance demarcation by quantifying structural similarities, such as root-mean-square deviation in predicted folds, to complement amino acid identity thresholds.[23]Virology
Virion structure
The orthoflavivirus virion is a spherical particle about 50 nm in diameter, enveloped by a host-derived lipid bilayer that confers stability and facilitates entry into host cells.[1] The envelope surrounds a nucleocapsid core, exhibiting pseudo-T=3 icosahedral symmetry without true spikes at neutral pH, which distinguishes it from other enveloped viruses.[24] The virion incorporates three structural proteins encoded by the viral genome: the capsid protein C (11 kDa), which forms a protein-RNA complex inside the envelope; the precursor membrane protein prM (26 kDa), which is cleaved to mature membrane protein M (8 kDa) during virion release; and the envelope glycoprotein E (50 kDa), the primary surface protein responsible for receptor binding and membrane fusion. These proteins assemble such that 180 copies of E form 90 dimers arranged in a characteristic "herringbone" pattern on the envelope surface, with M proteins intercalated beneath, while multiple C proteins associate with the genomic RNA to create the internal core.[25] Cryo-electron microscopy (cryo-EM) studies have provided high-resolution models of orthoflavivirus virions, revealing the E protein dimers tiling the outer surface in an icosahedral-like lattice and the condensed RNA genome packaged within the C protein shell.[24] For instance, reconstructions of mature dengue virus at 3.5 Å resolution demonstrate how E dimers lie flat against the lipid envelope, enabling efficient particle formation without protruding domains.[25] These structures highlight the virion's compact architecture, with the nucleocapsid core occupying the central volume and interacting minimally with the envelope in mature particles. Glycosylation of the E protein, typically at one or two N-linked sites depending on the virus species, plays a critical role in host cell attachment by modulating interactions with cellular receptors and influencing viral tropism.[26] This post-translational modification enhances E protein stability and facilitates binding to attachment factors such as lectins or glycosaminoglycans on target cells, thereby promoting efficient entry.[27]Genome organization
The genome of orthoflaviviruses consists of a positive-sense, single-stranded RNA molecule approximately 11 kb in length, ranging from 9.2 to 11.0 kb across species. It is capped at the 5' end with a type 1 cap structure (m⁷GpppAmpG) that facilitates translation by host machinery, and lacks a 3' poly(A) tail, instead featuring a conserved dinucleotide sequence (CU) at the terminus, often preceded by a polyuridine (poly-U) tract within the 3' untranslated region (UTR).[1][28] The genome is flanked by 5' and 3' UTRs of variable lengths (typically 95–150 nt at the 5' end and 400–700 nt at the 3' end), which contain conserved RNA sequence motifs critical for replication and packaging, though these regions exhibit species-specific variations.[1] The viral RNA harbors a single long open reading frame (ORF) exceeding 10,000 nucleotides, which encodes a polyprotein of about 3,400 amino acids. This polyprotein undergoes co- and post-translational processing by viral (NS2B-NS3 protease) and host proteases to yield 10 mature proteins: three structural components—the capsid protein (C, ~11 kDa), precursor membrane protein (prM, ~26 kDa, which is cleaved to mature membrane protein M, ~8 kDa), and envelope glycoprotein (E, ~50 kDa)—and seven non-structural proteins (NS1, ~46 kDa; NS2A, ~22 kDa; NS2B, ~14 kDa; NS3, ~70 kDa; NS4A, ~16 kDa; NS4B, ~27 kDa; and NS5, ~103 kDa).[1][29] The structural proteins form the virion core and envelope, while the non-structural proteins support RNA replication, modulation of host responses, and virion assembly.[1] A hallmark of orthoflavivirus genome organization is the presence of conserved functional motifs within the encoded proteins, notably the GDD amino acid sequence in the C-terminal RNA-dependent RNA polymerase (RdRp) domain of NS5, which forms the catalytic active site for de novo RNA synthesis.[30] Genomes within the same orthoflavivirus species typically share 70–90% nucleotide sequence identity, underscoring their genetic stability and evolutionary cohesion, with higher identity (>90%) often observed among strains of a single virus like dengue virus serotype 1.[31][32] This level of conservation facilitates antigenic cross-reactivity and informs taxonomic demarcation, where interspecies differences exceed these thresholds.[31]Replication and life cycle
Orthoflaviviruses initiate infection through attachment of the envelope (E) glycoprotein to host cell surface receptors, such as DC-SIGN (CD209) on dendritic cells and mannose receptors on macrophages, facilitating receptor-mediated endocytosis.[33] In the acidic environment of endosomes, conformational changes in the E protein dimers trigger fusion between the viral envelope and endosomal membrane, releasing the positive-sense single-stranded RNA genome into the cytoplasm.[34] This entry mechanism is conserved across orthoflaviviruses, including dengue and Zika viruses, and exploits host glycosaminoglycans and lectins for enhanced binding efficiency.[35] Upon uncoating, the viral RNA genome serves directly as mRNA for translation on endoplasmic reticulum (ER)-associated ribosomes, producing a single polyprotein precursor that encompasses structural (C, prM/M, E) and non-structural (NS1–NS5) proteins.[34] Cleavage of this polyprotein occurs co- and post-translationally by host signal peptidase for structural proteins and the viral NS2B-NS3 protease complex for non-structural regions, enabling functional maturation of viral components.[28] The structural proteins, briefly referenced from virion composition, interact with the nascent polyprotein to initiate downstream processes.[36] RNA replication takes place within specialized cytoplasmic vesicles derived from ER membranes, induced by viral non-structural proteins including NS4A and NS4B, forming a replication organelle that shields double-stranded RNA intermediates from innate immune detection.[37] The NS5 protein, acting as the RNA-dependent RNA polymerase (RdRp), synthesizes negative-sense RNA intermediates using the positive-sense genome as template, followed by production of new positive-sense genomic RNAs for packaging and further translation cycles.[34] Host factors like lipid metabolism pathways, including glycerophospholipid remodeling, support vesicle formation and polymerase activity, ensuring efficient amplification of the genome.[38] Assembly of progeny virions occurs at the ER, where the capsid (C) protein packages positive-sense RNA into immature nucleocapsids that bud into the ER lumen, acquiring a lipid envelope with prM and E proteins.[36] During transport through the secretory pathway to the Golgi apparatus, host furin protease cleaves prM to mature M protein, inducing E protein rearrangement into the infectious, spiky conformation essential for subsequent cell entry.[35] Fully mature virions are then released via exocytosis, completing the replication cycle while evading lysosomal degradation through pH-dependent maturation.[28]Molecular mechanisms
RNA secondary structure elements
The 5' untranslated region (UTR) of orthoflaviviruses spans approximately 100 nucleotides and adopts a conserved Y-shaped secondary structure formed by two principal stem-loop elements: stem-loop A (SLA) and stem-loop B (SLB). SLA, located at the 5' terminus, consists of a stable basal stem with an apical loop that serves as a promoter for RNA-dependent RNA polymerase recruitment, while SLB features a shorter stem and contributes to the overall branched architecture. This Y-shaped motif enables long-range base-pairing interactions between complementary sequences in the 5' and 3' UTRs, promoting genome cyclization necessary for efficient replication initiation. Furthermore, the structured 5' UTR supports an internal ribosome entry site (IRES)-like mechanism, facilitating cap-independent translation by recruiting host ribosomes directly to the start codon without reliance on the 5' cap structure.[39][40] In contrast, the 3' UTR is longer, ranging from 400 to 700 nucleotides across orthoflavivirus species, and folds into a complex array of motifs that enhance viral RNA stability and synthesis. Key elements include two tandem dumbbell structures (DB1 and DB2), each comprising paired stems connected by loops, which together form a compact, H-shaped domain critical for replication enhancer activity by interacting with viral and host proteins. These dumbbells are further stabilized by adjacent pseudoknot structures, where distal loops base-pair with stems to create tertiary folds that resist degradation and promote polymerase processivity. Upstream of the terminal poly-U/UA tract, a conserved hairpin (cHP) forms a stable stem-loop that anchors long-range interactions, bridging the 3' terminus to upstream regions and maintaining overall UTR integrity during the viral life cycle.[41][39][42] Experimental characterization of these RNA secondary structures has relied on techniques such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probing, which reveals nucleotide-level reactivity to map flexible loops and rigid stems, confirming the predicted Y-shape in the 5' UTR and pseudoknotted dumbbells in the 3' UTR for viruses like dengue and Zika. Complementary site-directed mutagenesis studies have disrupted specific base pairs—for instance, altering the SLA apical loop or cHP stem—resulting in severe defects in cyclization, translation, and replication efficiency, thereby validating the functional importance of these motifs without altering primary sequence conservation.[43][44]sfRNA production and roles
Subgenomic flaviviral RNA (sfRNA) is produced during orthoflavivirus infection through the incomplete degradation of the viral genomic RNA by the host 5'-3' exoribonuclease XRN1. This process begins when XRN1, which typically degrades uncapped mRNAs in a 5'-to-3' direction, encounters structured elements within the 3' untranslated region (UTR) of the genomic RNA. Specifically, XRN1 stalls at resistant RNA motifs, such as the dumbbell (DB) structures and the conserved hairpin (cHP), preventing complete exonucleolytic processing and yielding noncoding sfRNA fragments of approximately 300-500 nucleotides in length. These structures form compact folds, including ring-like helices and pseudoknots, that physically impede XRN1's helicase activity, ensuring sfRNA accumulation in infected cells.[45][46] sfRNAs exert multiple roles in modulating host responses to favor viral replication and pathogenesis. A key function is the inhibition of type I interferon (IFN) signaling, achieved by sfRNA binding directly to the RING domain of TRIM25, an E3 ubiquitin ligase essential for activating the viral RNA sensor RIG-I. This interaction disrupts TRIM25-mediated ubiquitination of RIG-I's CARD domains, thereby blocking downstream IFN-β production and attenuating innate antiviral immunity. In orthoflaviviruses like dengue virus, this mechanism enhances viral fitness by evading early immune detection.[47] Beyond immune evasion, sfRNA suppresses cytopathicity in mammalian host cells, promoting persistent infection without inducing rapid cell death. By inhibiting global XRN1 activity through prolonged enzyme association, sfRNA stabilizes host mRNAs and reduces apoptotic pathways, such as those involving caspase-3 and PI3K-AKT signaling, allowing sustained viral propagation. In mosquito vectors, sfRNA further enhances replication by binding to host DEAD-box helicases like DDX6 homologs, which facilitates viral transmission; for instance, in Zika virus, sfRNA interaction with these factors boosts infectivity in Aedes aegypti salivary glands.[48][49] sfRNA production and functions vary across orthoflavivirus species, reflecting adaptations to diverse hosts. Dengue virus, for example, generates multiple sfRNA species—up to three or four per serotype—due to duplicated xrRNA elements and DB structures in its 3' UTR, with the largest isoform often predominant and contributing to serotype-specific immune antagonism and vector competence. In contrast, tick-borne species like tick-borne encephalitis virus typically produce a single sfRNA, underscoring evolutionary divergences in RNA architecture and host interactions.[50][51]Evolution
Origins and diversification
Sequence-based molecular clock analyses estimate the divergence of vertebrate-infecting Orthoflavivirus lineages from ancestral insect-specific flaviviruses at approximately 85,000–120,000 years ago.[52] However, endogenous viral elements integrated in host genomes indicate the broader Flaviviridae family originated over 100 million years ago, suggesting deeper evolutionary roots for the group.[53] This origin aligns with the emergence of dual-host cycles involving arthropod vectors and vertebrates, evolving from viruses restricted to insect hosts that lacked the ability to replicate in mammalian cells. Phylogenetic studies show variable evidence for co-divergence with arthropod hosts in some lineages. Diversification within the genus has primarily been driven by high mutation rates characteristic of RNA viruses, varying by clade from approximately 10^{-5} to 10^{-4} substitutions per site per year, facilitated by the error-prone nature of the viral RNA-dependent RNA polymerase lacking proofreading activity.[54] This polymerase infidelity contributes to antigenic drift, allowing gradual accumulation of mutations in surface glycoproteins that enable immune evasion and adaptation to new hosts without major structural changes. Recombination events, while possible in co-infected cells, are rare in orthoflaviviruses due to their single-stranded RNA genome and segmented-like replication strategy, occurring at frequencies low enough to have minimal impact on overall phylogeny compared to point mutations.[55] Fossil evidence of mosquitoes preserved in amber, dating back approximately 100 million years, indicates the ancient presence of potential arthropod vectors capable of supporting transmission cycles, predating the recent diversification of vertebrate-infecting orthoflaviviruses.[56] Such records suggest that the ecological split between tick and mosquito vectors occurred long before the adaptation of orthoflaviviruses to distinct transmission modes.Phylogenetic analysis
Phylogenetic analyses of orthoflaviviruses have established the genus as comprising four principal clades delineated by host specificity and transmission ecology: the tick-borne clade, the mosquito-borne clade (subdivided into Japanese encephalitis virus and dengue virus supergroups), the no-known-vector clade, and the insect-specific clade. These groupings are inferred primarily from sequences of the highly conserved non-structural protein 5 (NS5) gene, which encodes the RNA-dependent RNA polymerase and exhibits sufficient variability to resolve deep evolutionary relationships while maintaining alignability across diverse taxa.[57] Maximum-likelihood and Bayesian phylogenetic trees based on NS5 nucleotide or amino acid sequences consistently recover these clades with strong statistical support, often exceeding 95% bootstrap values or 0.99 posterior probabilities for major nodes. For instance, the divergence between the tick-borne and mosquito-borne clades is marked by substantial genetic distance, with pairwise nucleotide identities averaging approximately 56% in the NS5 region, reflecting an early split in the genus's evolutionary history. The envelope (E) protein gene, while less conserved, complements NS5-based phylogenies by informing serological cross-reactivity and antigenic evolution, as it drives host immune recognition and is thus prioritized for studies of serocomplexes within clades.[57] Recent cryo-EM structures of NS5 in complex with stem-loop A (SLA) RNA from representative mosquito-borne orthoflaviviruses, such as dengue virus, reveal conserved interaction motifs essential for replication.[58]Taxonomy and classification
Species criteria and list
The International Committee on Taxonomy of Viruses (ICTV) defines species demarcation within the genus Orthoflavivirus using a multifaceted set of criteria, including nucleotide and deduced amino acid sequence data, antigenic characteristics, geographic association, vector association, host association, disease association, and ecological characteristics.[1] These criteria integrate genetic divergence with biological and epidemiological factors to distinguish species, particularly for closely related viruses that may share phylogenetic proximity.[1] In practice, viruses are often considered distinct species if they share less than approximately 84% nucleotide identity across the complete genome, alongside other criteria such as antigenic and ecological differences, with amino acid identity in the E protein also informing distinctions.[1] As of 2025, the ICTV recognizes 53 species in the genus Orthoflavivirus, reflecting ongoing genomic surveillance and taxonomic updates.[1] All species possess a positive-sense, single-stranded RNA genome of approximately 10,700–11,300 nucleotides, encoding a single polyprotein that is cleaved into structural and non-structural proteins.[1] Host ranges generally involve vertebrate amplification (such as mammals or birds) and arthropod transmission, though some species are restricted to insects or lack known vertebrate hosts.[1] Notably, the four antigenically distinct serotypes of dengue virus (DENV-1 through DENV-4) are unified as the single species Orthoflavivirus denguei due to their overlapping geographic distributions, shared vectors, and similar disease profiles in humans, despite nucleotide identities of 65–75% between serotypes.[1] The following table summarizes the recognized species, with approximate genome sizes (typically ~11 kb) and brief host range overviews based on established associations.| Species Name | Genome Size (nt) | Host Range Summary |
|---|---|---|
| Orthoflavivirus gadgetsense | ~10,800 | Birds, ticks; occasional mammals |
| Orthoflavivirus kyasanurense | ~10,800 | Monkeys, humans, ticks |
| Orthoflavivirus langatense | ~10,800 | Rodents, ticks; mild human infections |
| Orthoflavivirus loupingi | ~10,800 | Sheep, humans, ticks |
| Orthoflavivirus omskense | ~10,800 | Rodents, humans, ticks |
| Orthoflavivirus powassanense | ~10,800 | Small mammals, humans, ticks |
| Orthoflavivirus royalense | ~10,800 | Seabirds, ticks |
| Orthoflavivirus encephalitidis | ~10,900 | Mammals, birds, humans, ticks |
| Orthoflavivirus meabanense | ~10,800 | Seabirds, ticks |
| Orthoflavivirus saumarezense | ~10,800 | Seabirds, ticks |
| Orthoflavivirus tyuleniyense | ~10,800 | Seabirds, ticks |
| Orthoflavivirus kadamense | ~10,800 | Ruminants, ticks |
| Orthoflavivirus aroaense | ~10,800 | Birds, mosquitoes |
| Orthoflavivirus denguei | ~10,700–10,800 | Humans, primates, mosquitoes |
| Orthoflavivirus cacipacoreense | ~10,900 | Rodents, mosquitoes |
| Orthoflavivirus japonicum | ~11,000 | Birds, mosquitoes |
| Orthoflavivirus koutangoense | ~10,800 | Rodents, ticks |
| Orthoflavivirus murrayense | ~10,900 | Marsupials, mosquitoes |
| Orthoflavivirus louisense | ~10,800 | Birds, mosquitoes |
| Orthoflavivirus usutuense | ~11,000 | Birds, humans, mosquitoes |
| Orthoflavivirus nilense | ~10,800 | Birds, mosquitoes |
| Orthoflavivirus yaoundeense | ~10,900 | Primates, mosquitoes |
| Orthoflavivirus kokoberaorum | ~10,900 | Marsupials, mosquitoes |
| Orthoflavivirus bagazaense | ~10,800 | Birds, humans, mosquitoes |
| Orthoflavivirus ilheusense | ~10,900 | Primates, birds, mosquitoes |
| Orthoflavivirus israelense | ~10,900 | Birds, humans, mosquitoes |
| Orthoflavivirus ntayaense | ~10,900 | Birds, mosquitoes |
| Orthoflavivirus tembusu | ~10,900 | Ducks, mosquitoes |
| Orthoflavivirus zikaense | ~10,800 | Primates, humans, mosquitoes |
| Orthoflavivirus sepikense | ~10,800 | Humans, mosquitoes |
| Orthoflavivirus wesselsbronense | ~10,900 | Ruminants, humans, mosquitoes |
| Orthoflavivirus flavi | ~10,800 | Birds, mosquitoes |
| Orthoflavivirus kedougouense | ~10,800 | Primates, mosquitoes |
| Orthoflavivirus banziense | ~10,900 | Rodents, humans, mosquitoes |
| Orthoflavivirus boubouiense | ~10,800 | Rodents, mosquitoes |
| Orthoflavivirus edgehillense | ~10,900 | Marsupials, mosquitoes |
| Orthoflavivirus jugraense | ~10,800 | Unknown vertebrates, mosquitoes |
| Orthoflavivirus saboyaense | ~10,900 | Bats, mosquitoes |
| Orthoflavivirus ugandaense | ~10,900 | Birds, mosquitoes |
| Orthoflavivirus entebbeense | ~10,800 | Bats, mosquitoes |
| Orthoflavivirus yokoseense | ~10,800 | Bats, mosquitoes |
| Orthoflavivirus apoiense | ~10,700 | Insects only (no known vertebrate host) |
| Orthoflavivirus cowboneense | ~10,700 | Insects only (no known vertebrate host) |
| Orthoflavivirus jutiapaense | ~10,700 | Insects only (no known vertebrate host) |
| Orthoflavivirus modocense | ~10,800 | Rodents (no known arthropod vector) |
| Orthoflavivirus viejaense | ~10,800 | Rodents (no known arthropod vector) |
| Orthoflavivirus perlitaense | ~10,700 | Insects only (no known vertebrate host) |
| Orthoflavivirus bukalasaense | ~10,700 | Insects only (no known vertebrate host) |
| Orthoflavivirus careyense | ~10,700 | Insects only (no known vertebrate host) |
| Orthoflavivirus dakarense | ~10,800 | Rodents (no known arthropod vector) |
| Orthoflavivirus montanaense | ~10,800 | Rodents (no known arthropod vector) |
| Orthoflavivirus phnompenhense | ~10,700 | Insects only (no known vertebrate host) |
| Orthoflavivirus bravoense | ~10,700 | Insects only (no known vertebrate host) |