Flaviviruses (genus Orthoflavivirus) show remarkably broad cell tropism: insect-specific flaviviruses enter vertebrate cells, no-known-vector and tick-borne flaviviruses enter mosquito cells, and mosquito-borne flaviviruses enter tick cells, all with efficiency comparable to entry into natural host cells. This breadth implies that receptor engagement does not restrict flavivirus host range, yet the cellular receptors mediating this cross-phylum entry have not been identified for the mosquito-borne flaviviruses, and the host-range barriers act after entry. The problem asks for the molecular basis of this entry-independent host tropism: (i) identification of the receptor(s) — in vertebrate and arthropod cells — that mediate broad flavivirus entry, and a test of the conjecture that they are highly conserved molecules; (ii) molecular definition of the post-entry blocks that abort productive infection, exemplified by Yokose virus entering and replicating in mosquito cells without producing infectious particles; and (iii) the viral determinants (untranslated regions, nonstructural proteins) and evolutionary pathways by which single-host flaviviruses could acquire dual-host tropism. Recent work has identified receptors for some flaviviruses (LRP8 for tick-borne encephalitis and yellow fever viruses), but the mosquito-borne core and the genus-wide entry mechanism remain unresolved.

Background

The genus Flavivirus (renamed Orthoflavivirus in 2023) comprises enveloped, positive-sense RNA viruses divided by host range into mosquito-borne flaviviruses (MBFs, including dengue virus DENV, Zika virus ZIKV, West Nile virus WNV, Japanese encephalitis virus JEV, and yellow fever virus YFV), tick-borne flaviviruses (TBFs, including tick-borne encephalitis virus TBEV), insect-specific flaviviruses (ISFs, which infect only mosquitoes), and no-known-vector flaviviruses (NKVs, maintained in vertebrates such as bats). The virion surface bears one glycoprotein, E, which drives attachment and, after endocytosis, membrane fusion in the acidified endosome; the precursor membrane protein prM is cleaved during virion maturation, and correct prM processing and E stability are required for assembly of infectious particles. Flaviviruses enter cells by receptor-dependent endocytosis, and several candidate host molecules have been proposed: phosphatidylserine (PS) receptors such as TIM-1 and AXL, which capture the viral membrane in a process called apoptotic mimicry because the virion exposes PS like an apoptotic body; C-type lectins such as DC-SIGN; and numerous attachment factors (HSP70/90, laminin receptor, integrins, glycosaminoglycans). However, genetic removal of any single candidate fails to abolish infection, so no consensus internalization receptor exists for the major mosquito-borne flaviviruses.

The problem arises from an experimental observation that made this picture puzzling. Zhang et al. (2023) used the reporter viral particle (RVP) system — single-round particles in which a West Nile virus replicon carrying a GFP reporter is pseudotyped with the C-prM-E structural proteins of other flaviviruses — to show that ISFs enter vertebrate cells, MBFs enter tick cells, and NKVs and TBFs enter mosquito cells as efficiently as ZIKV enters its natural host cells. Removing all glycosylation from the E protein did not impair entry of MBFs or TBFs, ruling out universal glycan-binding proteins as the explanation. Using a reverse-genetics infectious clone, the same study found that Yokose virus (YOKV), a bat-associated NKV, enters and replicates its RNA in mosquito C6/36 cells but fails to produce infectious particles. The authors concluded that flavivirus host tropism is largely entry-independent: the cross-species barrier lies after entry, and the receptors that permit such broad entry — presumably highly conserved molecules, since the host range crosses animal phyla — remain unidentified. Earlier work from the same group (Zhang et al. 2022) had shown with chimeric viruses between a dual-host affiliated ISF (dISF — an insect-specific flavivirus phylogenetically nested within the mosquito-borne clade) and mosquito-borne flaviviruses (dISF–MBF chimeras) that the dISF Donggang virus enters vertebrate cells but cannot initiate replication there, and that swapping its untranslated regions (UTRs, the noncoding RNA termini that recruit host factors for genome replication) with those of ZIKV rescues replication — establishing UTR–host-factor interactions as one post-entry host-range determinant. Subsequent studies have begun to define receptors for parts of the genus: genome-wide screens identified LRP8 (an apolipoprotein E and reelin receptor of the LDL receptor family) as a receptor for TBEV and for YFV, and transferrin receptor 1 as a JEV entry factor; notably, apart from YFV (which is itself mosquito-borne), LRP8 was not used by the mosquito-transmitted orthoflaviviruses tested. For DENV and ZIKV, new candidates (LRP1, the mosquito midgut protein EPrRec, and entry via GAS6 — a soluble phosphatidylserine-binding protein that bridges virions to the AXL receptor) continue to appear without settling the question. What remains is the genus-wide picture Zhang et al. (2023) pointed to: which host molecules mediate broad flavivirus entry across hosts, what molecular step aborts infection of the wrong host after entry, and what it would take — in viral genetic terms — for a single-host flavivirus to evolve a dual-host transmission cycle.

Problem Statement

Given that entry is demonstrably not the host-range barrier for flaviviruses — every tested flavivirus group efficiently enters non-host cells — the problem is to establish the molecular basis of this entry-independent host tropism. Parts (1) and (2) jointly answer the central question posed by Zhang et al. (2023): what mediates flavivirus entry across hosts, and what blocks productive infection in the wrong host? Part (3) extends this question along the direction Zhang et al. (2023) itself proposes: what it would take, in viral genetic terms, for a single-host flavivirus to acquire dual-host tropism.

(1) Receptor identification. Identify the cellular receptor(s) that mediate entry of flaviviruses into both vertebrate and arthropod cells, in particular for the mosquito-borne flaviviruses (DENV, ZIKV, WNV, JEV) and for the observed efficient entry of ISFs into vertebrate cells and of NKVs and TBFs into mosquito cells; and determine whether a single highly conserved molecule, or a set of redundant factors (e.g., phosphatidylserine-receptor apoptotic mimicry plus protein receptors), accounts for the genus-wide breadth of entry.

(2) Post-entry blocks. Define the molecular step at which productive infection aborts in non-host cells, exemplified by Yokose virus in mosquito cells, where entry and RNA replication occur but no infectious particles are produced — for example, whether the block lies in E protein stability, prM processing, virion assembly, or another step — and which host or viral components (e.g., 3' non-coding region elements) determine it?

(3) Dual-host adaptation. Determine which viral genetic changes confer the ability to replicate productively in both a vertebrate and an arthropod host — the roles of UTR–host-factor interactions and nonstructural protein functions (e.g., NS4B/NS5 temperature sensitivity) — and thereby which evolutionary pathways could allow an insect-specific or vertebrate-specific flavivirus to acquire dual-host tropism, using engineered infectious clones across the genus.

Parts (1) and (2) are facets of a single objective — the molecular basis of flavivirus host tropism at and after the entry step — as framed by the entry-independent host-tropism conclusion of Zhang et al. (2023); part (3) is the extension proposed by Zhang et al. (2023), which anticipated that engineering infectious clones of ISFs and NKVs, together with other MBFs, will uncover the mechanism underlying the dual-host adaptation of MBFs. The answer is complete when all three parts are answered: the receptor(s) mediating broad entry are identified and validated in vertebrate and arthropod cells — either as one and the same highly conserved receptor used in both phyla, or as a defined set of factors that collectively accounts for entry across them; the post-entry restrictions that abort productive infection in non-host cells and their viral determinants are mechanistically defined, with Yokose virus in mosquito cells as the required worked exemplar; and the viral determinants of dual-host replication and the evolutionary pathway by which a single-host flavivirus could acquire dual-host tropism are identified. As a sufficiency boundary for parts (1) and (2), a general mechanism demonstrated on at least one representative of each virus group suffices, and an exhaustive definition for every cross-host combination is not required.

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