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

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

Aedes albopictus is a competent vector of five arboviruses affecting human health, greater Paris, France, 2023.

Aedes albopictus collected in 2023 in the greater Paris area (Île-de-France) were experimentally able to transmit five arboviruses: West Nile virus from 3 days post-infection (dpi), chikungunya virus and Usutu virus from 7 dpi, dengue virus and Zika virus from 21 dpi. Given the growing number of imported dengue cases reported in early 2024 in France, surveillance of Ae. albopictus should be reinforced during the Paris Olympic Games in July, when many international visitors including from endemic countries are expected.

E-M349E and NS2A/2B-P1(T) are compensatory mutations of rDTMUV-NS2AB-P1P1'(AA), which regain virus proliferation by enhancing the virus package and restoring NS2A/2B cleavage.

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. The genome of DTMUV is translated into a polyprotein, which is further cleaved into several protein by viral NS2B3 protease and host proteases. Crucially, the cleavage of the NS2A/2B precursor during this process is essential for the formation of replication complexes and viral packaging. Previous research has demonstrated that alanine mutations in NS2A/2B (P1P1' (AA)) result in an attenuated strain (rDTMUV-NS2A/2B-P1P1' (AA)) by disrupting NS2A/2B cleavage. In this study, we investigate the effects of the P1P1' (AA) mutation on the viral life cycle and explore compensatory mutations in rDTMUV-NS2A/2B-P1P1' (AA). Infected ducklings exhibit similar body weight gain and viral tissue loads to DTMUV-WT. Compensatory mutations E-M349E and P1(T) emerge, restoring proliferation levels to those of rDTMUV-WT. Specifically, E-M349E enhances viral packaging, while P1(T) reinstates NS2A/2B proteolysis in vitro. Thus, our findings reveal novel compensatory sites capable of restoring the attenuated DTMUV during polyprotein cleavage and packaging.

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

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

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

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

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

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

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

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

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

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

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

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

Characterisation of ten NS2B-NS3 proteases: Paving the way for pan-flavivirus drugs.

Flaviviruses can cause severe illness in humans. Effective and safe vaccines are available for some species; however, for many flaviviruses disease prevention or specific treatments remain unavailable. The viral replication cycle depends on the proteolytic activity of the NS2B-NS3 protease, which releases functional viral proteins from a non-functional polyprotein precursor, rendering the protease a promising drug target. In this study, we characterised recombinant NS2B-NS3 proteases from ten flaviviruses including three unreported proteases from the Usutu, Kyasanur forest disease and Powassan viruses. All protease constructs comprise a covalent Gly4-Ser-Gly4 linker connecting the NS3 serine protease domain with its cofactor NS2B. We conducted a comprehensive cleavage site analysis revealing areas of high conversion. While all proteases were active in enzymatic assays, we noted a 1000-fold difference in catalytic efficiency across proteases from different flaviviruses. Two bicyclic peptide inhibitors displayed anti-pan-flaviviral protease activity with inhibition constants ranging from 10 to 1000 nM.

Indirect interaction between an endemic and an invading pathogen: A case study of Plasmodium and Usutu virus dynamics in a shared bird host population.

Infectious disease agents can influence each other's dynamics in shared host populations. We consider such influence for two mosquito-borne infections where one pathogen is endemic at the time that a second pathogen invades. We regard a setting where the vector has a bias towards biting host individuals infected with the endemic pathogen and where there is a cost to co-infected hosts. As a motivating case study, we regard Plasmodium spp., that cause avian malaria, as the endemic pathogen, and Usutu virus (USUV) as the invading pathogen. Hosts with malaria attract more mosquitoes compared to susceptible hosts, a phenomenon named vector bias. The possible trade-off between the vector-bias effect and the co-infection mortality is studied using a compartmental epidemic model. We focus first on the basic reproduction number R0 for Usutu virus invading into a malaria-endemic population, and then explore the long-term dynamics of both pathogens once Usutu virus has become established. We find that the vector bias facilitates the introduction of malaria into a susceptible population, as well as the introduction of Usutu in a malaria-endemic population. In the long term, however, both a vector bias and co-infection mortality lead to a decrease in the number of individuals infected with either pathogen, suggesting that avian malaria is unlikely to be a promoter of Usutu invasion. This proposed approach is general and allows for new insights into other negative associations between endemic and invading vector-borne pathogens.

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

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

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

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

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

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

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.

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

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

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

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

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

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

Low-temperature culture enhances production of flavivirus virus-like particles in mammalian cells.

Flavivirus virus-like particles (VLPs) exhibit a striking structural resemblance to viral particles, making them highly adaptable for various applications, including vaccines and diagnostics. Consequently, increasing VLPs production is important and can be achieved by optimizing expression plasmids and cell culture conditions. While attempting to express genotype III (GIII) Japanese encephalitis virus (JEV) VLPs containing the G104H mutation in the envelope (E) protein, we failed to generate VLPs in COS-1 cells. However, VLPs production was restored by cultivating plasmid-transfected cells at a lower temperature, specifically 28 °C. Furthermore, we observed that the enhancement in JEV VLPs production was independent of amino acid mutations in the E protein. The optimal condition for JEV VLPs production in plasmid-transfected COS-1 cells consisted of an initial culture at 37 °C for 6 h, followed by a shift to 28 °C (37/28 °C) for cultivation. Under 37/28 °C cultivation conditions, flavivirus VLPs production significantly increased in various mammalian cell lines regardless of whether its expression was transiently transfected or clonally selected cells. Remarkably, clonally selected cell lines expressing flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. Binding affinity analyses using monoclonal antibodies revealed similar binding patterns for VLPs of genotype I (GI) JEV, GIII JEV, West Nile virus (WNV), and dengue virus serotype 2 (DENV-2) produced under both 37 °C or 37/28 °C cultivation conditions. In summary, our study demonstrated that the production of flavivirus VLPs can be significantly improved under 37/28 °C cultivation conditions without affecting the conformational structure of the E protein. KEYPOINTS: • Low-temperature culture (37/28 °C) enhances production of flavivirus VLPs. • Flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. • 37/28 °C cultivation did not alter the structure of flavivirus VLPs.

Prior dengue virus serotype 3 infection modulates subsequent plasmablast responses to Zika virus infection in rhesus macaques.

Immunodominant and highly conserved flavivirus envelope proteins can trigger cross-reactive IgG antibodies against related flaviviruses, which shapes subsequent protection or disease severity. This study examined how prior dengue serotype 3 (DENV-3) infection affects subsequent Zika virus (ZIKV) plasmablast responses in rhesus macaques (n = 4). We found that prior DENV-3 infection was not associated with diminished ZIKV-neutralizing antibodies or magnitude of plasmablast activation. Rather, characterization of 363 plasmablasts and their derivative 177 monoclonal antibody supernatants from acute ZIKV infection revealed that prior DENV-3 infection was associated with a differential isotype distribution toward IgG, lower somatic hypermutation, and lesser B cell receptor variable gene diversity as compared with repeat ZIKV challenge. We did not find long-lasting DENV-3 cross-reactive IgG after a ZIKV infection but did find persistent ZIKV-binding cross-reactive IgG after a DENV-3 infection, suggesting non-reciprocal cross-reactive immunity. Infection with ZIKV after DENV-3 boosted pre-existing DENV-3-neutralizing antibodies by two- to threefold, demonstrating immune imprinting. These findings suggest that the order of DENV and ZIKV infections has impact on the quality of early B cell immunity which has implications for optimal immunization strategies. IMPORTANCE: The Zika virus epidemic of 2015-2016 in the Americas revealed that this mosquito-transmitted virus could be congenitally transmitted during pregnancy and cause birth defects in newborns. Currently, there are no interventions to mitigate this disease and Zika virus is likely to re-emerge. Understanding how protective antibody responses are generated against Zika virus can help in the development of a safe and effective vaccine. One main challenge is that Zika virus co-circulates with related viruses like dengue, such that prior exposure to one can generate cross-reactive antibodies against the other which may enhance infection and disease from the second virus. In this study, we sought to understand how prior dengue virus infection impacts subsequent immunity to Zika virus by single-cell sequencing of antibody producing cells in a second Zika virus infection. Identifying specific qualities of Zika virus immunity that are modulated by prior dengue virus immunity will enable optimal immunization strategies.