Cutaneous events determining transmission of mosquito-borne orthoflaviviruses remain largely uncharacterized. Here, we report single-cell RNA-sequencing of skin from immunocompetent mice exposed to West Nile virus-infected mosquitoes, capturing early response at a critical transmission bottleneck. Skin-resident fibroblasts, keratinocytes, and myeloid cells were exposed to infectious saliva. At the bite site, neutrophils and mast cells diminished, while lymphoid cells augmented. Cell-cell communication analysis showed that structural skin cells activate immune signaling targeting myeloid populations, which signal to lymphoid cells. Transcriptional profiling revealed cell subtype-specific responses integrating immune activation, skin repair and metabolic remodeling. Using in vivo gene silencing, we demonstrated that fibroblast-expressed LRRC15 (leucine rich repeat-containing 15) functions as a restriction factor, limiting viral replication in skin, viral dissemination to draining lymph nodes, and disease severity. Collectively, our transcriptomic-resolution and functional analyses provide cellular and molecular understanding of bite-initiated arboviral transmission, establishing skin-resident fibroblasts as frontline defender cells. HIGHLIGHTS ### Competing Interest Statement The authors have declared no competing interest. Fondation pour la Recherche Médicale, SPF202110013925, ARF202309017577 Agence Nationale de la Recherche, ANR-20-CE15-0006, ANR-21-CE15-0041 EU Horizon, 101137006 PHC Siam, 49487UJ CIRM, EDUC4-12822

Ticks are obligate blood-feeding ectoparasites that harbor a wide diversity of microorganisms. Rhipicephalus sanguineus, the brown dog tick, is globally distributed and poses significant veterinary and public health concerns due to its close association with companion animals and its occasional infestation of humans. However, the virome of this species in Thailand remains poorly characterized. In this study, we employed DNA Nanoball sequencing to investigate the virome of 80 R. sanguineus ticks, grouped into five pools, collected from dogs in Chachoengsao Province, Thailand, in 2023. Three viruses were identified: Brown dog tick phlebovirus 2 (BDTPV2), Changping tick virus 2 (CpTV-2), and Bole tick virus 4 (BLTV4), all detected in male ticks. These results highlight the need for further investigation into the ecological roles and biological significance of these viruses. Overall, our findings provide an updated perspective on the R. sanguineus virome in Thailand and underscore the importance of continued surveillance of tick-associated viruses within the One Health framework.

Introducción: Los ortoflavivirus transmitidos por mosquitos, como el virus del Nilo Occidental (WNV) y el virus Zika (ZIKV), causan cientos de millones de infecciones anuales y aún carecemos de intervenciones ampliamente eficaces. La transmisión se inicia con la deposición del virus en la piel durante la picadura; pero en presencia de una potente respuesta antiviral local, se limita la infección. Aunque se sabe que la saliva del mosquito modula la inmunidad del hospedador, los factores virales implicados no están completamente definidos. El objetivo de este estudio fue identificar y caracterizar componentes virales salivarios conservados que potencien la transmisión de ortoflavivirus. Métodos: Identificamos ARN subgenómico no codificante (sfRNA) en la saliva de Culex quinquefasciatus infectados con WNV y Aedes aegypti infectados con ZIKV. El sfRNA estaba contenido en vesículas lipídicas distintas de los viriones y su secreción no requería infección del intestino medio. Análisis cuantitativos mostraron que concentraciones elevadas de sfRNA en saliva infecciosa se correlacionaban con mayor infección en células cutáneas humanas y explantes de piel, independientemente de los niveles de ARN genómico viral (gRNA). Resultados: Ensayos funcionales demostraron que la entrega de sfRNA aumentaba la infección in vitro, en explantes de piel humana y en un modelo murino, donde además agravaba la enfermedad y reducía la supervivencia. Mecánicamente, el sfRNA suprimió la respuesta temprana de interferón tipo I, inhibiendo selectivamente la señalización mediada por MDA5, sin afectar la vía de RIG-I. Estos efectos se observaron tanto para WNV como para ZIKV, lo que indica un papel conservado en la potenciación de la transmisión. Conclusiones: Este estudio establece al sfRNA salival como un factor conservado que favorece la transmisión de ortoflavivirus al modular la inmunidad innata del hospedador. Bloquear su secreción o función podría constituir una estrategia novedosa para el control de la transmisión de ortoflavivirus a nivel global.

Viruses exploit extracellular vesicles (EVs) to transfer infection-enhancing viral RNAs. However, mechanisms underlying viral RNA loading remain elusive. We leveraged our previous discovery that dengue virus secretes transmission-enhancing subgenomic flaviviral RNA (sfRNA) into mosquito salivary EVs to investigate viral RNA loading mechanism. We demonstrate that sfRNA alone promotes the secretion of sfRNA-containing EVs marked by the mosquito EV biogenesis protein AeSyntenin, by applying microscopy and viral genetic editing in in vitro and in vivo models. SfRNA via its stem loop structures interacts intracellularly with mosquito AeSyntenin and this interaction is selectively maintained within EVs as shown by complementary RNA-affinity chromatography and RNA immunoprecipitation, and AI-based prediction. Finally, we used systemic and salivary gland-specific protein depletion to establish a functional role for mosquito AeSyntenin in exosome production and salivary secretion of sfRNA. We propose that sfRNA binds AeSyntenin to drive its selective packaging and release into exosomes, elucidating a mechanism for viral RNA incorporation into EVs.

Numerous orthoflaviviruses transmitted through the bites of different mosquito species infect more than 500 million people annually. Skin infection at the bite site represents a critical and conserved step in transmission and a deeper understanding of this process will promote the design of broad-spectrum interventions. Here, we identify and characterize a transmission-enhancing viral factor in mosquito saliva that is shared across orthoflaviviruses. Saliva from West Nile virus-infected Culex and Zika virus-infected Aedes contains a viral non-coding RNA, subgenomic flaviviral RNA (sfRNA), within lipid vesicles distinct from virions. Higher concentration of sfRNA in infectious saliva positively correlates with infection intensity in human cells and skin explants. Early sfRNA delivery into transmission-relevant skin cell types and human skin explant demonstrate that sfRNA is responsible for the infection enhancement. Co-inoculation of sfRNA in a mouse model of transmission enhanced skin infection and worsened disease severity, supporting the role of salivary sfRNA as a transmission-enhancer. Mechanistically, salivary sfRNA attenuates early interferon response in human skin cells and skin explants by disrupting MDA5 signaling. Our results, derived from two distinct orthoflaviviruses and supported by prior studies, establish salivary sfRNA as a pan-orthoflavivirus transmission-enhancing factor driven by a conserved viral non-coding RNA. Factors in mosquito saliva are required for efficient Orthoflavivirus transmission. The authors report a viral RNA fragment in saliva that reduce early innate immune response to enhance transmission.

Many flaviviruses with high pandemic potential are transmitted through mosquito bites. While mosquito saliva is essential for transmission and represents a promising pan-flaviviral target, there is a dearth of knowledge on salivary metabolic transmission enhancers. Here, we show that extracellular vesicle (EV)-derived sphingomyelins in mosquito saliva reconfigure the human cell lipidome to increase viral protein levels, boosting skin infection and enhancing transmission for flaviviruses. Lipids within internalized mosquito EVs enhance infection in fibroblast and immune human primary cells for multiple flaviviruses. Mosquito EV lipids selectively increase viral translation by inhibiting infection-induced endoplasmic reticulum (ER)-associated degradation of viral proteins. Infection enhancement solely results from the sphingomyelins within salivary mosquito EVs that augment human cell sphingomyelin concentration. Finally, EV-lipid co-inoculation exacerbates disease severity in vivo in mouse transmission assays. By discovering and elucidating how metabolic components of mosquito saliva promote transmission of flaviviruses, our study unveils lipids as a new category of targets against vectored transmission.

Chikungunya virus (CHIKV) poses a significant challenge as there are currently no targeted antiviral drugs or vaccines to combat this infection. Here, we demonstrate that interferon-induced transmembrane proteins (IFITMs), including IFITM1, IFITM2, and IFITM3, which are interferon-stimulated genes (ISGs), inhibit CHIKV infection in human skin fibroblasts. Overexpression of IFITMs in cells restricts viral infection, whereas knockdown of IFITMs enhances viral infection. IFITMs overexpression causes a substantial upregulation of antiviral genes, namely TLR3, TLR7, TLR8, and TLR9, and their downstream signaling molecules such as TRADD, IRAK1, TRAF6, and MAP3K7, involved in TLRs signaling pathways. Furthermore, the DHX58 gene encoding the LGP2 protein, a negative regulator of RIG-I in RLRs signaling pathways, was downregulated in the overexpressed cells. Transcription factors including interferon regulatory factors (IRF) 3/5/7, which are downstream signaling components of both TLR and RLR signaling pathways, were also upregulated, resulting in enhanced IFNs signaling. IFITMs not only inhibits the early and late stages of viral infection but can also alter the antiviral innate-immune response to restrict CHIKV infection in human skin fibroblasts. Additionally, IFITMs exhibit their antiviral activity against Zika virus (ZIKV). Altogether, these results show the broad-spectrum antiviral property of IFITMs against arboviruses in foreskin cells.

Extracellular vesicles (EVs) are non-replicative, cell-derived membranous structures secreted by potentially all eukaryotic cells, playing a crucial role in intercellular communication. The study of EVs requires approaches and tools, which have predominantly been developed for mammalian models. Here, we undertook a multimodal characterization of mosquito EVs to provide a technical and knowledge foundation for their study. First, using a cell line model from Aedes aegypti and applying multiple analytical technologies (i.e., NTA, TEM, cryo-EM, and AFM), we observed that mosquito EVs range from 20 to 500 nm in diameter and that a majority are smaller than 100 nm. Second, we showed that smaller EVs are secreted in mosquito saliva. Third, we evaluated the capacity of differential centrifugation and size exclusion chromatography to separate mosquito EVs, revealing the strengths and weaknesses of each technology. Finally, we identified a mosquito homolog of CD63 as an extravesicular marker and the mosquito syntenin as a putative luminal marker. Overall, our results promote the development of tools and approaches for the study of mosquito EVs.

Numerous orthoflaviviruses transmitted through the bites of different mosquito species infect more than 500 million people annually. Bite-initiated skin infection represents a critical and conserved step in transmission and a deeper understanding of this process will promote the design of broad-spectrum interventions to address diverse orthoflavivirus health threats. Here, we identify and characterize a transmission-enhancing viral factor in mosquito saliva that is shared across orthoflaviviruses. Saliva of West Nile virus-infected Culex and Zika virus-infected Aedes contains a viral non-coding RNA, subgenomic orthoflaviviral RNA (sfRNA), within lipid vesicles distinct from virions. Higher concentration of sfRNA in infectious saliva positively correlates with infection intensity in human cells and skin explants. Early sfRNA delivery into transmission-relevant skin cell types and human skin explant demonstrate that sfRNA is responsible for the infection enhancement. Co-inoculation of sfRNA in a mouse model of transmission enhanced skin infection and worsened disease severity, evidencing the role of salivary sfRNA as a transmission-enhancer. Mechanistically, salivary sfRNA attenuates early interferon response in human skin cells and skin explants by altering MDA5-mediated signaling. Our results, derived from two distinct orthoflaviviruses and supported by prior studies, establish salivary sfRNA as a pan-orthoflavivirus transmission-enhancing factor driven by a conserved viral non-coding RNA. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Latin America has been experiencing an Oropouche virus (OROV) outbreak of unprecedented magnitude and spread since 2023-24 for unknown reasons. We aimed to identify risk predictors of and areas at risk for OROV transmission. METHODS:In this multidisciplinary, laboratory-based, modelling study, we retrospectively tested anonymised serum samples collected between 2001 and 2022 for studies on virus epidemiology and medical diagnostics in Bolivia, Brazil, Colombia, Costa Rica, Ecuador, and Peru with nucleoprotein-based commercial ELISAs for OROV-specific IgG and IgM antibodies. Serum samples positive for IgG from different ecological regions and sampling years were tested against Guaroa virus and two OROV glycoprotein reassortants (Iquitos virus and Madre de Dios virus) via plaque reduction neutralisation testing (PRNT) to validate IgG ELISA specificity and support antigenic cartography. Three OROV strains were included in the neutralisation testing, a Cuban OROV isolate from the 2023-24 outbreak, a contemporary Peruvian OROV isolate taken from a patient in 2020, and a historical OROV isolate from Brazil. We analysed the serological data alongside age, sex, cohort, and geographical residence data for the serum samples; reported OROV incidence data; and vector occurrence data to explore OROV transmission in ecologically different regions of Latin America. We used the MaxEnt machine learning methodology to spatially analyse and predict OROV infection risk across Latin America, fitting one model with presence-absence serological data (seropositive results were recorded as presence and seronegative results were recorded as absence) and one model with presence-only, reported incidence data from 2024. We computed marginal dependency plots, variable contribution, and permutation metrics to analyse the impact of socioecological predictors and fitted a generalised linear mixed-effects model with logit link and binary error structure to analyse the potential effects of age, sex, or cohort type bias and interactions between age or sex and cohort type in our serological data. We conducted antigenic cartography and evolutionary characterisations of all available genomic sequences for all three OROV genome segments from the National Center for Biotechnology Information, including branch-specific selection pressure analysis and the construction of OROV phylogenetic trees. FINDINGS:In total, 9420 serum samples were included in this study, representing 76 provinces in the six Latin American countries previously mentioned. The sex distribution across the combined cohorts was 48% female (4237 of 8910 samples with available data) and 52% male (4673 of 8910 samples) and the mean age was 29·5 years (range 0-95 years). The samples were collected from census-based cohorts, cohorts of healthy individuals, and cohorts of febrile patients receiving routine health care. The average OROV IgG antibody detection rate was 6·3% (95% CI 5·8-6·8), with substantial regional heterogeneity. The presence-absence, serology-based model predicted high-risk areas for OROV transmission in the Amazon River basin, around the coastal and southern areas of Brazil, and in parts of central America and the Caribbean islands, consistent with case data from the 2023-24 outbreak reported by the Pan American Health Organization. Areas with a high predicted risk of OROV transmission with the serology-based model showed a statistically significant positive correlation with state-level incidence rates per 100 000 people in 2024 (generalised linear model, p=0·0003). The area under the curve estimates were 0·79 (95% CI 0·78-0·80) for the serology-based model and 0·66 (95% CI 0·65-0·66) for the presence-only incidence-based model. Longitudinal diagnostic testing of serum samples from cohorts of febrile patients suggested constant circulation of OROV in endemic regions at varying intensity. Climate variables accounted for more than 60% of variable contribution in both the serology-based and incidence-based models. Antigenic cartography, evolutionary analyses, and in-vitro growth comparisons showed clear differentiation between OROV and its glycoprotein reassortants, but not between the three different OROV strains. PRNT titres of OROV-neutralising serum samples were strongly correlated between all three tested OROV isolates (r>0·83; p<0·0001) but were not correlated with the two glycoprotein reassortants. INTERPRETATION:Our data suggest that climatic factors are major drivers of OROV spread and were potentially exacerbated during 2024 by extreme weather events. OROV glycoprotein reassortants, but not individual OROV strains, probably have distinct antigenicity. Preparedness for OROV outbreaks requires enhanced diagnostics, surveillance, and vector control in current and future endemic areas, which could all be informed by the risk predictions presented in this Article. FUNDING:European Union. TRANSLATIONS:For the Spanish and Portuguese translations of the abstract see Supplementary Materials section.

Rabies is an acute and fatal form of encephalomyelitis caused by rabies virus (RABV) infection. RABV is present throughout the world, particularly in Asia and Africa, in host animals such as domestic dogs. Canine rabies is responsible for thousands human deaths despite existing preventable and prophylactic solutions. RABV is endemic to southern Africa and circulates, in addition to dogs, among wild mammals, livestock and domestic carnivores (e.g. cats), and accidentally in humans. Two different genetic variants of RABV are described in these African regions: Africa 1 lineage, subtype b (AF1b) belonging to the Cosmopolitan clade, and Africa 3 (AF3) clade, associated with the dog and the mongoose, respectively. However, information about the genetic diversity of RABV circulating in Zimbabwe remains limited. In order to bridge this knowledge gap, we analyzed RABV strains present in this country between 2014 and 2020. To this end, brain samples from 50 domestic and 24 wild animal species from different locations in Zimbabwe were collected by the country’s veterinary services. Seventy out of the 74 (94.6

Bat-borne severe acute respiratory syndrome-related coronaviruses (bat SARSr-CoVs), belonging to the Sarbecovirus subgenus of the Betacoronavirus genus pose a potential risk for zoonotic spillover. This study reports the complete genome sequences of bat sarbecoviruses from Nycteris macrotis and Rhinolophus simulator insectivorous bat species in Zimbabwe. Using genome walking and nested PCR approaches, six full viral genomes were amplified, sequenced and assembled: one from Nycteris macrotis (MAG1042) and five from Rhinolophus simulator bats species (MAG388, MAG562, MAG575, MAG850, and MAG859). Comparative genomic analysis revealed a high degree of sequence conservation (99.6-99.9%) among the Rhinolophus simulator-characterised sequences, while the Nycteris macrotis characterised sequence exhibited 96.7% identity with the Rhinolophus simulator consensus (MAG Cons) genomes, suggesting genus-specific evolution. Furthermore, the spike region exhibited notable divergence, with only 88% sequence identity between the two genera. A complete absence of ORF8, and truncation of ORF7b genes were observed, while the RNA dependent RNA polymerase genes were highly conserved across all strains. Phylogenetic analysis demonstrated distinct continental clustering, with the Zimbabwean genomic sequences forming a well-supported clade within the African lineage (Lineage 4). These newly characterized Zimbabwean sarbecoviruses sequences represent the most recent evolutionary branches within the Lineage 4-African clade, indicating ongoing viral diversification in local bat populations. These findings expand understanding of SARSr-CoV diversity in African bats and underscore the importance of geographical isolation in viral evolution.

Recent evidence of vertical transmission of Tonate virus (TONV) during early pregnancy and its association with fetal neurological anomalies highlights its potential public health threat. TONV is an understudied alphavirus endemic to French Guiana. The growing presence of Aedes (Ae.) albopictus in Europe raises concerns about its ability to transmit emerging arboviruses, including TONV. We assessed the vector competence of Ae. albopictus populations from mainland France and La Réunion Island via oral infections using different TONV doses. Both populations supported efficient viral replication, with infectious viral particles appearing in saliva by day 5 post-infection. Infection rate (IR), stepwise dissemination rate (sDR), and transmission efficiency (TE) increased with rising viral concentrations. At a viral concentration of 106 PFU/mL IR reached 80%, and TE at day 5 post-infection was 27% for the mainland strain and 37% for the La Réunion strain. Notably, TE declined over time in the mainland strain, while increasing progressively in the La Réunion population. Comparative infections with Chikungunya virus revealed that TONV was transmitted at similar or greater rates, confirming Ae. albopictus as a competent vector. Sequencing of mosquito organs revealed intra-host TONV genetic variability. A recurrent polymorphism at position 11,357 in the 3'UTR was detected in body tissues but not in heads of La Réunion mosquitoes, suggesting tissue-specific selection or bottlenecks. Our results demonstrate that Ae. albopictus from both tropical and temperate areas can efficiently transmit TONV and emphasize the importance of genomic surveillance to anticipate risk of its emergence in areas where this vector is established.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus with a mortality rate of up to 30%. First identified in China in 2009, it was later reported in other Asian countries, including Thailand in 2020. SFTSV has been detected in several tick species, including Rhipicephalus sanguineus , known for infesting dogs. We conducted a seroprevalence study of SFTSV in Bangkok and Nong Khai, Thailand, by analyzing 1162 human samples collected between 2019 and 2023. The testing method relied on IgG detection using ELISA and confirmed though a virus seroneutralization test. The results indicated that out of the participants, 12 (1.1%) tested positive for anti-SFTSV IgG antibodies; however, none exhibited positive results in the seroneutralization assay. Additionally, molecular detection of SFTSV, Crimean-Congo hemorrhagic fever (CCHF), Coxiella spp., Bartonella spp., and Rickettsia spp. was performed on 433 Rh. sanguineus ticks collected from 49 dogs in 2023 in Chachoengsao Province, Thailand. No evidence of these pathogens was found in ticks. These findings highlight the importance of exploring viral cross-reactivity. Furthermore, it is important to conduct additional studies to isolate SFTSV from animals and ticks in order to identify the potential transmission routes contributing to human and animal infections in Thailand.

Dengue virus (DENV) poses a global health threat, affecting millions individuals annually with no specific therapy and limited vaccines. Mosquitoes, mainly Aedes aegypti and Aedes albopictus worldwide, transmit DENV through their saliva during blood meals. In this study, we aimed to understand how Aedes mosquito saliva modulate skin immune responses during DENV infection in individuals living in mosquito-endemic regions. To accomplish this, we dissociated skin cells from Cambodian volunteers and incubated them with salivary gland extract (SGE) from three different mosquito strains: Ae. aegypti USDA strain, Ae. aegypti and Ae. albopictus wild type (WT) in the presence/absence of DENV. We observed notable alterations in skin immune cell phenotypes subsequent to exposure to Aedes salivary gland extract (SGE). Specifically, exposure lead to an increase in the frequency of macrophages expressing chemokine receptor CCR2, and neutrophils expressing CD69. Additionally, we noted a substantial increase in the percentage of macrophages that became infected with DENV in the presence of Aedes SGE. Differences in cellular responses were observed when Aedes SGE of three distinct mosquito strains were compared. Our findings deepen the understanding of mosquito saliva's role in DENV infection and skin immune responses in individuals regularly exposed to mosquito bites. This study provides insights into skin immune cell dynamics that could guide strategies to mitigate DENV transmission and other arbovirus diseases.

Mosquito saliva plays a determining role in flavivirus transmission. Here, we discover and elucidate how salivary lipids enhance transmission. Building upon our discovery of salivary extracellular vesicles (EV), we determined that lipids within mosquito EVs, and neither within human EVs nor virions, enhance infection for flaviviruses in primary cell types relevant for transmission. Mechanistically, mosquito EV-lipids specifically promote viral protein levels by reducing ER-associated degradation. Infection enhancement is caused by sphingomyelins within mosquito salivary EVs that elevate sphingomyelin concentration within host cells. Transmission assays showed that mosquito EV-lipids exacerbate disease severity. Our study reveals that EV-associated sphingomyelins within mosquito saliva enhance transmission for multiple flaviviruses by reconfiguring the host lipidome to promote viral protein levels and the resulting skin infection. Our findings open a new dimension centered on lipids in the interplay between hosts, mosquitoes and flaviviruses that determine transmission, unveiling lipids as a new pan-flavivirus target. Highlights ### Competing Interest Statement The authors have declared no competing interest.

Changes to our environment have led to the emergence of human pathogens such as chikungunya virus. Chikungunya virus infection is today a major public health concern. It is a debilitating chronic disease impeding patients' mobility, affecting millions of people. Disease development relies on skeletal muscle infection. The importance of skeletal muscle in chikungunya virus infection led to the hypothesis that it could serve as a viral reservoir and could participate to virus persistence. Here we questioned the interconnection between skeletal muscle cells metabolism, their differentiation stage and the infectivity of the chikungunya virus. We infected human skeletal muscle stem cells at different stages of differentiation with chikungunya virus to study the impact of their metabolism on virus production and inversely the impact of virus on cell metabolism. We observed that chikungunya virus infectivity is cell differentiation and metabolism-dependent. Chikungunya virus interferes with the cellular metabolism in quiescent undifferentiated and proliferative muscle cells. Moreover, activation of chikungunya infected quiescent muscle stem cells, induces their proliferation, increases glycolysis and amplifies virus production. Therefore, our results showed that Chikungunya virus infectivity and the antiviral response of skeletal muscle cells relies on their energetic metabolism and their differentiation stage. Then, muscle stem cells could serve as viral reservoir producing virus after their activation.

BACKGROUND:Zika virus (ZIKV) has spread to five of the six World Health Organization (WHO) regions. Given the substantial number of asymptomatic infections and clinical presentations resembling those of other arboviruses, estimating the true burden of ZIKV infections is both challenging and essential. Therefore, we conducted a systematic review and meta-analysis of seroprevalence studies of ZIKV IgG in asymptomatic population to estimate its global impact and distribution. METHODOLOGY/PRINCIPAL FINDINGS:We conducted extensive searches and compiled a collection of articles published from Jan/01/2000, to Jul/31/2023, from Embase, Pubmed, SciELO, and Scopus databases. The random effects model was used to pool prevalences, reported with their 95% confidence interval (CI), a tool to assess the risk of study bias in prevalence studies, and the I2 method for heterogeneity (PROSPERO registration No. CRD42023442227). Eighty-four studies from 49 countries/territories, with a diversity of study designs and serological tests were included. The global seroprevalence of ZIKV was 21.0% (95%CI 16.1%-26.4%). Evidence of IgG antibodies was identified in all WHO regions, except for Europe. Seroprevalence correlated with the epidemics in the Americas (39.9%, 95%CI:30.0-49.9), and in some Western Pacific countries (15.6%, 95%CI:8.2-24.9), as well as with recent and past circulation in Southeast Asia (22.8%, 95%CI:16.5-29.7), particularly in Thailand. Additionally, sustained low circulation was observed in Africa (8.4%, 95%CI:4.8-12.9), except for Gabon (43.7%), and Burkina Faso (22.8%). Although no autochthonous transmission was identified in the Eastern Mediterranean, a seroprevalence of 16.0% was recorded. CONCLUSIONS/SIGNIFICANCE:The study highlights the high heterogeneity and gaps in the distribution of seroprevalence. The implementation of standardized protocols and the development of tests with high specificity are essential for ensuring a valid comparison between studies. Equally crucial are vector surveillance and control methods to reduce the risk of emerging and re-emerging ZIKV outbreaks, whether caused by Ae. aegypti or Ae. albopictus or by the Asian or African ZIKV.