The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components.

During the life cycle of mosquito-borne flaviviruses, substantial subgenomic flaviviral RNA (sfRNA) is produced via incomplete degradation of viral genomic RNA by host XRN1. Zika virus (ZIKV) sfRNA has been detected in mosquito and mammalian somatic cells. Human neural progenitor cells (hNPCs) in the developing brain are the major target cells of ZIKV, and antiviral RNA interference (RNAi) plays a critical role in hNPCs. However, whether ZIKV sfRNA was produced in ZIKV-infected hNPCs as well as its function remains not known. In this study, we demonstrate that abundant sfRNA was produced in ZIKV-infected hNPCs. RNA pulldown and mass spectrum assays showed ZIKV sfRNA interacted with host proteins RHA and PACT, both of which are RNA-induced silencing complex (RISC) components. Functionally, ZIKV sfRNA can antagonize RNAi by outcompeting small interfering RNAs (siRNAs) in binding to RHA and PACT. Furthermore, the 3' stem loop (3'SL) of sfRNA was responsible for RISC components binding and RNAi inhibition, and 3'SL can enhance the replication of a viral suppressor of RNAi (VSR)-deficient virus in a RHA- and PACT-dependent manner. More importantly, the ability of binding to RISC components is conversed among multiple flaviviral 3'SLs. Together, our results identified flavivirus 3'SL as a potent VSR in RNA format, highlighting the complexity in virus-host interaction during flavivirus infection.IMPORTANCEZika virus (ZIKV) infection mainly targets human neural progenitor cells (hNPCs) and induces cell death and dysregulated cell-cycle progression, leading to microcephaly and other central nervous system abnormalities. RNA interference (RNAi) plays critical roles during ZIKV infections in hNPCs, and ZIKV has evolved to encode specific viral proteins to antagonize RNAi. Herein, we first show that abundant sfRNA was produced in ZIKV-infected hNPCs in a similar pattern to that in other cells. Importantly, ZIKV sfRNA acts as a potent viral suppressor of RNAi (VSR) by competing with siRNAs for binding RISC components, RHA and PACT. The 3'SL of sfRNA is responsible for binding RISC components, which is a conserved feature among mosquito-borne flaviviruses. As most known VSRs are viral proteins, our findings highlight the importance of viral non-coding RNAs during the antagonism of host RNAi-based antiviral innate immunity.

Gain without pain: adaptation and increased virulence of Zika virus in vertebrate host without fitness cost in mosquito vector.

IMPORTANCE: Previously, we modeled direct transmission chains of Zika virus (ZIKV) by serially passaging ZIKV in mice and mosquitoes and found that direct mouse transmission chains selected for viruses with increased virulence in mice and the acquisition of non-synonymous amino acid substitutions. Here, we show that these same mouse-passaged viruses also maintain fitness and transmission capacity in mosquitoes. We used infectious clone-derived viruses to demonstrate that the substitution in nonstructural protein 4A contributes to increased virulence in mice.

Lipid droplets in Zika neuroinfection: Potential targets for intervention?

Lipid droplets (LD) are evolutionarily conserved lipid-enriched organelles with a diverse array of cell- and stimulus-regulated proteins. Accumulating evidence demonstrates that intracellular pathogens exploit LD as energy sources, replication sites, and part of the mechanisms of immune evasion. Nevertheless, LD can also favor the host as part of the immune and inflammatory response to pathogens. The functions of LD in the central nervous system have gained great interest due to their presence in various cell types in the brain and for their suggested involvement in neurodevelopment and neurodegenerative diseases. Only recently have the roles of LD in neuroinfections begun to be explored. Recent findings reveal that lipid remodelling and increased LD biogenesis play important roles for Zika virus (ZIKV) replication and pathogenesis in neural cells. Moreover, blocking LD formation by targeting DGAT-1 in vivo inhibited virus replication and inflammation in the brain. Therefore, targeting lipid metabolism and LD biogenesis may represent potential strategies for anti-ZIKV treatment development. Here, we review the progress in understanding LD functions in the central nervous system in the context of the host response to Zika infection.

PKR-mediated stress response enhances dengue and Zika virus replication.

IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.

Development of disposable electrode for the detection of mosquito-borne viruses.

Development of disposable, rapid, and convenient biosensor with high sensitivity and reliability is the most desired method of viral disease prevention. To achieve this goal, in this work, a practical impedimetric biosensor has been implemented into a disposable electrode on a screen-printed carbon electrode (SPCE) for the detection of two mosquito-borne viruses. The biosensor fabrication has step-wisely carried out on the disposable electrode surface at room temperature: starting from conductive film formation, physical binding of the gold nanoparticles (AuNPs)-polyaniline (PAni) into the conductive film, and biofunctionalization. To get the maximum efficiency of the antibody, biotinylated antibody has been conjugated on the surface of AuNP-PAni/PAni-SPCE via the streptavidin-biotin conjugation method which is a critical factor for the high sensitivity. Using the antibody-antigen interaction, this disposable electrode has designed to detect mosquito-borne infectious viruses, Chikungunya virus (CHIKV), and Zika virus (ZIKV) separately in a wide linear range of 100 fg mL-1 to 1 ng mL-1 with a low detection limit of 1.33 and 12.31 fg mL-1 , respectively.

Redox Regulation and Metabolic Dependency of Zika Virus Replication: Inhibition by Nrf2-Antioxidant Response and NAD(H) Antimetabolites.

Viral infections alter host cell metabolism and homeostasis; however, the mechanisms that regulate these processes have only begun to be elucidated. We report here that Zika virus (ZIKV) infection activates the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2), which precedes oxidative stress. Downregulation of Nrf2 or inhibition of glutathione (GSH) synthesis resulted in significantly increased viral replication. Interestingly, 6-amino-nicotinamide (6-AN), a nicotinamide analog commonly used as an inhibitor of the pentose phosphate pathway (PPP), decreased viral replication by over 1,000-fold. This inhibition was neither recapitulated by the knockdown of PPP enzymes, glucose 6-phosphate dehydrogenase (G6PD), or 6-phosphogluconate dehydrogenase (6PGD), nor prevented by supplementation with ribose 5-phosphate. Instead, our metabolomics and metabolic phenotype studies support a mechanism in which 6-AN depletes cells of NAD(H) and impairs NAD(H)-dependent glycolytic steps resulting in inhibition of viral replication. The inhibitory effect of 6-AN was rescued with precursors of the salvage pathway but not with those of other NAD+ biosynthesis pathways. Inhibition of glycolysis reduced viral protein levels, which were recovered transiently. This transient recovery in viral protein synthesis was prevented when oxidative metabolism was inhibited by blockage of the mitochondrial pyruvate carrier, fatty acid oxidation, or glutaminolysis, demonstrating a compensatory role of mitochondrial metabolism in ZIKV replication. These results establish an antagonistic role for the host cell Nrf2/GSH/NADPH-dependent antioxidant response against ZIKV and demonstrate the dependency of ZIKV replication on NAD(H). Importantly, our work suggests the potential use of NAD(H) antimetabolite therapy against the viral infection. IMPORTANCE Zika virus (ZIKV) is a major public health concern of international proportions. While the incidence of ZIKV infections has declined substantially in recent years, the potential for the reemergence or reintroduction remains high. Although viral infection alters host cell metabolism and homeostasis to promote its replication, deciphering the mechanism(s) involved in these processes is important for identifying therapeutic targets. The present work reveals the complexities of host cell redox regulation and metabolic dependency of ZIKV replication. An antagonistic effect of the Nrf2/GSH/NADP(H)-dependent antioxidant response against ZIKV infection and an essential role of NAD(H) metabolism and glycolysis for viral replication are established for the first time. These findings highlight the potential use of NAD(H) antimetabolites to counter ZIKV infection and pathogenesis.

In Vitro and In Vivo Coinfection and Superinfection Dynamics of Mayaro and Zika Viruses in Mosquito and Vertebrate Backgrounds.

Globalization and climate change have contributed to the simultaneous increase and spread of arboviral diseases. Cocirculation of several arboviruses in the same geographic region provides an impetus to study the impacts of multiple concurrent infections within an individual vector mosquito. Here, we describe coinfection and superinfection with the Mayaro virus (Togaviridae, Alphavirus) and Zika virus (Flaviviridae, Flavivirus) in vertebrate and mosquito cells, as well as Aedes aegypti adult mosquitoes, to understand the interaction dynamics of these pathogens and effects on viral infection, dissemination, and transmission. Aedes aegypti mosquitoes were able to be infected with and transmit both pathogens simultaneously. However, whereas Mayaro virus was largely unaffected by coinfection, it had a negative impact on infection and dissemination rates for Zika virus compared to single infection scenarios. Superinfection of Mayaro virus atop a previous Zika virus infection resulted in increased Mayaro virus infection rates. At the cellular level, we found that mosquito and vertebrate cells were also capable of being simultaneously infected with both pathogens. Similar to our findings in vivo, Mayaro virus negatively affected Zika virus replication in vertebrate cells, displaying complete blocking under certain conditions. Viral interference did not occur in mosquito cells. IMPORTANCE Epidemiological and clinical studies indicate that multiple arboviruses are cocirculating in human populations, leading to some individuals carrying more than one arbovirus at the same time. In turn, mosquitoes can become infected with multiple pathogens simultaneously (coinfection) or sequentially (superinfection). Coinfection and superinfection can have synergistic, neutral, or antagonistic effects on viral infection dynamics and ultimately have impacts on human health. Here we investigate the interaction between Zika virus and Mayaro virus, two emerging mosquito-borne pathogens currently circulating together in Latin America and the Caribbean. We find a major mosquito vector of these viruses-Aedes aegypti-can carry and transmit both arboviruses at the same time. Our findings emphasize the importance of considering co- and superinfection dynamics during vector-pathogen interaction studies, surveillance programs, and risk assessment efforts in epidemic areas.

Pathogenicity and Structural Basis of Zika Variants with Glycan Loop Deletions in the Envelope Protein.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

Tracking the Replication-Competent Zika Virus with Tetracysteine-Tagged Capsid Protein in Living Cells.

Real-time imaging of viruses in living cells considerably facilitates the study of virus-host interactions. However, generating a fluorescently labeled recombinant virus is challenging, especially for Zika virus (ZIKV), which causes microcephaly in neonates. The monocistronic nature of the ZIKV genome represents a major challenge for generating a replication-competent genetically engineered ZIKV suitable for real-time imaging. Here, we generated a fluorescent ZIKV by introducing the biarsenical tetracysteine (TC) tag system. After separately inserting the TC tag at six sites in the capsid protein, we found that only when we inserted the TC tag at the site of amino acids 27/28 (AA27/28, or TC27) could the genetically engineered ZIKV be rescued. Importantly, the TC27 ZIKV is characterized as replication and infection competent. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the dynamic nuclear import behavior of the capsid protein. In addition, using the single-particle tracking technology, we acquired real-time imaging evidence that ZIKV moved along the cellular filopodia and entered into the cytoplasm via endocytosis. Thus, we provide a feasible strategy to generate a replication-competent TC-tagged ZIKV for real-time imaging, which should greatly facilitate the study of ZIKV-host interactions in living cells. IMPORTANCE Zika virus (ZIKV) is the mosquito-borne enveloped flavivirus that causes microcephaly in neonates. While real-time imaging plays a critical role in dissecting viral biology, no fluorescent, genetically engineered ZIKV for single-particle tracking is currently available. Here, we generated a replication-competent genetically engineered ZIKV by introducing the tetracysteine (TC) tag into its capsid protein. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the nuclear import behavior of the capsid protein and the endocytosis process of single ZIKV particle. Taken together, these results demonstrate a fluorescent labeling strategy to track the ZIKV-host interactions at both the protein level and the viral particle level. Our replication-competent TC27 ZIKV should open an avenue to study the ZIKV-host interactions and may provide applications for antiviral screening.

Neurogenesis and Viral Infection.

Neural stem cells (NSCs) are multipotent stem cells that reside in the fetal and adult mammalian brain, which can self-renew and differentiate into neurons and supporting cells. Intrinsic and extrinsic cues, from cells in the local niche and from distant sites, stringently orchestrates the self-renewal and differentiation competence of NSCs. Ample evidence supports the important role of NSCs in neuroplasticity, aging, disease, and repair of the nervous system. Indeed, activation of NSCs or their transplantation into injured areas of the central nervous system can lead to regeneration in animal models. Viral invasion of NSCs can negatively affect neurogenesis and synaptogenesis, with consequent cell death, impairment of cell cycle progression, early differentiation, which cause neural progenitors depletion in the cortical layer of the brain. Herein, we will review the current understanding of Zika virus (ZIKV) infection of the fetal brain and the NSCs, which are the preferential population targeted by ZIKV. Furthermore, the potential neurotropic properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may cause direct neurological damage, will be discussed.

Apoptosis during ZIKA Virus Infection: Too Soon or Too Late?

Cell death by apoptosis is a major cellular response in the control of tissue homeostasis and as a defense mechanism in the case of cellular aggression such as an infection. Cell self-destruction is part of antiviral responses, aimed at limiting the spread of a virus. Although it may contribute to the deleterious effects in infectious pathology, apoptosis remains a key mechanism for viral clearance and the resolution of infection. The control mechanisms of cell death processes by viruses have been extensively studied. Apoptosis can be triggered by different viral determinants through different pathways as a result of virally induced cell stresses and innate immune responses. Zika virus (ZIKV) induces Zika disease in humans, which has caused severe neurological forms, birth defects, and microcephaly in newborns during the last epidemics. ZIKV also surprised by revealing an ability to persist in the genital tract and in semen, thus being sexually transmitted. Mechanisms of diverting antiviral responses such as the interferon response, the role of cytopathic effects and apoptosis in the etiology of the disease have been widely studied and debated. In this review, we examined the interplay between ZIKV infection of different cell types and apoptosis and how the virus deals with this cellular response. We illustrate a duality in the effects of ZIKV-controlled apoptosis, depending on whether it occurs too early or too late, respectively, in neuropathogenesis, or in long-term viral persistence. We further discuss a prospective role for apoptosis in ZIKV-related therapies, and the use of ZIKV as an oncolytic agent.

Seroprevalence of dengue, Zika, chikungunya and Ross River viruses across the Solomon Islands.

Across the Pacific, and including in the Solomon Islands, outbreaks of arboviruses such as dengue, chikungunya, and Zika are increasing in frequency, scale and impact. Outbreaks of mosquito-borne disease have the potential to overwhelm the health systems of small island nations. This study mapped the seroprevalence of dengue, Zika, chikungunya and Ross River viruses in 5 study sites in the Solomon Islands. Serum samples from 1,021 participants were analysed by ELISA. Overall, 56% of participants were flavivirus-seropositive for dengue (28%), Zika (1%) or both flaviviruses (27%); and 53% of participants were alphavirus-seropositive for chikungunya (3%), Ross River virus (31%) or both alphaviruses (18%). Seroprevalence for both flaviviruses and alphaviruses varied by village and age of the participant. The most prevalent arboviruses in the Solomon Islands were dengue and Ross River virus. The high seroprevalence of dengue suggests that herd immunity may be a driver of dengue outbreak dynamics in the Solomon Islands. Despite being undetected prior to this survey, serology results suggest that Ross River virus transmission is endemic. There is a real need to increase the diagnostic capacities for each of the arboviruses to support effective case management and to provide timely information to inform vector control efforts and other outbreak mitigation interventions.

Hyperendemic Dengue and Possible Zika Circulation in the Westernmost Region of the Indonesian Archipelago.

The transmission of dengue and other medically important mosquito-borne viruses in the westernmost region of Indonesia is not well described. We assessed dengue and Zika virus seroprevalence in Aceh province, the westernmost area of the Indonesian archipelago. Serum samples collected from 199 randomly sampled healthy residents of Aceh Jaya in 2017 were analyzed for neutralizing antibodies by plaque reduction neutralization test (PRNT). Almost all study participants (198/199; 99.5%) presented with multitypic profiles of neutralizing antibodies to two or more DENV serotypes, indicating transmission of multiple DENV in the region prior to 2017. All residents were exposed to one or more DENV serotypes by the age of 30 years. The highest geometric mean titers were measured for DENV-4, followed by DENV-1, DENV-2 and DENV-3. Among a subset of 116 sera, 27 neutralized ZIKV with a high stringency (20 with PRNT90 > 10 and 7 with PRNT90 > 40). This study showed that DENV is hyperendemic in the westernmost region of the Indonesian archipelago and suggested that ZIKV may have circulated prior to 2017.

DDX50 Is a Viral Restriction Factor That Enhances IRF3 Activation.

The transcription factors IRF3 and NF-κB are crucial in innate immune signalling in response to many viral and bacterial pathogens. However, mechanisms leading to their activation remain incompletely understood. Viral RNA can be detected by RLR receptors, such as RIG-I and MDA5, and the dsRNA receptor TLR3. Alternatively, the DExD-Box RNA helicases DDX1-DDX21-DHX36 activate IRF3/NF-κB in a TRIF-dependent manner independent of RIG-I, MDA5, or TLR3. Here, we describe DDX50, which shares 55.6% amino acid identity with DDX21, as a non-redundant factor that promotes activation of the IRF3 signalling pathway following its stimulation with viral RNA or infection with RNA and DNA viruses. Deletion of DDX50 in mouse and human cells impaired IRF3 phosphorylation and IRF3-dependent endogenous gene expression and cytokine/chemokine production in response to cytoplasmic dsRNA (polyIC transfection), and infection by RNA and DNA viruses. Mechanistically, whilst DDX50 co-immunoprecipitated TRIF, it acted independently to the previously described TRIF-dependent RNA sensor DDX1. Indeed, shRNA-mediated depletion of DDX1 showed DDX1 was dispensable for signalling in response to RNA virus infection. Importantly, loss of DDX50 resulted in a significant increase in replication and dissemination of virus following infection with vaccinia virus, herpes simplex virus, or Zika virus, highlighting its important role as a broad-ranging viral restriction factor.

Vav Proteins in Development of the Brain: A Potential Relationship to the Pathogenesis of Congenital Zika Syndrome?

Zika virus (ZIKV) infection during pregnancy can result in a significant impact on the brain and eye of the developing fetus, termed congenital zika syndrome (CZS). At a morphological level, the main serious presentations of CZS are microcephaly and retinal scarring. At a cellular level, many cell types of the brain may be involved, but primarily neuronal progenitor cells (NPC) and developing neurons. Vav proteins have guanine exchange activity in converting GDP to GTP on proteins such as Rac1, Cdc42 and RhoA to stimulate intracellular signaling pathways. These signaling pathways are known to play important roles in maintaining the polarity and self-renewal of NPC pools by coordinating the formation of adherens junctions with cytoskeletal rearrangements. In developing neurons, these same pathways are adopted to control the formation and growth of neurites and mediate axonal guidance and targeting in the brain and retina. This review describes the role of Vavs in these processes and highlights the points of potential ZIKV interaction, such as (i) the binding and entry of ZIKV in cells via TAM receptors, which may activate Vav/Rac/RhoA signaling; (ii) the functional convergence of ZIKV NS2A with Vav in modulating adherens junctions; (iii) ZIKV NS4A/4B protein effects on PI3K/AKT in a regulatory loop via PPI3 to influence Vav/Rac1 signaling in neurite outgrowth; and (iv) the induction of SOCS1 and USP9X following ZIKV infection to regulate Vav protein degradation or activation, respectively, and impact Vav/Rac/RhoA signaling in NPC and neurons. Experiments to define these interactions will further our understanding of the molecular basis of CZS and potentially other developmental disorders stemming from in utero infections. Additionally, Vav/Rac/RhoA signaling pathways may present tractable targets for therapeutic intervention or molecular rationale for disease severity in CZS.

Host cytoskeletal vimentin serves as a structural organizer and an RNA-binding protein regulator to facilitate Zika viral replication.

Emerging microbe infections, such as Zika virus (ZIKV), pose an increasing threat to human health. Investigations on ZIKV replication have revealed the construction of replication complexes (RCs), but the role of cytoskeleton in this process is largely unknown. Here, we investigated the function of cytoskeletal intermediate filament protein vimentin in the life cycle of ZIKV infection. Using advanced imaging techniques, we uncovered that vimentin filaments undergo drastic reorganization upon viral protein synthesis to form a perinuclear cage-like structure that embraces and concentrates RCs. Genetic removal of vimentin markedly disrupted the integrity of RCs and resulted in fragmented subcellular dispersion of viral proteins. This led to reduced viral genome replication, viral protein production, and release of infectious virions, without interrupting viral binding and entry. Furthermore, mass spectrometry and RNA-sequencing screens identified interactions and interplay between vimentin and hundreds of endoplasmic reticulum (ER)-resident RNA-binding proteins. Among them, the cytoplasmic-region of ribosome receptor binding protein 1, an ER transmembrane protein that directly binds viral RNA, interacted with and was regulated by vimentin, resulting in modulation of ZIKV replication. Together, the data in our work reveal a dual role for vimentin as a structural element for RC integrity and as an RNA-binding-regulating hub during ZIKV infection, thus unveiling a layer of interplay between Zika virus and host cell.

Gain-of-function genetic screening identifies the antiviral function of TMEM120A via STING activation.

Zika virus (ZIKV) infection can be associated with neurological pathologies, such as microcephaly in newborns and Guillain-Barre syndrome in adults. Effective therapeutics are currently not available. As such, a comprehensive understanding of virus-host interactions may guide the development of medications for ZIKV. Here we report a human genome-wide overexpression screen to identify host factors that regulate ZIKV infection and find TMEM120A as a ZIKV restriction factor. TMEM120A overexpression significantly inhibits ZIKV replication, while TMEM120A knockdown increases ZIKV infection in cell lines. Moreover, Tmem120a knockout in mice facilitates ZIKV infection in primary mouse embryonic fibroblasts (MEF) cells. Mechanistically, the antiviral activity of TMEM120A is dependent on STING, as TMEM120A interacts with STING, promotes the translocation of STING from the endoplasmic reticulum (ER) to ER-Golgi intermediate compartment (ERGIC) and enhances the phosphorylation of downstream TBK1 and IRF3, resulting in the expression of multiple antiviral cytokines and interferon-stimulated genes. In summary, our gain-of-function screening identifies TMEM120A as a key activator of the antiviral signaling of STING.

Evidence against Zika virus infection of pets and peri-domestic animals in Latin America and Africa.

Decades after its discovery in East Africa, Zika virus (ZIKV) emerged in Brazil in 2013 and infected millions of people during intense urban transmission. Whether vertebrates other than humans are involved in ZIKV transmission cycles remained unclear. Here, we investigate the role of different animals as ZIKV reservoirs by testing 1723 sera of pets, peri-domestic animals and African non-human primates (NHP) sampled during 2013-2018 in Brazil and 2006-2016 in Côte d'Ivoire. Exhaustive neutralization testing substantiated co-circulation of multiple flaviviruses and failed to confirm ZIKV infection in pets or peri-domestic animals in Côte d'Ivoire (n=259) and Brazil (n=1416). In contrast, ZIKV seroprevalence was 22.2% (2/9, 95% CI, 2.8-60.1) in West African chimpanzees (Pan troglodytes verus) and 11.1% (1/9, 95% CI, 0.3-48.3) in king colobus (Colobus polycomos). Our results indicate that while NHP may represent ZIKV reservoirs in Africa, pets or peri-domestic animals likely do not play a role in ZIKV transmission cycles.