Dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) are medically important arboviruses causing severe human disease worldwide and in regions of the US, but optimal serologic diagnosis is lacking?a disconcerting problem for returning travelers, pregnant women, and those living in endemic regions. Since a large proportion of DENV, ZIKV, and CHIKV infections are asymptomatic, patients seeking medical care for acute infection only represent the ?tip of the iceberg?. Furthermore, existing serologic tests for DENV and ZIKV are compromised by cross-reactive antibody responses. Thus, the true population immunity and susceptibility to arboviral infections remains unclear. At the patient level, precise knowledge of recent and past arbovirus infection is needed to inform clinical decisions, whereas at the population level, precise knowledge of immunity and attack rates (including asymptomatic infection) is needed to prioritize interventions such as vaccination. Our long-term goal is to develop highly sensitive and specific oral fluid (OF) multiplex arbovirus diagnostics that pave the way for future OF point-of-care tests. Our objective here is to develop and validate tests for differential detection of IgG specific for ZIKV, DENV, and CHIKV infection using oral fluid (OF) specimens. Our hypothesis is that pathogen-specific IgG in OF will reflect the antibody (Ab) binding profile observed in serum, and that novel ZIKV-, DENV-, and CHIKV-specific antigens can be integrated into a multiplex Ab binding assay that confers adequate sensitivity and specificity for detecting convalescent phase arbovirus infection. The feasibility of our goal is supported by our published work demonstrating that OF hepatitis E virus (HEV) IgG assays perform equally well as blood based HEV diagnostics and by our preliminary data demonstrating the differential detection of DENV- and ZIKV-specific IgG in plasma and matched OF using novel recombinant antigens. To achieve our goal we will pursue two aims: 1) Build a well-characterized OF panel with matched plasma to develop OF arbovirus diagnostics; and 2) Optimize and validate the multiplex OF arbovirus Ab assays. For aim 1 we will leverage four ongoing studies to build a singular collection of matched OF and blood specimens for optimization and validation of arbovirus OF diagnostics: 1) a human DENV vaccination and post-vaccination challenge study; 2) the first human ZIKV challenge study; 3) a study of returning US travelers with DENV, ZIKV and CHIKV infections; and 4) a mother-infant cohort in Nicaragua where DENV, ZIKV and CHIKV are endemic. Our multi-disciplinary team has in-depth expertise in controlled human vaccine and epidemiologic arbovirus field studies, in novel arbovirus antigen development, arbovirus serodiagnostics, and in oral fluid diagnostics. Our approach is innovative because it represents the most comprehensive endeavor to produce a multiplex OF arbovirus Ab assay and to validate it using real-world specimens. The proposed research would revolutionize public health efforts by providing a practical tool to refine the spatial and temporal scales of arbovirus surveillance and prioritization of vaccine and vector control interventions for disease prevention.