The co-circulation of the Arboviruses Zika, Dengue and Chikungunya in the tropical and subtropical regions of the Americas, including Nicaragua, has resulted in an unprecedented epidemiological emergency. Secondary Dengue infection is a major risk factor for severe dengue disease due to cross-reactive antibodies, resulting in vascular leakage that can lead to shock. In 2015, yet another flavivirus, Zika, was introduced in the region. Zika has proven to be a major public health threat as it is linked to congenital birth defects and Guillain-Barré syndrome. The co-circulation of these viruses poses unique challenges in terms clinical management for differentiating the three illnesses and addressing long-term clinical consequences of ZIKV and CHIKV infection, as well as serological laboratory diagnosis and national surveillance programs and potential immune enhancement of flavivirus infection and disease.

This community based study addresses these challenges by leveraging two existing studies in Managua: the Pediatric Dengue Cohort Study (ongoing since 2004) and a hospital-based study (ongoing since 1998) as well as the extensive infrastructure already in place from previous and ongoing NIH-supported Zika, dengue, chikungunya, and influenza studies in Managua. Our projects and sites have demonstrated high-quality laboratory, epidemiological, clinical, and operational effectiveness, together with extensive community buy-in and support from the Nicaraguan Ministry of Health. Here, we propose to combine and expand the characterization of all three co- circulating arboviruses over the next 5 years.

SPECIFIC AIM 1. Epidemiological and clinical characterization of acute arboviral infection and risk factor analysis. There is an urgent need to better understand the natural history of ZIKV, especially during concomitant circulation of DENV and CHIKV. Similarly, little is known about the natural history of pediatric CHIKV. We hypothesize that arboviral co-infection and co-morbidities represent significant risk factors for pediatric arboviral infection, along with age, gender and household-related factors.

Aim 1.1. Epidemiology and spatial analysis of concurrent Zika, dengue and chikungunya. Aim 1.2. Natural history of pediatric Zika and chikungunya, including long-term follow-up. Aim 1.3. Risk factor analysis for infection outcome and disease severity.

SPECIFIC AIM 2. Develop and evaluate diagnostic tools for arboviral infection. Antibodies against the two flaviviruses, DENV and ZIKV, can be cross-reactive, especially in 2° flavivirus infections. We hypothesize that using novel, next-gen approaches (e.g., peptide arrays, envelope protein domains or mutants), it will be possible to develop reliable diagnostic tools (in collaboration with Drs. Aravinda de Silva, Daved Fremont and Ian Lipkin) to serologically distinguish DENV from ZIKV infection.

Aim 2.1. Establish and evaluate conventional serological assays (IgM, IgG, Inhibition ELISA; NS1 indirect ELISA). Aim 2.2. Evaluate next-gen serological assays for differential diagnosis of ZIKV and DENV infection.

SPECIFIC AIM 3. Study antibody-dependent enhancement and immune responses in DENV/ZIKV sequential infections. We hypothesize that sequential flavivirus infections (i.e. 2° flavivirus infection) may lead to immune enhancement and modulate clinical outcomes. We will analyze DENV-ZIKV (both primary and 2° DENV) and ZIKV-DENV infection(s).
Aim 3.1. Define DENV and ZIKV immune history and status in the Nicaraguan pediatric studies.

Aim 3.2. Characterize the effect of prior immunity to DENV/ZIKV on infection outcome, including inapparent, symptomatic and severe infection.
Aim 3.3. Characterize the quality, breadth and magnitude of antibody and B cell responses in sequential flavivirus infections, using antibody depletion assays and the Quad-Color FluoroSpot. Overall, we propose the epidemiological, clinical and immunological characterization of arboviroses in Nicaragua by continuing a successful and responsive program directed towards critical pandemic diseases.