Comprehensive analysis of early T cell responses to acute Zika Virus infection during the first epidemic in Bahia, Brazil.

BACKGROUND: In most cases, Zika virus (ZIKV) causes a self-limited acute illness in adults, characterized by mild clinical symptoms that resolve within a few days. Immune responses, both innate and adaptive, play a central role in controlling and eliminating virus-infected cells during the early stages of infection. AIM: To test the hypothesis that circulating T cells exhibit phenotypic and functional activation characteristics during the viremic phase of ZIKV infection. METHODS: A comprehensive analysis using mass cytometry was performed on peripheral blood mononuclear cells obtained from patients with acute ZIKV infection (as confirmed by RT-PCR) and compared with that from healthy donors (HD). The frequency of IFN-γ-producing T cells in response to peptide pools covering immunogenic regions of structural and nonstructural ZIKV proteins was quantified using an ELISpot assay. RESULTS: Circulating CD4+ and CD8+ T lymphocytes from ZIKV-infected patients expressed higher levels of IFN-γ and pSTAT-5, as well as cell surface markers associated with proliferation (Ki-67), activation ((HLA-DR, CD38) or exhaustion (PD1 and CTLA-4), compared to those from HD. Activation of CD4+ and CD8+ memory T cell subsets, including Transitional Memory T Cells (TTM), Effector Memory T cells (TEM), and Effector Memory T cells Re-expressing CD45RA (TEMRA), was prominent among CD4+ T cell subset of ZIKV-infected patients and was associated with increased levels of IFN-γ, pSTAT-5, Ki-67, CTLA-4, and PD1, as compared to HD. Additionally, approximately 30% of ZIKV-infected patients exhibited a T cell response primarily directed against the ZIKV NS5 protein. CONCLUSION: Circulating T lymphocytes spontaneously produce IFN-γ and express elevated levels of pSTAT-5 during the early phase of ZIKV infection whereas recognition of ZIKV antigen results in the generation of virus-specific IFN-γ-producing T cells.

Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope.

Antigenic similarities between Zika virus (ZIKV) and other flaviviruses pose challenges to the development of virus-specific diagnostic tools and effective vaccines. Starting with a DNA-encoded one-bead-one-compound combinatorial library of 508,032 synthetic, non-natural oligomers, we selected and characterized small molecules that mimic ZIKV epitopes. High-throughput fluorescence-activated cell sorter-based bead screening was used to select molecules that bound IgG from ZIKV-immune but not from dengue-immune sera. Deep sequencing of the DNA from the "Zika-only" beads identified 40 candidate molecular structures. A lead candidate small molecule "CZV1-1" was selected that correctly identifies serum specimens from Zika-experienced patients with good sensitivity and specificity (85.3% and 98.4%, respectively). Binding competition studies of purified anti-CZV1-1 IgG against known ZIKV-specific monoclonal antibodies (mAbs) showed that CZV1-1 mimics a nonlinear, neutralizing conformational epitope in the domain III of the ZIKV envelope. Purified anti-CZV1-1 IgG neutralized infection of ZIKV in cell cultures with potencies comparable to highly specific ZIKV-neutralizing mAbs. This study demonstrates an innovative approach for identification of synthetic non-natural molecular mimics of conformational virus epitopes. Such molecular mimics may have value in the development of accurate diagnostic assays for Zika, as well as for other viruses.

Prevalence of arboviruses in sickle cell disease patients from two different regions of Brazil, the North and Northeast.

Sickle Cell Disease (SCD) is a hereditary disease characterized by extravascular and intravascular hemolysis and clinical variability, from mild pain to potentially life-threatening. Arboviruses include mainly Zika (ZIKV), Chikungunya (CHKV), and Dengue (DENV) virus, and are considered a public and social health problem. The present cross-sectional observational study aimed to investigate the prevalence of arbovirus infection in SCD patients from two Brazilian cities, Salvador and Manaus located in Bahia and Amazonas states respectively. A total of 409 individuals with SCD were included in the study, and 307 (75.06 %) patients tested positive for DENV-IgG, 161 (39.36 %) for ZIKV-IgG, and 60 (14.67 %) for CHIKV-IgG. Only one individual was positive for DENV-NS1 and another for DENV-IgM, both from Salvador. No individuals had positive serology for ZIKV-IgM or CHIKV-IgM. Arbovirus positivity by IgG testing revealed that the SCD group presented high frequencies in both cities. Interestingly, these differences were only statistically significant for ZIKV-IgG (p = 0.023) and CHIKV-IgG (p = 0.005) among SCD patients from Manaus. The reshaping of arbovirus from its natural habitat by humans due to disorderly urban expansion and the ease of international Mobility has been responsible for facilitating the spread of vector-borne infectious diseases in humans. We found the need for further studies on arboviruses in this population to elucidate the real association and impact, especially in acute infection. We hope that this study will contribute to improvements in the personalized clinical follow-up of SCD patients, identifying the influence of arbovirus infection in severe disease manifestations.

Global seroprevalence of Zika virus in asymptomatic individuals: A systematic review.

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.

The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses.

Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology.

Harmonization of Zika serological assays and comparative evaluation of two commercial ZIKA IgG ELISA kits.

The interpretation for Zika virus serology results is challenging due to high antibody cross reactivity with other flaviviruses. This limits availability of reliable and accurate methods for serosurveillance studies to understand the disease burden. Therefore, we conducted study to harmonize anti-Zika IgG antibody detection assays with 1st WHO International Standard (16/352) and working standard (16/320) for anti-Zika virus antibody.Additionally, evaluated NuGenTMZIKA-IgG and NovaLisa®ZIKA virus IgG-Capture ELISA using a panel of 278 seraFurther, 106 samples positive for other-flavi viruses were taken for assessing cross-reactivity of the assay, all serums were further tested by Zika-PRNT. The results of this study indicates satisfactory performance of both the assays. Serological and neutralization assays were calibrated according to the international standards. This will help in understanding antibody dynamics in serosurveillance and vaccine studies. However the performance of the kits with possibilities of cross-reactivity will have to be verified by coupling ZIKV and DENV specific ELISA.

A Divalent Chikungunya and Zika Nanovaccine with Thermostable Self-Assembly Multivalent Scaffold LS-SUMO.

The convergence strategies of antigenic subunits and synthetic nanoparticle scaffold platform improve the vaccine production efficiency and enhance vaccine-induced immunogenicity. Selecting the appropriate nanoparticle scaffold is crucial to controlling target antigens immunologically. Lumazine synthase (LS) is an attractive candidate for a vaccine display system due to its thermostability, modification tolerance, and morphological plasticity. Here, the first development of a multivalent thermostable scaffold, LS-SUMO (SUMO, small ubiquitin-likemodifier), and a divalent nanovaccine covalently conjugated with Chikungunya virus E2 and Zika virus EDIII antigens, is reported. Compared with antigen monomers, LS-SUMO nanoparticle vaccines elicit a higher humoral response and neutralizing antibodies against both antigen targets in mouse sera. Mice immunized with LS-SUMO conjugates produce CD4+ T cell-mediated Th2-biased responses and promote humoral immunity. Importantly, LS-SUMO conjugates possess equivalent humoral immunogenicity after heat treatment. Taken together, LS-SUMO is a powerful biotargeting nanoplatform with high-yield production, thermal stability and opens a new avenue for multivalent presentation of various antigens.

Design of a multi-epitope Zika virus vaccine candidate - an in-silico study.

Zika virus (ZIKV), an RNA virus, rapidly spreads Aedes mosquito-borne sickness. Currently, there are neither effective vaccines nor therapeutics available to prevent or treat ZIKV infection. In this study, to address these unmet medical needs, we aimed to design B- and T-cell candidate multi-epitope-based subunit against ZIKV using an in silico approach. In this study we applied immunoinformatics, molecular docking, and dynamic simulation assessments targeting the most immunogenic proteins; the capsid (C), envelope (E) proteins and the non-stuctural protein (NS1), described in our previous study, and which predicted immunodominant B and T cell epitopes. The final non-allergenic and highly antigenic multi-epitope was constituted of immunogenic screened-epitopes (3 CTL and 3 HTL) and the ß-defensin as an adjuvant that have been linked using EAAAK, AAY, and GPGPG linkers, respectively. The final construct containing 143 amino acids was characterized for its allergenicity, antigenicity, and physiochemical properties; and found to be safe and immunogenic with a good prediction of solubility. The existence of IFN-γ epitopes asserts the capacity to trigger strong immune responses. Subsequently, the molecular docking among vaccine and immune receptors (TLR2/TLR4) was revealed with a good binding affinity with and stable molecular interactions. Molecular dynamics simulation confirmed the stability of the complexes. Finally, the construct was subjected to in silico cloning demonstrating the efficiently of its expression in E.coli. However, this study needs the experimental validation to demonstrate vaccine safety and efficacy.Communicated by Ramaswamy H. Sarma.

Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity.

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function.

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

Evaluation of Serologic Cross-Reactivity Between Dengue Virus and SARS-CoV-2 in Patients with Acute Febrile Illness - United States and Puerto Rico, April 2020-March 2021.

The diagnosis of dengue disease, caused by the dengue virus (DENV) (a flavivirus), often requires serologic testing during acute and early convalescent phases of the disease. Some symptoms of DENV infection, such as nonspecific fever, are similar to those caused by infection with SARS-CoV-2, the virus that causes COVID-19. In studies with few COVID-19 cases, positive DENV immunoglobulin M (IgM) results were reported with various serologic tests, indicating possible cross-reactivity in these tests for DENV and SARS-CoV-2 infections (1,2). DENV antibodies can cross-react with other flaviviruses, including Zika virus. To assess the potential cross-reactivity of SARS-CoV-2, DENV, and Zika virus IgM antibodies, serum specimens from 97 patients from Puerto Rico and 12 U.S.-based patients with confirmed COVID-19 were tested using the DENV Detect IgM Capture enzyme-linked immunosorbent assay (ELISA) (InBios International).* In addition, 122 serum specimens from patients with confirmed dengue and 121 from patients with confirmed Zika virus disease (all from Puerto Rico) were tested using the SARS-CoV-2 pan-Ig Spike Protein ELISA (CDC).† Results obtained for DENV, Zika virus IgM, and SARS-CoV-2 antibodies indicated 98% test specificity and minimal levels of cross-reactivity between the two flaviviruses and SARS-CoV-2. These findings indicate that diagnoses of dengue or Zika virus diseases with the serological assays described in this report are not affected by COVID-19, nor do dengue or Zika virus diseases interfere with the diagnosis of COVID-19.

One antibody to treat them all.

Conserved flavivirus protein holds potential as target for versatile vaccines and therapies.

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.

Current Understanding of the Role of T Cells in Chikungunya, Dengue and Zika Infections.

Arboviral infections such as Chikungunya (CHIKV), Dengue (DENV) and Zika (ZIKV) are a major disease burden in tropical and sub-tropical countries, and there are no effective vaccinations or therapeutic drugs available at this time. Understanding the role of the T cell response is very important when designing effective vaccines. Currently, comprehensive identification of T cell epitopes during a DENV infection shows that CD8 and CD4 T cells and their specific phenotypes play protective and pathogenic roles. The protective role of CD8 T cells in DENV is carried out through the killing of infected cells and the production of proinflammatory cytokines, as CD4 T cells enhance B cell and CD8 T cell activities. A limited number of studies attempted to identify the involvement of T cells in CHIKV and ZIKV infection. The identification of human immunodominant ZIKV viral epitopes responsive to specific T cells is scarce, and none have been identified for CHIKV. In CHIKV infection, CD8 T cells are activated during the acute phase in the lymph nodes/blood, and CD4 T cells are activated during the chronic phase in the joints/muscles. Studies on the role of T cells in ZIKV-neuropathogenesis are limited and need to be explored. Many studies have shown the modulating actions of T cells due to cross-reactivity between DENV-ZIKV co-infections and have repeated heterologous/homologous DENV infection, which is an important factor to consider when developing an effective vaccine.

Involvement of Th1Th17 Cell Subpopulations in the Immune Responses of Mothers Who Gave Birth to Children with Congenital Zika Syndrome (CZS).

High levels of T helper 17 cell (Th17)-related cytokines have been shown in acute Zika virus (ZIKV) infection. We hypothesized that the high levels of Th17-related cytokines, associated with a regulatory environment during pregnancy, create a favorable milieu for the differentiation of CD4+Th17 cells. We present data from a cross-sectional study on mothers who confirmed ZIKV infection by qRT-PCR and their children. We also recruited non-pregnant women infected with ZIKV in the same period. ZIKV infection occurred between 2015 and 2017. We collected samples for this study between 2018 and 2019, years after the initial infection. We highlight that, after in vitro stimulation with ZIKV CD4 megapool (ZIKV MP), we found a lower frequency of IL-17-producing CD4+ T cells (Th17), especially in the mothers, confirmed by the decrease in IL-17 production in the supernatant. However, a higher frequency of CD4+ IL-17+ IFN-γ+ T cells (Th1Th17) responding to the ZIKV MP was observed in the cells of the mothers and children but not in those of the non-pregnant women. Our data indicate that the priming of CD4 T cells of the Th1Th17 phenotype occurred preferentially in the mothers who gave birth to children with CZS and in the children.

Zika Virus Immunoglobulin G Seroprevalence among Young Adults Living with HIV or without HIV in Thailand from 1997 to 2017.

Zika virus (ZIKV) epidemiological data in Thailand are limited. We assessed ZIKV IgG seroprevalence among young adults during 1997-2017 and determined factors associated with ZIKV IgG seropositivity. This retrospective laboratory study included randomly selected subjects aged 18-25 years participating in large clinical studies conducted in Thailand during 1997-2017. Stored plasma samples were analyzed for ZIKV IgG using an ELISA test (Anti-Zika Virus IgG, EUROIMMUN, Lübeck, Germany). Sociodemographic, clinical and laboratory data were used in univariable and multivariable analyses to identify factors associated with ZIKV IgG positivity. Of the 1648 subjects included, 1259 were pregnant women, 844 were living with HIV and 111 were living with HBV. ZIKV IgG seroprevalence was similar among the HIV-infected and -uninfected pregnant women (22.8% vs. 25.8%, p-value = 0.335) and was overall stable among the pregnant women, with a 25.2% prevalence. Factors independently associated with ZIKV IgG positivity included an age of 23-25 years as compared to 18-20 years, an HIV RNA load below 3.88 log10 copies/mL and birth in regions outside northern Thailand. Our study shows that a large proportion of the population in Thailand probably remains susceptible to ZIKV infection, which could be the ground for future outbreaks. Continued surveillance of ZIKV spread in Thailand is needed to inform public health policies.

Zika virus baculovirus-expressed envelope protein elicited humoral and cellular immunity in immunocompetent mice.

Zika virus (ZIKV) is a mosquito-borne virus that has a high risk of inducing Guillain-Barré syndrome and microcephaly in newborns. Because vaccination is considered the most effective strategy against ZIKV infection, we designed a recombinant vaccine utilizing the baculovirus expression system with two strains of ZIKV envelope protein (MR766, Env_M; ZBRX6, Env_Z). Animals inoculated with Env_M and Env_Z produced ZIKV-specific antibodies and secreted effector cytokines such as interferon-γ, tumor necrosis factor-α, and interleukin-12. Moreover, the progeny of immunized females had detectable maternal antibodies that protected them against two ZIKV strains (MR766 and PRVABC59) and a Dengue virus strain. We propose that the baculovirus expression system ZIKV envelope protein recombinant provides a safe and effective vaccine strategy.

The seroepidemiology of dengue in a US military population based in Puerto Rico during the early phase of the Zika pandemic.

Understanding the burden and risk factors of dengue virus (DENV) infection in Puerto Rico is important for the prevention of dengue in local, traveler and military populations. Using sera from the Department of Defense Serum Repository, we estimated the prevalence and predictors of DENV seropositivity in those who had served in Puerto Rico, stratified by birth or prior residence ("birth/residence") in dengue-endemic versus non-endemic regions. We selected sera collected in early 2015 from 500 U.S. military members, a time-point also permitting detection of early cryptic Zika virus (ZIKV) circulation. 87.2% were born or resided in a DENV-endemic area before their military service in Puerto Rico. A high-throughput, flow-cytometry-based neutralization assay was employed to screen sera for ZIKV and DENV neutralizing antibodies, and confirmatory testing was done by plaque-reduction neutralization test (PRNT). We identified one Puerto Rico resident who seroconverted to ZIKV by June 2015, suggesting cryptic ZIKV circulation in Puerto Rico at least 4 months before the first reported cases. A further six PRNT-positive presumptive ZIKV infections which were resolved as DENV infections only by the use of paired sera. We noted 66.8% of the total study sample was DENV seropositive by early 2015. Logistic regression analysis indicated that birth/residence in a dengue non-endemic region (before military service in Puerto Rico) was associated with a lower odds of DENV exposure by January-June 2015 (aOR = 0.28, p = 0.001). Among those with birth/residence in a non-endemic country, we noted moderate evidence to support increase in odds of DENV exposure for each year of military service in Puerto Rico (aOR = 1.58, p = 0.06), but no association with age. In those with birth/residence in dengue-endemic regions (before military service in Puerto Rico), we noted that age (aOR = 1.04, p = 0.02), rather than duration of Puerto Rico service, was associated with dengue seropositivity, suggesting earlier lifetime DENV exposure. Our findings provide insights into the burden and predictors of DENV infection in local, traveler and military populations in Puerto Rico. Our study also highlights substantial PRNT ZIKV false-positivity when paired sera are not available, even during periods of very low ZIKV prevalence.