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.

Two-year Decay of Zika Virus Neutralizing Antibodies in People Living in an Endemic Region in Brazil.

It is currently not clear whether humoral immunity to Zika virus (ZIKV) elicited upon natural ZIKV infection is long-lasting. In addition, cross-reactivity of anti-ZIKV antibodies with antigenically related dengue viruses (DENV) may have biological implications in nonnaive individuals who subsequently acquire a heterotypic infection. Cross-reactive humoral immunity between ZIKV and DENV also complicates the interpretation of serological tests to evaluate previous exposure to either virus. Here, we have measured the 2-year decay of ZIKV neutralizing antibodies in people living in a ZIKV/DENV endemic area in Brazil who were identified as having an acute infection (group 1) or past (but recent) infection (group 2) with ZIKV in 2015-16. The titers of neutralizing antibodies to ZIKV decreased 9.1 and 2.3 times in groups 1 and 2, respectively. We also show that the plaque reduction neutralization assay (PRNT) is a reliable method to measure past exposure to ZIKV in coendemic areas.

Audiological follow-up of children with congenital Zika syndrome.

How does the auditory function of children with congenital Zika syndrome present during the first three years of life? To determine the auditory function of children with congenital Zika syndrome during the first three years of life and estimate the frequency and long-term presentation of hearing loss in this syndrome, an auditory assessment with screening and diagnostic tests was conducted. The screening test consisted of measuring the short latency ABR using click stimuli. If the ABR click indicated hearing loss, confirmation was obtained with a frequency-specific ABR (FS-ABR), in which the stimuli were tone bursts at frequencies of 500 and 2000 Hz by bone and air conduction. This case series included 107 children with confirmed congenital Zika syndrome, and the cumulative incidence of sensorineural hearing loss in the first three years of life was 9.3% (10/107). There were no cases of delayed-onset or progressive deficits in hearing. Early presentation of sensorineural hearing loss seems to occur with a higher frequency in children with congenital Zika syndrome than in the general population. Sensorineural hearing loss resulting from congenital Zika virus infection does not appear to present with delayed onset or with progressive deficits.

Self-Enriching Electrospun Biosensors for Impedimetric Sensing of Zika Virus.

Zika virus (ZIKV) infection is associated with the Guillain-Barré syndrome, and when non-vector congenital transmission occurs, fetal brain abnormalities are expected. After ZIKV infection, the blood, breast milk, and other body fluids contain low viral loads. Their detection is challenging as it requires the processing of larger input volumes of the clinical samples. Pre-enrichment is a valuable strategy to increase the analyte concentration. Therefore, the authors propose the use of a hierarchal composite polyaniline-(electrospun nanofiber) hydrogel mat (ENM) for the simultaneous enrichment and impedimetric sensing of ZIKV viral particles. The electrospinning conditions of polyvinyl alcohol and alginate, including blend formulation, were optimized through a factorial design. Disintegration and gelatinization were controlled via cross-linking to improve the hydrogel properties. Hierarchization was achieved by in situ chemical deposition of conductive polyaniline. The carboxyl groups of the ENM were used for the covalent immobilization of anti-ZIKV polyclonal antibodies used in the specific recognition of ZIKV within the medium of Vero cell culture. The specific capture and desorption of virions were studied at different pHs. ENMs were characterized by scanning electron microscopy and FTIR. Atomic force microscopy along with UV-vis and electrochemical impedance spectroscopies was used to monitor the antibody immobilization, ZIKV capture, and elution processes. Our results show that 14.2 mg (0.25 cm3) of ENM can capture 38.7 ± 2.5 µg of ZIKV with a desorption rate of 99.97% (38.29 ± 2.7 µg ZIKV), which is reusable for at least three times. Therefore, the capture capacity (micrograms of ZIKV captured per milligram of ENM) of polyaniline-hierarchized mats was 2.72 µg ZIKV/mg. The impedance LOD value was determined to be 2.76 µg of ZIKV particles (approximately 6.6 × 103 PFU/mL). As a result, we present a fast small-scale purification system that can simultaneously monitor ZIKV electrochemically and optically.

Identification of a Zika NS2B epitope as a biomarker for severe clinical phenotypes.

The identification of specific biomarkers for Zika infection and its clinical complications is fundamental to mitigate the infection spread, which has been associated with a broad range of neurological sequelae. We present the characterization of antibody responses in serum samples from individuals infected with Zika, presenting non-severe (classical) and severe (neurological disease) phenotypes, with high-density peptide arrays comprising the Zika NS1 and NS2B proteins. The data pinpoints one strongly IgG-targeted NS2B epitope in non-severe infections, which is absent in Zika patients, where infection progressed to the severe phenotype. This differential IgG profile between the studied groups was confirmed by multivariate data analysis. Molecular dynamics simulations and circular dichroism have shown that the peptide in solution presents itself in a sub-optimal conformation for antibody recognition, which led us to computationally engineer an artificial protein able to stabilize the NS2B epitope structure. The engineered protein was used to interrogate paired samples from mothers and their babies presenting Zika-associated microcephaly and confirmed the absence of NS2B IgG response in those samples. These findings suggest that the assessment of antibody responses to the herein identified NS2B epitope is a strong candidate biomarker for the diagnosis and prognosis of Zika-associated neurological disease.

NS1 glycoprotein detection in serum and urine as an electrochemical screening immunosensor for dengue and Zika virus.

The incidence of infection by the dengue virus (DENV) has grown dramatically, reaching 128 countries in tropical and subtropical regions worldwide, with a pattern of hyper-endemicity. DENV is a mosquito-borne disease having four serotypes, one or two circulating in epidemic outbreaks. The diagnosis of DENV is challenging mainly due to the circulation of new viruses with remarkable similarities, such as Zika (ZIKV) that may cause fetal microcephaly. DENV affects 390 million people per year, but these numbers may be higher due to the underreported and misclassified cases. Recently, the NS1 nonstructural protein has been described in serum and urine of DENV and ZIKV patients, suggesting its use as a biomarker for screening since a negative NS1 sample confirms the absence of these infections. Herein, a label-free immunosensor comprising an assembled nanostructured thin film of carbon nanotube-ethylenediamine is described. The advantage of in situ electrosynthesis of polymer film is to allow major control of thickness and conductivity, in addition to designing the reactive groups for functionalization. A quartz crystal microbalance system was used to estimate the thickness of the polymeric film obtained. The anti-NS1 monoclonal antibodies were immobilized to carbon nanotubes by covalent linkage, permitting a high stability during measurements. Analytical responses to NS1 were obtained by differential pulse voltammetry (DPV), showing a linear range from 20 to 800 ng mL-1 and reproducibility of 3.0%, with a limit of detection (LOD) of 6.8 ng mL- 1. This immunosensor was capable of detecting ZIKV and DENV NS1 in spiked urine and real serum in a clinical range.Graphical abstract.

A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein.

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube-polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

High Incidence of Zika or Chikungunya Infection among Pregnant Women Hospitalized Due to Obstetrical Complications in Northeastern Brazil-Implications for Laboratory Screening in Arbovirus Endemic Area.

The diagnostic of arbovirus-related obstetric complications in high-risk pregnancy and childbirth care is challenging, especially in endemic areas. We conducted a prospective study to track active or recent Zika (ZIKV), dengue (DENV), or chikungunya (CHIKV) virus infection among hospitalized pregnant women (PW) with obstetric complications in a hospital at the epicenter of Zika outbreak and ZIKV-related microcephaly in Brazil. Clinical data and blood samples were collected at enrollment and 10 days after the admission of study participants, between October 2018 and May 2019. Further clinical data were extracted from medical records. Samples were screened by molecular and serological tests. Out of 780 participants, 93.1% (95% CI: 91.1-94.7%) presented previous DENV exposure (IgG). ZIKV, CHIKV, and/or DENV laboratory markers of recent or active infection were detected in 130 PW, yielding a prevalence of 16.6% (95% CI: 14.2-19.5%); 9.4% (95% CI: 7.4-11.7%), 7.4% (95% CI: 5.7-9.7%), and 0.38% (95% CI: 0.1-1.2%) of CHIKV, ZIKV, and DENV infections, respectively. Most ZIKV infections were detected by molecular assays (89.6%), while CHIKV infections were detected by serology (95.9%). Our findings highlight the need for arbovirus infections screening in PW with obstetrical complications, potentially associated to these infections in endemic areas regardless of the signs or symptoms suggestive of arboviral disease.

The Prevalence of Adenoid Hypertrophy among Children with Zika Related Microcephaly.

Upper respiratory obstruction is a common sequela in children with Zika-related microcephaly (ZRM). As a cross-sectional analysis nested in a cohort study, this study aims to investigate the prevalence of adenoid hypertrophy (AH) in children with ZRM and symptoms of respiratory obstruction. The data were collected in the first three years of life from children with ZRM who were followed in two reference centers for otorhinolaryngological care of patients with congenital Zika syndrome. Out of 92 children with confirmed ZRM, 57 were evaluated by nasopharyngoscopy after presenting with upper respiratory obstruction symptoms. In this study, 31 of the 57 (54%) children with ZRM who were evaluated had obstructive AH. Thirteen children with obstructive AH were submitted to surgery, which resulted in the complete resolution of symptoms for 11, partial resolution in 1, and no improvement in 1. No evidence of direct involvement by Zika virus (ZIKV) infection in the adenoid tissues was demonstrated by histology or immunohistochemistry. Our results suggest that there is a high prevalence and early presentation of AH in children with ZRM, with consequent upper airway obstruction causing upper airway obstructive disorder, secretory otitis media, and dysphagia.

Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors.

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.