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Background: Zika virus (ZIKV) is a mosquito-borne virus that is also transmitted sexually; however, the epidemiological relevance of ZIKV sexual transmission in endemic regions is unclear.Methods: We performed a household-based serosurvey in Northeast Brazil to evaluate the differential exposure to ZIKV and chikungunya virus (CHIKV) among households. Individuals who participated in our previous arboviral disease cohort (indexes) were recontacted and enrolled, and their household members were newly enrolled.Results: The relative risk of sexual partners being ZIKV-seropositive when living with a ZIKV-seropositive index participant was significantly higher, whereas this was not observed among nonsexual partners of the index. For CHIKV, both sexual and nonsexual partner household members living with a CHIKV-seropositive index had a significantly higher risk of being seropositive. In the nonindex-based dyadic and generalized linear mixed model analyses, the odds of sexual dyads having a concordant ZIKV plaque reduction neutralization test result was significantly higher. We have also analyzed retrospective clinical data according to the participants' exposure to ZIKV and CHIKV.Conclusions: Our data suggest that ZIKV sexual transmission may be a key factor for the high ZIKV seroprevalence among households in endemic areas and raises important questions about differential disease from the 2 modes of transmission. [ABSTRACT FROM AUTHOR]

Laboratory confirmation of Zika virus (ZIKV) infection during pregnancy is challenging due to cross-reactivity with dengue virus (DENV) and limited knowledge about the kinetics of anti-Zika antibody responses during pregnancy. We described ZIKV and DENV serological markers and the maternal-fetal transfer of antibodies among mothers and neonates after the ZIKV microcephaly outbreak in Northeast Brazil (2016). We included 89 microcephaly cases and 173 neonate controls at time of birth and their mothers. Microcephaly cases were defined as newborns with a particular head circumference (2 SD below the mean). Two controls without microcephaly were matched by the expected date of delivery and area of residence. We tested maternal serum for recent (ZIKV genome, IgM and IgG3 anti-NS1) and previous (ZIKV and DENV neutralizing antibodies [NAbs]) markers of infection. Multiple markers of recent or previous ZIKV and DENV infection in mothers were analyzed using principal component analysis (PCA). At delivery, 5.6% of microcephaly case mothers and 1.7% of control mothers were positive for ZIKV IgM. Positivity for ZIKV IgG3 anti-NS1 was 8.0% for case mothers and 3.5% for control mothers. ZIKV NAbs was slightly higher among mothers of cases (69.6%) than that of mothers of controls (57.2%; p = 0.054). DENV exposure was detected in 85.8% of all mothers. PCA discriminated two distinct components related to recent or previous ZIKV infection and DENV exposure. ZIKV NAbs were higher in newborns than in their corresponding mothers (p<0.001). We detected a high frequency of ZIKV exposure among mothers after the first wave of the ZIKV outbreak in Northeast Brazil. However, we found low sensitivity of the serological markers to recent infection (IgM and IgG3 anti-NS1) in perinatal samples of mothers of microcephaly cases. Since the neutralization test cannot precisely determine the time of infection, testing for ZIKV immune status should be performed as early as possible and throughout pregnancy to monitor acute Zika infection in endemic areas. [ABSTRACT FROM AUTHOR]

Anti-Flavivirus antibodies are highly cross-reactive and may facilitate Zika virus (ZIKV) infection through the antibody-dependent enhancement (ADE) mechanism. We demonstrate that dengue-specific antibodies enhance the infection of a primary Brazilian ZIKV isolate in a FcγRII-expressing K562 cell line. In addition, we demonstrate that serum samples from dengue-immune pregnant women enhanced ZIKV infection. These findings highlight the need for epidemiological studies and animal models to further confirm the role of ADE in the development of congenital and neurological complications associated with ZIKV infections. [ABSTRACT FROM AUTHOR]

Background: Maternal-fetal transferred dengue virus (DENV)-specific antibodies have been implicated in the immunopathogenesis of dengue during infancy.Methods: A prospective birth cohort was established in a dengue-endemic area in the Northeast Region of Brazil. DENV-specific immunoglobulin G (IgG) and DENV-1-4 serotype-specific neutralizing antibody (NAb) levels were assessed in 376 paired maternal and umbilical cord blood samples. The kinetics of enhancing activity by maternally acquired DENV antibodies was determined in serum samples from children enrolled in the cohort.Results: Mothers were mostly immune to DENV-3 alone (53.7%) or combined with DENV-4 (30.6%). Levels of DENV-specific IgG, DENV-3 NAbs, and DENV-4 NAbs were significantly higher in newborns than in their respective mothers. Mothers immune to a single serotype transferred greater levels of DENV-specific IgG (P = .02) and DENV-3 NAbs (P = .04) than mothers immune to multiple DENV serotypes. Maternally acquired DENV-3 NAbs disappeared in >90% of the children by the age of 4 months. The peak enhancing activity was detected by the age of 2 months (P < .0001) and rapidly declined by the age of 4 months (P = .0035).Conclusions: Unlike Asian infants, the enhancing activity of DENV infection by maternally transferred DENV antibodies occurs at earlier ages in Brazilian children. These findings might explain the low occurrence of severe dengue among infants in our setting. [ABSTRACT FROM AUTHOR]

Background: Dengue virus (DENV) was reintroduced into Brazil in 1986 and by 1995 it had spread throughout the country. In 2007 the number of dengue hemorrhagic fever (DHF) cases more than doubled and a shift in the age distribution was reported. While previously the majority of DHF cases occurred among adults, in 2007 53% of cases occurred in children under 15 years old. The reasons for this shift have not been determined. Methods and Findings: Age stratified cross-sectional seroepidemiologic survey conducted in Recife, Brazil in 2006. Serostatus was determined by ELISA based detection of Dengue IgG. We estimated time-constant and time-varying forces of infection of DENV between 1986 and 2006. We used discrete-time simulation to estimate the accumulation of monotypic and multitypic immunity over time in a population previously completely susceptible to DENV. We projected the age distribution of population immunity to dengue assuming similar hazards of infection in future years. The overall prevalence of DENV IgG was 0.80 (n = 1427). The time-constant force of infection for the period was estimated to be 0.052 (95% CI 0.041, 0.063), corresponding to 5.2% of susceptible individuals becoming infected each year by each serotype. Simulations show that as time since re-emergence of dengue goes by, multitypic immunity accumulates in adults while an increasing proportion of susceptible individuals and those with monotypic immunity are among young age groups. The median age of those monotypically immune can be expected to shift from 24 years, 10 years after introduction, to 13 years, 50 years after introduction. Of those monotypically immune, the proportion under 15 years old shifts from 27% to 58%. These results are consistent with the dengue notification records from the same region since 1995. Interpretation: Assuming that persons who have been monotypically exposed are at highest risk for severe dengue, the shift towards younger patient ages observed in Brazil can be partially explained by the accumulation of multitypic immunity against DENV-1, 2, and 3 in older age groups, 22 years after the re-introduction of these viruses. Serotype specific seroepidemiologic studies are necessary to accurately estimate the serotype specific forces of infection. Author Summary: The spread of dengue virus is a major public health problem. Though the burden of dengue has historically been concentrated in Southeast Asian countries, Brazil has become the country that reports the largest number of cases in the world. While prior to 2007 the disease affected mostly adults, during the 2007 epidemic the number of dengue hemorrhagic fever cases more than doubled, and over 53% of cases were in children under 15 years of age. In this paper, we propose that the conditions for the shift were being set gradually since the re-introduction of dengue in 1986 and that they represent the transition from re-emergence to hyperendemicity. Using data from an age stratified seroprevalence study conducted in Recife, we estimated the force of infection (a measure of transmission intensity) between 1986–2006 and used these estimates to simulate the accumulation of immunity since the re-emergence. As the length of time that dengue has circulated increases, adults have a lower probability of remaining susceptible to primary or secondary infection and thus, cases become on average younger. If in fact the shift represents the transition from re-emergence to hyperendemicity, similar shifts are likely to be observed in the rest of Brazil, the American continent and other regions where transmission emerges. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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.

Laboratory confirmation of Zika virus (ZIKV) infection during pregnancy is challenging due to cross-reactivity with dengue virus (DENV) and limited knowledge about the kinetics of anti-Zika antibody responses during pregnancy. We described ZIKV and DENV serological markers and the maternal-fetal transfer of antibodies among mothers and neonates after the ZIKV microcephaly outbreak in Northeast Brazil (2016). We included 89 microcephaly cases and 173 neonate controls at time of birth and their mothers. Microcephaly cases were defined as newborns with a particular head circumference (2 SD below the mean). Two controls without microcephaly were matched by the expected date of delivery and area of residence. We tested maternal serum for recent (ZIKV genome, IgM and IgG3 anti-NS1) and previous (ZIKV and DENV neutralizing antibodies [NAbs]) markers of infection. Multiple markers of recent or previous ZIKV and DENV infection in mothers were analyzed using principal component analysis (PCA). At delivery, 5.6% of microcephaly case mothers and 1.7% of control mothers were positive for ZIKV IgM. Positivity for ZIKV IgG3 anti-NS1 was 8.0% for case mothers and 3.5% for control mothers. ZIKV NAbs was slightly higher among mothers of cases (69.6%) than that of mothers of controls (57.2%; p = 0.054). DENV exposure was detected in 85.8% of all mothers. PCA discriminated two distinct components related to recent or previous ZIKV infection and DENV exposure. ZIKV NAbs were higher in newborns than in their corresponding mothers (p<0.001). We detected a high frequency of ZIKV exposure among mothers after the first wave of the ZIKV outbreak in Northeast Brazil. However, we found low sensitivity of the serological markers to recent infection (IgM and IgG3 anti-NS1) in perinatal samples of mothers of microcephaly cases. Since the neutralization test cannot precisely determine the time of infection, testing for ZIKV immune status should be performed as early as possible and throughout pregnancy to monitor acute Zika infection in endemic areas., Competing Interests: The authors have declared that no competing interests exist.

Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.

Background: Dengue affects nearly 400 million people annually worldwide and considered one of the most serious health threats in tropical and subtropical countries., Objective: To analyze the occurrence of dengue infection among the parturient who have formed the baseline of an ongoing birth cohort study in the city of Recife, Northeast of Brazil., Study Design: From March 2011 to May 2012, we recruited 417 parturients with low-risk pregnancies at maternity ward who agreed to a follow-up of their babies. Dengue infection was accessed through DENV RT-PCR and anti-dengue antibodies (IgM and IgG). The prevalence of IgG antibodies in the parturients and their concepts were determined. The concordance among the pairs was tested using Kappa. The association of recent infection (IgM and/or DENV RT-PCR positive) with the maternal characteristics and clinical features of the neonates was analyzed through logistic regression., Results: The prevalence of IgG antibodies in the maternal and cord blood samples was 95.1% (95% CI: 92.6-96.9%) and 95.8% (95% CI: 93.4-97.5%), respectively, with high agreement between maternal-cord pairs (Kappa=0.93). The prevalence of recent infection was 10.6% (95% CI: 7.9-14.2%) in the parturients. Reported fever during pregnancy was associated to recent infection (p=0.023)., Conclusion: The data draw attention for the high frequency of anti-dengue antibodies in the women studied and for the high occurrence of infection during pregnancy in this region of Brazil. There is a need for further studies to better characterize dengue infection in pregnant women., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Four genetic polymorphisms located at the promoter (C-257T) and coding regions of CFH gene (exon 2 G257A, exon 14 A2089G and exon 19 G2881T) were investigated in 121 dengue patients (DENV-3) in order to assess the relationship between allele/haplotypes variants and clinical outcomes. A statistical value was found between the CFH-257T allele (TT/TC genotypes) and reduced susceptibility to severe dengue (SD). Statistical associations indicate that individuals bearing a T allele presented significantly higher protein levels in plasma. The -257T variant is located within a NF-κB binding site, suggesting that this variant might have effect on the ability of the CFH gene to respond to signals via the NF-κB pathway. The G257A allelic variant showed significant protection against severe dengue. When CFH haplotypes effect was considered, the ancestral CG/CG promoter-exon 2 SNP genotype showed significant risk to SD either in a general comparison (ancestral × all variant genotypes), as well as in individual genotypes comparison (ancestral × each variant genotype), where the most prevalent effect was observed in the CG/CG × CA/TG comparison. These findings support the involvement of -257T, 257A allele variants and haplotypes on severe dengue phenotype protection, related with high basal CFH expression., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated, and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes, but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum-neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil, which is associated with increasing disease severity.

Background: We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples., Methodology/principal Findings: The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity: classic dengue fever or dengue hemorrhagic fever (DHF) samples were compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2-3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels., Conclusions/significance: Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection.