Multiomics Approach Reveals Serum Biomarker Candidates for Congenital Zika Syndrome.

The Zika virus (ZIKV) can be vertically transmitted, causing congenital Zika syndrome (CZS) in fetuses. ZIKV infection in early gestational trimesters increases the chances of developing CZS. This syndrome involves several pathologies with a complex diagnosis. In this work, we aim to identify biological processes and molecular pathways related to CZS and propose a series of putative protein and metabolite biomarkers for CZS prognosis in early pregnancy trimesters. We analyzed serum samples of healthy pregnant women and ZIKV-infected pregnant women bearing nonmicrocephalic and microcephalic fetuses. A total of 1090 proteins and 512 metabolites were identified by bottom-up proteomics and untargeted metabolomics, respectively. Univariate and multivariate statistical approaches were applied to find CZS differentially abundant proteins (DAP) and metabolites (DAM). Enrichment analysis (i.e., biological processes and molecular pathways) of the DAP and the DAM allowed us to identify the ECM organization and proteoglycans, amino acid metabolism, and arachidonic acid metabolism as CZS signatures. Five proteins and four metabolites were selected as CZS biomarker candidates. Serum multiomics analysis led us to propose nine putative biomarkers for CZS prognosis with high sensitivity and specificity.

Proteomics and Metabolomics in Congenital Zika Syndrome: A Review of Molecular Insights and Biomarker Discovery.

Zika virus (ZIKV) infection can be transmitted vertically, leading to the development of congenital Zika syndrome (CZS) in infected fetuses. During the early stages of gestation, the fetuses face an elevated risk of developing CZS. However, it is important to note that late-stage infections can also result in adverse outcomes. The differences between CZS and non-CZS phenotypes remain poorly understood. In this review, we provide a summary of the molecular mechanisms underlying ZIKV infection and placental and blood-brain barriers trespassing. Also, we have included molecular alterations that elucidate the progression of CZS by proteomics and metabolomics studies. Lastly, this review comprises investigations into body fluid samples, which have aided to identify potential biomarkers associated with CZS.

The Hip of Children with Congenital Zika Syndrome: A Prospective Observational Study.

OBJECTIVE: To assess the clinical and radiographic characteristics of hip joint deformities in children with congenital Zika syndrome (CZS), and the evolution of hip joint deformities in affected infants for the first 3 years of life. STUDY DESIGN: This prospective observational study evaluated orthopedic clinical examinations performed every 3 months to assess hip flexion and extension, lateral and medial rotation, and abduction and adduction, as well as lower limb muscle length and tone. The biannual radiograph comprised anteroposterior panoramic pelvic radiographs with the lower limbs in extension. Percentage of migration was used as a radiographic study tool to measure and evaluate linear hip displacement. RESULTS: From November 2018 to March 2020, we followed 30 children with CZS, of whom 15 (50%) had normal pelvic radiographs on admission; 5 (33.3%) developed hip displacement by the second radiograph examination. During follow-up radiographic examinations, 20 of the 30 children (66.7%) were diagnosed with hip displacement and/or dislocation of at least 1 side, and 10 of the 30 (33.3%) remained normal. Among 30 affected patients, 13 (43.3%) had hip displacement on the right side and 9 (30%) on the left side. Logistic regression analysis revealed that spasticity (P = .0033; OR, 15.9) and ophthalmologic abnormalities (P = .0163; OR, 16.9) were associated with hip dislocation during follow-up. CONCLUSIONS: Pelvic radiographic follow-up for all children with CZS will complement physical examination, diagnosis, and monitoring for hip joint deformities.

Global RNAseq of ocular cells reveals gene dysregulation in both asymptomatic and with Congenital Zika Syndrome infants exposed prenatally to Zika virus.

In 2015, Brazil reported an outbreak identified as Zika virus (ZIKV) infection associated with congenital abnormalities. To date, a total of 86 countries and territories have described evidence of Zika infection and recently the appearance of the African ZIKV lineage in Brazil highlights the risk of a new epidemic. The spectrum of ZIKV infection-induced alterations at both cellular and molecular levels is not completely elucidated. Here, we present for the first time the gene expression responses associated with prenatal ZIKV infection from ocular cells. We applied a recently developed non-invasive method (impression cytology) which use eye cells as a model for ZIKV studies. The ocular profiling revealed significant differences between exposed and control groups, as well as a different pattern in ocular transcripts from Congenital Zika Syndrome (CZS) compared to ZIKV-exposed but asymptomatic infants. Our data showed pathways related to mismatch repair, cancer, and PI3K/AKT/mTOR signaling and genes probably causative or protective in the modulation of ZIKV infection. Ocular cells revealed the effects of ZIKV infection on primordial neuronal cell genes, evidenced by changes in genes associated with embryonic cells. The changes in gene expression support an association with the gestational period of the infection and provide evidence for the resulting clinical and ophthalmological pathologies. Additionally, the findings of cell death- and cancer-associated deregulated genes raise concerns about the early onset of other potential pathologies including the need for tumor surveillance. Our results thus provide direct evidence that infants exposed prenatally to the Zika virus, not only with CZS but also without clinical signs (asymptomatic) express cellular and molecular changes with potential clinical implications.

The burden of global outbreaks: Photos of the daily lives of children with congenital Zika syndrome during the COVID-19 pandemic.

INTRODUCTION: In Brazil, more than 3500 children with congenital Zika syndrome (CZS) face difficulties participating in activities of daily living, which may be aggravated by health emergencies, such as the COVID-19 pandemic. Participation could be defined as the individual's involvement in daily life situations, and participation restrictions are problems that may arise in involvement in everyday situations. AIM: To explore the daily lives of children with CZS during the COVID-19 pandemic using photographic narratives captured by mothers and discuss possible strategies to improve participation results. METHODS: In this participatory action research, seven young Brazilian mothers acted as co-researchers using photovoice to describe the experiences of their children with CZS (from 2 to 5 years old). Also, mothers contributed to validate the contents. The research was conducted online and included the following steps: pilot study, recruitment, individualized training, sociodemographic interview, photovoice training, photo taking, focus group for contextualization, data transcription and analysis and validation of analyses by the mothers. RESULTS: Content analysis revealed five categories that influenced the participation of the children: participation preferences, family relationships, access to healthcare, access to education and social isolation. Regarding participation preferences, mothers reported their children's desire to play with peers and family members and have autonomy. Mothers described the family environment as a happy, peaceful and safe place for the children. Lack of therapy was perceived to negatively impact the health of children; thus, treatments were considered essential for child development. Access to education included accessibility of remote education and a perceived lack of infrastructure and pedagogical preparation. Last, social isolation due to COVID-19 directly affected the daily lives and behaviour of the children, interrupting therapies and medical appointments. CONCLUSION: The photos and narratives captured several aspects of the daily lives of children with CZS impacted by the COVID-19 pandemic, reinforcing the importance of considering the negative effects of social isolation and offering education and social assistance to promote participation and integral health. PATIENT/PUBLIC CONTRIBUTION: Consistent with a participatory action research framework, Mothers acted as co-researchers and participated in all stages of the research, especially in validating the data analysed by the researchers.

A combination of two human monoclonal antibodies limits fetal damage by Zika virus in macaques.

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.

Prevalence of sleep disorders in children with Congenital Zika Syndrome.

Studies have reported that children with Congenital Zika Syndrome (CZS) experience changes in their sleep patterns, which can result in mood disturbances, behavioral issues and delays in growth and development. This systematic review synthesized the available evidence on the prevalence of sleep disorders in children with CZS. Eligible studies were those with an observational design that reported sleep disorders in children with CZS using validated questionnaires, polysomnography/electroencephalographic recording or parent/caregiver reports. Searches were conducted in PubMed, Web of Science, SCOPUS and Embase, as well as a gray literature search using Google Scholar. The Freeman-Tukey double-arcsine transformation with a random-effects model was used to estimate the pooled prevalence of sleep disorders with a 95% confidence interval (CI). Five studies were included and data from 340 Brazilian children with CZS were analyzed. The overall prevalence of sleep disorders was 27.4% (95% CI 16.7-39.4), without differences among studies using validated questionnaires (29.4%, 95% CI 21.4-37.8) or report from parents and caregivers (27.4%, 95% CI 11.5-47.0). Sleep disorders are prevalent in children with CZS, impacting their development and quality of life. It is critical to examine the quality of sleep in these children to develop appropriate interventions that can mitigate these issues.

The article discusses a systematic review of studies that have explored the prevalence of sleep disorders in children with Congenital Zika Syndrome (CZS), a condition caused by the Zika virus. The study found that children with CZS often experience changes in their sleep patterns, which can lead to mood disturbances, behavioral issues and delays in growth and development. The review included five studies with a total of 340 Brazilian children with CZS, and the overall prevalence of sleep disorders was found to be 27.4%. This indicates that sleep disorders are prevalent in children with CZS and can significantly impact their development and quality of life. The authors suggest that further research is needed to develop appropriate interventions to mitigate these issues.

Evaluation and monitoring of eye findings in children exposed to Zika virus during gestation: 3 years of follow-up.

INTRODUCTION: Congenital Zika syndrome (CZS) is a recently described disease. Our main objective was to evaluate and monitor, over 3 years, the ophthalmoscopic findings in children exposed to zika virus (ZIKV) during gestation. METHODS: This prospective observational study was conducted in Rio de Janeiro, Brazil, between April 2016 and May 2019. We evaluated two groups with exanthema serving as a proxy for viremia: (i) children whose mothers had exanthema during pregnancy and (ii) children who had microcephaly without maternal exanthema during pregnancy. We performed indirect ophthalmoscopy at recruitment and every 6 months thereafter. We also tested the association between ocular findings with maternal exanthema, microcephaly, CZS and maternal infection confirmed by reverse transcriptase quantitative polymerase chain reaction and gender. RESULTS: Of the 72 children included, 16 (22.2%) had optic nerve and/or retinal lesions. All 16 had CZS and 15 (93.7%) had microcephaly (14 at birth and 1 postnatally). The child with postnatally acquired microcephaly was born to a mother without exanthema during pregnancy. Fifty-six (77.8%) of the 72 children were followed for a median time of 24 months and none exhibited differences between admission and follow-up examinations. After logistic regression, only microcephaly at birth was associated with eye abnormalities (odds ratio, 77.015; 95% confidence interval, 8.85-670.38; p < 0.001). CONCLUSION: We observed that there was no progression of the lesions over the follow-up period. We also showed that the eye findings were associated only with microcephaly at birth. Attention should be paid to all children born during a ZIKV epidemic, regardless of maternal exanthema and/or microcephaly at birth.

Zika Virus Infection of Pregnant Ifnar1-/- Mice Triggers Strain-Specific Differences in Fetal Outcomes.

Zika virus (ZIKV) is a flavivirus that causes a constellation of adverse fetal outcomes collectively termed congenital Zika syndrome (CZS). However, not all pregnancies exposed to ZIKV result in an infant with apparent defects. During the 2015 to 2016 American outbreak of ZIKV, CZS rates varied by geographic location. The underlying mechanisms responsible for this heterogeneity in outcomes have not been well defined. Therefore, we sought to characterize and compare the pathogenic potential of multiple Asian-/American-lineage ZIKV strains in an established Ifnar1-/- pregnant mouse model. Here, we show significant differences in the rate of fetal demise following maternal inoculation with ZIKV strains from Puerto Rico, Panama, Mexico, Brazil, and Cambodia. Rates of fetal demise broadly correlated with maternal viremia but were independent of fetus and placenta virus titer, indicating that additional underlying factors contribute to fetal outcome. Our results, in concert with those from other studies, suggest that subtle differences in ZIKV strains may have important phenotypic impacts. With ZIKV now endemic in the Americas, greater emphasis needs to be placed on elucidating and understanding the underlying mechanisms that contribute to fetal outcome. IMPORTANCE Zika virus (ZIKV) transmission has been reported in 87 countries and territories around the globe. ZIKV infection during pregnancy is associated with adverse fetal outcomes, including birth defects, microcephaly, neurological complications, and even spontaneous abortion. Rates of adverse fetal outcomes vary between regions, and not every pregnancy exposed to ZIKV results in birth defects. Not much is known about how or if the infecting ZIKV strain is linked to fetal outcomes. Our research provides evidence of phenotypic heterogeneity between Asian-/American-lineage ZIKV strains and provides insight into the underlying causes of adverse fetal outcomes. Understanding ZIKV strain-dependent pathogenic potential during pregnancy and elucidating underlying causes of diverse clinical sequelae observed during human infections is critical to understanding ZIKV on a global scale.

African-Lineage Zika Virus Replication Dynamics and Maternal-Fetal Interface Infection in Pregnant Rhesus Macaques.

Following the Zika virus (ZIKV) outbreak in the Americas, ZIKV was causally associated with microcephaly and a range of neurological and developmental symptoms, termed congenital Zika syndrome (CZS). The viruses responsible for this outbreak belonged to the Asian lineage of ZIKV. However, in vitro and in vivo studies assessing the pathogenesis of African-lineage ZIKV demonstrated that African-lineage isolates often replicated to high titers and caused more-severe pathology than Asian-lineage isolates. To date, the pathogenesis of African-lineage ZIKV in a translational model, particularly during pregnancy, has not been rigorously characterized. Here, we infected four pregnant rhesus macaques with a low-passage-number strain of African-lineage ZIKV and compared its pathogenesis to those for a cohort of four pregnant rhesus macaques infected with an Asian-lineage isolate and a cohort of mock-inoculated controls. The viral replication kinetics for the two experimental groups were not significantly different, and both groups developed robust neutralizing antibody titers above levels considered to be protective. There was no evidence of significant fetal head growth restriction or gross fetal harm at delivery (1 to 1.5 weeks prior to full term) in either group. However, a significantly higher burden of ZIKV viral RNA (vRNA) was found in the maternal-fetal interface tissues of the macaques exposed to an African-lineage isolate. Our findings suggest that ZIKV of any genetic lineage poses a threat to pregnant individuals and their infants. IMPORTANCE ZIKV was first identified in 1947 in Africa, but most of our knowledge of ZIKV is based on studies of the distinct Asian genetic lineage, which caused the outbreak in the Americas in 2015 to 2016. In its most recent update, the WHO stated that improved understanding of African-lineage ZIKV pathogenesis during pregnancy must be a priority. The recent detection of African-lineage isolates in Brazil underscores the need to understand the impact of these viruses. Here, we provide the first comprehensive assessment of African-lineage ZIKV infection during pregnancy in a translational nonhuman primate model. We show that African-lineage isolates replicate with kinetics similar to those of Asian-lineage isolates and can infect the placenta. However, there was no evidence of more-severe outcomes with African-lineage isolates. Our results highlight both the threat that African-lineage ZIKV poses to pregnant individuals and their infants and the need for epidemiological and translational in vivo studies with African-lineage ZIKV.

Urological outcomes in children with congenital Zika syndrome: The experience of a cohort in Campina Grande, Brazil.

OBJECTIVE: To describe the urological outcomes in children with congenital Zika syndrome (CZS) and investigate the relationship between clinical and urological findings in this population. METHODS: This cross-sectional study involved children with CZS followed up by a referral centre for children with microcephaly in the state of Paraiba in northeast Brazil. The urological evaluation included clinical history, urine culture results, ultrasonography of the urinary tract, and urodynamic evaluation, following the protocol proposed by Costa Monteiro et al. (2017). Descriptive statistical analysis was performed in addition to association and correlation tests, considering clinical and urodynamic variables. RESULTS: Among the 88 children with CZS (35.5 ± 5.5 months), 97.7% had microcephaly, and 51% presented urinary tract infection (UTI) confirmed with clinical history and lab tests. The number of confirmed UTI episodes varied from one to 14 per child. The urodynamic evaluation confirmed the presence of an overactive bladder in 78 children and incomplete voiding in 50. Urodynamic findings were associated with the number of confirmed UTI episodes, child's sex, and actual weight, in addition to the use of anticonvulsant and myorelaxant drugs. CONCLUSIONS: UTIs were confirmed in most children. Other urological outcomes observed were overactive bladder and low bladder capacity, which were associated with the number of confirmed UTI episodes, use of anticonvulsants and myorelaxants, and the child's sex and weight. These are treatable conditions, and it is paramount that paediatricians, neonatologists, and infectious disease specialists are aware of them to make clinical decisions and help reduce the risk of renal damage and other morbidities.

Immunological imbalance in microcephalic children with congenital Zika virus syndrome.

Microcephalic children due congenital Zika virus syndrome (CZS) present neurological symptoms already well described. However, several other alterations can also be observed. Here, we aimed to evaluate the immune system of microcephaly CZS children. We showed that these patients have enlarged thymus, spleen and cervical lymph nodes, analysed by ultrasound and compared to the reference values for healthy children. In the periphery, they have an increase in eosinophil count and morphological alterations as hypersegmented neutrophils and atypical lymphocytes, even in the absence of urinary tract infections, parasitological infections or other current symptomatic infections. Microcephalic children due CZS also have high levels of IFN-γ, IL-2, IL-4, IL-5 and type I IFNs, compared to healthy controls. In addition, this population showed a deficient cellular immune memory as demonstrated by the low reactivity to the tuberculin skin test even though they had been vaccinated with BCG less than 2 years before the challenge with the PPD. Together, our data demonstrate for the first time that CZS can cause alterations in primary and secondary lymphoid organs and also alters the morphology and functionality of the immune system cells, which broadens the spectrum of CZS symptoms. This knowledge may assist the development of specific therapeutic and more efficient vaccination schemes for this population of patients.

Two-year follow-up of children with congenital Zika syndrome: the evolution of clinical patterns.

The aim of the study was to describe neurological manifestations in children with congenital Zika syndrome (CZS) in the first 2 years of age. In this prospective observational study, children with CZS treated at a university hospital received a neurological assessment and were evaluated using two neurodevelopmental scales (the Denver II test and the assessment of gross motor development of the World Health Organization) by a pediatric neurologist on admission to the study and at 4, 8, 12, 18, and 24 months of age. The data collected were stored in Microsoft Excel version 14.6.3. Thirty-eight children (27 males and 11 females; a median age of 4.3 months (interquartile range (IQR): 1.6-11.4)) with CZS were evaluated. Irritability was present in 50% and 27% of the children at 8 months and 24 months, respectively. Axial hypertonia was highly prevalent at 4 months (77%), with a decrease to 50% at 24 months. At all ages, spastic tetraparesis was the most common motor abnormality (> 80%). Twenty-seven (71%) participants were diagnosed with epilepsy, and the median age at seizure onset was 6 months (IQR: 3.5-8). The most frequent types of seizures were focal seizures and spasms, with spasms being the most frequent in the first year of life (52%) and focal crises being the most frequent in the second year of life (50%).Conclusion: This study allowed observation of neurological abnormalities over time, the evolution of epileptic manifestations, and recognition of new patterns of clinical neurological abnormalities, helping clinicians to recognize CZS earlier, minimizing the impact of new outbreaks. What is Known: • Clinical patterns of SZC patients at pre-established ages or date of data collection • More frequent studies with data collection of clinical-radiological features of patient's over his first year of life What is New: • Comprehensive clinical neurological progression data regarding CZS in the first 2 years of life, recognizing patterns • Hypothesis including a new CZS spectrum with milder clinical-radiological features.

Population-based surveillance for congenital zika virus syndrome: a latent class analysis of recorded cases from 2015-2018.

OBJECTIVE: This study aims to describe clinical findings and determine the medium-term survival of congenital zika syndrome (CZS) suspected cases. METHODS: A retrospective cohort study using routine register-based linked data. It included all suspected cases of CZS born in Brazil from January 1, 2015, to December 31, 2018, and followed up from birth until death, 36 months, or December 31, 2018, whichever came first. Latent class analysis was used to cluster unconfirmed cases into classes with similar combinations of anthropometry at birth, imaging findings, maternally reported rash, region, and year of birth. Kaplan-Meier curves were plotted, and Cox proportional hazards models were fitted to determine mortality up to 36 months. RESULTS: We followed 11,850 suspected cases of CZS, of which 28.3% were confirmed, 9.3% inconclusive and 62.4% unconfirmed. Confirmed cases had almost two times higher mortality when compared with unconfirmed cases. Among unconfirmed cases, we identified three distinct clusters with different mortality trajectories. The highest mortality risk was observed in those with abnormal imaging findings compatible with congenital infections (HR = 12.6; IC95%8.8-18.0) and other abnormalities (HR = 11.6; IC95%8.6-15.6) compared with those with normal imaging findings. The risk was high in those with severe microcephaly (HR = 8.2; IC95%6.4-10.6) and macrocephaly (HR = 6.6; IC95%4.5-9.7) compared with normal head size. CONCLUSION: Abnormal imaging and head circumference appear to be the main drivers of the increased mortality among suspected cases of CZS. We suggest identifying children who are more likely to die and have a greater need to optimise interventions and resource allocation regardless of the final diagnoses.

My child is growing and now? Exploring the environmental needs of children with congenital Zika syndrome according to their caregivers' perceptions.

INTRODUCTION: Promoting social inclusion of children with congenital Zika virus syndrome (CZS) is challenging, mostly, when there is a transport problem, low access to information and a long distance between the house and health services. Participation can be understood as involvement in a life situation and is strongly influenced by physical, social and attitudinal environmental factors; however, was still little explored in the case of children with CZS. In this sense, this study aimed to explore the perception of caregivers about the environmental needs of children with CZS, differentiating barriers and facilitators. METHODS: This is qualitative research. Thematic analysis was used to identify the environmental needs perceived by caregivers of children with CZS. The patient public involvement (PPI) approach was incorporated with the purpose of validating the data analysis performed by the researchers. After this step, the data were categorized in terms of barriers and facilitators and validated by the group of researchers. RESULTS: A relevant environmental need reported by caregivers as a barrier was social support for children with CZS. Ableism was also evidenced as an important attitudinal barrier. Health services were essential for the lives of children with CZS and the availability of auxiliary devices as facilitators of participation. Environmental factors related to medication and food routines were, for the most part, facilitators. CONCLUSION: This study contributes to critical approaches to the impacts linked to environmental factors of children with CZS, recognition of these children is an evolving process and fundamental to basic rights for adequate living in society. The data point to the need to implement public policies aimed at children with CZS, as well as the availability of qualified professionals to apply family-centred care and skills-focused management. Building friendly environments that promote broad social participation will contribute to the healthy growth of children with CZS. PATIENT OR PUBLIC CONTRIBUTION: Six caregivers (20% of the caregivers) as part of the PPI approach were contacted and participated in individual virtual meetings to discuss the results of the thematic analysis regarding the environmental needs of children with CZS.

Congenital Zika syndrome and living conditions in the largest city of northeastern Brazil.

BACKGROUND: The Zika virus (ZIKV) epidemic hit Brazil in 2015 and resulted in a generation of children at risk of congenital Zika syndrome (CZS). The social vulnerability of certain segments of the population contributed to the disproportional occurrence of CZS in the Brazilian Northeast, the poorest region in the country. Living conditions are essential factors in understanding the social determination of CZS, which is embedded in a complex interaction between biological, environmental, and social factors. Salvador, the biggest city in the region, played a central role in the context of the epidemic and was a pioneer in reporting the ZIKV infection and registering a high number of cases of CZS. The aim of the study was identifying the incidence and spatial distribution pattern of children with CZS in the municipality of Salvador, according to living conditions. METHODS: This is an ecological study that uses the reported cases of ZIKV and CZS registered in the epidemiological surveillance database of the Municipal Secretariat of Health of the city of Salvador between August of 2015 and July of 2016. The neighborhoods formed the analysis units and the thematic maps were built based on the reported cases. Associations between CZS and living conditions were assessed using the Kernel ratio and a spatial autoregressive linear regression model. RESULTS: Seven hundred twenty-six live births were reported, of which 236 (32.5%) were confirmed for CZS. Despite the reports of ZIKV infection being widely distributed, the cases of CZS were concentrated in poor areas of the city. A positive spatial association was observed between living in places with poorer living conditions and births of children with CZS. CONCLUSIONS: This study shows the role of living conditions in the occurrence of births of children with CZS and indicates the need for approaches that recognize the part played by social inequalities in determining CZS and in caring for the children affected.

Zika Virus Infection in the Developing Mouse Produces Dramatically Different Neuropathology Dependent on Viral Strain.

Zika virus (ZIKV) infection during pregnancy has been causally linked to a constellation of neurodevelopmental deformities in the fetus resulting in a disease termed congenital Zika syndrome (CZS). Here we detail how ZIKV infection produces extensive neuropathology in the developing mouse brain and spinal cord of both sexes. Surprisingly, neuropathology differs depending on viral strain with a French Polynesian isolate producing primarily excitotoxicity and a Brazilian isolate being almost exclusively apoptotic but occurring over a prolonged period that is more likely to produce severe hypoplasia. We also show exposure can produce a characteristic pattern of infection that mirrors neuropathology and ultimately results in gross morphological deformities strikingly similar to CZS. This research provides a valuable mouse model mirroring the clinical course of disease that can be used to test potential therapies to improve treatment and gain a better understanding of the disabilities associated with CZS.SIGNIFICANCE STATEMENT Zika virus (ZIKV) infection during pregnancy has been causally linked to a constellation of neurodevelopmental deformities in the fetus resulting in a disease termed congenital Zika syndrome. Despite its devastating effects, very little is known about how ZIKV infection produces fetal neuropathology. Here we detail the temporal progression of ZIKV infection in the mouse brain and spinal cord resulting in massive neurodegeneration of infected regions. We also report a ZIKV strain from a region of Brazil with high levels of microcephaly (abnormally small head circumference) produces particularly devastating neuropathology.

Prolonged Maternal Zika Viremia as a Marker of Adverse Perinatal Outcomes.

Whether prolonged maternal viremia after Zika virus infection represents a risk factor for maternal-fetal transmission and subsequent adverse outcomes remains unclear. In this prospective cohort study in French Guiana, we enrolled Zika virus-infected pregnant women with a positive PCR result at inclusion and noninfected pregnant women; both groups underwent serologic testing in each trimester and at delivery during January-July 2016. Prolonged viremia was defined as ongoing virus detection >30 days postinfection. Adverse outcomes (fetal loss or neurologic anomalies) were more common in fetuses and neonates from mothers with prolonged viremia (40.0%) compared with those from infected mothers without prolonged viremia (5.3%, adjusted relative risk [aRR] 7.2 [95% CI 0.9-57.6]) or those from noninfected mothers (6.6%, aRR 6.7 [95% CI 3.0-15.1]). Congenital infections were confirmed more often in fetuses and neonates from mothers with prolonged viremia compared with the other 2 groups (60.0% vs. 26.3% vs. 0.0%, aRR 2.3 [95% CI 0.9-5.5]).

Two Sides of a Coin: a Zika Virus Mutation Selected in Pregnant Rhesus Macaques Promotes Fetal Infection in Mice but at a Cost of Reduced Fitness in Nonpregnant Macaques and Diminished Transmissibility by Vectors.

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Aedes aegypti mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics.IMPORTANCE Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with ZIKV with the mutation in nonpregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in nonpregnant hosts.

Early maternal Zika infection predicts severe neonatal neurological damage: results from the prospective Natural History of Zika Virus Infection in Gestation cohort study.

OBJECTIVE: To define the prevalence of adverse outcomes of maternal infection in a large cohort of ZIKV-infected Brazilian women and their infants. DESIGN: Prospective population-based cohort study. SETTING: Ribeirão Preto's region's private and public health facilities. POPULATION: Symptomatic ZIKV-infected mothers and their infants. METHODS: Prenatal/early neonatal data were obtained for all mother-child pairs. A subgroup of infants had cranial ultrasonography, eye fundoscopy, hearing and neurological examinations and Bayley III screening tests within 3 months of age. MAIN OUTCOME MEASURES: Prevalence of pregnancy losses and anomalies detected at birth or within 3 months according to the gestational age of infection. RESULTS: Overall, 511 ZIKV-infected women were identified from a total of 1116 symptomatic women; as there were two twins, there were a total of 513 fetuses included. Of these, 13 (2.5%; 95% CI 1.5-4.3) presented with major signs of congenital Zika syndrome (CZS). Of the 511 women, there were 489 livebirths and 24 (4.7%) pregnancy losses (20 miscarriages and four stillbirths). ZIKV-related anomalies occurred in the offspring of 42/511 (8.2%) mothers. Microcephaly or other CNS malformations were diagnosed in 1/4 (25.0%) stillbirths and in 19/489 (3.9%; 95% CI 2.5-5.9) of the liveborn infants. Fetal abnormalities were 14.0 (95% CI 7.6-26.0) times more likely with gestational infection occurring in ≤11 weeks. On follow up of 280 asymptomatic infants, 2/155 (1.3%) had eye abnormalities, 1/207 (0.5%) had CNS imaging findings and 16/199 (8%) presented neurological alert signs. CONCLUSIONS: This prospective population-based study represents the largest Brazilian cohort study of ZIKV in pregnancy. Congenital anomalies potentially associated with CZS are less frequent than previously thought. There is a strong association between the gestational age of infection (≤11 weeks) and a poorer early infant prognosis. A notable proportion of apparently asymptomatic newborns can present with subclinical findings within 3 months of age. TWEETABLE ABSTRACT: ZIKV and pregnancy: adverse outcomes are less common, more prevalent for first-trimester infections, and potentially subclinical.