research studies on zika

Volume 30, Number 2—February 2024

Evidence of Zika Virus Reinfection by Genome Diversity and Antibody Response Analysis, Brazil

Cite This Article

We generated 238 Zika virus (ZIKV) genomes from 135 persons in Brazil who had samples collected over 1 year to evaluate virus persistence. Phylogenetic inference clustered the genomes together with previously reported ZIKV strains from northern Brazil, showing that ZIKV has been remained relatively stable over time. Temporal phylogenetic analysis revealed limited within-host diversity among most ZIKV-persistent infected associated samples. However, we detected unusual virus temporal diversity from > 5 persons, uncovering the existence of divergent genomes within the same patient. All those patients showed an increase in neutralizing antibody levels, followed by a decline at the convalescent phase of ZIKV infection. Of interest, in 3 of those patients, titers of neutralizing antibodies increased again after 6 months of ZIKV infection, concomitantly with real-time reverse transcription PCR re-positivity, supporting ZIKV reinfection events. Altogether, our findings provide evidence for the existence of ZIKV reinfection events.

Zika virus (ZIKV) is an arthropodborne virus belonging to the Flaviviridae family. Virions are enveloped for a single-stranded positive-sense RNA genome of ≈10.8-kb ( 1 ). ZIKV is transmitted through the bite of infected Aedes spp. mosquitoes, mainly A. aegypti , which are widely distributed throughout the tropical and subtropical regions of the world.

In 2015, a large ZIKV epidemic was documented in Brazil, resulting in an estimated 440,000–1.3 million cases ( 2 ). Of great concern, the epidemic was preceded by a dramatic increase in the number of congenital anomalies, including newborn microcephaly ( 3 , 4 ). However, since the largest outbreak in 2015, ZIKV has decreased its circulation; novel cases are only sporadically reported ( 5 ).

ZIKV infections are usually asymptomatic, although a small proportion of persons may experience mild symptoms such as fever, rash, nonpurulent conjunctivitis, muscle pain, and joint pain. During pregnancy, ZIKV infection may result in microcephaly and other congenital abnormalities in the developing fetus ( 3 ). Suspected cases are diagnosed by detection of viral RNA in blood and urine during the acute phase of the disease and in other body fluids with variable frequency and duration by reverse transcription PCR (RT-PCR) ( 6 ). As previously reported, ZIKV infection may result in a persistent viral infection, as demonstrated by the prolonged detection of viral RNA in semen; the longest detection was up to 370 days after symptom onset ( 7 ). Virus compartmentalization and persistence are common features of ZIKV infection; however, the clinical and immunological aspects of ZIKV persistence, reactivation, and reinfection are still unknown.

ZIKV phylogenetic studies have described the circulation of 2 distinct African and Asian lineages ( 8 ). The initial genetic analysis of the first ZIKV isolates from Brazil revealed the circulation of the Asian genotype during the 2015–2016 epidemic ( 9 ). Asian-derived strains that currently circulate in the Americas are now named ZIKV American strains and are well known for their capacity to infect neuronal progenitor cells, disrupting cell development, proliferation, and differentiation ( 10 , 11 ). Because the genomic replication of ZIKV is based on an error-prone RNA-dependent RNA polymerase (RdRp), which leads to nucleotide misincorporation during viral replication, ZIKV infection behaves as viral populations composed of genetically related sequences, similar to other RNA virus infections. As the viral replication progresses in an infected person, mutations start to accumulate, resulting in more heterogeneous viral genomic populations. Those viral population clouds are the foundation of the quasispecies theory, which posits that RNA viruses produce larger, highly variable population clouds that can evade the host immune system more efficiently ( 12 ). Furthermore, accumulating data show that viral cloud variability is able to interfere with disease progression ( 13 , 14 ). In this context, next-generation sequencing (NGS) provides a powerful tool to gain a deeper understanding of viral diversity by increasing the depth of sequencing coverage, defined as the number of reads for a given nucleotide). Therefore, the assessment of viral diversity is key to better understand virulence, evolution, and host-specific adaptations providing a direct translational information to mitigate effects of viral pathogens.

In this study, we deployed an NGS protocol to gain insight into the genetic diversity of ZIKV in naturally infected patients. Specifically, we used a previously established cohort study conducted in northern Brazil to assess virus diversity from patients with prolonged ZIKV infection ( 15 – 17 ). Since 2016, we have observed limited virus diversity and decreasing ZIKV transmission over the years, which was likely because of population immunity elicited during the first outbreak waves. We also found that virus diversity was limited in longitudinally sequenced samples from persons persistently infected with ZIKV, indicating restrained evolutionary rates and selection pressures acting on RNA arthropod-borne viruses; our results were consistent with previously published findings ( 18 , 19 ). However, we also detected the existence of divergent genomes within the same patient in a small number of samples analyzed; those participants responded to infection with alterations in neutralizing antibodies levels concomitantly with ZIKV redetection by real-time RT-PCR (rRT-PCR) several months after the initial virus exposure.

The study protocol and procedures have been reviewed and approved by the World Health Organization Research Ethics Review Committee (protocol ID: ERC.0002786); Brazilian National Research Ethics Commission (CAAE: 62.518.016.6.1001.0008); Institutional Ethics and Research Committee of the Evandro Chagas National Institute of Infectious Diseases, Fiocruz, Rio de Janeiro (CAAE: 62.518.016.6.2002.5262); Institutional Ethics and Research Committee of the Aggeu Magalhães Research Center, Fiocruz, Recife (CAAE: 62.518.016.6.2001.5190) and Institutional Ethics and Research Committee of the Tropical Medicine Foundation, Manaus, Amazonas (CAAE: 62.518.016.6.2003.0005).

Study Participants and Specimen Collection

Participants comprised men and women > 18 years of age with a confirmed diagnosis of ZIKV infection by RT-PCR, as described previously ( 15 , 16 , 20 ). Participants were persons with symptomatic cases diagnosed at the study collaborating clinics (index case-patients) and their asymptomatic or symptomatic household and sexual contacts. After ZIKV infection confirmation performed 48 hours after study recruitment, we collected other specimens at established intervals, or visits ( Table 1 ), and routinely tested for molecular screening using a multiplex rRT-PCR assay to detect ZIKV, dengue virus, and chikungunya virus.

NGS and Analysis

We processed all specimens with a positive ZIKV rRT-PCR result, defined as a cycle threshold (Ct) value < 38, using a previously established NGS protocol ( 21 ). For this study, we processed plasma, urine, and semen samples (semen is more frequently associated with persistence). For sequencing, we first obtained a complementary DNA employing the ProtoScript II Reverse transcription kit (New England Biolabs, https://www.neb.com ) and a set of random primers (random sequence [d(N)6]). We obtained whole-genome amplicons from a multiplex PCR reaction using a set of ZIKV designed primers, as described by Quick et al. ( 21 ); we purified amplicons using the Q5 High-Fidelity DNA Polymerase kit (New England Biolabs) and performed library preparation with the Nextera XT Library Prep kit (Illumina, https://www.illumina.com ) using 2 ng of DNA. We sequenced the obtained libraries using the MiSeq Reagent kit version 3 (Illumina) on an Illumina MiSeq. We processed raw fastq data to generate consensus files (base calls only at regions with > 5×) and to call SNVs and iSNVs (only regions with a coverage depth of > 100×) using ViralFlow version 0.0.6 ( 22 ) and a reference ZIKV genome (GenBank accession no. KX197192.1).

Phylogenetic and Bayesian Analysis

The new genomic sequences reported in this study were initially submitted to a genotyping analysis using the ZIKV typing tool ( http://genomedetective.com/app/typingtool/zika ). We aligned genomic data generated in this study (238 genomes with coverage breadth > 70 and average coverage depth of 100×) with a worldwide dataset of ZIKV genome sequences (n = 840 for all known ZIKV genotypes and n = 481 for ZIKV American strains). We aligned sequences using MAFFT ( https://mafft.cbrc.jp/alignment/software ) and inferred a preliminary maximum-likelihood tree using IQ-TREE version 2 ( http://www.iqtree.org ). Before conducting temporal analysis, we assessed our dataset for molecular clock signal in TempEst version 1.5.3 ( http://tree.bio.ed.ac.uk/software/tempest ) after removing any potential outliers that might violate the molecular clock assumption. To estimate a time-calibrated phylogeny, we used the Bayesian software package BEAST version 1.10.4 (https://beast.community) with the Bayesian skyline tree prior with an uncorrelated relaxed clock and the lognormal distribution. We ran analyses in duplicate in BEAST for 100 million Markov chain Monte Carlo (MCMC) steps, sampling parameters, and trees every 10,000th step. We checked convergence of MCMC chains using Tracer version 1.7.1 (https://beast.community/tracer). We summarized maximum clade credibility trees using TreeAnnotator (https://beast.community/treeannotator) after discarding 10% as burn-in. We submitted the genomes from this study to the Genome Detective for the analysis of the mutational pattern profile using the annotated genome aligner AGA ( https://www.genomedetective.com/app/aga ). We plotted results in R Studio version 4.2.1 ( https://posit.co ) using the Lollipop plot.

ZIKV Neutralization Assays

We measured ZIKV neutralizing antibody titers by a high-throughput ZIKV 50% microneutralization assay (MN 50 ), using a wild-type live virus as described previously ( 22 ). We defined seropositivity as a titer > 1:10.

Cohort Definition and Sample Assessment for NGS

During June 2017–June 2019, our study recruited a total of 255 persons with ZIKV-confirmed infection in Manaus, Brazil. Among the participants, 99% were enrolled within 1 week after the onset of illness. For this study, genomic analysis included 135 persons with confirmed ZIKV infection experiencing rash, itching, fever, and arthralgia; mean age was 38.27 ( + 12.97) years ( Table 2 ). Of those 135 persons, 56 participants had > 1 sample available, defined as a different specimen at the same visit (5/135) or any specimen at a different study visit (51/135). We sequenced those samples, which yielded a total of 238 ZIKV genomes with a median genome coverage breadth of 90%. Most of these genomes were obtained from plasma and urine samples; a minor proportion (n = 20) were obtained from semen specimens.

Phylogenetic Characterization

Figure 1 . Genomic epidemiology of Zika virus strains obtained from study participants in northern Brazil and reference sequences. A) Time-scaled maximum clade credibility tree of Zika virus Asian lineage in Brazil, including...

Figure 2 . Single-nucleotide variants per gene for Zika virus strains obtained from study participants in northern Brazil. Amino acid changes in the polyprotein are allocated along the genome. Only mutations that appear...

Initially, our objective was to thoroughly characterize ZIKV circulating from northern Brazil. We observed that all ZIKV strains circulating in Manaus since the beginning of the outbreak, including our newly generated genomes, grouped together in a unique clade within the ZIKV Asian lineage ( Appendix 1 Figure 1). In addition, our maximum clade credibility (MCC) tree clustered our generated ZIKV genomes together with viral strains previously isolated in northern Brazil ( Figure 1 , panel A). From this analysis, we estimated the time of the most recent common ancestor (tMRCA) occurred in late March 2014 (95% highest posterior density range January–August 2014) ( Figure 1 , panel B). We also explored the collective mutational pattern found in the consensus genomes obtained in this study. Most of the mutations were observed in nonstructural protein (NS) 1 protein (5 in total) and NS5, which also has 5 mutations, although with a lower frequency than NS1 ( Figure 2 ).

Figure 3 . Notified Zika virus cases per week in Manaus municipality, Brazil, 2016–2019, from the Brazilian Ministry of Health. Shading reflects each epidemic year. Data source: https://datasus.saude.gov.br/informacoes-de-saude-tabnet .

Next, we searched for the total number of ZIKV cases reported in Manaus from DATASUS ( https://datasus.saude.gov.br/informacoes-de-saude-tabnet ), the national health information system that compiles clinical and laboratory-confirmed cases across all the states of Brazil. Our analysis revealed that the initial occurrence of ZIKV cases in Manaus dates to 2016, a significant surge of 6,033 cases that marked the peak of the ZIKV epidemic in northern Brazil. However, after the initial surge in 2016, subsequent waves experienced a significant decrease in the overall number of reported ZIKV cases. That downward trend persisted and reached a notable low point in 2019, when only 126 cases were documented ( Figure 3 ). We concluded that the ZIKV circulating strains in northern Brazil exhibited stability over time, undergoing minimal mutations, contributing to the decline of the epidemic.

ZIKV Within-Host Genetic Diversity

Figure 4 . Phylogenetic analysis of study participants persistently infected with Zika virus, Brazil. A) Maximum-likelihood phylogenetic tree of persistent samples. The phylogenetic tree shows all 10 participants with confirmed persistent infection. Boldface...

We followed our study protocol, specifically designed to investigate the persistence of ZIKV in body fluids, to assess virus diversity among persons who remained persistently infected. Although there is no consensus in the literature, we defined ZIKV persistence as any participant with ZIKV-positive rRT-PCR detection within 30 days after its initial ZIKV confirmation. By applying this criterion, we identified 10 patients who had > 1 positive persistent sample from plasma, urine, or semen. Individual temporal phylogenetic analysis grouped those ZIKV-persistent genomes into 2 major clades. For 5 of the patients, all their samples grouped into a single clade in the tree ( Figure 4 , panel A); those clusters indicated limited viral diversity and maintenance of a single viral lineage through time in these persistently infected persons, independent of the type of specimen analyzed. Because ZIKV neutralizing antibodies (ZIKV-NAb) are highly protective and increasing titers from acute to convalescent phase are usually linked to viral clearance, we then assessed the levels of ZIKV-NAb. Our results showed that almost all the 10 persistently ZIKV-infected participants responded with higher levels (>2,000) of ZIKV-NAb by 30 days after disease onset ( Figure 4 , panel B), indicating a strong neutralizing antibody response at the convalescent phase. Those results eliminated the possibility of a dysregulated immune response as a cause of persistent ZIKV infections.

Figure 5 . Maximum-likelihood phylogenetic tree supporting Zika virus reinfection among study participants in northern Brazil. The tree shows the 5 participants with divergent samples in which coinfection by different ZIKV genomes was...

Figure 6 . Zika virus rRT-PCR results from plasma, urine, and semen (when applicable) specimens supporting reinfection among female (A) and male (B) study participants in northern Brazil. Each square represents an analyzed...

Our phylogenetic analysis also showed 5 participants with ZIKV genomes clustering in distinct clades or subclades in the tree ( Figure 4 , panel A; Figure 5 ; Appendix 1 Figure 2), which suggests the presence of divergent viral genomes within the same participant over time. Those participants had highly supported minor variants (approximate likelihood ratio test >70%) that were not consistently found among all samples from the same person and showed no consistent pattern of minor variant sites accumulation over time ( Appendix 2 ). We hypothesize that the presence of such temporally divergent ZIKV genomes in the same person suggests a reinfection event by a distinct ZIKV clade. Thus, to further assess whether those participants were reinfected, we checked their rRT-PCR results. We observed that 1 participant (ID251064) had a continuous rRT-PCR–positive result up to 8 days after the initial ZIKV infection; viral RNA was not detected until study visit 8 (61–90 days after disease onset), when a ZIKV rRT-PCR result was again positive in plasma ( Figure 6 ). Two participants (ID251069 and ID151035) tested positive for ZIKV RNA in either plasma or urine for up to 21 days after ZIKV confirmation. Those participants then remained ZIKV-negative for 10 months but returned to positivity at the last study visit, performed 311–360 days after disease onset ( Figure 6 ). The rRT-PCR–positive samples indicating reinfection exhibited the highest degree of divergence in terms of the ZIKV genome compared with the acute phase–sequenced samples obtained from the same participants ( Figure 5 ).

Figure 7 . Zika virus neutralizing antibody titers from acute and convalescent serum samples supporting reinfection among 5 study participants in northern Brazil. A) Participant ID151006; B) participant ID151026; C) participant ID151035; D)...

Because reports on the genomic characteristics of ZIKV isolates from 2017 onward in northern Brazil are lacking, we conducted a complementary analysis of our own dataset that revealed the presence of these exact genomes associated with reinfection within the population. Of note, we observed the presence of these same genomes in multiple samples from our cohort ( Appendix 1 Figure 2), providing strong evidence that the viruses were circulating both temporally and geographically. Finally, to support the assumption of reinfection, we analyzed the levels of ZIKV-NAb at 7, 30, 180, and 360 days after disease onset, assuming that antibody titers would vary among initial infection and reinfection, mirroring rRT-PCR results. We observed that all but 1 participant (ID251069) responded with increased levels of ZIKV-NAb at the convalescent phase of the disease (30 days after symptom onset) ( Figure 7 ). At 180 days after onset we observed a decay in ZIKV-NAb levels at an interval when the primary infection was already cleared. All 3 potentially reinfected participants (ID251069, ID151035, ID251064) responded with a second increase in the levels of ZIKV-NAb at the last interval analyzed ( Figure 7 ), which was preceded by viral RNA redetection in plasma or urine specimens. We also discarded other arbovirus infections as an inducer of ZIKV-NAb response because our study protocol was based on a validated multiplex rRT-PCR and none of the participants tested positive for either dengue or chikungunya virus. Other well-known circulating arboviruses in northern Brazil are Oropouche virus (OROV) ( 23 ) and yellow fever virus (YFV) ( 24 ). To date, no documented reports have indicated that OROV is capable of eliciting a ZIKV-specific antibody response. Furthermore, most study participants had prior YFV vaccination, so it is unlikely that they had become infected; thus, we have effectively ruled out YFV as a potential confounding factor in relation to the antibody response associated with ZIKV reinfection. Of note, none of the 3 potential reinfection cases reported symptoms typically associated with ZIKV infection, as confirmed through a comprehensive anamnesis conducted during each study visit at our study clinic. Collectively, our data strongly support the occurrence of reinfection events in at least 3 healthy persons residing in a ZIKV-endemic area in Brazil.

Given the number of ZIKV cases registered at the peak of the 2016 epidemic in the northern and other regions of Brazil ( 5 ), added to the risk for new outbreaks, it is critical to study ZIKV evolution and its potential for adaptation to vertebrate hosts. Moreover, virus persistence may exert high evolutive pressures that contribute to virus evolution and transmission. In our study, we showed that the obtained ZIKV genomes clustered together with other ZIKV Asian strains previously isolated from northern Brazil, suggesting that this strain persisted locally through natural transmission and was kept circulating among humans until August 2019 or later. We also found that the temporal circulation of ZIKV in Manaus started a descending curve, supported by a decreasing number of cases registered after the peak of the epidemic in 2016. Thus, based on a seroprevalence study from northeastern Brazil showing that the ZIKV antibody prevalence reached a peak of 63% from 2015 to 2016 ( 25 ), in addition to other studies showing a high ( >60%) seroprevalence of ZIKV antibodies in the general population ( 26 – 28 ), we hypothesize that, within a single year, community immunity was enough to constrain virus circulation. In fact, our findings are consistent with a lower reproduction number (R0) since late 2016 in Salvador ( 25 ), corroborating mathematical modeling studies showing that ZIKV epidemics would be over in 3 years from its introduction in 2016 ( 29 ).

Long-term cohort studies can provide longitudinal data on individual virus diversity, virus evolution, clinical symptoms, and immunological outcomes, and so are crucial to better understanding ZIKV natural history. We found evidence of limited virus diversity over time from persistently ZIKV-infected persons, a feature that has also been observed by other independent studies ( 18 , 19 ). Thus, we can suggest that the evolutionary rates and selection pressures acting on ZIKV are moderate, affecting virus evolution and adaptation to local populations. In fact, similar to a previous report ( 30 ), we estimated the ZIKV whole-genome evolutionary rate at around 1.18 × 10 −3 substitutions/site/year. Arboviruses primarily spread through horizontal transmission between arthropod vectors and vertebrate hosts. As a result, virus evolution is restricted by the need for optimal replication in one host, which may compromise their adaptation in the other ( 31 ), contributing to the short-term and long-term reduced number of adaptative mutations observed. In addition, various factors, including the short duration and low viremia observed in naturally infected persons ( 32 ), contribute to limiting ZIKV diversity. Consequently, our findings indicate that ZIKV displayed a relatively stable genome evolution over time and did not undergo rapid changes or diversification during the epidemic in northern Brazil.

The most notable finding of our study is the identification of reinfection events, which is highly intriguing. Given that the ZIKV epidemic in Brazil originated from a single virus strain, and combined with the observation that the virus has remained relatively stable over time, tracking reinfections becomes a challenging task. Complicating matters further, most infections are asymptomatic or cause only mild symptoms, such as fever, rash, and itching ( 33 ). As a result, persons who have been potentially reinfected may have gone unnoticed, especially considering that mild symptoms often do not prompt persons to seek medical attention. Although reinfections are extremely difficult to confirm when there are only very similar phylogenetically strains causing an outbreak, we detected divergent viruses in ZIKV-infected persons who provided longitudinal samples, which suggests a subsequent and distinct infection event.

Monitoring community virus circulation plays a crucial role in confirming infections within a population. As extensively explored for several other viruses ( 34 ), mapping diversity in a community can provide valuable information for confirming infection cases and understanding the dynamics of an outbreak. Thus, by sequencing viral genomes, it is possible to identify an specific strain or variant of the virus present in an individual or a community ( 35 ). Despite detecting the presence of these same reinfection-associated ZIKV genomes in other participants of our cohort, temporally and geographically confirming the circulation of these genomes in that population, there is a notable absence of independent studies validating the presence of these genomes at the time we detected potential reinfection cases. Most of the investigations from other groups were conducted during the early stages of the ZIKV outbreak; therefore, the literature lacks reports that describe the characteristics of the viruses circulating from late 2017 onward.

Confirmation of reinfection events based solely on molecular detection may introduce uncertainties because of the possibility of cross-contamination during sample processing. To address this concern, we conducted an assessment of ZIKV antibody response. By measuring the levels of ZIKV-NAb over time, we can add a deeper understanding of ZIKV infection dynamics, immune response effectiveness, and the potential for future reinfections ( 36 ). Here, we observed that 3 persons responded with a second increase in ZIKV-NAb levels, which was temporarily associated with rRT-PCR positivity at a late time point after the initial infection. We discarded other arbovirus infections as a cause of secondary ZIKV-NAb increase because all the participants from our cohort were tested in a multiplex rRT-PCR and none of them were positive for dengue or chikungunya virus. We also discarded YFV infection and YFV vaccination because our study participants were previously vaccinated against YFV. We also assumed that these reinfection events were very mild, mostly manifesting as an asymptomatic disease, because no symptoms were reported.

Our findings hold significant implications for public health, epidemiology, clinical practice, and diagnostics. However, the frequency of reinfections during the latest ZIKV outbreaks remains uncertain. Our study emphasizes the critical role of ongoing genomic surveillance in viral infections to enhance public health interventions. Therefore, we underscore the necessity of implementing continuous surveillance strategies, which are vital for monitoring the evolutionary changes of viruses over time and gaining a comprehensive understanding of arbovirus diversity.

Dr. Castilho has a PhD in tropical infectious diseases and works at the Tropical Medicine Foundation Doctor Heitor Vieira Dourado, Manaus, Brazil. Her research interests include arbovirus and epidemiology.

Acknowledgments

We thank the ZIKABRA Study Team: André Luiz de Abreu, Ximena Pamela Diaz Bermudez, Patrícia Brasil, Carlos Alexandre Antunes Brito, Tatiana Jorge Fernandes, Ndema Habib, Marcus Vinicius Guimarães Lacerda, Cristina Pimenta, Lydie Trautman.

Complete data set is available upon request. All the ZIKV genomes generated in this study are public available at the European Nucleotide Archive ( https://www.ebi.ac.uk/ena/browser/home ) under project no. PRJEB63302.

The research leading to these results received funding from the Wellcome Trust (grant no. 206522/Z/17/Z); UNDP-UNFPA-UNICEF-WHO-World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP), a cosponsored program executed by the World Health Organization (WHO) (WHO-SRH/HRP; grant nos. 2017/720873-0 and 2017/731359-0); Brazilian Ministry of Health (Convênio 837059/2016, Processo 25000162039201616); US National Institute of Allergy and Infectious Diseases of the National Institutes of Health: (award no. R21AI139777); and the Henry M. Jackson Foundation for the Advancement of Military Medicine (prime award no. W81XWH-18-2-0040). G.L.W. is supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico through their productivity research fellowships (no. 303902/2019-1). The funders had no role in the study design, data collection, analysis, the decision to publish, or the preparation of the manuscript.

The authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated.

Author contributions: A.M.B.F., M.G., V.F., L.C.J.A., L.C.M., and C.D. performed sequencing assays and data analysis, M.C.C., C.H.A.B.-M., and A.M-N. laboratory procedures and clinical data collection; G.A.C., E.K., and N.B. performed data analysis and clinical support; R.L.B. performed microneutralization assays, K.M. performed study supervision and data analysis, G.L.W. performed phylogenetic analysis and manuscript editing, R.F.O.F. conceived and supervised the study and wrote the manuscript. All authors reviewed and approved the final version of the manuscript. The authors declare they have no conflict of interests, and the funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.

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• Figure 1 . Genomic epidemiology of Zika virus strains obtained from study participants in northern Brazil and reference sequences. A) Time-scaled maximum clade credibility tree of Zika virus Asian lineage in Brazil,...
• Figure 2 . Single-nucleotide variants per gene for Zika virus strains obtained from study participants in northern Brazil. Amino acid changes in the polyprotein are allocated along the genome. Only mutations that...
• Figure 3 . Notified Zika virus cases per week in Manaus municipality, Brazil, 2016–2019, from the Brazilian Ministry of Health. Shading reflects each epidemic year. Data source: https://datasus.saude.gov.br/informacoes-de-saude-tabnet.
• Figure 4 . Phylogenetic analysis of study participants persistently infected with Zika virus, Brazil. A) Maximum-likelihood phylogenetic tree of persistent samples. The phylogenetic tree shows all 10 participants with confirmed persistent infection....
• Figure 5 . Maximum-likelihood phylogenetic tree supporting Zika virus reinfection among study participants in northern Brazil. The tree shows the 5 participants with divergent samples in which coinfection by different ZIKV genomes...
• Figure 6 . Zika virus rRT-PCR results from plasma, urine, and semen (when applicable) specimens supporting reinfection among female (A) and male (B) study participants in northern Brazil. Each square represents an...
• Figure 7 . Zika virus neutralizing antibody titers from acute and convalescent serum samples supporting reinfection among 5 study participants in northern Brazil. A) Participant ID151006; B) participant ID151026; C) participant ID151035;...
• Table 1 . Study visits and sample collection for participants in study of Zika virus reinfection, Brazil
• Table 2 . Characteristics of participants included in study of genomic analysis of Zika virus reinfection, Brazil

DOI: 10.3201/eid3002.230122

Original Publication Date: January 17, 2024

1 These authors contributed equally to this article.

Table of Contents – Volume 30, Number 2—February 2024

Please use the form below to submit correspondence to the authors or contact them at the following address:

Rafael F.O. Franca, Fundacao Oswaldo Cruz Virology and Experimental Therapy, Av. Prof. Moraes Rego s/n Recife/PE 50740-465, Recife 50740465, Brazil

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Open Access

Peer-reviewed

Research Article

Characteristics, risk factors, and outcomes related to Zika virus infection during pregnancy in Northeastern Thailand: A prospective pregnancy cohort study, 2018–2020

Roles Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation Thailand Ministry of Public Health, Department of Disease Control, Nonthaburi, Thailand

Roles Conceptualization, Data curation, Formal analysis, Methodology, Project administration

Affiliation Thailand Ministry of Public Health–US Centers for Disease Control and Prevention Collaboration, Nonthaburi, Thailand

Roles Writing – original draft, Writing – review & editing

Affiliation US Centers for Disease Control and Prevention, Division of Global Health Protection, Atlanta, Georgia, United States of America

Roles Investigation, Resources, Validation

Roles Investigation, Methodology, Resources, Validation

Roles Investigation, Supervision, Validation

Affiliations Thailand Ministry of Public Health–US Centers for Disease Control and Prevention Collaboration, Nonthaburi, Thailand, US Centers for Disease Control and Prevention, Division of Global Health Protection, Atlanta, Georgia, United States of America

Roles Data curation, Formal analysis, Validation, Visualization

Roles Conceptualization, Funding acquisition, Project administration, Supervision

Roles Data curation, Project administration, Supervision, Validation

Roles Data curation, Supervision

Roles Conceptualization, Funding acquisition, Methodology, Project administration, Resources

Roles Conceptualization, Methodology

Roles Investigation

Affiliation Thailand Ministry of Public Health, Department of Medical Services, Nonthaburi, Thailand

Roles Methodology

Affiliation Bueng Kan Provincial Public Health Office, Bueng Kan, Thailand

Affiliation Bueng Kan Hospital, Bueng Kan, Thailand

Affiliation Mukdahan Provincial Public Health Office, Mukdahan, Thailand

Affiliation Mukdahan Hospital, Mukdahan, Thailand

Roles Investigation, Methodology

Roles Conceptualization, Formal analysis, Methodology, Writing – review & editing

•  [ ... ],

Roles Writing – review & editing

• [ view all ]
• [ view less ]
• Jurai Wongsawat, 
• Somsak Thamthitiwat, 
• Victoria J. Hicks, 
• Sumonmal Uttayamakul, 
• Phanthaneeya Teepruksa, 
• Pongpun Sawatwong, 
• Beth Skaggs, 
• Philip A. Mock, 
• John R. MacArthur, 

• Published: May 17, 2024
• https://doi.org/10.1371/journal.pntd.0012176
• Reader Comments

In response to the 2015–2016 Zika virus (ZIKV) outbreak and the causal relationship established between maternal ZIKV infection and adverse infant outcomes, we conducted a cohort study to estimate the incidence of ZIKV infection in pregnancy and assess its impacts in women and infants.

Methodology/Principal findings

From May 2018-January 2020, we prospectively followed pregnant women recruited from 134 participating hospitals in two non-adjacent provinces in northeastern Thailand. We collected demographic, clinical, and epidemiologic data and blood and urine at routine antenatal care visits until delivery. ZIKV infections were confirmed by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Specimens with confirmed ZIKV underwent whole genome sequencing.

Among 3,312 women enrolled, 12 (0.36%) had ZIKV infections, of which two (17%) were detected at enrollment. Ten (83%, 3 in 2 nd and 7 in 3 rd trimester) ZIKV infections were detected during study follow-up, resulting in an infection rate of 0.15 per 1,000 person-weeks (95% CI: 0.07–0.28). The majority (11/12, 91.7%) of infections occurred in one province. Persistent ZIKV viremia (42 days) was found in only one woman. Six women with confirmed ZIKV infections were asymptomatic until delivery. Sequencing of 8 ZIKV isolates revealed all were of Asian lineage. All 12 ZIKV infected women gave birth to live, full-term infants; the only observed adverse birth outcome was low birth weight in one (8%) infant. Pregnancies in 3,300 ZIKV-rRT-PCR-negative women were complicated by 101 (3%) fetal deaths, of which 67 (66%) had miscarriages and 34 (34%) had stillbirths. There were no differences between adverse fetal or birth outcomes of live infants born to ZIKV-rRT-PCR-positive mothers compared to live infants born to ZIKV-rRT-PCR-negative mothers.

Conclusions/Significance

Confirmed ZIKV infections occurred infrequently in this large pregnancy cohort and observed adverse maternal and birth outcomes did not differ between mothers with and without confirmed infections.

Author summary

Zika virus (ZIKV) is an emerging infectious pathogen that was elevated to a Public Health Emergency of International Concern in 2016, due to its possible association with congenital microcephaly, a serious birth defect for infants. We launched a prospective cohort study of pregnant women to look for evidence of ZIKV infection during pregnancy. We enrolled participants in two provinces in northeastern Thailand during May 2018-January 2020. We found a low frequency of ZIKV infection (12/3312, 0.36% by rRT-PCR assay) during the study; two infections were detected at enrollment, while ten were detected later. Six of 12 ZIKV-confirmed women were asymptomatic throughout the study. Most (11/12, 92%) Zika cases occurred in one province. All 12 women with ZIKV delivered normal live births; only one infant had low birth weight and no infants had microcephaly. The eight ZIKV isolates available for whole genome sequencing were of the Asian lineage. At enrollment, the median Dengue virus IgG titer was significantly higher in ZIKV-confirmed than non-ZIKV-confirmed women. Identifying the factors associated with neonatal outcomes after ZIKV exposure in utero would be made easier with additional research studies.

Citation: Wongsawat J, Thamthitiwat S, Hicks VJ, Uttayamakul S, Teepruksa P, Sawatwong P, et al. (2024) Characteristics, risk factors, and outcomes related to Zika virus infection during pregnancy in Northeastern Thailand: A prospective pregnancy cohort study, 2018–2020. PLoS Negl Trop Dis 18(5): e0012176. https://doi.org/10.1371/journal.pntd.0012176

Editor: Elvina Viennet, Australian Red Cross Lifelood, AUSTRALIA

Received: October 20, 2023; Accepted: April 29, 2024; Published: May 17, 2024

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This project received funding provided by the U.S. government through the Office of Infectious Diseases, Bureau for Global Health, U.S. Agency for International Development, from the Zika Emergency Fund under the terms of an Interagency Agreement with the U.S. Centers for Disease Control and Prevention (CoAg/IAA 16CGH1613816), and also from the U.S. Centers for Disease Control and Prevention (CoAg/ 1U01GH002084). Subsequently, the Thailand MOPH – U.S. CDC collaboration unit utilized this funding to provide financial support for this study. The funders played no role in the design of the study, data collection and analysis, decision to publish, or preparation of the manuscript. The following authors received compensation from the project for their part-time hours dedicated to research activities: JW, SU, AV, PD, KS, CW, PS, BP, and PS. The following authors received partial support for their salaries: ST, PT, PS, BS, PAM, JRM, IS, PS, PK, and EB. DJ, the Project Coordinator at one of provincial sites, received a full salary from the project.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Zika virus (ZIKV) is an emerging infectious pathogen that has risen to the highest level of importance in public health. ZIKV is a single-stranded ribonucleic acid (RNA) virus and is a member of the Flavivirus family that is primarily spread by mosquitoes [ 1 ]. ZIKV is closely related to other mosquito-borne flaviviruses including dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus, and yellow fever virus. Patients with acute ZIKV infection often present with similar symptoms as those infected with other arboviruses, including DENV and chikungunya virus (CHIKV). Although most patients with acute ZIKV infection are asymptomatic, some patients present with mild fever, rash, arthralgia, and conjunctivitis [ 2 ].

Researchers first isolated ZIKV from the serum of a Ugandan rhesus monkey in 1947 [ 3 ]. Though serological and entomological evidence indicated that ZIKV had spread widely throughout Asia and Africa, until 2007, only a few human Zika cases were reported [ 4 ]. The two major genetic lineages for ZIKV, Asian and African, were named after the distribution of the virus in these two continents [ 5 ]. The first major ZIKV outbreaks were reported in 2007 on Yap Island in the Federated States of Micronesia and in Gabon in Central Africa [ 6 , 7 ]. ZIKV outbreaks in the Pacific region later occurred in close succession in French Polynesia (2013–14), New Caledonia (2014), the Cook Islands (2014), and Easter Island (2014) [ 8 , 9 ].

In February 2016, the World Health Organization (WHO) declared a Public Health Emergency of International Concern (PHEIC) in response to an increasing ZIKV outbreak in Latin America [ 10 – 12 ]. Although the link between ZIKV infection and newly reported clusters of microcephaly and other neurological disorders was unclear [ 13 ], the WHO called for increased Zika surveillance and research related to ZIKV, microcephaly, and other neurological disorders worldwide. In addition, new public health recommendations were issued to help prevent ZIKV infection in pregnant women and the general population [ 13 , 14 ]. Scientists have since established a causal relationship between maternal Zika virus infection during pregnancy and adverse birth outcomes in infants [ 14 , 15 ].

Before 2016, there had been sporadic Zika cases reported in Thailand, though researchers have recently suggested that the virus could have been circulating in the country since 2002 [ 16 , 17 ]. While some Zika case reports had been previously published, there were gaps in scientific understanding surrounding ZIKV infection in pregnancy in Thailand and Southeast Asia.

In response to the PHEIC declaration, the Thailand Ministry of Public Health (MOPH) and the United States Centers for Disease Control and Prevention (CDC) launched a study to investigate maternal ZIKV infection in Thailand. The Pregnancy Outcomes After Acute Zika Virus Infection in Northeastern Thailand (PrOZINT) study was a prospective cohort study designed to follow pregnant women and their newborns over time. Study objectives were to (1) estimate the incidence of ZIKV infection in pregnant women and their newborns; (2) assess the risk of adverse maternal, fetal, and infant outcomes associated with maternal ZIKV infection during pregnancy; and (3) assess the modifiers of the risk of adverse outcomes in pregnant women and infants after maternal ZIKV infection, such as gestational age at infection, previous exposure to flaviviruses, the presence of ZIKV infection symptoms, and co-infections with other flaviviruses. In this analysis, we describe the incidence of ZIKV infection and the characteristics, risk factors, and pregnancy, fetal, and infant outcomes related to ZIKV infections in a cohort of pregnant women. The results from the follow-up and developmental milestones of the infant cohort will be reported separately.

Ethics statement

The protocol was reviewed by the Thailand Ministry Of Public Health Ethical Committee (FWA 00001953, approval number Ref.no.22/2560) and the Institutional Review Boards of both provincial hospitals (Bueng Kan hospital, approval number BKHEC2018-06 and Mukdahan hospital, approval number MEC 05/61). Written informed consents were obtained from all participants. For participants aged 15–17 years, written informed consents were also obtained from their parent or legal guardian.

Study setting

The PrOZINT study was conducted in the Bueng Kan and Mukdahan provinces of northeastern Thailand. These two provinces were selected due to the number of Zika cases reported via routine Zika surveillance in 2016 [ 18 ]. Both provinces are located on the border with Laos, separated by approximately 290 kilometers. Bueng Kan has a total population of 424,091 inhabitants living in eight districts. Mukdahan has a population of 353,174 living in seven districts [ 19 ]. Both provinces have a tropical climate with wet and dry seasons. Annual rainfall averages are around 2365 millimeters (mm) in Bueng Kan and 1547mm in Mukdahan [ 20 , 21 ].

Study population and enrollment

From May 2018 to January 2020, pregnant women were consecutively approached for enrollment during regular antenatal visits at 134 study sites, which included the provincial hospitals and all district hospitals in Bueng Kan and Mukdahan provinces. Study staff administered an eligibility screening questionnaire and interested women signed a written informed consent form to enroll in the study. Minors (aged 15–17 years) were asked to sign an informed assent along with the consent of her parent or legal guardian. Participants were then followed from study enrollment until the end of pregnancy, with clinical touchpoints occurring during routine antenatal care (ANC) and sick visits.

Inclusion criteria

Study staff recruited women aged 15–49 years who were visiting a participating health facility for confirmation of pregnancy or routine ANC. During the first year of the study, the eligibility criteria were confirmed pregnancy, the willingness to deliver at a participating hospital, and the intention of attending routine ANC and well-child checkups. Women with ectopic, molar, or multiple pregnancies were excluded. Women presenting to a health facility in active labor without a history of ANC were excluded from the study because it was not possible to obtain their clinical histories and records of any prior ZIKV testing. Without their previous clinical and testing information, the study would be unable to assess which outcomes of these women were ZIKV-related and the duration of viremia for any women who tested positive for ZIKV would also be unknown. Women unwilling to consent to infant or maternal laboratory testing and follow-up were not enrolled into the study.

At the beginning of the study, we enrolled pregnant women in any trimester. A review of the 2018 study data showed frequent blighted anembryonic pregnancies had occurred in several participants who were in their first trimester, resulting in several adverse events reported to the Thailand MOPH Ethical Committee. All women with this adverse pregnancy outcome had tested negative for ZIKV using rRT-PCR, and thus the causes of blighted anembryonic pregnancies for each participant were determined to be unrelated to ZIKV. In addition, the observed rate of blighted anembryonic pregnancies observed in the cohort was comparable to the background rate in this geographic area. Due to the frequent occurrences of non-ZIKV–related blighted anembryonic pregnancy, the inclusion criteria were then subsequently modified, and so only women with a gestational age ≥12 weeks were enrolled in the study. The amendment revising the inclusion criteria was approved by the Thailand Ministry Of Public Health Ethical Committee, and starting in mid-2019, only participants in their second or third trimester were enrolled in the study.

Data collection

At enrollment and during each of the routine ANC visits, study staff administered standardized questionnaires about ZIKV risk factors, symptom history (including rash, fever, arthralgia, and conjunctivitis), and any medical history changes since the last visit. Routine ANC visits in Thailand take place at approximately weeks 12, 18, 24, 30, and 36 of gestation. Information was also abstracted from medical charts regarding routine patient history interviews and physical examinations. Variables collected included maternal demographics, risk factors and behavioral factors (such as location spent during daytime and evening and personal protective measures) for acquiring vector-borne diseases, parity, ANC clinical data, laboratory tests, in utero ultrasound, and clinical and laboratory data. If there was an unscheduled visit (i.e., non-ANC visit), study staff administered a questionnaire to assess for signs and symptoms related to ZIKV infection and pregnancy complications.

Maternal EDTA blood, clotted blood, and urine specimens were collected at all visits, including enrollment, routine ANC, unscheduled visits, and delivery ( S1 Fig ). Study staff collected 3 cubic centimeters (cc) of whole blood, 3 cc serum, and 20 cc urine from participants at enrollment, during each routine ANC visit, and each unscheduled visit to test for evidence of past or current ZIKV, DENV, and CHIKV, infection/exposure. Ultrasounds, amniocenteses, and lumbar punctures were performed if clinically indicated and according to Thailand’s guidelines [ 22 ].

ZIKV-positive women (defined as those with detectable ZIKV RNA by rRT-PCR) received follow-up prenatal assessments, clinical care, and psychosocial support according to Thailand’s guidelines for diagnosis and care of pregnant women with ZIKV infection. ZIKV-positive women were asked to return to the hospital at two-week intervals for clinical examinations and blood and urine sample collection, until two consecutive samples were negative for ZIKV by rRT-PCR. After that time, the normal frequency of routine ANC visits resumed.

The study staff administered labor and delivery case report forms to and collected whole blood, serum, and urine from mothers at delivery or within the first 96 hours after delivery. Infant EDTA blood and urine were collected at delivery as a part of the infant cohort follow-up procedures. Neonatal examination data were abstracted, including measurements of the head circumference, weight, and length, infant APGAR scores, a Dubowitz examination score (a method to estimate an infant’s gestational age by combined physical and neuromuscular assessment), and cranial ultrasound findings.

Laboratory testing

Due to the serological cross-reactivity among flaviviruses circulating in Thailand, we then use molecular test to confirm ZIKV infection in pregnant women. In addition, to better understand background of possibility of any flavivirus infections among each enrolled pregnant women, especially if occurred between their ANC (study) visits, the serological tests were also performed at every visit.

Maternal and infant blood specimens were tested for ZIKV, CHIKV, and DENV RNA using the CDC Trioplex rRT-PCR assay [ 23 ]. Maternal urine specimens were tested for ZIKV RNA using single-plex rRT-PCR with ZIKV primers and probes as below:

Zika1087 5’-CCGCTGCCCAACACAAG-3’ Zika1108FAM 5’-AGCCTACCTTGACAAGCAGTCAGACACTCAA-3’ Zika1163c 5’-CCACTAACGTTCTTTTGCAGACAT-3’

For both blood and urine specimens, nucleic acid was extracted by MagNaPure 24 nucleic acid extraction system (Roche, USA) with MagNa Pure 24 Total NA Isolation Kit (Cat. No. 07628036001). Trioplex rRT-PCR assays were performed as previously described [ 23 ]. A positive result was defined as cycle threshold (Ct) < 38. Singleplex rRT-PCR assays were performed as previously described [ 24 ]; specimens with Ct < 38 were considered positive for ZIKV. Whole genome sequencing was conducted on ZIKV from maternal rRT-PCR positive EDTA blood specimens by next generation sequencing using the MiSeq (Illumina, USA) platform to determine the ZIKV genotype [ 25 ].

Serologic testing was done by commercial enzyme-linked immunosorbent assay (ELISA) (InBios International, USA) according to manufacturer recommendations; results were read at 450 nm using a microplate reader (Thermo Scientific Multiskan FC, USAorSpectraMax, Molecular Devices, USA). Maternal serum specimens collected at enrollment, routine ANC visits, sick visits, and at delivery were tested for IgM antibodies against ZIKV and DENV using the Zika virus IgM capture ELISA and DENV IgM Capture ELISA, respectively. ( S2 Fig ) We defined a positive IgM result for ZIKV and DENV as having an immune status ratio (ISR) of ≥ 1.9, and ≥ 2.84, respectively [ 26 – 28 ].

IgG antibodies for ZIKV and DENV were tested in all maternal sera collected at study enrollment, unscheduled visits, and delivery. IgG antibodies were also tested in maternal sera from women who were positive for ZIKV or DENV by IgM ELISA and/or Trioplex rRT-PCR, using the ZIKV IgG Capture ELISA and DENV IgG-ELISA. IgG positive ISR for ZIKV and DENV were ≥ 1.1 and ≥ 2.84, respectively [ 29 – 31 ]. Women with clinical signs and symptoms of CHIKV-like infection and/or who were positive for CHIKV by TrioplexrRT-PCR were tested for CHIKV IgM and IgG antibodies using CHIK IgM ELISA and CHIKIgG ELISA, with an ISR of ≥ 1.1 for both tests [ 32 , 33 ].

Case definitions-

Acute zikv infection..

We classified maternal Zika cases in accordance with Thai and the World Health Organization’s guidelines [ 22 , 34 ]. Per these guidelines, a ZIKV case was as follows:

Laboratory confirmation of recent ZIKV infection is defined as:

• the presence of ZIKV RNA or antigens in serum or other samples (e.g. saliva, tissues, urine, whole blood), or
• the presence of IgM antibodies against ZIKV and a PRNT90 for ZIKV with titre ≥20 and ZIKV PRNT90 titre ratio ≥ 4 compared to other flaviviruses; and exclusion of other flaviviruses [ 34 ].

For the present analysis, we defined ZIKV infections only according to the presence or absence of ZIKV RNA, as we did not perform PRNT 90 for ZIKV. Confirmed acute ZIKV infection was defined as the detection of ZIKV RNA in a laboratory specimen by rRT-PCR (blood or urine). For DENV and CHIKV, confirmed acute infection was also defined as the detection of viral RNA by Trioplex rRT-PCR in blood specimens.

Maternal outcomes.

Maternal death was defined as the death of a woman while pregnant or within 42 days of termination of pregnancy, irrespective of the duration and site of the pregnancy, from any cause related to or aggravated by the pregnancy or its management, but not from accidental or incidental causes. Maternal complications during pregnancy e.g., preeclampsia, gestational diabetes, etc. and route of deliveries were also collected. Obstetrical complications were defined as intra- and post-partum complications or any adverse events, e.g., high blood pressure, premature rupture of membranes, placenta previa, hemorrhage, thromboembolism, etc.

Fetal outcomes.

Intrauterine growth restriction (IUGR), or the condition in which a fetus did not achieve the expected in utero growth potential due to genetic or environmental factors, was defined as an estimated fetal weight below the 10th percentile. Moderate Fetal Growth Restriction (FGR) was defined as birth weight in the 3rd to 10th percentiles, and severe FGR as less than the 3rd percentile [ 35 ]. Spontaneous abortion or miscarriage was defined as a fetus at less than 20 weeks with no signs of life prior to the complete expulsion or extraction from the mother with confirmation that the fetus showed no signs of life after delivery and could not be resuscitated. Stillbirth was defined as a fetus at or after 20 weeks with no signs of life prior to the complete expulsion or extraction from the mother with confirmation that the fetus showed no signs of life after delivery and could not be resuscitated [ 36 , 37 ].

Birth outcomes.

Preterm, or premature, birth was defined as birth of a liveborn infant less than 37 weeks gestation, as calculated by gestational age by a pregnancy-dating ultrasound performed at <28 weeks gestation. The birth timeframe for extreme preterm was less than 28 weeks, very preterm was 28 to less than 32 weeks, and late preterm was 32 to less than 37 weeks [ 38 , 39 ]. Neonatal death was defined as the death of an infant at days 0–27 of life; a death occurring between days 0–6 was defined as an early neonatal death [ 40 ]. Small for gestational age (SGA) was defined as an infant with a birth weight below the 10th percentile for gestational age [ 41 ].

Congenital microcephaly for term or mature neonates (born at 37 weeks gestation) was defined as measured head circumference (HC) within 24–72 hours of birth that is less than the 3 rd percentile on the Thai child growth chart (which is based on WHO Child Growth Standards) or more than 2 standard deviations (SD) below the median for gestational age and sex on Intergrowth-21 Standard for pregnant women with confirmed gestational age [ 42 – 45 ]. Congenital microcephaly for preterm neonates (born at less than 37 weeks of gestation) was defined as measured HC within 24–72 hours of birth that is less than the 3 rd percentile for gestational age and sex on the Fenton Preterm Growth Chart. Congenital microcephaly for a pregnancy loss was defined as prenatal HC that is more than 3 SD below the mean on prenatal ultrasound or postnatal HC that is less than the 3rd percentile [ 46 ]. Suspected cases of Congenital Zika Syndrome (CZS) were defined as liveborn infant, fetal death or stillbirth with congenital microcephaly or any congenital malformation of the central nervous system (e.g. hypertonia, eye abnormalities, abnormal hearing test, etc.) or congenital contractures (e.g., club foot, arthrogryposis, etc.) [ 47 ].

A composite variable was developed to account for any adverse fetal or birth outcome, defined as one or more of the following: IUGR, preterm birth, low birth weight, SGA, congenital anomaly (e.g., microcephaly, intracranial calcification documented by postnatal cranial ultrasound), or neonatal death.

Data management and statistical analysis

Study staff collected information on hardcopy forms and/or tablets and then entered the data into a secure, password-protected Microsoft Access database for data management at the study sites (Microsoft Corporation, Redmond, Washington, USA). Quality control measures were established. Records were routinely reviewed to determine the accuracy of data entry, and discrepancies were resolved by review and re-entry of source documents. Duplicates were checked and removed and data backups were conducted frequently in accordance with data management standard operating procedures.

Sample size was calculated based on an estimated ZIKV infection prevalence of 5% for pregnant women in Thailand [ 18 ] and ±2.5% precision of the prevalence estimate. We used a Bayesian sample size calculation [ 48 ] which took into account uncertainties of the prevalence (0–10%), sensitivity (88.1–99.9%), and specificity (93–99.9%) of EUROIMMUN AG serology testing [ 49 ]. The cohort sample size needed was calculated to be 3,148 to detect the prevalence of ZIKV infection. If we were to assume a loss to follow up of 20%, then the sample size needed at enrollment is 3,478.

Categorical variables were summarized using counts and percentages, and means and standard deviations, or the medians and interquartile ranges were calculated for continuous variables. Group comparisons were carried out using chi-square or Fisher’s exact test for proportions, as appropriate, and the t-test or Wilcoxon test for continuous variables. Household income was converted to 2017 US dollar (USD) using the 2017 yearly average exchange rates (35.372 Baht to 1 USD) from the US Internal Revenue Service [ 50 ]. Exact binomial 95% confidence intervals (95% CIs) were calculated for prevalence estimates for ZIKV, DENV, and CHIKV infections. The incidence rates were calculated as the number of rRT-PCR test positive infections divided by the number of person-weeks of follow-up among those testing negative at enrollment. The estimated time of infection was defined as the midpoint between the date of the last test-negative and first test-positive visits. We requested ZIKV-positive women to return to the hospital at two-weeks intervals, until a ZIKV-positive woman had two consecutive ZIKV tests that were PCR negative. We considered the duration of ZIKV infection to be the period from the estimated time of infection until the 1st negative PCR test result. The person-weeks of follow-up were calculated from the date of enrollment to the estimated date of infection for the test positive, or to the last test date for the uninfected. We calculated 95% CIs for incidence estimates using the exact Poisson method.

Risk ratios (RR) were used as the measure of effect for the composite fetal and birth adverse outcomes. Adjusted RR (aRR) for other significant risk factors with p-values < 0.1 were estimated using log binomial regression [ 51 ]. Risk factors considered included demographics, behaviors, pregnancy history, the presence of Zika symptoms, prior infections or immunizations, and co-infections with other flaviviruses. Final variables included in the model were determined using a backwards stepdown procedure at p ≤ 0.05. Subjects with missing data were excluded in the analysis.

All statistical analyses were performed using SAS (Version 9.4; SAS Institute, Cary, North Carolina, USA) and Stata (Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC).

Overview of maternal cohort

An overview of the prospective maternal cohort and pregnancy outcomes in this study is provided in Fig 1 ; a total of 3,312 pregnant women were enrolled. Among the 12 ZIKV-positive women, all delivered live infants; one infant was found to have an adverse fetal outcome, specifically low birth weight (2,420 grams), resulting in a total of one (8%) adverse outcome in this group.

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1 Note: One maternal participant died during childbirth. The newborn born to this deceased mother survived and is counted in the infant birth numbers.

https://doi.org/10.1371/journal.pntd.0012176.g001

Incidence of ZIKV and other infections

Overall, 3,300 ZIKV-negative pregnant women were enrolled. Of these, 3,111 (94%) completed follow up, including 3,010 (91%) who had live births (2,707 term, 303 preterm) and 101 (3%) fetal losses (67 miscarriages, 34 stillbirths) ( Fig 1 ). There were 373 infants with adverse birth outcomes born to mothers in this group, resulting in a total of 474 (15%) total adverse outcomes in this group.

From May 2018—January 2020, 12 (0.36%) of 3,312 pregnant women enrolled in the study were ZIKV rRT-PCR positive. For two women, the ZIKV rRT-PCR positive results were detected upon enrollment, while 10 (3 in 2 nd and 7 in 3 rd trimester) were detected later during the follow-up period (incidence rate of 0.15 per 1,000 person-weeks (95% CI: 0.07, 0.28)). At enrollment, 2/3312 (0.06%) participants were ZIKV-positive. The incidence rate of ZIKV infection for women enrolled based on the initial eligibility criteria including all gestational ages was ([5/48341.7]*1000 = 0.103 per 1000 weeks (95% CI: 0.034, 0.24) and ([5 /16475.4]*1000 = 0.303 per 1000 weeks (95% CI: 0.098, 0.71) (IRR = 2.93 (95% CI: 0.68, 12.75), p = 0.10) on the revised inclusion criteria of gestational age ≥12 weeks.

Ten rRT-PCR confirmed dengue infections were detected during follow-up among ZIKV rRT-PCR negative women. Dengue infection at enrollment was 1/3312 or 0.03% (95% CI: 0.0007, 0.17), with nine incident infections for an incidence rate of 0.14 per 1,000 person-weeks (95% CI: 0.06, 0.26). Chikungunya infection at enrollment was 0/3312 or 0%, with ten incident infections detected for an incidence rate of 0.15 per 1,000 person-weeks (95% CI: 0.07, 0.28). None of the confirmed Zika infections had a positive rRT-PCR result for CHIKV or DENV detected during the study.

Distribution of ZIKV

Among the 12 ZIKV-positive women, 11 were diagnosed in Bueng Kan Province and only one was detected in Mukdahan ( p = 0.002) ( Table 1 ). More Zika cases (n = 4) were detected in Seka District, located in the eastern part of Bueng Kan province. Nine Zika cases were detected between May to October, which is the rainy season in northeastern Thailand ( Fig 2 ).

https://doi.org/10.1371/journal.pntd.0012176.g002

https://doi.org/10.1371/journal.pntd.0012176.t001

Cohort baseline characteristics

Baseline demographic, behavioral, and clinical characteristics of the 3,312 pregnant women enrolled in the study were examined (Tables 1 and 2 ). Overall, the average age was 26.6 (SD 6.7) years, and most women completed secondary education (n = 1995, 60%). When comparing women with and without confirmed ZIKV, women with ZIKV infection were more likely to be from Bueng Kan (p = 0.002) ( Table 1 ). All other demographic, behavioral, and clinical characteristics examined were similar between groups. Long-sleeved shirts and long pants significantly differed among the two groups.

https://doi.org/10.1371/journal.pntd.0012176.t002

Clinical and immunological characteristics

Symptoms and flavivirus-related immunological status of the 12 ZIKV-positive women throughout their pregnancies are shown in S3 Fig . Five (42%) of the 12 confirmed ZIKV infections were detected during the second trimester and the remaining 7 were detected during the third trimester. Two confirmed ZIKV infections detected at enrollment had a gestational age of around 16–17 weeks (Cases 4 and 5). Whereas, among the 10 incident Zika cases detected after enrollment, the gestational age at the estimated time of infection was 28.5 (IQR 20–33) weeks.

Among the women with confirmed ZIKV infection, six (50%) had ZIKV detected in both blood and urine, four (33%) had ZIKV detected only in blood, and two (17%) had ZIKV detected only in urine. Persistent ZIKV viremia was observed in one Zika case (Case 8, only the first rRT-PCR is shown in S3 Fig ). The first positive rRT-PCR test for Case 8 was on October 15, 2019, and her last positive rRT-PCR test was 42 days later, on November 26, 2019, with five positive rRT-PCR tests during the interval. Two women with ZIKV infection had one positive rRT-PCR result late in their pregnancy but no further ZIKV rRT-PCR testing because they delivered in non-study hospitals. ZIKV was detected in eight women who subsequently had negative rRT-PCR results at all subsequent testing time points. One Zika case had a ZIKV rRT-PCR test positive in blood but negative in urine at testing timepoint 3, followed by a negative rRT-PCR test result in blood and a positive rRT-PCR test result in urine at testing timepoint 4, followed by a negative rRT-PCR test result in both blood and urine specimens at testing timepoint 5. Among the women with confirmed ZIKV infection, nine had at least one positive ZIKV IgM ELISA result at some point during follow-up (Cases 3–8 and 10–12, S3 Fig ). Cases 1, 2 and 9 had negative ZIKV IgM ELISA results. Cases 4–6 sero-reverted (from positive to negative) at least once on ZIKV IgM ELISA.

Over the course of the study ( S3 Fig ), six of the women with confirmed ZIKV infection (Cases 1,2,4,5,7,9) were asymptomatic at enrollment and continued to be asymptomatic for the duration of follow-up.

Four (33%) cases were symptomatic during the visit when ZIKV was detected (Cases 3, 6, 10, and 11), while three (25%) cases reported symptoms prior to the visit when ZIKV was detected (Cases 8, 11, and 12); one case (Case 11) reported symptoms both prior to and during the visit when ZIKV was detected. Symptoms reported included rash, myalgia, and arthralgia. Of the 12 confirmed Zika cases, two (17%) reported more than one Zika-like symptom at the time of the visit in which the positive specimen was collected (Cases 6 and 10). None of the women with confirmed ZIKV infection reported having a previous history of ZIKV, DENV, JEV, or CHIKV infections ( Table 2 ). No statistical differences were seen between pregnant women with or without confirmed ZIKV infection in whether they had positive dengue IgG result at enrollment (p>0.999). However, median DENV IgG titers at baseline were higher (p = 0.042) among women with confirmed ZIKV infection when compared to women without confirmed ZIKV infection.

Contribution of ZIKV strains

Whole genome sequencing was performed on ZIKV isolates from eight cases, and all were of Asian lineage.

Outcomes of mothers and infants with and without ZIKV infection during pregnancy

Of the 3,312 women enrolled in the study, a total of 3,123 women completed follow-up. No differences were detected between women with and without confirmed ZIKV infection in terms of pregnancy, fetal, or birth outcomes ( Table 3 ). No obstetric complications or adverse fetal outcomes, such as fetal loss or neonatal death, occurred in the women with confirmed ZIKV infection. One maternal death occurred in a subject who did not have ZIKV infection; this woman experienced an amniotic fluid embolism. A total of 3,022 live births occurred in the maternal cohort, 12 (0.4%) of which occurred from women with confirmed ZIKV infection. The median birthweight of the cohort was 3,030 grams, with most infants at normal birthweight. ( Table 3 )

https://doi.org/10.1371/journal.pntd.0012176.t003

Among 12 infants born to mothers with confirmed ZIKV infection, neither microcephaly ( S4A and S4B Fig ) nor SGA was found.

In a multivariable analysis examining factors related to adverse infant outcomes, which included fetal loss and any adverse fetal or birth outcomes among live births, the only factors that were independently associated with adverse infant outcomes were age, education, travel to another province, and previous pregnancy (See Table 4 for aRR and confidence intervals). In the multivariable analysis, 1.2% (37/3123) of the data were excluded due to missing data. In our study population, maternal ZIKV infection was not associated with adverse infant outcomes.

https://doi.org/10.1371/journal.pntd.0012176.t004

Overall results and comparisons to results from other studies

Since the ZIKV PHEIC declaration (February to November 2016), numerous studies of pregnant women and infants around the world have reported varying rates of incidence and of symptoms related to ZIKV infections during pregnancy. From May 2018 to January 2020, this large, prospective cohort study in northeastern Thailand found that ZIKV infection was infrequently detected among pregnant women and resulted in mild symptoms and no serious adverse maternal or birth outcomes were detected. The incidence of ZIKV infection among pregnant women attending ANC visits between May 2018 to January 2020 in our cohort was <0.5% which is lower than that reported from many other prospective pregnancy cohort studies conducted previously during 2016–2017. For example, higher rates were observed among symptomatic women attending an acute febrile illness clinic in Rio de Janeiro (53%) during September 2015 to May 2016 [ 52 ], as well as women with or without symptoms included in systematic population-based monitoring in French Guiana (19%) during February to June 2016 [ 53 ], and attending clinics for high-risk pregnancies in Sao Paulo (7.7%) during March 2016 –August 2017 [ 54 ], and ANC in Peru (3.2%) during May to July 2016 [ 55 ], and Honduras (2%) during July 2016-December 2016 [ 56 ]. A study among symptomatic and asymptomatic pregnant women from Mexico during July 2016 –August 2017 revealed a similar cumulative incidence (0.31) as in our study [ 57 ].

Different factors across studies in terms of populations, inclusion criteria (e.g., existence of symptoms), timing (year) of the studies, in addition to local epidemics, likely contributed to differences in incidence rates in these cohorts. However, the low incidence in our study is consistent with relatively small numbers of ZIKV infections in Thailand at the height of the ZIKV outbreak in 2016 and 2017.[ 58 ] We also observed a higher incidence in Bueng Kan province than in the neighboring Mukdahan province, which may suggest that the Zika activity in Thailand was more localized and sporadic. There remains limited evidence as to why ZIKV incidence is relatively low in Southeast Asia and why large-scale outbreaks of ZIKV, like those observed in the Pacific and the Americas, have not been seen in Asia [ 16 , 59 ].

Laboratory testing considerations

Immunological cross-reactivity between the flaviviruses, especially between DENV and ZIKV, is an issue concerning serological diagnosis [ 3 , 4 ]. Besides misdiagnosis as other flavivirus infections, possible explanations may include virus factors (e.g., low pathogenicity of indigenous ZIKV strain, low ZIKV viremia), host factors (e.g., ZIKV protection from previous natural or acquired immunity from other flavivirus infections or vaccinations), and environmental factors (e.g., mosquito transmissibility, no natural reservoir of ZIKV) [ 60 – 65 ]. More research is needed to better understand questions related to these factors.

ZIKV infection in Southeast Asia has been generally characterized by asymptomatic or mild disease, with symptoms mainly consisting of fever, muscle pain, joint pain and sometimes rash or headache; more serious symptoms have been reported less commonly [ 60 – 65 ]. Individuals may not seek care for mild symptoms and patients with symptoms. We found that only two (17%) ZIKV-positive pregnant women in our cohort reported more than one Zika-like symptom at the time of ZIKV detection. In 2016–2017, during the peak of the outbreak, two cases of microcephaly and/or CNS malformations cases suggestive of congenital ZIKV infections or potentially associated with ZIKV infection were reported in Thailand [ 58 ].

Birth and neonatal outcomes in the cohort

No serious adverse birth outcomes were found in ZIKV-infected pregnant women in this study. Many studies from the Americas have reported a causal link between ZIVK infection and congenital microcephaly. In a longitudinal study in Rio de Janeiro, pregnant women with acute ZIKV infection were followed-up and 42% of 117 infants born to 116 ZIKV-positive women had abnormal clinical or brain imaging findings or both, including four infants with microcephaly [ 52 ]. A prospective cohort study of women with high-risk pregnancies in São Paulo State found that infants that were ZIKV-RT-PCR positive in the first 10 days of life had a five-fold increased risk of microcephaly overall and a ten-fold increased risk of disproportionate microcephaly [ 54 ]. However, other studies did not find evidence of vertical transmission, fetal anomalies, or congenital disease [ 57 , 60 , 66 , 67 ]. In our study, only one of the ZIKV-positive mothers delivered a low-birth weight infant, while all the other ZIKV-positive mothers delivered normal live births; no congenital microcephaly nor other abnormalities were reported.

We also found no significant differences in adverse neonatal outcomes among pregnant women with confirmed ZIKV infection compared with uninfected women. No significant differences in incidence or outcomes were detected before and after the change in eligibility criteria based on gestational age in our study. Other studies in Southeast Asia have also found no association between ZIKV infection during pregnancy and adverse neonatal outcomes, including microcephaly [ 68 , 69 ] There is still limited understanding about why the pathogenicity of ZIKV infection in Southeast Asia appears to be less severe than ZIKV infection in the Americas.

Strain virulence and prior infection

We performed whole genome sequencing of ZIKV isolates taken from three-quarters of the Zika cases in our study and all were of the Asian lineage strain. Previous studies suggest that the African-lineage ZIKV strains cause more profound cell death in human neural progenitor (hNP) cells and neurons than Asian lineage ZIKV, while Asian lineage ZIKV isolates impair the proliferation and migration of hNP cells, and neuron maturation [ 70 , 71 ]. It has also been suggested that the virus circulating in Southeast Asia produces low levels of viremia [ 60 , 72 ] It is possible the strain of ZIKV was a contributing factor for the overall mild ZIKV outcomes of infants in our cohort who were born to mothers with confirmed ZIKV infection during pregnancy.

Whether or not a previous infection by ZIKV or another flavivirus such as DENV protects against or enhances secondary infection by a heterologous Flavivirus is under debate [ 73 – 76 ]. One study found more than 95% of pregnant Thai women had neutralizing antibodies to at least one of the four dengue virus serotypes [ 77 ]. In Thailand, where DENV is endemic and where there are active JEV vaccination campaigns with high coverage, our study revealed no statistical difference in confirmed ZIKV infection in women whether or not they had positive dengue IgG results at enrollment. However, the higher DENV IgG titers among women with than without confirmed ZIKV infection observed in our study may warrant more research to support this finding, as the interaction between previous and current flaviviruses infections is still debated. Elapsed time after previous flavivirus infection could play a role in risk of acquired infection and severity of clinical presentation of subsequent flavivirus exposure, but further study is needed. The relationship between strain virulence and prior flavivirus infection should be explored further.

Characteristics of women with and without ZIKV infection and infant outcomes

Demographic, behavioral, and baseline clinical characteristics were mostly similar between participants with and without ZIKV infection; however, working in an agricultural occupation was also associated with increased risk of ZIKV infections. Implementing measures to reduce transmission, especially among those most at risk for infection, and educating clinicians and communities about limiting exposure during periods when ZIKV is circulating can help prevent infections. Most Zika cases in our study occurred during the rainy season and were detected in specific geographic locations. Strengthening Zika surveillance and limiting travel to ZIKV-affected areas can improve early detection and prevention of further transmission. In addition, local authorities should implement strategies to prevent or inhibit mosquito breeding to control their populations in these areas [ 78 ].

Previous studies support a possible correlation between prolonged ZIKV viremia in pregnant women and adverse neonatal outcomes [ 79 – 81 ]. Prolonged ZIKV viremia (42 days) was documented in only one Zika case in our study which occurred during the second trimester and the woman had a normal live birth.

Congenital ZIKV infection has been associated with serious birth defects and other complications during pregnancy. In addition, microcephaly and neurodevelopmental delays can first present years after congenital ZIKV exposure [ 82 , 83 ] Although we found low prevalence of ZIKV infection in our cohort, pregnant women in Thailand may still be at continued risk for ZIKV infection. A report of ZIKV seroprevalence in pregnant women attending antenatal care clinics in one hospital from May to October 2019 in Bangkok, Thailand, revealed that approximately 60% of pregnant Thai women did not have detectable levels of neutralizing antibodies against ZIKV [ 84 ]. Similarly, a post-outbreak study conducted in two communities in Southern Thailand also reported a low prevalence (12.8%) of ZIKV neutralizing antibodies in pregnant women [ 85 ]. Continued surveillance for adverse infant outcomes and neurodevelopmental delays among children would support estimating the burden of congenital ZIKV infections in Southeast Asia.

Limitations

There were several limitations to this study. The small number of ZIKV infections detected in this study may have limited our ability to detect factors associated with ZIKV infection. The sample size calculation was based on a higher expected prevalence. The small number of ZIKV infections should be taken into account when interpreting comparisons between groups. Second, we did not enroll pregnant women during the first trimester after the first year thus we may have missed some ZIKV infections that could have occurred during this early period of gestation. Previous studies have found that maternal ZIKV infections during the first trimester can result in more adverse outcomes than infections occurring in the final two trimesters [ 86 – 89 ]. By not enrolling women under 12 weeks of gestation, there could have been selection bias in our study as we missed all women who potentially had a ZIKV infection early in pregnancy. Our study was limited to participants who were been attending ANC visits, and therefore we do not have data about women who did not routinely engage with the public health system during pregnancy. Our study was performed in areas where other flaviviruses like DENV were co-circulating and the cross reactivity of DENV and ZIKV infection was a challenge for interpreting the DENV and ZIKV serology [ 90 ]. We did not perform a confirmatory plaque reduction neutralization test, which is used to resolve false-positive IgM antibody results caused by non-specific reactivity [ 90 , 91 ]. RT-PCR was used to confirm ZIKV infection to avoid challenges in interpreting positive test results [ 92 , 93 ]. Also, there may have been some imperfect recall regarding symptoms occurring between ANC visits. Finally, our study took place during 2018 to 2020, after Zika cases had begun to decline globally [ 82 ] and our findings may differ from those from studies conducted at the peak of the 2016 ZIKV outbreak.

This study was the first and largest prospective cohort study to investigate maternal and birth outcomes in Thailand. We found a low incidence of ZIKV and no differences in adverse maternal and birth outcomes associated with confirmed maternal ZIKV infection. Our findings are relevant for researchers, public health officials, and providers who study and provide services to pregnant women and infants in Thailand and Southeast Asia. However, more research is warranted to investigate whether the ZIKV virus strain, previous exposure to other flaviviruses, or timing of ZIKV infection during pregnancy may play a role in maternal and neonatal outcomes in Thailand.

Supporting information

S1 fig. molecular testing algorithm for maternal specimens..

https://doi.org/10.1371/journal.pntd.0012176.s001

S2 Fig. Serology testing algorithm for maternal specimens.

https://doi.org/10.1371/journal.pntd.0012176.s002

S3 Fig. Symptoms by flavivirus type-related immunological status in the 12 pregnant women with confirmed ZIKV infection.

https://doi.org/10.1371/journal.pntd.0012176.s003

S4 Fig. Head circumference measures at birth among infants born to mothers with and without confirmed ZIKV infection during pregnancy in the study.

https://doi.org/10.1371/journal.pntd.0012176.s004

S1 Data. Dictionary for the data sets.

https://doi.org/10.1371/journal.pntd.0012176.s005

S2 Data. Data for table 1 , 2 , 3 , and 4 .

https://doi.org/10.1371/journal.pntd.0012176.s006

S3 Data. Data for S3 Fig .

https://doi.org/10.1371/journal.pntd.0012176.s007

S4 Data. Data for S4 Fig .

https://doi.org/10.1371/journal.pntd.0012176.s008

Acknowledgments

We would like to fully acknowledge the contributions of Sunanta Henchaichon, Suwit Somponpun (Jack), Toni Whistler, and Chris Gregory from the CDC. We acknowledge technical and administrative support from Rick Brown and Phiangjai Boonsuk at WHO Thailand. We also acknowledge Maneeratt Sermsakul, Pawita Suwanwatthana, Ravee Nitiyanontakij, and Nichaya Praditsupat Bamrasnaradura Infectious Diseases Institute for their excellent coordinating ethical and laboratory issues. We also acknowledge clinicians and staff from the Bueng Kansite: Chatchawal Ritthiti, Wisanu Wittayabamrung, Sucheewa Rachuso, Chaiyaporn Songpraserdjarern, Panjit Jittungboonya, Supattarawadee Poompuen, Wanna Jiranapakul, Patcharaporn Junloon, Onnapa Satjapakasit, Ratchaneewan Kongburan, Nadtaya Mills, Surapong Laksawut, Prasert Ditsomboon, Charoon Surarak, Pramote Srikaew, and Kritsanapong Chumphon. We also acknowledge clinicians and staff from the Mukdahan site: Patcharawadee Sridapan, Chanya Jamnansiri, Sopon Nilkumhang, Kantinan Mahasuvirachai, Piyapong Bamroong, Panwad Uthiyo, Irada Sirikridsada, and Jiathip Luangrungrot. Lastly, we thank all participants in this study. We would also like to acknowledge technical support from the WHO Thailand office.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention, the U.S. Agency for International Development or the U.S. Government.

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Zika virus, like other members of the flavivirus family including dengue and West Nile virus, is most typically transmitted to humans through the bite of infected Aedes aegypti mosquitoes. Less commonly, Zika virus can be spread from person to person through sexual intercourse. Most people who become infected with Zika virus do not become sick; but about 1 in 5 people may develop such symptoms as fever, rash, and conjunctivitis (reddened eyes). People who become infected while pregnant may transmit the virus to the fetus, which can result in very serious birth defects, including microcephaly (unusually small head).  

Zika virus was discovered in the Zika forest in Uganda in 1947. In 2015, cases were reported in Brazil and an outbreak of Zika virus disease followed in South and Central America as well as the Caribbean. The first cases of locally transmitted Zika virus in the continental United States were confirmed in Florida in July 2016. No cases of mosquito-transmitted Zika virus have been detected in the United States since 2018. 

NIAID supports research to better understand Zika virus, the disease it causes, and ways to combat it, including research on diagnostics to rapidly determine if someone is or has been infected with Zika and to distinguish from other flaviviruses. 

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• Zika virus is transmitted primarily by Aedes mosquitoes, which bite mostly during the day.
• Most people with Zika virus infection do not develop symptoms; those who do typically have symptoms including rash, fever, conjunctivitis, muscle and joint pain, malaise and headache that last for 2–7 days.
• Zika virus infection during pregnancy can cause infants to be born with microcephaly and other congenital malformations as well as preterm birth and miscarriage.
• Zika virus infection is associated with Guillain-Barré syndrome, neuropathy and myelitis in adults and children.
• In February 2016, WHO declared Zika-related microcephaly a Public Health Emergency of International Concern (PHEIC), and the causal link between the Zika virus and congenital malformations was confirmed. WHO declared the end of the PHEIC in November of the same year.
• Although cases of Zika virus disease declined from 2017 onwards globally, transmission persists at low levels in several countries in the Americas and other endemic regions.

Zika virus is a mosquito-borne virus first identified in Uganda in 1947 in a Rhesus macaque monkey followed by evidence of infection and disease in humans in other African countries in the 1950s.

From the 1960s to 1980s, sporadic human infections were detected across Africa and Asia. However, since 2007 outbreaks of Zika virus disease have been recorded in Africa, the Americas, Asia and the Pacific.

In outbreaks over the last decade Zika virus infection was found to be associated with increased incidence of Guillain-Barré syndrome. When Zika virus emerged in the Americas, with a large epidemic in Brazil in 2015, an association between Zika virus infection and microcephaly (smaller than normal head size) was first described; there were similar findings in French Polynesia upon retrospective review. From February to November 2016, WHO declared a Public Health Emergency of International Concern (PHEIC) regarding microcephaly, other neurological disorders and Zika virus, and the causal link between Zika virus and congenital malformations was soon confirmed (1,2) . Outbreaks of Zika virus disease were identified throughout most of the Americas and in other regions with established Aedes aegypti mosquitos. Infections were detected in travellers from active transmission areas and sexual transmission was confirmed as an alternate route of Zika virus infection.

Cases of Zika virus disease globally declined from 2017 onwards; however, Zika virus transmission persists at low levels in several countries in the Americas and in other endemic regions. In addition, the first local mosquito-transmitted Zika virus disease cases were reported in Europe in 2019 and Zika virus outbreak activity was detected in India in 2021. To date, a total of 89 countries and territories have reported evidence of mosquito transmitted Zika virus infection; however, surveillance remains limited globally. 

• Zika epidemiology update (February 2022)
• History of Zika virus

Most people infected with Zika virus do not develop symptoms. Among those who do, they typically start 3–14 days after infection, are generally mild including rash, fever, conjunctivitis, muscle and joint pain, malaise and headache, and usually last for 2 – 7 days. These symptoms are common to other arboviral and non-arboviral diseases; thus, the diagnosis of Zika virus infection requires laboratory confirmation.

Complications

Zika virus infection during pregnancy is a cause of microcephaly and other congenital malformations in the infant, including limb contractures, high muscle tone, eye abnormalities and hearing loss. These clinical features are collectively referred to as congenital Zika syndrome.

The risk of congenital malformations following infection in pregnancy remains unknown; an estimated 5–15% of infants born to women infected with Zika virus during pregnancy have evidence of Zika-related complications (3) . Congenital malformations occur following both symptomatic and asymptomatic infection. Zika infection in pregnancy can also cause complications such as fetal loss, stillbirth and preterm birth.  

Zika virus infection can also cause Guillain-Barré syndrome, neuropathy and myelitis, particularly in adults and older children.

Research is ongoing to investigate the risk and effects of Zika virus infection on pregnancy outcomes, strategies for prevention and control, and effects of infection on other neurological disorders in children and adults.

• Questions and answers: Zika virus and complications

Transmission

Zika virus is primarily transmitted by infected mosquitoes of the  Aedes  ( Stegomyia ) genus, mainly  Aedes aegypti , in tropical and subtropical regions.  Aedes  mosquitoes usually bite during the day. These mosquitoes also transmit dengue, chikungunya and urban yellow fever.

Zika virus is also transmitted from mother to fetus during pregnancy, as well as through sexual contact, transfusion of blood and blood products, and possibly through organ transplantation.

Infection with Zika virus may be suspected based on symptoms of persons living in or visiting areas with Zika virus transmission and/or  Aedes  mosquito vectors. A diagnosis of Zika virus infection can only be confirmed by laboratory tests of blood or other body fluids, and it must be differentiated from cross-reactive related flaviviruses such as dengue virus, to which the patient may have been exposed or previously vaccinated.

• Laboratory testing for Zika virus and dengue virus infections

There is no specific treatment available for Zika virus infection or disease.

People with symptoms such as rash, fever or joint pain should get plenty of rest, drink fluids, and treat symptoms with antipyretics and/or analgesics. Nonsteroidal anti-inflammatory drugs should be avoided until dengue virus infections are ruled out because of bleeding risk. If symptoms worsen, patients should seek medical care and advice.

Pregnant women living in areas with Zika transmission or who develop symptoms of Zika virus infection should seek medical attention for laboratory testing, information, counselling and other clinical care.  

No vaccine is yet available for the prevention or treatment of Zika virus infection. Development of a Zika vaccine remains an active area of research.

Mosquito bites

Protection against mosquito bites during the day and early evening is a key measure to prevent Zika virus infection, especially among pregnant women, women of reproductive age and young children.

Personal protection measures include wearing clothing (preferably light-coloured) that covers as much of the body as possible; using physical barriers such as window screens and closed doors and windows; and applying insect repellent to skin or clothing that contains DEET, IR3535 or icaridin according to the product label instructions.

Young children and pregnant women should sleep under mosquito nets if sleeping during the day or early evening. Travellers and those living in affected areas should take the same basic precautions described above to protect themselves from mosquito bites.

Aedes  mosquitoes breed in small collections of water around homes, schools and work sites. It is important to eliminate these mosquito breeding sites, including covering water storage containers, removing standing water in flowerpots, and cleaning up trash and used tires. Community initiatives are essential to support local government and public health programs to reduce mosquito breeding sites. Health authorities may also advise use of larvicides and insecticides to reduce mosquito populations and disease spread.

• Vector control operations framework for Zika virus

Prevention of sexual transmission

For regions with active transmission of Zika virus, all people with Zika virus infection and their sexual partners (particularly pregnant women) should receive information about the risks of sexual transmission of Zika virus.

WHO recommends that sexually active men and women be counselled and offered a full range of contraceptive methods to be able to make an informed choice about whether and when to become pregnant in order to prevent possible adverse pregnancy and fetal outcomes.

Women who have had unprotected sex and do not wish to become pregnant due to concerns about Zika virus infection should have ready access to emergency contraceptive services and counselling. Pregnant women should practice safer sex (including correct and consistent use of condoms) or abstain from sexual activity for at least the entire duration of pregnancy.

For regions with no active transmission of Zika virus, WHO recommends practicing safer sex or abstinence for a period of three months for men and two months for women who are returning from areas of active Zika virus transmission to prevent infection of their sex partners. Sexual partners of pregnant women living in or returning from areas where local transmission of Zika virus occurs should practice safer sex or abstain from sexual activity throughout pregnancy.

• Prevention of sexual transmission of Zika virus

WHO response

WHO supports countries to conduct surveillance and control of arboviruses through the implementation of the Global Arbovirus Initiative , which is aligned with and expands upon recommendations laid out in the Zika Strategic Response Plan .

WHO responds to Zika in the following ways:

• supporting countries in the confirmation of outbreaks through its collaborating network of laboratories;
• providing technical support and guidance to countries for the effective management of mosquito-borne disease outbreaks;
• reviewing the development of new tools, including insecticide products and application technologies;
• formulating evidence-based strategies, policies, and outbreak management plans;
• providing technical support and guidance to countries for the effective management of cases and outbreaks;
• supporting countries to improve their reporting systems;
• providing training on clinical management, diagnosis and vector control at the regional level with some of its collaborating centres; and
• publishing guidelines and handbooks on epidemiological surveillance, laboratory, clinical case management and vector control for Member States.
• de Araújo TVB, Ximenes RA de A, Miranda-Filho D de B, et al. Association between microcephaly, Zika virus infection, and other risk factors in Brazil: Final report of a case-control study. Lancet Infect Dis . 3099(17)30727-2
• Krauer F, Riesen M, Reveiz L, et al. Zika Virus Infection as a Cause of Congenital Brain Abnormalities and Guillain–Barré Syndrome: Systematic Review. PLoS Med . 2017;14(1). doi:10.1371/journal.pmed.10022
• Musso D, Ko AI, Baud D. Zika Virus Infection – After the Pandemic. N Engl J Med . 2019;381(15). doi:10.1056/nejmra1808246

Zika virus disease

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• 21 October 2023

Scientists deliberately gave women Zika — here’s why

• Mariana Lenharo

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For the first time, scientists have deliberately infected people with Zika virus to learn whether such a strategy could help to test vaccines against the pathogen.

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Remembering Zika: Parents offered their kids for studies, then say they were forgotten

Mariana Lenharo

Rochelle dos Santos embraces her daughter, who was born with microcephaly in 2016 after dos Santos contracted Zika during her pregnancy in midwest Brazil. Ueslei Marcelino/Undark Magazine hide caption

Rochelle dos Santos embraces her daughter, who was born with microcephaly in 2016 after dos Santos contracted Zika during her pregnancy in midwest Brazil.

Rochelle dos Santos learned that her daughter would probably be born with microcephaly — a condition where a baby's head is much smaller than expected — when she was seven months pregnant. It was 2016 and Brazil was going through an unprecedented microcephaly outbreak associated with the mosquito-borne virus Zika. After the baby was born and the diagnosis of congenital Zika syndrome was confirmed, several researchers approached dos Santos to see if she'd join relevant clinical studies. Eager to understand her daughter's condition, she agreed.

Dos Santos says she was surprised to learn through a social media post last year that an international study that she participated in had been published in the journal Brain & Development . The study took over a year to be completed, and dos Santos had taken her daughter multiple times to the hospital for evaluations. As the head of an association for families of children affected by Zika in Goiás state in midwest Brazil, dos Santos wanted to share the findings with the other caregivers. She says she had to reach out directly to Hélio van der Linden, a neurologist at the Dr. Henrique Santillo State Center for Rehabilitation and Readaptation who authored the study in partnership with researchers in Brazil and the United States, to ask that a copy be shared with her. But she says he told her there was no point because it was written in English.

"Of course, we get upset," she recalled in her native Portuguese. "We want to have this feedback and better understand this situation that is new for everyone." Dos Santos — who noted that while she speaks only a little English, her husband reads and speaks English capably — said she feels used and that many other families share the same sentiment. "We know that COVID is now the priority," dos Santos adds, "but our children are still here, they still have needs."

The study's author sent her the article, and dos Santos says her husband translated it for her — though she adds that she was also asked by van der Linden not to share it. (Van der Linden told Undark by email that while he did point out to dos Santos that the article was written in English, his main concern was running afoul of the journal's publishing rules. His request not to share it, he added, was for social media posts. "There was no problem in sharing the article with other mothers," he wrote, "but I believe this wasn't clear to the mother of the patient.")

Children with congenital Zika syndrome face numerous health issues , all originating from the peculiar way in which Zika attacks the developing brain. In addition to the condition's most pronounced feature — reduced head size — many have rigid muscles, difficulty swallowing and breathing and problems with the retina and optic nerve, as well as other symptoms that emerge as the children grow. "The doctors say that only time will tell how our children will be tomorrow," dos Santos says, "because there are no adults with this syndrome."

Dos Santos is not the only caregiver who felt left behind by scientists. Family groups like the one she heads have sprung up across the country, and members are increasingly at odds with the scientists who have used their children for research. The grandmother and caretaker of a boy with congenital Zika syndrome, Alessandra Hora dos Santos (no relation to Rochelle), launched one of these associations in Alagoas state in northeast Brazil in 2017. She says that lately she has been declining requests to participate in new studies — although such invitations are becoming rare because there haven't been new outbreaks of the syndrome since 2016 — and she noticed that other families are doing the same.

Scientists who conducted the studies on Zika during the peak and the aftermath of the outbreak admit that communicating the results to families is not always effective, and that it was not the top priority during the Zika crisis. In the rush to collect data, not all researchers took the time to explain in detail what their projects were about and set clear expectations. Busy caretakers, on the other hand, were hardly able to carefully read the informed consent forms they were signing to authorize investigators to collect data from their children. Over the last few years, these families have demanded to participate more actively in the scientific discussion around Zika.

"We feel diminished," says Alessandra Hora dos Santos. "It's like we were lab rats. They come in nicely, collect information, collect exams on the child, and in the end, we don't know of any results. It's like we are being used without even knowing why that is being done."

Left: Dos Santos helps her daughter, pictured, with physical therapy. Many children with congenital Zika disorder are physically disabled. Right: Due to difficulties swallowing, dos Santos' daughter uses a feeding tube, which is attached to her wheelchair. Ueslei Marcelino/Undark Magazine hide caption

Left: Dos Santos helps her daughter, pictured, with physical therapy. Many children with congenital Zika disorder are physically disabled. Right: Due to difficulties swallowing, dos Santos' daughter uses a feeding tube, which is attached to her wheelchair.

Left: Dos Santos' daughter uses leg braces to help her stand. Other symptoms of the disorder emerge as children grow. Right: Dos Santos guides her daughter to create a finger painting. "Our children are still here, they still have needs," she says. Ueslei Marcelino/Undark Magazine hide caption

Left: Dos Santos' daughter uses leg braces to help her stand. Other symptoms of the disorder emerge as children grow. Right: Dos Santos guides her daughter to create a finger painting. "Our children are still here, they still have needs," she says.

A scramble for Zika research participants

By the time physicians started to notice a surge in microcephaly in Brazil in mid-2015, researchers had to scramble to design studies, get funding and conduct analyses. Eventually, scientists from multiple institutions coalesced in the Microcephaly Epidemic Research Group (MERG). They began the research efforts even before the link with Zika had been established and had a crucial role in guiding public health strategies to tackle the epidemic. "There was a lot of pressure coming from the media and the health ministry," says infectious disease expert Demócrito de Barros Miranda-Filho, a member of MERG and a professor at the University of Pernambuco. "We had to develop all the projects from scratch and submit them to the ethics committees within a deadline," he says, adding that there was also pressure to give answers to the families.

One of the group's concerns was to immediately share individual results of tests and clinical evaluations that could directly impact the child's treatment. But when it comes to the general findings at the end of the study, says Miranda-Filho, the researchers didn't properly communicate them to the participants.

The Troubled History Of Vaccines And Conflict Zones

"It is very complex to decode biological questions and put them into a more understandable language," says Thália Velho Barreto de Araújo, an epidemiologist at the Federal University of Pernambuco and a member of MERG. "We haven't figured out a way to do that yet, and we would need research resources to get advice for transforming technical language into something palatable." Ricardo Arraes de Alencar Ximenes, an epidemiologist at both the University of Pernambuco and the Federal University of Pernambuco, notes that one of the obstacles to develop well-thought-out communication strategies is getting dedicated funding.

Physician Camila Ventura, one of the coordinators of an ambitious project with the goal of evaluating the neurodevelopment of about 200 children with congenital Zika syndrome over five years, says she is familiar with the families' demands and agrees with them. But there are other obstacles beyond adequate funding, she says. For example, with funding from the United States National Institutes of Health, the project is being developed at the Altino Ventura Foundation, a Brazilian health nonprofit, in partnership with the U.S. research organization RTI International. Because the project is done in partnership with other organizations, Ventura says it's not solely up to her to provide this feedback.

"This criticism applies to our own institution and I try my best to push for these answers" from our research partners, says Ventura. "The mothers see that we're collecting data and they want to know: What about my kid?" she adds. "Is he getting better?"

Van der Linden wrote that when he invites a family to participate in a study, he tries to make it clear that the goal is to better understand the condition and that the findings might not benefit the participants themselves. "I explain that after the study is done, there won't be a 'result.' Sincerely, I don't offer or promise to call each one to explain the details, etc. I always make it clear that it is for science," he wrote to Undark by email. "I believe there might have been an over-expectation, or an unrealistic expectation of something that was never promised."

Soraya Fleischer, an anthropologist at the University of Brasília who coordinates a research project on the impact of Zika on the lives of families, says it's also important to consider what these mothers mean when they ask for study results. "For the researchers, the result is what is published in a well-qualified scientific journal or goes into their resume," she says. But for the families, says Fleischer, sometimes the result is a simple blood test that confirms that the child's disabilities were caused by Zika — an important document that grants access to certain social benefits reserved for children with the syndrome, which can be difficult to get via the public health system.

Not every parent has had a bad experience with Zika researchers. Jaqueline Silva de Oliveira, the mother of a 5-year-old girl with congenital Zika syndrome, says that whenever she needs these types of reports in order to claim social benefits, she reaches out to the scientist who enrolled her family in a genetics study. The girl's twin brother was not affected by Zika, which caught the attention of a group at the Human Genome and Stem Cell Research Center at the University of São Paulo that wanted to try to identify potential protective genes.

"I participated to be able to help prevent other children from having microcephaly," says de Oliveira. She says she can't explain in her words what the results of the study were and she didn't receive a document describing them. But overall, she thinks having participated in the study was a positive experience. She continues to have a connection with the researchers, and they helped her find a neurologist, one of the best in the state, she says, who managed to control her daughter's epilepsy crises. "I helped the researcher on the study," she says, "and when I needed it, she helped me."

Caregivers struggle with research fatigue and trust

During the initial 2015 Zika outbreak and the years that followed, participation in the Brazilian Zika studies could be difficult. Luciana Lira, a medical anthropologist at the Federal University of Pernambuco, recalls accompanying two mothers to an event in 2018 in Recife, in Pernambuco state, one of the epicenters of the congenital Zika syndrome outbreak. The event was organized by a local university and an association for families of children with rare diseases. While the other mothers attended talks and participated in conversation circles, the mothers of children with congenital Zika syndrome were directed to a hall where researchers organized a task force to collect blood for a research project.

On that occasion, Lira says she watched while a nurse approached a mother to participate in the study. The mother "was so agitated that, when the nurse approached her and started explaining the study, she clearly wasn't paying full attention because there were more urgent things to deal with. Her daughter was having a crying fit, she had to fix her feeding tube, all of that," says Lira. "Then she agreed to participate, signed a paper and that's it. This type of situation has become very commonplace."

The researcher behind the project was Nilson Antonio de Assunção, a chemistry professor at the Federal University of São Paulo who was then studying the biochemical characteristics of blood among children with Zika. The study hasn't been published yet, de Assunção says, adding that he is aware that some families don't fully understand the purpose of his research when they agree to participate. "They get nervous because they are at an event, these are humble people, their children are crying and they end up not understanding very well what we're explaining."

De Assunção says there isn't much to be done about creating better strategies to communicate with families of children participating in studies. "I have been noticing this distrust in families," he says, "but those who end up losing are the families themselves." He says that he has previously tried to explain and educate the population about his work. "No matter what you do," he adds, "there will always be this distrust."

Lira and her colleagues have been observing the relationship between caregivers of children with Zika and biomedical scientists in Recife. Silvana Matos, also an anthropologist at the Federal University of Pernambuco, says that initially the caregivers welcomed the attention from scientists because they wanted to understand what had happened to their children. "The thing they complained the most about, right after this initial period," she says, "was that the test results never came back to them and the researchers, from Brazil or abroad, never reached out again to tell them what happened."

The families' experiences with the medical trials made them wary of researchers more broadly. By the time the anthropologists started working with the families in late 2016, they had to redesign their work to deal with this research fatigue and gain trust, says Lira. The families "had been overwhelmed both by scientists trying to collect organic samples, and by journalists and researchers wanting to interview them," says Fleischer. "There was an eagerness to learn what was happening" among the scientists and journalists, she adds, and the families "were the source."

Lira spent several months following caregivers around before doing any interviews. Fleischer, who is not based in Recife, decided to come back to the city several times over the years to revisit the families and show them what had been produced with the data they had collected before — for example, an article or a newspaper story. Realizing that the caretakers were too busy to read long articles, Fleischer's group created a blog to publish short stories about life with Zika that they would print out and distribute to the participants during their visits. The fact that the researchers kept coming back and reporting what they were doing made the families feel respected, according to Fleischer, and it was essential to build trust.

Dos Santos, left, with her daughters. Dos Santos says she feels used and that many other families share the same sentiment. Medical anthropologist Luciana Lira says the families became overwhelmed by scientists and journalists, and that she had to change her approach to gain the families' trust. Ueslei Marcelino/Undark Magazine hide caption

Dos Santos, left, with her daughters. Dos Santos says she feels used and that many other families share the same sentiment. Medical anthropologist Luciana Lira says the families became overwhelmed by scientists and journalists, and that she had to change her approach to gain the families' trust.

What researchers owe study participants

In Brazil, the ethical and legal framework for research involving human subjects was established in 1996 through a resolution by the Brazilian National Council of Health. To conduct a study involving human subjects in Brazil, researchers have to submit their proposal to a research ethics committee, much like in the U.S. Every research organization may constitute its own committee, which responds to the National Commission for Research Ethics (CONEP, by its Portuguese acronym).

Before entering a study, participants must sign a free and informed consent form, a document that describes the study, its goals and possible risks and benefits of participating. According to the commission, the document should be written in clear and accessible language.

The need to share the findings with participants, which is at the core of the caregivers' complaints, is not directly covered by the 1996 resolution. But the current ethical norms , in force since 2012, do state that research findings should be communicated to the community if there's a potential to benefit the population, notes biologist Maria Mercedes Bendati, who retired from the municipal health department of Porto Alegre, in southern Brazil, in 2017 and is a CONEP member. "It already says that it is important to give this feedback," she says. The next step, she adds, is to implement the requirement "and make it very clear in the academic education of the researchers that they should fulfill their social role, and know that the research implies giving these answers to the participants."

Bendati participated in the Pan American Health Organization Zika Ethics Consultation in April 2016, which originated an ethics guidance on key issues raised by the Zika outbreak.

Florencia Luna, the chair of the Zika Ethics Consultation, says the goal of the guidance was precisely to prevent situations like the ones the caregivers described. "We were very concerned about doing this research at that moment in the middle of the outbreak. So it's a little bit like now, with COVID," she says. "Even if you want to do [research] fast and quick, and you should do it like that, that doesn't mean you have to avoid ethical standards."

Luna, who is also the director of the bioethics program at the Latin American Faculty of Social Sciences in Argentina, believes that returning to the participants with the results is an ethical obligation. "Personally, I do think it is very important to come back and tell the good or the bad news," she says, especially with Zika, which involves mothers and babies with health conditions. "At least to send them a letter, to call them on the phone," she adds. "Maybe not to make them go to the clinic because it would be too burdensome for them, but there are other ways where you can communicate nowadays, with smartphones, with the internet."

According to the International Ethical Guidelines for Health-related Research Involving Humans, a 2016 document prepared by the Council for International Organizations of Medical Sciences in collaboration with the World Health Organization, researchers "should engage potential participants and communities in a meaningful participatory process" which includes the dissemination of the study's results.

Despite such guidelines, not communicating results to participants is seen by some researchers as business as usual. Carl Elliott, an expert in bioethics and a professor of philosophy at the University of Minnesota, says the situation narrated by Rochelle dos Santos, where the investigator hesitated to send her the study for which her daughter had collaborated, didn't surprise him.

"If I were the research subject or the mother of the research subject, it would offend me and I think justifiably," he says. "That said, I think the vast majority of research subjects don't do that sort of follow-up. They don't ask or are not even particularly interested in the papers." Elliot says he doesn't think the investigator gave the right response, but he imagines he was probably surprised by the request.

In any case, Elliott says he believes that, if a participant actively asks, the researcher must provide the results: "It's shameful that it takes so much effort, and often money, for the public to get access to the results of scientific studies published in the medical literature."

Bioethics expert Carl Elliot says that the situation Rochelle dos Santos (pictured) described, where the investigator hesitated to send her the study for which her daughter had collaborated, didn't surprise him. Ueslei Marcelino/Undark Magazine hide caption

Bioethics expert Carl Elliot says that the situation Rochelle dos Santos (pictured) described, where the investigator hesitated to send her the study for which her daughter had collaborated, didn't surprise him.

Caregivers speak up

In September 2018, the Brazilian caregivers' discontent culminated at the annual Congress of the Brazilian Society of Tropical Medicine in Recife. That year, the program included several sessions about congenital Zika syndrome. According to a paper written by Lira, none of the families' associations had been invited.

During one of the sessions on the main stage, Germana Soares, the mother of a boy with congenital Zika syndrome and the president of one of the largest family associations, requested to speak. She read aloud a letter to the event's organizers. "We believe there is a lack of empathy and sensitivity to our reality, and a lack of respect in the fact that we were underestimated. As if we — the mothers, relatives and caretakers — would lack the understanding to participate in a technical event to discuss a topic that is of our biggest interest," the letter stated. "Are we mothers so ignorant, without the least bit of education, that we cannot understand a scientific article or a lecture? Or should the researchers be the ones to use a language that is more comprehensible? Are we totally wrong to demand a discussion about ethics in biomedical research? Are we just numbers?"

The organizers were apparently caught by surprise, as Soares' speech wasn't in the program. One of the speakers at the session called Sinval Pinto Brandão Filho, the president of the Society, to ask him what to do about it. He advised him to let Soares speak. "Our organization welcomes this debate with great satisfaction because we study the tropical diseases, in terms of controlling them," says Brandão Filho, adding that every year the Brazilian Society of Tropical Medicine invites patients of neglected diseases to a public forum during the congress to discuss the problems they face. "I see this as something specific that was immediately recognized that it should be more sensitively incorporated into the tribute session."

Today, only sporadic cases of congenital Zika syndrome still occur, which makes it difficult to get funding for research, scientists say. The research focus has shifted to COVID-19, but the Zika health emergency might have left a legacy when it comes to research ethics.

"My personal reflection about the Zika experience in ethics committees is that perhaps there should have been a dialogue with the researchers to ask them how the findings would be shared with the participants," says Bendati. "When it comes to COVID-19, the CONEP is now being very clear on the need for a proposal of feedback to be given to participants." Learning from the mistakes of Zika might have contributed to this evolution, Bendati adds.

Luna says she's aware that sometimes ethics are viewed as an obstacle to science. Tracking down the participants can be difficult, and researchers who might have moved on to another project often lack the time and the energy to pursue it. "But it's part of what we have to do in order to build trust, to continue working," she says. "If not, these women will not collaborate in any other research in their lives because they were disappointed."

Mariana Lenharo is a science and health journalist whose writing has appeared in Scientific American , Mother Jones , Elemental , BBC News Brazil, among other publications. She is currently based in São Paulo, Brazil.

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Large Scandinavian study surprises: no advantage of ‘no-touch’ vein harvesting over conventional technique for coronary bypass

Key take-aways 

• The SWEDEGRAFT registry-based trial showed that no-touch harvesting was not superior to the conventional technique in reducing vein graft failure or improving clinical outcomes after coronary artery bypass grafting. 
• There were more early and late leg wound complications with the no-touch technique. 
• The trial results do not support the routine use of the no-touch technique. 

London, United Kingdom – 31 August 2024: No-touch graft harvesting did not appear to be beneficial compared with the conventional technique for coronary artery bypass grafting (CABG), according to late-breaking research presented in a Hot Line session today at ESC Congress 2024. 1  

“For CABG surgery to be successful in treating ischaemic heart disease, the graft should provide sufficient myocardial blood supply. However, graft failure, where the graft becomes blocked, occurs in up to half of patients within 10 years. 2 Trials have suggested that using a ‘no-touch’ technique to harvest the saphenous vein graft, where the vein is harvested with part of the surrounding tissue, may reduce graft failure compared with the conventional open technique where the vein is stripped of surrounding tissue prior to grafting. 3,4 We conducted the SWEDEGRAFT trial, designed to reflect routine clinical practice, to compare no-touch and conventional harvesting in terms of short-term graft failure and long-term outcomes and found no benefit with the no-touch technique,” explained study chair and presenter, Professor Stefan James from Uppsala University, Uppsala, Sweden. 

SWEDEGRAFT was a registry-based, randomised trial designed to investigate whether a strategy of no-touch vein grafts was superior to the conventional vein graft technique in patients undergoing CABG. 5 Patients under 80 years old were enrolled who were scheduled for first-time isolated non-emergent CABG with at least one saphenous vein graft. Patients were randomly assigned in a 1:1 ratio to vein graft harvest using the no-touch technique or the conventional technique. 

The primary endpoint was the proportion of patients with graft failure defined as: graft occluded or stenosed >50% on coronary computed tomography angiography at 2 years after CABG, clinically driven coronary angiography demonstrating an occluded or stenosed >50% vein graft, or death within 2 years. Secondary endpoints included major adverse cardiovascular events (MACE, defined as all-cause death, myocardial infarction or any repeat revascularisation during follow-up) and post-operative leg wound complications. 

In total, 902 participants were randomised at all eight surgical sites in Sweden and one Danish site. The mean age was 67 years and 88% were male. The mean European System for Cardiac Operative Risk Evaluation (EuroSCORE) II score was 1.6. In 53% of cases, CABG was performed as an elective procedure.  

There was no significant difference in the primary endpoint of graft failure within 2 years, which occurred in 19.8% of patients in the no-touch group and 24.0% of patients in the conventional group (difference −4.3%; 95% confidence interval [CI] −10.1 to 1.6; p=0.15). There were no significant differences in any of the three individual components of the primary endpoint.  

In addition, the incidence of MACE was similar in the no-touch and conventional groups at mean follow-up of 52 months (12.6% vs. 9.9%, respectively; hazard ratio 1.30; 95% CI 0.87–1.93; p=0.195). However, there were significantly more leg wound complications in patients randomised to no-touch vs. conventional grafts at 3 months (24.7% vs. 13.8%, respectively; difference 10.9%; 95% CI 5.7–16.1) and at 2 years (49.6% vs. 25.2%, respectively; difference 24.4%; 95% CI 17.7–31.1). 

Summing up, Professor James said: “We have shown that the no-touch technique was not superior to the conventional technique in reducing graft failure or improving clinical events after CABG and was associated with more early and late leg wound complications. There was surprisingly good longevity of conventional vein grafts, reflecting the high quality of surgical and medical standards in Scandinavia. Our study highlights the importance of independent research where dedicated clinicians collaborate in large all-comer randomised controlled trials to determine the best treatment for the benefit of our patients. Results from this pragmatic registry trial do not support the routine use of no-touch harvesting and our findings should be considered in future guidelines.” 

Notes to editor

This press release accompanies both a presentation and an ESC press conference at ESC Congress 2024. It does not necessarily reflect the opinion of the European Society of Cardiology. 

ESC Press Office Tel: +33 (0)6 61 40 18 84   Email: [email protected]

The hashtag for ESC Congress 2024 is  #ESCCongress  

Follow us on X  @ESCardioNews   

Journalists are invited to become accredited and  register here . 

Check out the  ESC Media and Embargo Policy . 

Funding : The trial was supported by the Swedish Research Council, Swedish Heart and Lung Foundation, Health Research Foundation of Central Denmark Region. 

Study sponsor: Dept. of Surgical Sciences Uppsala University, Sweden  Academic research organisation: Uppsala Clinical Research Center, Uppsala University, Sweden. 

Disclosures: Stefan James reports no conflicts of interest of relevance for the trial. 

References and notes 

1‘SWEDEGRAFT - No-touch vein grafts in coronary artery bypass grafting’ will be discussed during Hot Line 4 on Saturday 31 August in room London. 

2Goldman S, Zadina K, Moritz T, et al. Long-term patency of saphenous vein and left internal mammary artery grafts after coronary artery bypass surgery: results from a Department of Veterans Affairs Cooperative Study. J Am Coll Cardiol. 2004;44:2149–2156. 

3Sousa DSR, Dashwood MR, Tsui JCS, et al. Improved patency in vein grafts harvested with surrounding tissue: results of a randomized study using three harvesting techniques. Ann Thorac Surg. 2002;73:1189–1195.  

4Tian M, Wang X, Sun H, et al. No-touch versus conventional vein harvesting techniques at 12 months after coronary artery bypass grafting surgery: multicenter randomized, controlled trial. Circulation. 2021;144:1120–1129. 

5Ragnarsson S, Janiec M, Modrau IS, et al. No-touch saphenous vein grafts in coronary artery surgery (SWEDEGRAFT): Rationale and design of a multicenter, prospective, registry-based randomized clinical trial. Am Heart J. 2020;224:17–24. 

About ESC Congress 2024 

It is the world’s largest gathering of cardiovascular professionals, disseminating ground-breaking science both onsite in London and online – from 30 August to 2 September. Explore the  scientific programme . More information is available from the ESC Press Office at [email protected]. 

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Rising resting heart rate over the years linked to shorter lifespan, study show

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New research uncovers a direct link between heart rate changes and longevity, revealing that even a slight increase in resting heart rate can significantly raise mortality risk.

A study published in the journal Scientific Reports reveals that a significant increase in resting heart rate over the years can significantly increase the risk of mortality in both men and women.

Heart rate is a measure of cardiac cycle frequency that reflects cardiovascular health, metabolic rate, and autonomic nervous system activity. Across mammalian species, larger body sizes are associated with lower heart rates and longer life spans.

A significant induction in human lifespan has been observed over the past two centuries, primarily due to improvements in medical science and social care. However, there remains a concern that the global rise in obesogenic environment may challenge further increase in lifespan.

Previously, the Framingham Heart Study observed higher mortality rates in cardiovascular disease-free individuals with faster heart rates. This study was pivotal in highlighting the potential risks associated with elevated heart rates, yet it did not explore the effects of changes in heart rate over time.

In the current study, scientists have determined the association between resting heart rate and lifespan within human populations. They also explored whether an increase in heart rate over the years correlates with a higher mortality risk. This research marks an important step in exploring a potential causal relationship between heart rate changes and lifespan.

Study design

Data from three population-based studies, including the Paris Prospective Study I, the Whitehall I Study, and the Framingham Heart Study, was analyzed to explore the association between resting heart rate and lifespan.

The studies employed linear regression to confirm the inverse relationship between resting heart rate and lifespan and Cox proportional hazards regression to assess mortality risk associated with changes in heart rate over time.

Appropriate statistical analyses were carried out to determine the association between changes in heart rate over the years and mortality risk. The results were adjusted for several classical risk factors, including age, body mass index, smoking, physical activity, diabetes, systolic blood pressure, and cholesterol levels.

Paris Perspective Study I

This study recruited a total of 7,976 healthy French men (aged 42 to 53 years) between 1967 and 1972. A total of 2,387 deaths were reported by December 1993.

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The analysis controlling for potential confounding factors revealed a significant inverse association between resting heart rate and lifespan.

Whitehall I study

This study recruited a total of 1,226 healthy British men (aged 52 ± 7 years) between 1967 and 1969. A total of 977 deaths were reported by 2008.

The analysis of study data revealed a significant inverse association between resting heart rate and lifespan. However, it is essential to note that changes in heart rate over time were not analyzed in this cohort, limiting the ability to generalize these findings fully.

Framingham Heart Study

This study recruited a total of 4,001 US women (aged 47 ± 16 years) and 3,299 men (aged 45 ± 15 years) between 1971 and 1975. A total of 2,409 deaths were reported among women over a follow-up period of 33 years. Among men, a total of 2,172 deaths were reported over a follow-up period of 31 years.

The findings revealed an inverse association between resting heart rate and lifespan in both men and women. Interestingly, women experienced a 5-year longer lifespan than men for each category of resting heart rate.

Resting heart rate vs. mortality risk

The scientists examined the associations between changes in resting heart rates over 5-year and 8-year periods and mortality risks among 5,589 male participants in the Paris Prospective Study I and 3,299 male and 4,001 female participants in the Framingham Heart Study, respectively.

The analysis controlling for known risk factors and resting heart rate revealed that an increase in resting heart rate over the years can significantly increase mortality rates in both men and women. Specifically, a 10 bpm increase in resting heart rate over 5 years in the Paris Prospective Study I was associated with a 20% higher mortality risk. Similarly, in the Framingham Heart Study, a 10 bpm increase over 8 years led to a 13% higher risk of mortality in men and a 9% higher risk in women.

Study significance

The study has analyzed data from three large-scale populations from the US, UK, and France and found that resting heart rate is inversely associated with lifespan and that an increase in resting heart rate over the years can significantly increase mortality risk in both men and women.

Mammals with larger body sizes generate more heat as a function of their body mass. Similarly, they lose more heat as a result of their body's surface area. Smaller mammals with higher body surface-to-mass ratio, on the other hand, conserve body temperature by increasing their metabolic rate, which in turn determines their heart rate. Thus, heart rate may serve as a surrogate marker for total energy expenditure. This concept of total energy expenditure throughout life may partly explain the inverse association between resting heart rate and lifespan, as larger organisms tend to have lower heart rates and longer lifespans.

In humans, the possibility of reducing heart rate to extend lifespan has been explored primarily in patients with serious illnesses. For instance, beta-adrenergic receptor blockers, which reduce heart rate, have been shown to decrease mortality risk in post-myocardial infarction patients with heart failure. However, no studies have yet investigated whether reducing heart rate can extend lifespan in healthy human populations.

Overall, the study findings highlight that regular heart rate measurement could be a simple and straightforward approach for monitoring human health and lifespan. The results suggest that heart rate might be a target for public health interventions, particularly in environments where unhealthy lifestyle factors contribute to rising heart rates. Further research is needed to explore the potential benefits of heart rate reduction in healthy individuals.

• Gaye B. 2024. Association between change in heart rate over years and life span in the Paris Prospective 1, the Whitehall 1, and Framingham studies. Scientific Reports. https://www.nature.com/articles/s41598-024-70806-8

Posted in: Men's Health News | Medical Research News | Women's Health News

Tags: Autonomic Nervous System , Blood , Blood Pressure , Body Mass Index , Cardiovascular Disease , Cholesterol , Diabetes , Frequency , Heart , Heart Failure , Heart Rate , heat , Mortality , Myocardial Infarction , Nervous System , Physical Activity , Public Health , Receptor , Research , Smoking , Social Care

Dr. Sanchari Sinha Dutta

Dr. Sanchari Sinha Dutta is a science communicator who believes in spreading the power of science in every corner of the world. She has a Bachelor of Science (B.Sc.) degree and a Master's of Science (M.Sc.) in biology and human physiology. Following her Master's degree, Sanchari went on to study a Ph.D. in human physiology. She has authored more than 10 original research articles, all of which have been published in world renowned international journals.

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Simple blood test could predict a person’s heart disease risk 30 years out, study finds

A new approach to a routine blood test could predict a person’s 30-year risk of heart disease, research published Saturday in the New England Journal of Medicine found. 

Doctors have long assessed their patients’ risk for cardiovascular disease by using a blood test to look at cholesterol levels, focusing particularly on LDL or “bad” cholesterol . But limiting blood testing to just cholesterol misses important — and usually silent — risk factors, experts say.

“We have other biomarkers that tell us about other kinds of biological problems our patients who are destined to have cardiovascular disease are likely to have,” said lead study author Dr. Paul Ridker, director of the Center for Cardiovascular Disease Prevention at Brigham and Women’s Hospital in Boston.

Ridker and his team found that in addition to LDL cholesterol, two other markers — a type of fat in the blood called lipoprotein (a), or Lp(a), and an indicator of inflammation — are important predictors of a person's risk of heart attack , stroke and coronary heart disease. 

The findings were also presented Saturday at the European Society of Cardiology Congress 2024 in London.

In the study, the researchers analyzed data from nearly 30,000 U.S. women who were part of the Women’s Health Study . On average, the women were 55 years old when they enrolled in the years 1992 through 1995. About 13% — roughly 3,600 participants — had either a heart attack or stroke, had surgery to fix a narrowed or blocked artery, or died from heart disease over the 30-year follow-up period. 

Though the research was done in women, Ridker said the findings would likely also apply to men.

Still, the focus on women was on purpose, he said. “This is a largely preventable disease, but women tend to be under treated and underdiagnosed.” 

All of the women had blood tests done at the beginning of the study to measure their LDL cholesterol, Lp(a) and C-reactive protein levels, a marker of inflammation in the body.

These measurements, individually as well as together, appeared to predict a woman’s heart health over the next three decades, the study found.

Women with the highest levels of LDL cholesterol had a 36% higher risk for heart disease compared with those with the lowest levels. The highest levels of Lp(a) indicated a 33% elevated risk, and those with the highest levels of CRP were 70% more at risk for heart disease. 

When the three were looked at together, women who had the highest levels were 1.5 times more likely to have a stroke and over three times more likely to develop coronary heart disease over the next 30 years compared with women with the lowest levels.  

All of the markers have been individually linked to higher risk of heart disease, but “all three represent different biological processes. They tell us why someone is actually at risk,” Ridker said.

Intervening early 

Traditional risk factors for heart disease include obesity, diabetes, high blood pressure and high cholesterol levels . Testing for Lp(a) and CRP can reveal less obvious risk factors.

“You can have no traditional risk factors and just by having that high Lp(a), you are at higher risk,” said Dr. Rachel Bond, system director of women’s heart health at Dignity Health in Arizona, who was not involved with the study. 

Bond said everyone should get their Lp(a) tested once in their lives. If they have elevated levels at any point, they will for life. There is one caveat: Post-menopausal women can develop high Lp(a) and may want to have their levels tested again at that time, Bond said. 

On the other hand, LDL cholesterol and CRP levels fluctuate throughout a person’s life. Ridker supports doctors running the three-pronged blood test when patients are in their 30s or 40s, to catch potentially overlooked risk factors early, when there is time to intervene. 

Although exercising, eating well and not smoking are all important, people with already elevated levels of Lp(a), LDL and CRP will likely require medication, said Dr. Steven Nissen, chief academic officer of the Heart, Vascular and Thoracic Institute at the Cleveland Clinic, who was not involved with the study. 

“We can’t expect lifestyle interventions are going to do the job alone for most people,” Nissen said. 

The study had several limitations that future research may address, including a lack of racial and ethnic diversity, which plays an important role in a person’s risk for heart disease. Nearly all of the participants — 94% — were white.

Nissen also noted that the study stopped measuring Lp(a) levels once they passed a certain threshold.

“The highest levels of lipoprotein (a) in this study weren’t even high enough to reach the clinical threshold at which a patient would be treated,” he said. “It tends to underestimate the risk of lipoprotein (a).”

Dr. Kunihiro Matsushita, a professor of epidemiology at Johns Hopkins Bloomberg School of Public Health, who specializes in cardiology, said that while inflammation is definitely important, “that doesn’t mean CRP is the best marker for predicting cardiovascular disease risk.” 

“Using three biomarkers is interesting, but the choice of which biomarkers these are can be explored further,” said Matsushita, who also wasn't involved with the new research. 

He added that testing for inflammation, LDL and Lp(a) is particularly important for people who are traditionally thought of as low risk for heart disease, including women, young people and those of East Asian descent. 

Ridker agreed.

“Physicians will not treat things they don’t measure,” he said. 

Kaitlin Sullivan is a contributor for NBCNews.com who has worked with NBC News Investigations. She reports on health, science and the environment and is a graduate of the Craig Newmark Graduate School of Journalism at City University of New York.

The universe had a secret life before the Big Bang, new study hints

The secrets of black holes and dark matter could lie before the Big Bang, a new study of "bouncing" cosmology hints.

The Big Bang may not have been the beginning of the universe, according to a theory of cosmology that suggests the universe can “bounce” between phases of contraction and expansion. If that theory is true, then it could have profound implications about the nature of the cosmos, including two of its most mysterious components: black holes and dark matter.

With this in mind, a recent study suggests that dark matter could be composed of black holes formed during a transition from the universe's last contraction to the current expansion phase, which occurred before the Big Bang. If this hypothesis holds, the gravitational waves generated during the black hole formation process might be detectable by future gravitational wave observatories, providing a way to confirm this dark matter generation scenario.

Observations of stellar movements in galaxies and the cosmic microwave background — an afterglow of the Big Bang — indicate that about 80% of all matter in the universe is dark matter , a substance that doesn't reflect, absorb or emit light. Despite its abundance, scientists have not yet identified what dark matter is made of.

In the new study, researchers explored a scenario where dark matter consists of primordial black holes formed from density fluctuations that occurred during the universe's last contraction phase, not long before the period of expansion that we observe now. They published their findings in June in the Journal of Cosmology and Astroparticle Physics .

The bouncing cosmos

The traditional cosmological view of the universe suggests that it started from a singularity, followed by a short period of extremely rapid expansion, called inflation. However, the authors behind the new study analyzed a more exotic theory, known as non-singular matter bouncing cosmology, which posits that the universe first underwent a contraction phase. This phase ended with a rebound due to the increasing density of matter, leading to the Big Bang and the accelerated expansion we observe today.

Related: The universe could stop expanding 'remarkably soon', study suggests

In this bouncing cosmology, the universe contracted to a size about 50 orders of magnitude smaller than it is today. After the rebound, photons and other particles were born, marking the Big Bang. Near the rebound, the matter density was so high that small black holes formed from quantum fluctuations in the matter’s density, making them viable candidates for dark matter.

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"Small primordial black holes can be produced during the very early stages of the universe, and if they are not too small, their decay due to Hawking radiation [a hypothetical phenomenon of black holes emitting particles due to quantum effects] will not be efficient enough to get rid of them, so they would still be around now," Patrick Peter , director of research at the French National Centre for Scientific Research (CNRS), who was not involved in the study, told Live Science in an email. "Weighing more or less the mass of an asteroid , they could contribute to dark matter, or even solve this issue altogether."

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The scientists' calculations show that this universe mode's properties, such as the curvature of space and the microwave background, match current observations, supporting their hypothesis.

To further test their predictions, the researchers hope to make use of next-generation gravitational wave observatories.The scientists calculated the properties of the gravitational waves produced during black hole formation in their model and found that they could be detected by upcoming gravitational observatories like the Laser Interferometer Space Antenna (LISA ) and the Einstein Telescope . These detections could confirm whether primordial black holes are indeed dark matter; however, it could take more than a decade before either facility sees first light.

"This work is important in the sense that it provides a natural way of forming small yet still present black holes forming dark matter in a framework which is not the usual one based on inflation," Peter said. "Other works currently investigate the behavior of such tiny black holes around stars, potentially leading to a way of detecting them in the future."

Andrey got his B.Sc. and M.Sc. degrees in elementary particle physics from Novosibirsk State University in Russia, and a Ph.D. in string theory from the Weizmann Institute of Science in Israel. He works as a science writer, specializing in physics, space, and technology. His articles have been published in  Elements ,  N+1 , and  AdvancedScienceNews .

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What is Actually Happening to Antarctica’s ‘Doomsday Glacier?’ — A New Study Offers An Entirely New Perspective

What we are seeing with Thwaites Glacier right now is a disaster in slow motion.

Antarctica’s Thwaites Glacier got its nickname, the “ Doomsday Glacier ”, for its potential to flood coastlines around the world if it collapsed. It is already contributing about 4 percent of annual sea-level rise as it loses ice, and one theory suggests the glacier could soon begin to collapse into the ocean like a row of dominoes.

But is that kind of rapid collapse really as likely as feared? A new study of Thwaites Glacier’s susceptibility to what’s known as marine ice cliff instability offers some hope. But the findings don’t mean Thwaites is stable.

Polar scientist Mathieu Morlighem , who led the study, explains the results.

Why is the Thwaites Glacier so important?

Thwaites Glacier drains a huge area of Antarctica’s ice sheet — about 74,000 square miles (192,000 square kilometers), an expanse bigger than Florida. If a snowflake falls within that drainage system, it will eventually end up as part of an iceberg in the ocean off Thwaites.

The bedrock under Thwaites Glacier sits below sea level and slopes downward going inland , so the glacier gets deeper toward the interior of the ice sheet. Once the glacier begins losing more ice than it gains from new snowfall and starts to retreat, it’s very hard to slow it down because of this slope. And Thwaites is already retreating at an accelerating rate as the climate warms.

A cross-section showing an ice shelf and inward-sloping bedrock.

Thwaites Glacier holds enough ice to raise global sea level by more than 2 feet (0.65 meters). Once Thwaites starts to destabilize, it also will destabilize neighboring glaciers . So, what happens to Thwaites affects all of the West Antarctic Ice Sheet, and that affects sea-level rise along coastlines everywhere.

What is marine ice cliff instability?

Marine ice cliff instability is a relatively new concept proposed by scientists in the past decade.

Many of the glaciers around Antarctica have huge floating extensions called ice shelves that buttress the glacier and slow its ice flow into the ocean. With climate warming, we have seen some of these floating extensions collapse, sometimes very rapidly , in the span of a few weeks or months.

The front of Thwaites’ floating ice shelf is over 200 feet (60 meters) tall in places. It becomes higher closer to land.

If Thwaites’ ice shelf were to collapse, it would expose a very tall ice cliff facing the ocean along its 75-mile (120-kilometer) front . There is only so much force that ice can sustain, so if the cliff is too tall, it will collapse into the ocean.

Once that happens, a new ice cliff farther back would be exposed, and the new cliff would be even taller because it is farther inland. The theory of marine ice cliff instability suggests that if the cliffs collapse quickly enough, there could be a domino effect of ever-higher ice cliffs collapsing one after the other.

However, no one has observed marine ice cliff instability in action. We don’t know if it will happen because a lot depends on how quickly the ice collapses.

Watching the Larsen B ice shelf collapse over less than six weeks in 2002. Once the ice shelf was gone, glaciers it had buttressed began flowing several times faster into the ocean. AGU.

What did you discover about the risk to Thwaites?

When the theory of marine ice cliff instability was first introduced, it used a rough approximation of how ice cliffs might collapse once the ice shelf was gone.

Studies since then have determined that ice cliffs won’t fail systematically until the ice is about 442 feet (135 meters) high. Even at that point, they would fail more slowly than projected until they became much taller.

We used three high-resolution models to explore what this new physical understanding of ice cliff instability would mean for Thwaites Glacier this century.

Our results show that if Thwaites’ entire ice shelf collapsed today, its ice front would not rapidly retreat inland due to marine ice cliff instability alone. Without the ice shelf, the glacier’s ice would flow much faster toward the ocean, thinning the front of the glacier. As a result, the ice cliffs wouldn’t be as high.

We found that Thwaites would remain fairly stable at least through 2100. We also simulated an ice shelf collapse in 50 years, when the glacier’s grounding line — where its grounded ice meets the ocean — would have retreated deeper inland. Even then, we found that marine ice cliff instability alone would not cause a rapid retreat.

Satellite data shows Antarctica losing ice mass since 2002. The area with the fastest ice loss includes Thwaites Glacier. NASA.

The results call into question some recent estimates of just how fast Thwaites might collapse. That includes a worst-case scenario that the Intergovernmental Panel on Climate Change mentioned in its latest assessment report but labelled as “low likelihood.”

Thwaites is the glacier everyone is worried about. If you model the entire ice sheet, this is where marine ice cliff instability starts and where it propagates far inland . So, if Thwaites isn’t as vulnerable to ice cliff failure as we thought, that’s a good sign for the entire ice sheet.

However, marine ice cliff instability is only one mechanism of ice loss. This finding doesn’t mean Thwaites is stable.

What else is causing glaciers to retreat at an accelerating rate?

There are many processes that make the Antarctic ice sheet unstable, some of them very well understood.

Ice-ocean interactions explain most of the recent ice mass loss so far. Antarctica is a very cold place , so atmospheric warming isn’t having a large effect yet. But warm ocean currents are getting under the ice shelves, and they are thinning the ice from below , which weakens the ice shelves. When that happens, the ice streams flow faster because there is less resistance.

Ocean-bottom water temperatures reach above freezing under parts of the Thwaites ice shelf. Thwaites Glacier is outlined in dashes, with colors showing how fast the ice flows. Ocean areas in gray are too shallow to affect the glacial undersides.

Over the past few decades , the Amundsen Sea sector, where Thwaites and Pine Island glaciers are located, has seen an intrusion of warm water from the Antarctic Circumpolar Current, which has been melting the ice from below .

What does climate change have to do with it?

Antarctica can seem like a faraway place, but human activities that warm the planet — such as burning fossil fuels — are having dramatic effects at the poles. Ice loss contributes to sea-level rise, affecting coastal regions around the world.

People’s choices today will determine how quickly the water rises.

This article was originally published on The Conversation by Mathieu Morlighem at the Dartmouth College . Read the original article here .

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A Glimmer of Hope: Recent Updates and Future Challenges in Zika Vaccine Development

The emergence and rapid spread of Zika virus (ZIKV) on a global scale as well as the establishment of a causal link between Zika infection and congenital syndrome and neurological disorders triggered unprecedented efforts towards the development of a safe and effective Zika vaccine. Multiple vaccine platforms, including purified inactivated virus, nucleic acid vaccines, live-attenuated vaccines, and viral-vectored vaccines, have advanced to human clinical trials. In this review, we discuss the recent advances in the field of Zika vaccine development and the challenges for future clinical efficacy trials. We provide a brief overview on Zika vaccine platforms in the pipeline before summarizing the vaccine candidates in clinical trials, with a focus on recent, promising results from vaccine candidates that completed phase I trials. Despite low levels of transmission during recent years, ZIKV has become endemic in the Americas and the potential of large Zika outbreaks remains real. It is important for vaccine developers to continue developing their Zika vaccines, so that a potential vaccine is ready for deployment and clinical efficacy trials when the next ZIKV outbreak occurs.

1. Introduction

Zika virus (ZIKV), a member of the Flaviviridae family ( Flavivirus genus), is a positive, single-stranded, enveloped RNA virus primarily transmitted to humans through the bite of infected Aedes aegypti mosquitoes [ 1 , 2 ]. ZIKV was initially isolated in 1947 as part of routine surveillance investigations of the yellow fever transmission cycle carried out by the Rockefeller Institute in the Zika forest of Uganda [ 1 ]. During the 50 years following its discovery, only few sporadic Zika cases associated with mild, self-limiting febrile disease were detected in humans in Africa and in some Asian countries [ 3 , 4 , 5 ]. The first large outbreak of Zika disease in humans was documented in 2007 on the Pacific Island of Yap, Federated States of Micronesia [ 6 ]. Seven years later, additional ZIKV outbreaks in humans were also registered on other Pacific Islands, including French Polynesia [ 7 , 8 , 9 ]. In early 2015, a large outbreak of Zika disease was registered in Brazil [ 10 , 11 , 12 ] and ZIKV spread rapidly throughout the Americas [ 13 , 14 ]. Until then, ZIKV disease was considered a benign viral infection with minor health consequences, but a causal link between ZIKV infection and clusters of microcephaly and neurological disorders was later recognized [ 15 , 16 , 17 ], leading the WHO to declare the ZIKV outbreak as a Public Health Emergency of International Concern in February 2016 [ 18 ].

Much has been learned during the ZIKV outbreak in the Americas. Multiple, previously unknown routes of human-to-human ZIKV transmission have been identified, including via sexual [ 19 , 20 , 21 , 22 , 23 , 24 ], intrauterine, and intrapartum transmission [ 16 , 17 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ], and via blood transfusion [ 8 , 32 , 33 ]. Evidence from epidemiological studies has also shown that ZIKV infection sporadically triggers Guillain–Barré syndrome, a rare but serious autoimmune-mediated attack of healthy peripheral neurons and glial cells, leading to ascending paralysis and polyneuropathy and a small number of cases of confirmed viral encephalitis. Cases of ZIKV-associated Guillain–Barré syndrome have been reported in Brazil, Colombia, French Polynesia, and several other countries [ 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. However, perhaps the most dramatic ZIKV feature discovered during the Americas outbreak has been the impact of ZIKV infections on pregnant women and their infants. Fetal microcephaly linked to Zika infections during pregnancy was first reported in Brazil [ 16 , 17 , 29 , 31 ]. Later, Zika-related microcephaly was also identified in other South and Central American countries, and in retrospective analyses of the French Polynesia outbreak [ 41 , 42 , 43 , 44 , 45 , 46 ]. Other Zika-associated congenital disorders have also been identified and include intrauterine growth restriction, fetal demise, cerebral calcifications, sensorineural hearing loss, visual impairment [ 26 , 28 , 30 , 47 , 48 , 49 , 50 ], and delayed cognitive development. However, the full spectrum of clinical presentations of congenital Zika syndrome is yet to be described.

Intense research efforts on the underlying pathogenic mechanisms of Zika infections have also been made during the 2015–2016 Zika outbreak. Within a relatively short period of time, models of Zika disease and pathogenesis in mice and non-human primates have been established, including using female pregnant animals (reviewed in [ 51 ]). These studies have revealed ZIKV tropism to maternal and fetal tissues, including a broad range of placental cell types and the developing fetal human brain [ 25 , 26 , 27 , 52 , 53 , 54 ]. Prolonged persistence of ZIKV genome in several body tissues and fluids, including the male and female reproductive tract, has also been identified [ 25 , 55 , 56 ]. The close antigenic relationship that results in considerable serological cross-reactivity between ZIKV and other flaviviruses, particularly dengue viruses (DENV), has also been a topic of intense research. The fact that poorly neutralizing cross-reactive antibodies can potentially mediate antibody-dependent enhancement (ADE) of Zika or dengue infections has been extensively explored using in vitro and ex vivo experimental approaches, animal models, and epidemiological analyses [ 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 ]. Antibody responses to DENV have been shown to be highly cross-reactive to ZIKV and vice-versa. In fact, several analyses have shown that these Zika and dengue cross-reactive antibodies are able to promote either enhancement of or protection against subsequent flavivirus infections, an effect that seems to depend on the antibody levels at the time of infection [ 57 , 58 , 59 ]. However, it is still unknown how these Zika and dengue antibody interactions will impact future vaccination efforts.

All the aforementioned aspects of the recent Zika outbreaks highlight the urge for the development of a safe and effective vaccine against Zika. Since 2016, a number of candidates using multiple vaccine platforms have been developed and have shown promising results in preclinical testing. In this review, we discuss the advances in the development of these vaccines. Recent results of phase I clinical trials on Zika vaccine candidates are summarized and discussed. In addition, we address important remaining challenges for the clinical development and future deployment of Zika vaccines.

2. Zika Virus Vaccine Development

The emergence and rapid spread of Zika virus throughout the Americas in 2015 as well as the establishment of causal link between Zika infection and congenital syndrome and neurological disorders triggered unprecedented efforts towards the development of a Zika vaccine. Over the past 5 years, multiple vaccine platform technologies have been used in the design of vaccine prototypes ( Figure 1 ), and 20 are currently in clinical evaluation in humans ( Table 1 ). The accelerated development of multiple Zika vaccine candidates have benefited from over three decades of extensive research on flavivirus immunity, flavivirus vaccine studies, and the quick response of the research community in generating knowledge on Zika biology, pathogenesis, and animal models.

Zika vaccine development. ( A ) Number of Zika vaccine candidates tested in preclinical studies using mice models and non-human primates, and in phase I and II clinical trials in humans; ( B ) phase I and II clinical trial sites. Created with BioRender.com.

Zika vaccine candidates in clinical trials.

NIAID, National Institute of Allergy and Infectious Diseases; WRAIR, Walter Reed Army Institute of Research (WRAIR), BIDMC, Beth Israel Deaconess Medical Center; VRC, Vaccine Research Center; ZIKV, Zika Virus; CHIKV, Chikungunya virus.

The use of traditional methods, such as the production of empirical, live-attenuated vaccines by repeated passages of virus in cell culture or in animals, or conventional viral inactivation methods have been proven efficacious for the development of multiple flavivirus vaccines. Safe and efficacious vaccines for the prophylaxis of yellow fever virus (YFV), tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus (JEV) have been administered to human populations (reviewed at [ 66 ]). The live-attenuated YFV 17D vaccine has been shown to be highly effective and is currently administered to individuals aged 9 months or older, living in or traveling to endemic countries. This vaccine has led to significant reduction in the number of cases and deaths by YFV in the past 80 years. Another licensed, live-attenuated vaccine based on the SA 14-14-2 strain of the JEV has also been extensively used in China and other Asian countries to protect against infections by JEV in children and adults. Inactivated vaccines are also available to prevent JEV and TBEV-induced disease in endemic countries and have been shown to be safe and to engender protection. In addition to these traditional vaccine approaches, a JEV-YFV chimera vaccine developed using innovative molecular biology clone technologies for viral attenuation has been licensed for use in humans. A single dose of this chimera JEV-YFV vaccine has been shown to be safe and to induce sustained antibody responses in clinical trials. In addition, the vast experience in the development of tetravalent DENV vaccines [ 67 ] has also contributed to the development of Zika vaccines. There is one DENV vaccine approved for use in several countries (Sanofi-Dengvaxia) that is based on YFV/DENV chimeras. However, the deployment of this vaccine requires confirmation of a previous DENV infection prior to immunization [ 68 ]. Other dengue vaccine candidates include a vaccine based on chimeras with an attenuated DENV-2 strain (Takeda-TDV) that have completed a phase III trial, and a vaccine based on a 30-nucleotide deletion in the 3′ untranslated region (UTR) to reduce the efficiency of the viral replication, which is currently concluding its phase III trial in Brazil (Butantan-TDV).

To date, there is no single approved therapeutic or vaccine against Zika, but lessons from these positive stories in flavivirus vaccinology has prompted optimism in the development of Zika vaccines. The WHO vaccine development technology roadmap lays out two priority scenarios for Zika vaccine deployment: epidemic and endemic uses [ 69 ]. Despite low levels of transmission during recent years, ZIKV has become endemic in the Americas and the threat of large Zika outbreaks remains. Mass Zika vaccination targeting pregnant women and women of childbearing age populations during outbreaks would likely prevent prenatal Zika infection and thus congenital Zika syndrome cases. In the case of endemic usage, routine Zika immunization of children and adults living in or traveling to endemic countries would allow the establishment of population immunity and aid the prevention of both prenatal Zika infection in pregnant women and other Zika-related neurological complications in adults.

Numerous Zika vaccine platforms have been developed and tested in preclinical and clinical studies. These vaccines include classical vaccine designs (inactivated virus vaccines and live-attenuated vaccines) and novel vaccine technologies (viral-vectored vaccines and nucleic acid vaccines). Whole, inactivated viruses are prepared by exposing virus particles to chemical agents or heat. Virion then becomes noninfectious and thus unable to cause disease [ 70 ]. Live-attenuated vaccine approaches can be developed by different methods to attenuate virions until the virus effectively loses its pathogenic properties. These classical vaccine approaches have been proven to be safe and cost-effective, but they also have some disadvantages. Inactivated vaccines are considered safer than live vaccines, thus potentially offering a higher chance of being used by high-priority groups, such as pregnant women. However, chemical inactivation can alter the molecular structure of antigens and thereby negatively influence vaccine immunogenicity. In addition, inactivated vaccines usually require the use of adjuvants to stimulate robust immune responses. On the other hand, live-attenuated vaccines usually induce a more potent immune response than inactivated vaccines, as they induce innate responses as well as B- and T-cell responses against structural and nonstructural antigens, affording vaccinated individuals with long-term protection. However, live-attenuated vaccines are not administered to individuals with weakened immune systems and their use is also not recommended in pregnant populations unless their benefits outweigh the risks associated with vaccination [ 70 , 71 ]. Platforms such as Virus-like Particles (VLPs) have also been developed as an alternative to classical vaccine designs. VLP-based vaccines are nonreplicating structures composed of viral structural proteins that resemble virions. This vaccine strategy is considered safer than live-attenuated and inactivated virus vaccine approaches since the potential for reversion to virulence and incomplete inactivation are eliminated. Recent preclinical data have shown that VLP-based vaccine candidates elicit strong neutralizing antibody responses in mice [ 72 , 73 , 74 ]. Specifically, VLPs displaying the third domain (DIII) of the ZIKV envelop (E) protein were able to elicit antibodies that neutralize ZIKV without enhancing DENV infection in vitro [ 72 , 73 ]. If results are confirmed in larger animal models, this vaccine platform might constitute a good candidate for further clinical trials in humans.

Viral-vectored and nucleic acid vaccines are developed based on the coding sequence information of the viral genome alone through genetic engineering techniques [ 71 ]. With the aid of recombinant DNA techniques, nucleic acid and viral vectors are engineered to express the antigens of interest. Replication-deficient forms of this vaccine design are safe and relatively stable. Viral-vectored vaccines produce protein antigens endogenously and thus induce potent humoral and cellular immune responses. One disadvantage of this approach is the potential to induce antivector immunity, potentially reducing the immunogenicity of later inoculations using the same viral vector. DNA and mRNA vaccines are based on the delivery of gene coding specific antigens. Following uptake into cells, DNA constructs are transcribed and translated into target proteins by host cells. Constructs of mRNA work in a similar manner, but bypass the transcription step [ 75 ]. Nucleic acid vaccine designs can induce potent humoral and cellular responses, especially when the antigens are engineered to target the MHCII processing compartment [ 76 , 77 ], but one limitation of this approach is the limited efficiency of cellular uptake of nucleic acids. Often, nucleic acid vaccines require additional delivery devices to facilitate nucleic acid entry into cells [ 71 , 75 ]. To date, approximately 90 studies have been published reporting data on preclinical testing of Zika vaccine candidates using mice and non-human primate models ( Figure 1 ). Several of these vaccine approaches showed promising results in animal models and advanced to clinical trials in humans.

3. Zika Virus Vaccines Candidates in Clinical Trials

As of September 2020, 20 phase I clinical trials have been registered in the clinicaltrials.gov platform ( Table 1 ). Of those, eight phase I clinical trials have their interim or final results published in peer-reviewed journals ( Table 2 ). So far, only one vaccine has advanced to phase II trials. The vast majority of the phase I trials were carried out in nonendemic areas of the United States and Europe. Four phase I clinical trials were conducted in Puerto Rico, an area with active Zika virus transmission. Phase II clinical trial sites include endemic and nonendemic areas of Zika transmission, including several sites in Latin America ( Figure 1 ).

Zika vaccine candidates with results of their phase I clinical trials published in peer-reviewed journals.

NIAID, National Institute of Allergy and Infectious Diseases; WRAIR, Walter Reed Army Institute of Research (WRAIR), BIDMC, Beth Israel Deaconess Medical Center; VRC, Vaccine Research Center; ZIKV, Zika Virus; JEV, Japanese Encephalitis Virus; IM, intramuscular.

3.1. DNA Vaccines

Three different Zika DNA vaccines have entered phase I clinical trials ( Table 1 ). The first report of a Zika vaccine candidate in phase I clinical trial was published in 2017 (NCT02809443) [ 82 ], only 2 years after the first Zika outbreak in Latin America. This open label clinical trial evaluated the safety and immunogenicity of a synthetic DNA vaccine encoding the pre-membrane (prM) and E proteins of Zika (GLS-5700). GLS-5700 was administered via intradermal injection followed by electroporation, in a prime-boost regimen consisting of three doses of vaccine. The vaccine was tested in healthy, dengue-seronegative adults. Results from this trial showed that the DNA vaccine was well-tolerated and did not elicit any severe adverse effect in the volunteers. All participants developed ZIKV-binding antibody responses. Around 60% of participants developed ZIKV-neutralizing antibody response, but at low titers (1:18 to 1:317) and that did not correlate with vaccine dose. Participants also developed moderate T-cell response after vaccination. Importantly, this immune response was protective in both in vitro and in vivo (adoptive transfer) challenge models [ 82 ]. Another phase I trial designed to evaluate safety, tolerability, and immunogenicity of GLS-5700 in dengue-seropositive adults has been completed (NCT02887482), but results are yet to be published.

Results from two other phase I clinical trials assessing safety and immunogenicity of Zika DNA vaccine candidates were jointly published in late 2018 [ 81 ]. In these trials, two vaccine formulations were independently tested: one wild-type ZIKV DNA vaccine formulation (VRC 5283) and one chimera JEV/ZIKV DNA vaccine (VRC 5288). Both vaccine constructs express ZIKV E antigen (French Polynesian strain), but in the JEV/ZIKV chimera, the stem and transmembrane regions of the E protein of ZIKV were modified to encode JEV sequences. In addition, in both vaccines the prM was comprised of analogous sequence from JEV to improve particle secretion. No information about previous flavivirus profile of the volunteers was provided in these publications. Different vaccine doses and inoculation methods (intramuscularly using a needle syringe or a needle-free device) were tested ( Table 2 ). Both DNA vaccine constructs were safe and well-tolerated and showed no severe adverse outcomes in healthy adult volunteers. VRC5283 showed the greatest immunogenicity given in split doses via needle-free injection All participants in the VRC5283 trial had detectable ZIKV antibody responses as well as ZIKV-neutralizing antibody, and showed CD4 and CD8 T-cell responses of the greatest magnitude when compared to VRC 5288 [ 81 ]. VRC5283 has advanced to an international placebo-controlled phase II trial. The efficacy of a three-dose (0, 4, and 8 weeks) vaccination regimen of VRC5283 administered via needle-free delivery with the Stratis device (NCT03110770) is being assessed in adults and adolescents residing in flavivirus endemic and nonendemic areas.

3.2. Purified, Inactivated Virus Vaccines

Four different Zika inactivated vaccines have entered phase I clinical trials ( Table 1 ): two of those have had their results recently published and the other two have completed phase I trial, but results are yet to be announced. In 2018, Modjarrad and colleagues [ 80 ] published the interim, aggregated results of three phase I clinical trials of a purified, formalin-inactivated Zika vaccine (ZPIV) (NCT02963909, NCT02952833, and NCT02937233). In these trials, healthy adults randomly received 5 µg of either ZIPV with alum adjuvant or saline placebo intramuscularly at weeks 0 and 4. All participants in these trials were initially reported to be flavivirus-naïve, but some participants were later found to be flavivirus positive in a post hoc analysis. ZPIV vaccine caused only minor to moderate reactogenicity. By day 57 of follow-up, more than 90% of the participants seroconverted to Zika neutralizing antibodies. However, 15% of those have geometric mean titers (GMT) lower than 100. Adoptive transfer of purified IgG collected at day 57 of follow-up provided robust protection against viremia in ZIKV-challenged mice [ 80 ].

In 2020, Stephenson and colleagues [ 79 ] published additional results for this vaccine in a trial comparing different doses and vaccination schedules of the ZPIV vaccine (NCT02937233). The focus of this trial was on vaccination schedule. In addition to testing a two-dose ZPIV schedule at weeks 0 and 4, which is a standard regimen for purified inactivated flavivirus vaccines, authors also examined the safety and immunogenicity of a single-dose regimen and an accelerated two-dose regimen with ZPIV given at weeks 0 and 2. Volunteers have no known history of previous flavivirus infection or vaccination. High antibody titers were observed when using the standard dose regimen. However, at week 28, a positive response was observed in only 13% of the participants who received ZPIV via the standard regimen ( n = 8) and in none of the participants who received ZPIV via the accelerated ( n = 7) or single-dose ( n = 10) regimens ( Table 2 ). Importantly, ZIKV-neutralizing antibody levels declined to a GMT of less than 100 by week 16. This short durability of antibody response is probably related to the lack of induction of robust T cell responses. Of note, the accelerated regimen elicited similar peak of ZIKV-neutralizing antibody titers when compared to the standard regimen, but this peak concentration was achieved sooner. However, the antibody response of both regimens was not durable [ 79 ]. Additional trials of the ZPIV vaccine have been completed and include testing two doses of ZPIV and a late ZPIV boost (NCT02963909), as well as higher ZPIV doses (NCT02952833) in healthy, flavivirus-naïve adult individuals. Another phase I trial is assessing ZPIV immunogenicity in healthy adults residing in a flavivirus endemic area (NCT03008122) ( Table 1 ).

Recently, Han and colleagues [ 78 ] reported the results of a phase I clinical trial on the safety, dose ranging, and immunogenicity (NCT03343626) of a different purified, formalin-inactivated, alum-adjuvated whole Zika virus vaccine candidate (PIZV). So far, this is the first large phase I trial that compared vaccine immunogenicity in flavivirus-naïve versus flavivirus-primed volunteers ( Table 2 ). Authors tested three doses (2, 5, and 10 µg) of this vaccine formulation and all were reported to be safe and well-tolerated in healthy adults with and without preexisting exposure to other flaviviruses throughout 57 weeks of follow-up. A two-dose regimen of the vaccine administered 28 days apart elicited a robust, dose-dependent Zika virus-neutralizing antibody response in 100% of the flavivirus-naïve participants. Neutralizing antibody GMT among flavivirus-naïve individuals remained >3000 in the 10 µg vaccine group by study week 57. A similar dose-dependent neutralizing antibody response was also noted among flavivirus-primed volunteers, but GMTs did not increase following second dose and tended to be lower than those in the flavivirus-naïve group [ 78 ].

3.3. mRNA Vaccines

Two mRNA vaccine candidates, named mRNA-1325 (NCT03014089) and mRNA-1893 (NCT04064905), have entered phase I clinical trials ( Table 1 ). These vaccine constructs incorporate prM/E genes of ZIKV, but differ with respect to the signal peptide at the amino terminus of prM. mRNA-1325 expresses the signal sequence from human IgE upstream of prM [ 83 ]. Both vaccine constructs were immunogenic and protected against Zika infection in multiple mouse models. Interim phase I data for mRNA-1893 were press released in April 2020 by the sponsors (ModernaTX Inc., Cambridge, MA, United States) [ 84 ]. A two-dose (10 or 30 µg) vaccination schedule of mRNA-1893 given 28 days apart was well-tolerated in healthy adults with and without preexisting exposure to other flaviviruses. No serious vaccine-related adverse events were reported by volunteers. Both 10 and 30 µg dose levels were immunogenic. All flavivirus-seronegative individuals seroconverted to Zika-neutralizing antibodies in the 30 µg dose group after the second vaccination. In the group consisting of flavivirus-primed participants, 75% achieved a four-fold boost in preexisting neutralizing titers following a second vaccination at the 30 µg dose. These results are yet to be published in peer-reviewed journals.

3.4. Live Attenuated Vaccines

Several strategies to develop a live-attenuated Zika vaccine have been employed, but despite promising preclinical data only one live-attenuated Zika vaccine candidate has entered phase I clinical trial so far (NCT03611946). Live-attenuated vaccine approaches include chimeric flavivirus constructs expressing ZIKV prM/E in the genetic background of YFV, JEV, or DENV [ 85 , 86 , 87 , 88 ] and nucleotide deletion in the 3′ UTR [ 89 , 90 ]. These vaccines have been proved immunogenic and protective in both mice and non-human primate models [ 85 , 86 , 87 , 88 , 89 , 90 ]. The live-attenuated chimeric rZIKV/D4D30-713 vaccine has completed phase I clinical trial in healthy, flavivirus-naïve adults (NCT03611946). This vaccine expresses ZIKV prM-E proteins in the genomic backbone of DENV-4 and encodes a 30-nucleotide deletion in the 3′ UTR to attenuate viral replication. Preclinical and phase I data on this vaccine have not yet been reported.

3.5. Viral-Vectored Vaccines

Different viral-vectored vaccines expressing ZIKV prM/E proteins have entered phase I clinical trials ( Table 1 ). These viral vectors include adenovirus and measles vaccine virus as the delivery platform for ZIKV antigens. Preclinical evaluation of a recombinant measles Schwarz vaccine (MV-ZIKA) vector expressing ZIKV prM-E reduced plasma viremia and viral load in distinct organs and in the placenta, preventing fetal infection [ 91 ]. MV-ZIKA has completed phase I clinical trial, but results are yet to be published (NCT02996890). Another similar measles-based vaccine (MV-ZIKV-RSP) has entered phase I trials and the study is currently recruiting participants in Austria (NCT04033068). A replication-deficient vaccine candidate based on chimpanzee adenovirus (ChAdOx1) encoding ZIKV prM-E has also proved protective against ZIKV challenge in mice. This vaccine elicited long-lasting anti-envelope immunity in mice with no evidence of enhancement of dengue virus in vitro [ 92 ]. ChAdOx1 has entered two phase I clinical trials to determine its safety and immunogenicity in healthy adults when administered as a standalone vaccine or coadministered with a Chikungunya virus (CHIKV) vaccine (NCT04015648 and NCT04440774). Immunization with a replication-incompetent human adenovirus type 26 (AD26)-vectored vaccine encoding ZIKV M and E proteins (Ad26.ZIKV.001) induced robust ZIKV binding and neutralizing antibody responses as well as cellular responses in mice and non-human primates during preclinical evaluation [ 93 ]. A single dose of Ad26.ZIKV.001 provided full protection against Zika challenge in several mice models and non-human primates. This vaccine has advanced to phase I clinical trial (NCT03356561).

4. Challenges in Late Stage Development of Zika Vaccines and Future Perspectives

Despite the fast-paced start and promising phase I clinical trial results, several challenges for the late development of ZIKV vaccine candidates still remain. Further development of Zika vaccines will require efficacy studies in phase III trials and these trials face major challenges. Typical flavivirus vaccine efficacy trials require the detection of a large number of virologically confirmed cases and comparison of the number of cases or the viremia levels between immunized and control groups. However, in order to compensate for the high proportion of asymptomatic/unapparent ZIKV infections, these studies would need to enroll a relatively large number of volunteers as compared to dengue vaccine studies. In addition, following the explosive 2015/2016 outbreaks, ZIKV transmission has been dramatically reduced. Thus, in the context of low level of transmission rates, the number of volunteers required would increase even further, making the assessment of clinical vaccine efficacy even more difficult [ 94 ]. In addition, the ultimate goal of a Zika vaccine is the prevention of congenital disease; measuring this effect directly in a phase III trial would be extremely difficult and costly. Therefore, it is crucial to better characterize the pathogenic mechanisms involved in Zika congenital disease in order to identify and validate correlates of immunity to be employed in the evaluation of the efficacy of vaccine candidates. It is also critical to take into consideration the challenge of developing a business model to attract enough investment in order to continue developing a Zika vaccine and, subsequently, maintain a sustained production of it. As with several other newly emerging pathogens that have threatened human health on a global scale, efforts for vaccine development usually decline when the epidemic wanes. Robust public and private funding must continue to be provided if we are to support phase III clinical trials of Zika vaccine candidates. One possible strategy to reduce the cost of phase III trials would be to have the trials and study sites pre-planned and ready to start on short notice when a new outbreak emerges.

Substantial concern also exists regarding the interactions between preexisting flavivirus immunity and the potential for vaccine-mediated ADE of disease. There has been a growing body of work supporting the idea that the balance between the concentration of cross-reactive antibodies at the time of infection and the interval between two infections are important factors influencing protection against or enhancement of flavivirus disease. In humans, enhancement of dengue disease has been shown to occur within a narrow range of preexisting DENV antibody concentration [ 95 ]. In Zika, high titers of preexisting DENV antibodies have been associated with protection against subsequent ZIKV infections in humans [ 58 ]. Recent evidence from experimental [ 59 ] and epidemiological [ 57 ] studies also suggests that low Zika antibody levels may affect clinical outcome of future dengue disease in humans by increasing the risk for severe dengue. How immunity to Zika vaccine candidates might shape future flavivirus epidemics needs to be addressed. Regardless of the platform, most candidates in phase I trials have focused on vaccine strategies targeting antigens (e.g., E and/or prM) that are known to elicit cross-reactive antibody responses. Ideal vaccine candidates should elicit a threshold of cross-reactive antibody concentration that is sufficient to effectively neutralize virus infection, thus providing protection and avoiding vaccine-mediated ADE effects. Of note, most phase I trials have tested the safety and immunogenicity of their vaccine candidates in naïve and previously exposed flavivirus individuals. Long-term analysis of the persistence of vaccine-elicited antibody response and interactions with other flavivirus will be critical to address potential risk for vaccine-mediate enhancement of subsequent flavivirus disease in humans. The possibility of vaccine-mediated enhancement of disease would require careful deployment of the eventual Zika vaccine to be performed only in the age groups with high DENV seroprevalence or only in individuals with a confirmed DENV infection.

Finally, an ideal Zika vaccine needs to be safe for at-risk populations, including women of childbearing age, pregnant women, young children, elderly, and immune-compromised individuals. To achieve such a broad population spectrum, multiple vaccine platforms may be required. In addition, a Zika vaccine should not induce Guillain–Barré-like neurological side effects or congenital malformations in fetuses. Most of the Zika vaccine candidates currently in human trials have been shown to protect fetus against ZIKV challenge and prevent neurological, placental, and testis damage from it in mice and non-human primate models, but if this will hold true in humans still needs to be determined in clinical trials.

5. Concluding Remarks

A number of Zika vaccine candidates have completed phase I trial and have been shown to be safe, well tolerated, and immunogenic in healthy adult volunteers. These viable Zika vaccine candidates should continue their development. ZIKV has become endemic and new cases of Zika congenital syndrome continue to be reported in endemic areas. It is very likely that we will face another large Zika epidemic in the next 10–15 years as herd immunity decay. Therefore, it is critical for Zika vaccine developers to be ready to activate phase III trials and ramp up vaccine production in case another epidemic emerges. Vaccine developers should also consider coordinating strategies to use Dengue and Zika vaccines to maximize the immunogenicity of both vaccines and to reduce the risks associated with deleterious DENV and ZIKV cross-reactive antibody interactions. Moreover, the coordinated use of both vaccines may also lead to a more attractive business model.

Author Contributions

P.M.S.C. and E.T.A.M. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

This research was funded by CuraZika Alliance Fund.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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