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Mosquito-borne Zika virus (ZIKV) from sub-Saharan Africa has recently gained attention due to its epidemic potential and its capacity to be highly teratogenic. To improve our knowledge on currently circulating strains of African ZIKV, we conducted protein sequence alignment and identified contemporary West Africa NS1 (NS1CWA) protein as a highly conserved viral protein. Comparison of NS1CWA with the NS1 of the historical African ZIKV strain MR766 (NS1MR766), revealed seven amino acid substitutions. The effects of NS1 mutations on protein expression, virus replication, and innate immune activation were assessed in human cells using recombinant NS1 proteins and a chimeric viral clone MR766 with NS1CWA replacing NS1MR766. Our data indicated higher secretion efficiency of NS1CWA compared to NS1MR766 associated with a change in subcellular distribution. A chimeric MR766 virus with NS1CWA instead of authentic protein displayed a greater viral replication efficiency, leading to more pronounced cell death compared to parental virus. Enhanced viral growth was associated with reduced activation of innate immunity. Our data raise questions of the importance of NS1 protein in the pathogenicity of contemporary ZIKV from sub-Saharan Africa and point to differences within viral strains of African lineage. Author summary: Mosquito-borne Zika virus (ZIKV) of African lineage has the potential to cause epidemics alongside a high risk of fetal pathogenicity. Improved surveillance has enabled a deeper understanding of the molecular characteristics of currently circulating viral strains from sub-Saharan Africa. A remarkable conservation of ZIKV NS1 protein has been identified between recently isolated viral strains from West Africa, with data indicating that contemporary African NS1 is secreted efficiently from human cells. The protein has been shown to enhance viral replication, associated with a reduced activation of innate immune responses. The NS1 protein might therefore play a major role in the pathogenicity of contemporary ZIKV from sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) has become a global health problem over the past decade due to the extension of the geographic distribution of the Asian/American genotype. Recent epidemics of Asian/American ZIKV have been associated with developmental disorders in humans. There is mounting evidence that African ZIKV may be associated with increased fetal pathogenicity necessitating to pay a greater attention towards currently circulating viral strains in sub-Saharan Africa. Here, we generated an infectious molecular clone GUINEA-18 of a recently transmitted human ZIKV isolate from West Africa, ZIKV-15555. The available infectious molecular clone MR766MC of historical African ZIKV strain MR766-NIID was used for a molecular clone-based comparative study. Viral clones GUINEA-18 and MR766MC were compared for their ability to replicate in VeroE6, A549 and HCM3 cell lines. There was a lower replication rate for GUINEA-18 associated with weaker cytotoxicity and reduced innate immune system activation compared with MR766MC. Analysis of chimeric viruses between viral clones stressed the importance of NS1 to NS4B proteins, with a particular focus of NS4B on GUINEA-18 replicative properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. GUINEA-18 was greatly efficient in inhibiting stress granule assembly in A549 cells subjected to a physiological stressor, with NS1 to NS4B proteins also being critical in this process. The impact of these GUINEA-18 proteins on viral replicative abilities and host-cell responses to viral infection raises the question of the role of nonstructural proteins in the pathogenicity of currently circulating ZIKV in sub-Saharan Africa. Author summary: Most studies with the objective to understand the biology of Zika virus (ZIKV) were carried out using the epidemic Asian genotype of this pathogen. It is currently being discussed whether ZIKV of African genotype may have epidemic potential associated a high risk of fetal pathogenicity. It is thus urgent to improve our knowledge on recently isolated ZIKV strains from West Africa. In this study, we used the sequence of a viral strain from an individual infected by ZIKV in Guinea in 2018 to generate an infectious molecular clone. Analysis of this viral clone highlighted the preponderant role of NS1 to NS4B proteins in virus replication and cell interactions with a particular focus on ZIKV-specific stress granule formation blocking activity. We believe that our data will improve our understanding of the biology of contemporary West Africa ZIKV, opening perspectives towards a better understanding of the pathogenicity of African viral strains. [ABSTRACT FROM AUTHOR]

Background: Zika virus (ZIKV) has spread to five of the six World Health Organization (WHO) regions. Given the substantial number of asymptomatic infections and clinical presentations resembling those of other arboviruses, estimating the true burden of ZIKV infections is both challenging and essential. Therefore, we conducted a systematic review and meta-analysis of seroprevalence studies of ZIKV IgG in asymptomatic population to estimate its global impact and distribution. Methodology/Principal findings: We conducted extensive searches and compiled a collection of articles published from Jan/01/2000, to Jul/31/2023, from Embase, Pubmed, SciELO, and Scopus databases. The random effects model was used to pool prevalences, reported with their 95% confidence interval (CI), a tool to assess the risk of study bias in prevalence studies, and the I2 method for heterogeneity (PROSPERO registration No. CRD42023442227). Eighty-four studies from 49 countries/territories, with a diversity of study designs and serological tests were included. The global seroprevalence of ZIKV was 21.0% (95%CI 16.1%-26.4%). Evidence of IgG antibodies was identified in all WHO regions, except for Europe. Seroprevalence correlated with the epidemics in the Americas (39.9%, 95%CI:30.0–49.9), and in some Western Pacific countries (15.6%, 95%CI:8.2–24.9), as well as with recent and past circulation in Southeast Asia (22.8%, 95%CI:16.5–29.7), particularly in Thailand. Additionally, sustained low circulation was observed in Africa (8.4%, 95%CI:4.8–12.9), except for Gabon (43.7%), and Burkina Faso (22.8%). Although no autochthonous transmission was identified in the Eastern Mediterranean, a seroprevalence of 16.0% was recorded. Conclusions/Significance: The study highlights the high heterogeneity and gaps in the distribution of seroprevalence. The implementation of standardized protocols and the development of tests with high specificity are essential for ensuring a valid comparison between studies. Equally crucial are vector surveillance and control methods to reduce the risk of emerging and re-emerging ZIKV outbreaks, whether caused by Ae. aegypti or Ae. albopictus or by the Asian or African ZIKV. Author summary: Zika virus (ZIKV) remains an important public health concern. Estimating the true burden of the disease is a major challenge, often underestimated due to the substantial number of asymptomatic infections. Consequently, seroprevalence studies are valuable for determining the geographic extent of the virus, measuring levels of human immunity, and assessing potential infection-related risks. Thus, we conducted a systematic review and meta-analysis of the literature addressing the seroprevalence of ZIKV in asymptomatic individuals worldwide. The overall seroprevalence of ZIKV IgG antibodies was 21.0%, calculated based on 84 studies published between January 2000 and July 2023. Antibodies have been found in all WHO regions, but none in Europe, with rates ranging from 8.4% in Africa to 39.9% in America. Further standardized surveillance studies are needed to understand immunity per region and over time, evaluate vector dynamics, and assess the risk of future outbreaks. [ABSTRACT FROM AUTHOR]

Background: Mosquito-borne arboviruses are expanding their territory and elevating their infection prevalence due to the rapid climate change, urbanization, and increased international travel and global trade. Various significant arboviruses, including the dengue virus, Zika virus, Chikungunya virus, and yellow fever virus, are all reliant on the same primary vector, Aedes aegypti. Consequently, the occurrence of arbovirus coinfection in mosquitoes is anticipated. Arbovirus coinfection in mosquitoes has two patterns: simultaneous and sequential. Numerous studies have demonstrated that simultaneous coinfection of arboviruses in mosquitoes is unlikely to exert mutual developmental influence on these viruses. However, the viruses' interplay within a mosquito after the sequential coinfection seems intricated and not well understood. Methodology/principal findings: We conducted experiments aimed at examining the phenomenon of arbovirus sequential coinfection in both mosquito cell line (C6/36) and A. aegypti, specifically focusing on dengue virus (DENV, serotype 2) and Zika virus (ZIKV). We firstly observed that DENV and ZIKV can sequentially infect mosquito C6/36 cell line, but the replication level of the subsequently infected ZIKV was significantly suppressed. Similarly, A. aegypti mosquitoes can be sequentially coinfected by these two arboviruses, regardless of the order of virus exposure. However, the replication, dissemination, and the transmission potential of the secondary virus were significantly inhibited. We preliminarily explored the underlying mechanisms, revealing that arbovirus-infected mosquitoes exhibited activated innate immunity, disrupted lipid metabolism, and enhanced RNAi pathway, leading to reduced susceptibility to the secondary arbovirus infections. Conclusions/significance: Our findings suggest that, in contrast to simultaneous arbovirus coinfection in mosquitoes that can promote the transmission and co-circulation of these viruses, sequential coinfection appears to have limited influence on arbovirus transmission dynamics. However, it is important to note that more experimental investigations are needed to refine and expand upon this conclusion. Author summary: Arboviruses coinfection in mosquitoes can occur either simultaneously or sequentially. Simultaneous coinfection of arboviruses in mosquitoes is unlikely to exert mutual developmental influence on these viruses, resulting in a single mosquito bite may transmit multiple arboviruses to humans, thereby facilitating the spread of these pathogens. However, the interplay between viruses within a mosquito after the sequential coinfection is intricated and not well understood. We present results from DENV (serotype 2) and ZIKV sequential coinfection both in mosquito cell line (C6/36) and Aedes aegypti. Our results demonstrated that both mosquito cells and adult mosquitoes could be sequentially coinfected, but prior infection with one arbovirus significantly reduced susceptibility to a secondary arbovirus infection. Moreover, upon sequential coinfection with DENV and ZIKV through oral feeding, the transmission potential of A. aegypti for the ZIKV was aborted. We preliminarily explored the underlying mechanism of this phenomenon and observed that arbovirus-infected mosquitoes exhibited activated innate immunity, disrupted lipid metabolism, and enhanced RNAi pathway, all of which contributed to the reduced susceptibility to the secondary arbovirus infections. In the epidemiology of mosquito borne diseases, our findings indicates that, in contrast to simultaneous arbovirus coinfection in mosquitoes that can promote the transmission and co-circulation of these viruses, sequential coinfection appears to have limited influence on arbovirus transmission dynamics. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) transmission from humans to mosquitoes is a poorly documented, but critical component of DENV epidemiology. Magnitude of viremia is the primary determinant of successful human-to-mosquito DENV transmission. People with the same level of viremia, however, can vary in their infectiousness to mosquitoes as a function of other factors that remain to be elucidated. Here, we report on a field-based study in the city of Iquitos, Peru, where we conducted direct mosquito feedings on people naturally infected with DENV and that experienced mild illness. We also enrolled people naturally infected with Zika virus (ZIKV) after the introduction of ZIKV in Iquitos during the study period. Of the 54 study participants involved in direct mosquito feedings, 43 were infected with DENV-2, two with DENV-3, and nine with ZIKV. Our analysis excluded participants whose viremia was detectable at enrollment but undetectable at the time of mosquito feeding, which was the case for all participants with DENV-3 and ZIKV infections. We analyzed the probability of onward transmission during 50 feeding events involving 27 participants infected with DENV-2 based on the presence of infectious virus in mosquito saliva 7-16 days post blood meal. Transmission probability was positively associated with the level of viremia and duration of extrinsic incubation in the mosquito. In addition, transmission probability was influenced by the day of illness in a non-monotonic fashion; i.e., transmission probability increased until 2 days after symptom onset and decreased thereafter. We conclude that mildly ill DENV-infected humans with similar levels of viremia during the first two days after symptom onset will be most infectious to mosquitoes on the second day of their illness. Quantifying variation within and between people in their contribution to DENV transmission is essential to better understand the biological determinants of human infectiousness, parametrize epidemiological models, and improve disease surveillance and prevention strategies.

BackgroundAfter the unprecedented Zika virus (ZIKV) outbreak in the western hemisphere from 2015-2018, Aedes aegypti and Ae. albopictus are now well established primary and secondary ZIKV vectors, respectively. Consensus about identification and importance of other secondary ZIKV vectors remain. This systematic review aims to provide a list of vector species capable of transmitting ZIKV by reviewing evidence from laboratory vector competence (VC) studies and to identify key knowledge gaps and issues within the ZIKV VC literature.MethodsA search was performed until 15th March 2022 on the Cochrane Library, Lilacs, PubMed, Web of Science, WHOLIS and Google Scholar. The search strings included three general categories: 1) "ZIKA"; 2) "vector"; 3) "competence", "transmission", "isolation", or "feeding behavior" and their combinations. Inclusion and exclusion criteria has been predefined and quality of included articles was assessed by STROBE and STROME-ID criteria.FindingsFrom 8,986 articles retrieved, 2,349 non-duplicates were screened by title and abstracts,103 evaluated using the full text, and 45 included in this analysis. Main findings are 1) secondary vectors of interest include Ae. japonicus, Ae. detritus, and Ae. vexans at higher temperature 2) Culex quinquefasciatus was not found to be a competent vector of ZIKV, 3) considerable heterogeneity in VC, depending on the local mosquito strain and virus used in testing was observed. Critical issues or gaps identified included 1) inconsistent definitions of VC parameters across the literature; 2) equivalency of using different mosquito body parts to evaluate VC parameters for infection (mosquito bodies versus midguts), dissemination (heads, legs or wings versus salivary glands), and transmission (detection or virus amplification in saliva, FTA cards, transmission to neonatal mice); 3) articles that fail to use infectious virus assays to confirm the presence of live virus; 4) need for more studies using murine models with immunocompromised mice to infect mosquitoes.ConclusionRecent, large collaborative multi-country projects to conduct large scale evaluations of specific mosquito species represent the most appropriate approach to establish VC of mosquito species.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a survey of DENV susceptibility using a panel of seven field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain. Author summary: African populations of the mosquito Aedes aegypti are usually thought to be less likely to get infected by flaviviruses compared to Ae. aegypti mosquitoes found outside Africa. While this has been well-demonstrated for Zika virus, it is not clear if the same is true for dengue virus, which is the most common flavivirus in humans. Studying this is complicated by the strain diversity of dengue virus, including four main genetic types, potentially causing different interactions. In this study, we compared several mosquito populations and found that, in general, African mosquitoes were less likely to get infected by dengue virus compared to mosquitoes from outside Africa. However, in some cases, African mosquitoes were just as or even more likely to get infected. The specific strain of dengue virus also influenced how likely African mosquitoes were to get infected, showing that the relationship between African mosquitoes and dengue virus is complex. [ABSTRACT FROM AUTHOR]

The 2015–17 Zika virus (ZIKV) epidemic in the Americas subsided faster than expected and evolving population immunity was postulated to be the main reason. Herd immunization is suggested to occur around 60–70% seroprevalence, depending on demographic density and climate suitability. However, herd immunity was only documented for a few cities in South America, meaning a substantial portion of the population might still be vulnerable to a future Zika virus outbreak. The aim of our study was to determine the vulnerability of populations to ZIKV by comparing the environmental suitability of ZIKV transmission to the observed seroprevalence, based on published studies. Using a systematic search, we collected seroprevalence and geospatial data for 119 unique locations from 37 studies. Extracting the environmental suitability at each location and converting to a hypothetical expected seroprevalence, we were able to determine the discrepancy between observed and expected. This discrepancy is an indicator of vulnerability and divided into three categories: high risk, low risk, and very low risk. The vulnerability was used to evaluate the level of risk that each location still has for a ZIKV outbreak to occur. Of the 119 unique locations, 69 locations (58%) fell within the high risk category, 47 locations (39%) fell within the low risk category, and 3 locations (3%) fell within the very low risk category. The considerable heterogeneity between environmental suitability and seroprevalence potentially leaves a large population vulnerable to future infection. Vulnerability seems to be especially pronounced at the fringes of the environmental suitability for ZIKV (e.g. Sao Paulo, Brazil). The discrepancies between observed and expected seroprevalence raise the question: "why did the ZIKV epidemic stop with large populations unaffected?". This lack of understanding also highlights that future ZIKV outbreaks currently cannot be predicted with confidence. Author summary: After the ZIKV epidemic in the Americas, it remains unclear if and when a resurgence of the ZIKV could occur. We used publicly available data on environmental suitability of transmission as well as seroprevalence data to estimate future vulnerability to ZIKV outbreaks. Our results show a considerable discrepancy between the observed seroprevalence, from past exposure to the virus on one hand, and the environmental suitability, which raised the question why the epidemic subsided before reaching the expected herd immunization threshold in many locations. This lack of understanding also highlights that future ZIKV outbreaks currently cannot be predicted with confidence. Although we cannot provide an answer to the question why the epidemic subsided when it did, we present a better quantification and geospatial mapping of the potential vulnerability to future outbreaks, which will be crucial for decision makers to prepare for future outbreaks. [ABSTRACT FROM AUTHOR]

The distribution and intensity of viral diseases transmitted by Aedes aegypti mosquitoes, including dengue, have rapidly increased over the last century. Here, we study dengue virus (DENV) transmission across the ecologically and demographically distinct regions or Ecuador. We analyzed province-level age-stratified dengue incidence data from 2000–2019 using catalytic models to estimate the force of infection of DENV over eight decades. We found that provinces established endemic DENV transmission at different time periods. Coastal provinces with the largest and most connected cities had the earliest and highest increase in DENV transmission, starting around 1980 and continuing to the present. In contrast, remote and rural areas with reduced access, like the northern coast and the Amazon regions, experienced a rise in DENV transmission and endemicity only in the last 10 to 20 years. The newly introduced chikungunya and Zika viruses have age-specific distributions of hospital-seeking cases consistent with recent emergence across all provinces. To evaluate factors associated with geographic differences in DENV transmission potential, we modeled the DENV vector risk using 11,693 Aedes aegypti presence points to the resolution of 1 hectare. In total, 56% of the population of Ecuador, including in provinces identified as having increasing DENV transmission in our models, live in areas with high risk of Aedes aegypti, with population size, trash collection, elevation, and access to water as important determinants. Our investigation serves as a case study of the changes driving the expansion of DENV and other arboviruses globally and suggest that control efforts should be expanded to semi-urban and rural areas and to historically isolated regions to counteract increasing dengue outbreaks. Author summary: The factors driving the increasing burden of arboviruses like dengue virus are not fully understood. In this study, we measured changes in dengue virus transmission intensity and arbovirus disease risk across Ecuador, an ecologically and demographically diverse South American country. We found that differences in the distribution of dengue cases could be explained by changes in transmission of dengue virus over time: transmission was limited to coastal provinces with large cities between 1980–2000, expanding thereafter to higher elevation areas and ecologically suitable but previously geographically and socially isolated provinces. We also used species distribution mapping to show that both urban and rural areas in Ecuador are at medium to high risk for Aedes aegypti presence, with population size, trash removal, elevation, and access to water as strong predictors. Our investigation reveals changes driving the expansion of dengue virus and other arboviruses globally and provides an approach for identifying areas at early stages of establishing endemic transmission that should be targeted for intense preventative efforts to avert future epidemics. [ABSTRACT FROM AUTHOR]

Flaviviruses such as Zika and dengue viruses are persistent health concerns in endemic regions worldwide. Efforts to combat the spread of flaviviruses have been challenging, as no antivirals or optimal vaccines are available. Prevention and treatment of flavivirus-induced diseases require a comprehensive understanding of their life cycle. However, several aspects of flavivirus biogenesis, including genome packaging and virion assembly, are not well characterized. In this study, we focused on flavivirus capsid protein (C) using Zika virus (ZIKV) as a model to investigate the role of the externally oriented α3 helix (C α3) without a known or predicted function. Alanine scanning mutagenesis of surface-exposed amino acids on C α3 revealed a critical CN67 residue essential for ZIKV virion production. The CN67A mutation did not affect dimerization or RNA binding of purified C protein in vitro. The virus assembly is severely affected in cells transfected with an infectious cDNA clone of ZIKV with CN67A mutation, resulting in a highly attenuated phenotype. We isolated a revertant virus with a partially restored phenotype by continuous passage of the CN67A mutant virus in Vero E6 cells. Sequence analysis of the revertant revealed a second site mutation in the viral membrane (M) protein MF37L, indicating a genetic interaction between the C and M proteins of ZIKV. Introducing the MF37L mutation on the mutant ZIKV CN67A generated a double-mutant virus phenotypically consistent with the isolated genetic revertant. Similar results were obtained with analogous mutations on C and M proteins of dengue virus, suggesting the critical nature of C α3 and possible C and M residues contributing to virus assembly in other Aedes-transmitted flaviviruses. This study provides the first experimental evidence of a genetic interaction between the C protein and the viral envelope protein M, providing a mechanistic understanding of the molecular interactions involved in the assembly and budding of Aedes-transmitted flaviviruses. Author summary: Zika virus and other disease-causing flaviviruses constitute a significant threat to global human health. The development of effective antiviral strategies targeting flaviviruses has been hindered by a lack of evidence regarding their life cycle, particularly virion assembly. Here, we identified a critical Asn 67 residue in the α3 helix of the C protein mediating ZIKV assembly. We also isolated a compensatory mutation in the M protein, MF37L, that rescues the assembly defect caused by the CN67A mutation, revealing the first evidence of a genetic interaction between C and M protein. Using further molecular genetic analyses, we show that analogous resides in C and M proteins are also required for dengue virus (DENV) assembly, indicating a functional role of this interaction that is conserved among flaviviruses. The data reported here contribute to an increasing collection of evidence pointing toward an interaction between capsid and the external structural proteins of flaviviruses. Our findings describing the genetic interaction between C and M observed in cryo-EM structures but experimentally unproven significantly advance our understanding of Aedes-transmitted flavivirus assembly, which will be instrumental in designing assembly-specific flavivirus therapeutics and antivirals. [ABSTRACT FROM AUTHOR]

Introduction: Zika virus infection during pregnancy causes fetal microcephaly and brain damage. Congenital Zika syndrome (CZS) is characterized by systemic involvement with diffuse muscle impairment, a high frequency of arthrogryposis, and microphthalmia. Cardiac impairment in CZS has rarely been evaluated. Our study assessed morphology and biventricular cardiac function in children with CZS and advanced neurological dysfunction. Methods: This cross-sectional study was conducted on 52 children with CZS (Zika group; ZG) and 25 healthy children (control group; CG) in Paraiba, Brazil. Clinical evaluation, electrocardiogram (EKG), and transthoracic echocardiogram (TTE) were performed on all children. Additionally, troponin I and natriuretic peptide type B (BNP) levels, the degree of cerebral palsy, and neuroimaging findings were assessed in the ZG group. Results: The median age of the study population was 5 years in both groups, and 40.4% (ZG) and 60% (CG) were female. The most prevalent electrocardiographic alteration was sinus arrhythmia in both the ZG (n = 9, 17.3%) and CG (n = 4, 16%). The morphological parameters adjusted for Z score were as follows: left ventricular (LV) end-diastolic diameter in ZG: -2.36 [-5.10, 2.63] vs. CG: -1.07 [-3.43, 0.61], p<0.001); ascending aorta (ZG: -0.09 [-2.08, 1.60] vs. CG: 0.43 [-1.47, 2.2], p = 0.021); basal diameter of the right ventricle (RV) (ZG: -2.34 [-4.90, 0.97] vs. CG: -0.96 [-2.21, 0.40], p<0.01); and pulmonary artery dimension (ZG: -2.13 [-5.99, 0.98] vs. CG: -0.24 [-2.53, 0.59], p<0.01). The ejection fractions (%) were 65.7 and 65.6 in the ZG and CG, respectively (p = 0.968). The left atrium volume indices (mL/m2) in the ZG and CG were 13.15 [6.80, 18.00] and 18.80 [5.90, 25.30] (p<0.01), respectively, and the right atrium volume indices (mL/m2) were 10.10 [4.90, 15.30] and 15.80 [4.10, 24.80] (p<0.01). The functional findings adjusted for Z score were as follows: lateral systolic excursion of the mitral annular plane (MAPSE) (ZG: 0.36 [-2.79, 4.71] vs. CG: 1.79 [-0.93, 4.5], p = 0.001); tricuspid annular plane systolic excursion (TAPSE) (ZG: -2.43 [-5.47, 5.09] vs. CG: 0.07 [-1.98, 3.64], p<0.001); and the S' of the RV (ZG: 1.20 [3.35, 2.90] vs. CG: -0.20 [-2.15, 1.50], p = 0.0121). No differences in biventricular strain measurements were observed between the groups. Troponin I and BNP levels were normal in in the ZG. Grade V cerebral palsy and subcortical calcification were found in 88.6% and 97.22% of children in the ZG group, respectively. Conclusion: A reduction in cardiac dimensions and functional changes were found in CZS patients, based on the TAPSE, S' of the RV, and MAPSE, suggesting the importance of cardiac evaluation and follow-up in this group of patients. Author summary: Congenital Zika syndrome (CZS) occurs in the fetuses of mothers infected with the Zika virus during pregnancy, especially in the third trimester. The main findings are microcephaly and other neurological defects; however, several systemic effects have also been reported. Cardiac manifestations have rarely been evaluated. In this study, we compared cardiac anatomy and function between 52 children with CZS and 25 healthy children using electrocardiograms, echocardiograms, and biomarkers. We also assessed the degree of cerebral palsy and neuroimaging findings in the affected group. The hearts of the CZS patients were smaller and demonstrated functional alterations in comparison with those of the control individuals. Neurological impairment in the CZS patients was severe. The early detection of cardiac alterations highlights the need for cardiac evaluation and follow-up in this patient group. [ABSTRACT FROM AUTHOR]

Zika virus can infect the fetus through the placental barrier, causing ZIKV congenital syndrome and even miscarriage, which can cause great harm to pregnant women and infants. Currently, there is no vaccine and drug available to combat the Zika virus. In this study, we designed a fusion protein named EDIII-Fc, including the EDIII region of Zika E protein and human IgG Fc fragment, and obtained 293T cells that stably secreted EDIII-Fc protein using the lentiviral expression system. Mice were immunized with the EDIII-Fc protein, and it was observed that viral replication was significantly inhibited in the immunized mice compared to non-immunized mice. In rhesus macaques, we found that EDIII-Fc effectively induce the secretion of neutralizing antibodies and T cell immunity. These experimental data provide valid data for further use of Zika virus E protein to prepare an effective, safe, affordable Zika vaccine. Author summary: Zika virus (ZIKV) is a member of the Flavivirus genus. ZIKV could be transmitted through mosquito bites. In the population, ZIKV can be transmitted through mother-to-child transmission, sexual transmission, and blood transmission. Clinical symptoms of ZIKV infection in adults include rash, fever, arthralgia, and conjunctivitis, and neurological symptoms in those with severe infection, manifesting as Guillain-Barre syndrome (GBS). ZIKV can infect the fetus through the placental barrier, causing ZIKV congenital syndrome and even miscarriage. ZIKV causes fatal damage to the central nervous system of infants. Due to the lack of effective drugs, vaccination is the main strategy to against ZIKV. Conventional vaccines against the Zika virus have the potential to cause antibody-dependent enhancement (ADE). In this study, the authors designed a fusion protein named EDIII-Fc, including the EDIII region of Zika E protein and human IgG Fc fragment. EDIII protein has been shown to be incapable of causing ADE. Mice and rhesus monkeys were immunized with EDIII-Fc. EDIII-Fc could induce protective antibodies in mice and rhesus monkeys and protect mice against the challenge of ZIKV. These experimental data provide valid data for further use of ZIKV E protein to prepare an effective, safe, affordable Zika vaccine. [ABSTRACT FROM AUTHOR]

Background: The co-circulation of flaviviruses in tropical regions has led to the hypothesis that immunity generated by a previous dengue infection could promote severe disease outcomes in subsequent infections by heterologous serotypes. This study investigated the influence of antibodies generated by previous Zika infection on the clinical outcomes of dengue infection. Methodology/Principal findings: We enrolled 1,043 laboratory confirmed dengue patients and investigated their prior infection to Zika or dengue. Severe forms of dengue disease were more frequent in patients with previous Zika infection, but not in those previously exposed to dengue. Conclusions/Significance: Our findings suggest that previous Zika infection may represent a risk factor for subsequent severe dengue disease, but we did not find evidence of antibody-dependent enhancement (higher viral titer or pro-inflammatory cytokine overexpression) contributing to exacerbation of the subsequent dengue infection. Author summary: Although dengue is a disease known for years in the world and has been affecting several continents, some aspects remain unclear. One of them is about the possible factors that may influence the development of severe forms of the disease. Much has been discussed about the influence of a previous dengue episode, but the global spreading of other flaviviruses to areas where dengue was already circulating has aroused interest regarding a role like the other dengue serotypes. In this sense, the recent spreading of the Zika virus has become a factor of interest. In this study, the prior Zika virus infection was associated with a higher frequency of more severe forms in subsequent dengue. Preliminary findings did not suggest that the mechanism is the same one triggered in secondary dengue, known as antibody-dependent enhancement. These findings are a stimulus to develop further research that can understand the potential mechanisms involved in the pathogenesis. [ABSTRACT FROM AUTHOR]

Since emerging in French Polynesia and Brazil in the 2010s, Zika virus (ZIKV) has been associated with fetal congenital disease. Previous studies have compared ancestral and epidemic ZIKV strains to identify strain differences that may contribute to vertical transmission and fetal disease. However, within-host diversity in ZIKV populations during vertical transmission has not been well studied. Here, we used the established anti-interferon treated Rag1-/- mouse model of ZIKV vertical transmission to compare genomic variation within ZIKV populations in matched placentas, fetal bodies, and fetal brains via RNASeq. At early stages of vertical transmission, the ZIKV populations in the matched placentas and fetal bodies were similar. Most ZIKV single nucleotide variants were present in both tissues, indicating little to no restriction in transmission of ZIKV variants from placenta to fetus. In contrast, at later stages of fetal infection there was a sharp reduction in ZIKV diversity in fetal bodies and fetal brains. All fetal brain ZIKV populations were comprised of one of two haplotypes, containing either a single variant or three variants together, as largely homogenous populations. In most cases, the dominant haplotype present in the fetal brain was also the dominant haplotype present in the matched fetal body. However, in two of ten fetal brains the dominant ZIKV haplotype was undetectable or present at low frequencies in the matched placenta and fetal body ZIKV populations, suggesting evidence of a strict selective bottleneck and possible selection for certain variants during neuroinvasion of ZIKV into fetal brains. Author summary: Zika virus (ZIKV) is known to cause congenital disease in humans through vertical transmission of the virus from mothers to fetuses during pregnancy. How the virus populations change during this process is currently not well understood. To evaluate differences in ZIKV populations during vertical transmission, we infected pregnant mice with ZIKV and sequenced the viral populations from matched placentas and fetal bodies at early stages of vertical transmission, and matched placentas, fetal bodies, and fetal brains at late stages of vertical transmission when the virus enters the fetal brain. We determined that there is little restriction of ZIKV diversity during transmission from the placenta to the fetus at early stages. However, there was a sharp decrease in ZIKV diversity in both fetal bodies and fetal brains at late stages. Two predominant viral variants were found across all fetal brains and most fetal bodies at late stages, suggesting there may be possible selection for certain variants during neuroinvasion of ZIKV into fetal brains. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.

The importance of mosquitoes in human pathogen transmission has motivated major research efforts into mosquito biology in pursuit of more effective vector control measures. Aedes aegypti is a particular concern in tropical urban areas, where it is the primary vector of numerous flaviviruses, including the yellow fever, Zika, and dengue viruses. With an anthropophilic habit, Ae. aegypti prefers houses, human blood meals, and ovipositioning in water-filled containers. We hypothesized that this relatively simple ecological niche should allow us to predict the impacts of insecticidal control measures on mosquito populations. To do this, we use Skeeter Buster 2 (SB2), a stochastic, spatially explicit, mechanistic model of Ae. aegypti population biology. SB2 builds on Skeeter Buster, which reproduced equilibrium dynamics of Ae. aegypti in Iquitos, Peru. Our goal was to validate SB2 by predicting the response of mosquito populations to perturbations by indoor insecticidal spraying and widespread destructive insect surveys. To evaluate SB2, we conducted two field experiments in Iquitos, Peru: a smaller pilot study in 2013 (S-2013) followed by a larger experiment in 2014 (L-2014). Here, we compare model predictions with (previously reported) empirical results from these experiments. In both simulated and empirical populations, repeated spraying yielded substantial yet temporary reductions in adult densities. The proportional effects of spraying were broadly comparable between simulated and empirical results, but we found noteworthy differences. In particular, SB2 consistently over-estimated the proportion of nulliparous females and the proportion of containers holding immature mosquitoes. We also observed less temporal variation in simulated surveys of adult abundance relative to corresponding empirical observations. Our results indicate the presence of ecological heterogeneities or sampling processes not effectively represented by SB2. Although additional empirical research could further improve the accuracy and precision of SB2, our results underscore the importance of non-linear dynamics in the response of Ae. aegypti populations to perturbations, and suggest general limits to the fine-grained predictability of its population dynamics over space and time.

Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested to play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried out an experimental infection in wild-caught Artibeus lituratus bats and sampled several free-living neotropical bats across three countries of the region. Experimental ZIKV infection was performed in wild-caught adult bats (4 females and 5 males). The most relevant findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggested to be caused by ZIKV; however, since these were wild-caught bats, we cannot exclude other agents. Detection of ZIKV by qPCR was observed at low concentrations in only two urine samples in two inoculated animals. All other animals and tissues tested were negative. Finally, no virus-neutralizing antibodies were found in any animal. To determine ZIKV infection in nature, the blood of a total of 2056 bats was sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%), Anoura sp. (14%), and Molossus sp. (13.7%). No sample of any tested species was positive for ZIKV by qPCR. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics. Author summary: In previous works in 2008–2009, we found the presence of antibodies against flaviviruses, and viral RNA was detected in Neotropical chiropterans in Mexico, which led us to support the hypothesis that these animals could be reservoirs of flaviviruses. As controversial opinions have been exposed and based on a previous (2019) experimental ZIKV infection experiment conducted at Colorado State University using adult Artibeus males from a captive colony, in this work, we also experimentally infected adult Artibeus males complementarily adding females and using wild-caught animals instead of laboratory bats. We also monitored a diverse range of natural bat populations in Latin America for the presence of viral RNA against ZIKV in blood. A plaque reduction seroneutralization test was used for the detection of antibodies against ZIKV. Similar to the previous work, we found histopathological alterations in male testicles but also in the ovaries and oviducts of females, as well as gliosis and multifocal necrosis in pyramidal neurons and Purkinge cells of inoculated animals. Only two urine samples from inoculated animals showed viral RNA. Additionally, leukopenia and lymphoid follicular splenic hyperplasia were evidenced. In contrast to what was reported, no neutralizing antibodies against ZIKV were detected in any sample. Viral RNA within the blood was not present in any of the 2056 bat samples collected in French Guiana, Peru and Costa Rica and proceeding from 34 bat genera. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and might not have an important role in ZIKV transmission dynamics. [ABSTRACT FROM AUTHOR]

Since its detection in 2015 in Brazil, Zika virus (ZIKV) has remained in the spotlight of international public health and research as an emerging arboviral pathogen. In addition to single infection, ZIKV may occur in co-infection with dengue (DENV) and chikungunya (CHIKV) viruses, with whom ZIKV shares geographic distribution and the mosquito Aedes aegypti as a vector. The main mosquito immune response against arboviruses is RNA interference (RNAi). It is unknown whether or not the dynamics of the RNAi response differ between single arboviral infections and co-infections. In this study, we investigated the interaction of ZIKV and DENV, as well as ZIKV and CHIKV co-infections with the RNAi response in Ae. aegypti. Using small RNA sequencing, we found that the efficiency of small RNA production against ZIKV -a hallmark of antiviral RNAi—was mostly similar when comparing single and co-infections with either DENV or CHIKV. Silencing of key antiviral RNAi proteins, showed no change in effect on ZIKV replication when the cell is co-infected with ZIKV and DENV or CHIKV. Interestingly, we observed a negative effect on ZIKV replication during CHIKV co-infection in the context of Ago2-knockout cells, though his effect was absent during DENV co-infection. Overall, this study provides evidence that ZIKV single or co-infections with CHIKV or DENV are equally controlled by RNAi responses. Thus, Ae. aegypti mosquitoes and derived cells support co-infections of ZIKV with either CHIKV or DENV to a similar level than single infections, as long as the RNAi response is functional. Author summary: Zika virus (ZIKV) is a mosquito-borne human-pathogenic arbovirus of the Flaviviridae family, genus Flavivirus. Other arboviruses, including dengue (DENV) or chikungunya (CHIKV) virus, can occur in the same regions as ZIKV and are also transmitted by Aedes aegypti. Notably, it has been shown that these viruses can co-infect this mosquito, andco-transmission occurs. Such processes may add to the serious public health issues already linked to those pathogens. Arbovirus infections in mosquitoes are controlled through an immune response called RNA interference (RNAi). It is however unknown whether immune responses changs when a mosquito is exposed to a co-infection of ZIKV with either DENV or CHIKV. In this study, we provide evidence that ZIKV co-infections with CHIKV or DENV are similarly well controlled by RNAi as single infections. These findings give new insights into the dynamics of arboviral co-infections in mosquito vectors that increase our understanding of co-infection scenarios during arbovirus outbreaks. [ABSTRACT FROM AUTHOR]

Knowledge regarding the frequency of ocular abnormalities and abnormal visual function in children exposed to Zika virus (ZIKV) in utero but born without congenital Zika syndrome (CZS) is limited. We hypothesized that children exposed to ZIKV in utero born without CZS may have visual impairments in early childhood. We performed ophthalmic examination between 16 and 21 months of age and neurodevelopment assessment at 24 months of age with the Mullen Scales of Early Learning test (MSEL) on children enrolled in a cohort born to women pregnant during and shortly after the ZIKV epidemic in Nicaragua (2016–2017). ZIKV exposure status was defined based on maternal and infant serological testing. Visual impairment was defined as abnormal if the child had an abnormal ophthalmic exam and/or low visual reception score in the MSEL assessment. Of 124 children included in the analysis, 24 (19.4%) were classified as ZIKV-exposed and 100 (80.6%) unexposed according to maternal or cord blood serology. Ophthalmic examination showed that visual acuity did not differ significantly between groups, thus, 17.4% of ZIKV-exposed and 5.2% of unexposed had abnormal visual function (p = 0.07) and 12.5% of the ZIKV-exposed and 2% of the unexposed had abnormal contrast testing (p = 0.05). Low MSEL visual reception score was 3.2-fold higher in ZIKV-exposed than unexposed children, but not statistically significant (OR 3.2, CI: 0.8–14.0; p = 0.10). Visual impairment (a composite measure of visual function or low MESL visual reception score) was present in more ZIKV-exposed than in unexposed children (OR 3.7, CI: 1.2, 11.0; p = 0.02). However, the limited sample size warrants future investigations to fully assess the impact of in utero ZIKV exposure on ocular structures and visual function in early childhood, even in apparently healthy children. Author summary: Vertical transmission of Zika virus (ZIKV) during pregnancy has the potential to cause vision abnormalities even in the absence of overt ocular structural abnormalities or congenital Zika syndrome. We were able to classify infants' ZIKV exposure status by applying a serological algorithm of Zika antibodies in maternal and umbilical cord blood samples. We evaluated the ophthalmological abnormalities in children born in Nicaragua during the American ZIKV epidemic of 2015–2017 with no clinical abnormality detected at birth. We performed eye examinations on the children at 16–21 months of age, and neurocognitive testing by the Mullens Scales of Early Learning (MSEL) was performed at 24 months of age. Visual impairment, a composite measure of abnormal visual function and/or visual reception score was significantly more common in ZIKV-exposed than in unexposed children. We found a nonsignificant trend for a higher proportion of children exposed to ZIKV in utero had abnormal visual function and abnormal contrast testing than unexposed children. The visual reception score from the MSEL was also higher in Zika-exposed children, but these relationships did not reach statistical significance. These impairments could have implications for later academic performance, specifically reading. Early intervention may limit the morbidity associated with congenital ZIKV exposure. [ABSTRACT FROM AUTHOR]

Since Zika virus (ZIKV) emerged as a global human health threat, numerous studies have pointed to Aedes aegypti as the primary vector due to its high competence and propensity to feed on humans. The majority of vector competence studies have been conducted between 26-28°C, but arboviral extrinsic incubation periods (EIPs), and therefore transmission efficiency, are known to be affected strongly by temperature. To better understand the relationship between ZIKV EIPs and temperature, we evaluated the effect of adult mosquito exposure temperature on ZIKV infection, dissemination, and transmission in Ae. aegypti at four temperatures: 18°C, 21°C, 26°C, and 30°C. Mosquitoes were exposed to viremic mice infected with a 2015 Puerto Rican ZIKV strain, and engorged mosquitoes were sorted into the four temperatures with 80% RH and constant access to 10% sucrose. ZIKV infection, dissemination, and transmission rates were assessed via RT-qPCR from individual mosquito bodies, legs and wings, and saliva, respectively, at three to five time points per temperature from three to 31 days, based on expectations from other flavivirus EIPs. The median time from ZIKV ingestion to transmission (median EIP, EIP50) at each temperature was estimated by fitting a generalized linear mixed model for each temperature. EIP50 ranged from 5.1 days at 30°C to 24.2 days at 21°C. At 26°C, EIP50 was 9.6 days. At 18°C, only 15% transmitted by day 31 so EIP50 could not be estimated. This is among the first studies to characterize the effects of temperature on ZIKV EIP in Ae. aegypti, and the first to do so based on feeding of mosquitoes on a live, viremic host. This information is critical for modeling ZIKV transmission dynamics to understand geographic and seasonal limits of ZIKV risk; it is especially relevant for determining risk in subtropical regions with established Ae. aegypti populations and relatively high rates of return travel from the tropics (e.g. California or Florida), as these regions typically experience cooler temperature ranges than tropical regions.

BackgroundZika virus (ZIKV) is an arthropod-borne flavivirus transmitted by Aedes mosquitoes. Aedes albopictus is an important vector of ZIKV worldwide. To date, most experiments have focused on the vertical transmission of ZIKV in Ae. aegypti, while studies on Ae. albopictus are very limited. To explore vertical transmission in Ae. albopictus, a series of laboratory studies were carried out.Methodology/principal findingsIn this study, Ae. albopictus were blood-fed with ZIKV-infectious blood, and the ovaries and offspring viral infection rates were analyzed by reverse transcription PCR (RT-PCR), real-time reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC). ZIKV was detected in the ovaries and oviposited eggs in two gonotrophic cycles. The minimum filial egg infection rates in two gonotrophic cycles were 2.06% and 0.69%, and the effective population transmission rate was 1.87%. The hatching, pupation, and emergence rates of infected offspring were not significantly different from those of uninfected offspring, indicating that ZIKV did not prevent the offspring from completing the growth and development process. ZIKV was detected in three of thirteen C57BL/6 suckling mice bitten by ZIKV-positive F1 females, and the viremia persisted for at least seven days.Conclusions/significanceZIKV can be vertically transmitted in Ae. albopictus via transovarial transmission. The vertical transmission rates in F1 eggs and adults were 2.06% and 1.87%, respectively. Even though the vertical transmission rates were low, the female mosquitoes infected via the congenital route horizontally transmitted ZIKV to suckling mice through bloodsucking. This is the first experimental evidence of offspring with vertically transmitted ZIKV initiating new horizontal transmission. The present study deepens the understanding of the vertical transmission of flaviviruses in Aedes mosquitoes and sheds light on the prevention and control of mosquito-borne diseases.

Early detection of Zika virus (ZIKV) transmission within geographic regions informs implementation of community mitigation measures such as vector reduction strategies, travel advisories, enhanced surveillance among pregnant women, and possible implementation of blood and organ donor screening or deferral. Standardized, comparative assessments of ZIKV assay and testing lab performance are important to develop optimal approaches to ZIKV diagnostic testing and surveillance. We conducted an expanded blinded panel study to characterize and compare the analytical performance of fifteen diagnostic and blood screening ZIKV NAT assays, including detection among single- and multiplex assays detecting ZIKV, dengue virus (DENV) and chikungunya virus (CHIKV). A 300 member blinded panel was constructed, consisting of 11 serial half-log dilutions ranging from ~104 to 10−1 genome equivalents/mL in 25 replicates each of the Tahitian Asian ZIKV isolate in ZIKV-negative human serum. Additionally, clinical samples from individuals with DENV-like syndrome or suspected ZIKV infection in Brazil were evaluated. The majority of assays demonstrated good specificity. Analytical sensitivities varied 1–2 logs, with a substantially higher limit of detection (LOD) in one outlier. Similar analytical sensitivity for ZIKV RNA detection in singleplex and multiplex assays of the Grifols and ThermoFisher tests were observed. Coefficient of Assay Efficiency (CE), calculated to characterize assays' RNA extraction and amplification efficiency, ranged from 0.13 for the Certest VIASURE multiplex and 0.75 for the Grifols multiplex assays. In general, assays using transcription mediated amplification (TMA) technology had greater CE compared to assays using conventional PCR technology. Donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, primarily attributable to higher sample input volumes. However, ideal assays to maximize sensitivity and throughput may not be a viable option in all contexts, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection, particularly in resource constrained settings. Author summary: Having standardized, comparative studies of Zika virus (ZIKV) tests are important for implementing optimal diagnostic testing and disease surveillance. Detection of ZIKV nucleic acid is ideal for confirming infection; however, knowing the sensitivity and specificity of a test is needed for selecting the right test in a given laboratory. We looked at the analytical performance of 15 diagnostic and blood screening ZIKV molecular tests, including tests that detect ZIKV only and tests that detect ZIKV plus other arboviruses. We found donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, mostly due to the higher sample input volumes. We also found no difference in analytical sensitivity between tests that only detected ZIKV compared to tests that detected ZIKV and related viruses. The ideal molecular test will depend on the testing context, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection. [ABSTRACT FROM AUTHOR]

RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015–2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced transmission during the epidemic. Author summary: Zika virus was introduced to the Western Hemisphere, spread rapidly, and led to the 2015–2016 Zika epidemic and a rise in congenital microcephaly. It remains unclear whether Zika virus evolved to become more transmissible directly before or during the epidemic. To investigate whether Zika evolved to become more transmissible, we engineered a library of recombinant viruses that represent twelve major Zika virus lineages that circulated throughout the Americas. We measured the replicative fitness of each of these lineages by infecting live mosquitoes and human cells that are relevant for disease or transmission. We found that two of the lineages, one that dominated Central America and another that existed mostly in the Caribbean, appear to replicate more efficiently in human cells. While the fitness changes do not appear to have significant effects on the 2015–2016 Zika epidemic, our analysis suggests Zika virus evolved at least twice during this outbreak. Monitoring the phenotypic evolution during the course of an outbreak can help control spread and mitigate disease. We believe this framework can be applied to study phenotypic evolution during future epidemics caused by emerging RNA viruses. [ABSTRACT FROM AUTHOR]

Zika virus infection is associated with the development of Guillain-Barré syndrome (GBS), a neurological autoimmune disorder caused by immune recognition of gangliosides and other components at nerve membranes. Using a high-throughput ELISA, we have analyzed the anti-glycolipid antibody profile, including gangliosides, of plasma samples from patients with Zika infections associated or not with GBS in Salvador, Brazil. We have observed that Zika patients that develop GBS present higher levels of anti-ganglioside antibodies when compared to Zika patients without GBS. We also observed that a broad repertoire of gangliosides was targeted by both IgM and IgG anti-self antibodies in these patients. Since Zika virus infects neurons, which contain membrane gangliosides, antigen presentation of these infected cells may trigger the observed autoimmune anti-ganglioside antibodies suggesting direct infection-induced autoantibodies as a cause leading to GBS development. Collectively, our results establish a link between anti-ganglioside antibodies and Zika-associated GBS in patients.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus linked to devastating neurologic diseases. Immune responses to flaviviruses may be pathogenic or protective. Our understanding of human immune responses to ZIKV in vivo remains limited. Therefore, we performed a longitudinal molecular and phenotypic characterization of innate and adaptive immune responses during an acute ZIKV infection. We found that innate immune transcriptional and genomic responses were both cell type- and time-dependent. While interferon stimulated gene induction was common to all innate immune cells, the upregulation of important inflammatory cytokine genes was primarily limited to monocyte subsets. Additionally, genomic analysis revealed substantial chromatin remodeling at sites containing cell-type specific transcription factor binding motifs that may explain the observed changes in gene expression. In this dengue virus-experienced individual, adaptive immune responses were rapidly mobilized with T cell transcriptional activity and ZIKV neutralizing antibody responses peaking 6 days after the onset of symptoms. Collectively this study characterizes the development and resolution of an in vivo human immune response to acute ZIKV infection in an individual with pre-existing flavivirus immunity.

The gold-standard method for quantifying neutralizing antibody responses to many viruses, including dengue virus (DENV), is the plaque reduction neutralization test (PRNT, also called the immunofocus reduction neutralization test). The PRNT conducted on 96-well plates is high-throughput and requires a smaller volume of antiserum than on 6- or 24-well plates, but manual plaque counting is challenging and existing automated plaque counters are expensive or difficult to optimize. We have developed Viridot (Viridot package), a program for R with a user interface in shiny, that counts viral plaques of a variety of phenotypes, estimates neutralizing antibody titers, and performs other calculations of use to virologists. The Viridot plaque counter includes an automatic parameter identification mode (misses

Background: Zika virus (ZIKV) infection has potential result in severe birth effects. An improved understanding of global trend and regional differences is needed. Methods: Annual ZIKV infection episodes and incidence rates were collected from Global Burden of Disease Study 2019. Episodes changes and estimated annual percentage changes (EAPCs) of age-standardized incidence rate (ASR) were calculated. Top passenger airport-pairs were obtained from the International Air Transport Association to understand places susceptible to imported ZIKV cases. Results: Globally, the ASR increased by an average of 72.85% (95%CI: 16.47% to 156.53%) per year from 2011 to 2015 and subsequently decreased from 20.25 per 100,000 in 2015 to 3.44 per 100,000 in 2019. Most of ZIKV infections clustered in Latin America. The proportion of episodes in Central and Tropical Latin America decreased in 2019 with sporadic episodes elsewhere. High Socio-Demographic Index (SDI) regions had more episodes in 2019 than in 2015. Additionally, 15–49 years group had the largest proportion of episodes, females had a higher number of episodes, and a higher incidence rate of 70 plus group was observed in males than females. Certain cities in Europe, North America and Latin America/Caribbean had a high population mobility in ZIKV outbreak areas considered a high risk of imported cases. Conclusions: ZIKV infection is still a public health threat in Latin America and Caribbean and high SDI regions suffered an increasing trend of ZIKV infection. Interventions such as development of surveillance networks and vector-control should be attached to ZIKV control in these key regions. Reproductive suggestions should be taken to reduce ZIKV-related birth defects for the people of reproductive age who are facing a higher threat of ZIKV infection, especially females. Moreover, surveillance of travellers is needed to reverse the uptrends of travel-related imported ZIKV infection. More studies focusing on ZIKV should be performed to make targeted and effective prevention strategies in the future. Author summary: Zika virus (ZIKV) infection is a mosquito-borne illness and has potential result in severe birth effects. Currently, ZIKV is still causing an unprecedented ongoing epidemic in Latin America and threatening North America and potentially the rest of the world. This is the first study to assess the global landscape, long-term trends and regional differences in the incidence of ZIKV infection using the data from Global Burden of Disease (GBD) Study 2019, including the description of ZIKV infection episodes by different sex and by different year group, as well as the relationship between international travellers and imported ZIKV cases. Our study can not only serve as complement to previous studies, but also provide a more comprehensive perspective of global ZIKV infection prevention strategies. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher's exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV.

Background: Colombia has one of the highest burdens of arboviruses in South America. The country was in a state of hyperendemicity between 2014 and 2016, with co-circulation of several Aedes-borne viruses, including a syndemic of dengue, chikungunya, and Zika in 2015. Methodology/Principal findings: We analyzed the cases of dengue, chikungunya, and Zika notified in Colombia from January 2014 to December 2018 by municipality and week. The trajectory and velocity of spread was studied using trend surface analysis, and spatio-temporal high-risk clusters for each disease in separate and for the three diseases simultaneously (multivariate) were identified using Kulldorff's scan statistics. During the study period, there were 366,628, 77,345 and 74,793 cases of dengue, chikungunya, and Zika, respectively, in Colombia. The spread patterns for chikungunya and Zika were similar, although Zika's spread was accelerated. Both chikungunya and Zika mainly spread from the regions on the Atlantic coast and the south-west to the rest of the country. We identified 21, 16, and 13 spatio-temporal clusters of dengue, chikungunya and Zika, respectively, and, from the multivariate analysis, 20 spatio-temporal clusters, among which 7 were simultaneous for the three diseases. For all disease-specific analyses and the multivariate analysis, the most-likely cluster was identified in the south-western region of Colombia, including the Valle del Cauca department. Conclusions/Significance: The results further our understanding of emerging Aedes-borne diseases in Colombia by providing useful evidence on their potential site of entry and spread trajectory within the country, and identifying spatio-temporal disease-specific and multivariate high-risk clusters of dengue, chikungunya, and Zika, information that can be used to target interventions. Author summary: Dengue, chikungunya, and Zika are diseases transmitted to humans by the bite of infected Aedes mosquitoes. Between 2014 and 2016 chikungunya and Zika viruses started causing outbreaks in Colombia, one of the countries historically most affected by dengue. We used case counts of the diseases by municipality and week to study the spread trajectory of chikungunya and Zika within Colombia's territory, and to identify space-time high-risk clusters, i.e., the areas and time periods that dengue, chikungunya, and Zika were more present. Chikungunya and Zika spred similarly in Colombia, but Zika spread was faster. The Atlantic coast, a famous touristic destination in the country, was likely the place of entry of chikungunya and Zika in Colombia. The south-western region was identified as a high-risk cluster for all three diseases in separate and simultaneously. This region has a favorable climate for the Aedes mosquitoes and other characteristics that facilitate the diseases' transmission, such as social deprivation and high population mobility. Our results provide useful information on the locations that should be prioritized for interventions to prevent the entry of new diseases transmitted by Aedes and to reduce the burden of dengue, chikungunya and Zika where they are established. [ABSTRACT FROM AUTHOR]

Countermeasures against Zika virus (ZIKV), including vaccines, are frequently tested in nonhuman primates (NHP). Macaque models are important for understanding how ZIKV infections impact human pregnancy due to similarities in placental development. The lack of consistent adverse pregnancy outcomes in ZIKV-affected pregnancies poses a challenge in macaque studies where group sizes are often small (4–8 animals). Studies in small animal models suggest that African-lineage Zika viruses can cause more frequent and severe fetal outcomes. No adverse outcomes were observed in macaques exposed to 1x104 PFU (low dose) of African-lineage ZIKV at gestational day (GD) 45. Here, we exposed eight pregnant rhesus macaques to 1x108 PFU (high dose) of African-lineage ZIKV at GD 45 to test the hypothesis that adverse pregnancy outcomes are dose-dependent. Three of eight pregnancies ended prematurely with fetal death. ZIKV was detected in both fetal and placental tissues from all cases of early fetal loss. Further refinements of this exposure system (e.g., varying the dose and timing of infection) could lead to an even more consistent, unambiguous fetal loss phenotype for assessing ZIKV countermeasures in pregnancy. These data demonstrate that high-dose exposure to African-lineage ZIKV causes pregnancy loss in macaques and also suggest that ZIKV-induced first trimester pregnancy loss could be strain-specific. Author summary: Although pregnant rhesus macaques are susceptible to infection with Zika virus (ZIKV), fetal phenotypes can be subtle and variable. Most macaque studies of ZIKV have involved infection with Asian-lineage viruses because these viruses caused Western Hemisphere outbreaks beginning in 2015. African-lineage ZIKV yields more severe adverse fetal outcomes in small animal models. Here, we provide evidence that pregnant macaques infected late in the first trimester using a high dose of African-lineage ZIKV have frequent ZIKV-associated pregnancy loss. This severe phenotype establishes a new model for evaluating countermeasures and reinforces the idea that African-lineage ZIKV infection may be a cause of pregnancy loss in areas where it is endemic depending on the amount of transmitted virus during infection. [ABSTRACT FROM AUTHOR]

Mosquito-borne arboviruses are an important cause of morbidity and mortality in the Caribbean. In Puerto Rico, chikungunya, dengue, and Zika viruses have each caused large outbreaks during 2010–2022. To date, the majority of control measures to prevent these diseases focus on mosquito control and many require community participation. In 2018, the U.S. Centers for Disease Control and Prevention launched the COPA project, a community-based cohort study in Ponce, Puerto Rico, to measure the impact of novel vector control interventions in reducing arboviral infections. Randomly selected households from 38 designated cluster areas were offered participation, and baseline data were collected from 2,353 households between May 2018 and May 2019. Household-level responses were provided by one representative per home. Cross-sectional analyses of baseline data were conducted to estimate 1) the association between arboviral risk perception and annual household expenditure on mosquito control, and 2) the association between arboviral risk perception and engagement in ≥3 household-level risk reduction behaviors. In this study, 27% of household representatives believed their household was at high risk of arboviruses and 36% of households engaged in at least three of the six household-level preventive behaviors. Households where the representative perceived their household at high risk spent an average of $35.9 (95% confidence interval: $23.7, $48.1) more annually on mosquito bite prevention compared to households where the representative perceived no risk. The probability of engaging in ≥3 household-level mosquito-preventive behaviors was 10.2 percentage points greater (7.2, 13.0) in households where the representatives perceived high risk compared to those in which the representatives perceived no risk. Paired with other research, these results support investment in community-based participatory approaches to mosquito control and providing accessible information for communities to accurately interpret their risk. Author summary: Mosquito-borne disease is an important cause of illness and death in the Caribbean, including Puerto Rico. Most tactics to prevent these diseases rely on stopping mosquito bites, either by reducing the mosquito population or creating barriers between mosquitos and humans. These methods vary in the degree of community involvement required. This study used data collected from 2,353 households in Ponce, Puerto Rico from May 2018 to May 2019 to understand how household perception of risk of contracting these diseases related to 1) the amount of money households spent annually to prevent mosquito bites, and 2) the number of activities their household engaged in to prevent mosquito bites. We found that 27% of households perceived themselves at high risk of contracting these diseases, and 36% of households engaged in at least three activities to reduce their risk. On average, households that perceived themselves at high risk spent more money on mosquito bite prevention and engaged in more activities to prevent mosquito bites, compared to households that perceived no risk. Paired with other research in this area, these results support investment in community-based approaches to mosquito control and ensuring that communities have accessible information to understand their risk of mosquito-borne disease. [ABSTRACT FROM AUTHOR]

Timely, accurate, and comparative data on human mobility is of paramount importance for epidemic preparedness and response, but generally not available or easily accessible. Mobile phone metadata, typically in the form of Call Detail Records (CDRs), represents a powerful source of information on human movements at an unprecedented scale. In this work, we investigate the potential benefits of harnessing aggregated CDR-derived mobility to predict the 2015-2016 Zika virus (ZIKV) outbreak in Colombia, when compared to other traditional data sources. To simulate the spread of ZIKV at sub-national level in Colombia, we employ a stochastic metapopulation epidemic model for vector-borne diseases. Our model integrates detailed data on the key drivers of ZIKV spread, including the spatial heterogeneity of the mosquito abundance, and the exposure of the population to the virus due to environmental and socio-economic factors. Given the same modelling settings (i.e. initial conditions and epidemiological parameters), we perform in-silico simulations for each mobility network and assess their ability in reproducing the local outbreak as reported by the official surveillance data. We assess the performance of our epidemic modelling approach in capturing the ZIKV outbreak both nationally and sub-nationally. Our model estimates are strongly correlated with the surveillance data at the country level (Pearson's r = 0.92 for the CDR-informed network). Moreover, we found strong performance of the model estimates generated by the CDR-informed mobility networks in reproducing the local outbreak observed at the sub-national level. Compared to the CDR-informed networks, the performance of the other mobility networks is either comparatively similar or substantially lower, with no added value in predicting the local epidemic. This suggests that mobile phone data captures a better picture of human mobility patterns. This work contributes to the ongoing discussion on the value of aggregated mobility estimates from CDRs data that, with appropriate data protection and privacy safeguards, can be used for social impact applications and humanitarian action. Author summary: Human mobility plays a key role in the spread of many infectious diseases. Integrating this variable into spatial epidemic models can provide valuable insights for epidemic preparedness and response. Yet, there are numerous limitations and pitfalls often driven by data scarcity, especially in developing countries. To improve our understanding of the potential benefits of different human mobility data for outbreak prediction, in this work we focused on the aggregated mobility patterns derived from Call Detail Records (CDRs) data in comparison to more traditional data, including census data and mathematical mobility models. Using the 2015–2016 Zika virus (ZIKV) outbreak in Colombia as a case study, we employed a stochastic metapopulation model for vector-borne disease to simulate the ZIKV spread at the sub-national level in Colombia and assess the performance of each mobility network in capturing the ZIKV outbreak both nationally and sub-nationally. We found evidence that the population movements derived from aggregated CDRs data better capture the mobility and mixing patterns relevant to predict the local spread of ZIKV infections. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne flavivirus with maternal infection associated with preterm birth, congenital malformations, and fetal death, and adult infection associated with Guillain-Barré syndrome. Recent widespread endemic transmission of ZIKV and the potential for future outbreaks necessitate the development of an effective vaccine. We developed a ZIKV vaccine candidate based on virus-like-particles (VLPs) generated following transfection of mammalian HEK293T cells using a plasmid encoding the pre-membrane/membrane (prM/M) and envelope (E) structural protein genes. VLPs were collected from cell culture supernatant and purified by column chromatography with yields of approximately 1-2mg/L. To promote increased particle yields, a single amino acid change of phenylalanine to alanine was made in the E fusion loop at position 108 (F108A) of the lead VLP vaccine candidate. This mutation resulted in a modest 2-fold increase in F108A VLP production with no detectable prM processing by furin to a mature particle, in contrast to the lead candidate (parent). To evaluate immunogenicity and efficacy, AG129 mice were immunized with a dose titration of either the immature F108A or lead VLP (each alum adjuvanted). The resulting VLP-specific binding antibody (Ab) levels were comparable. However, geometric mean neutralizing Ab (nAb) titers using a recombinant ZIKV reporter were significantly lower with F108A immunization compared to lead. After virus challenge, all lead VLP-immunized groups showed a significant 3- to 4-Log10 reduction in mean ZIKV RNAemia levels compared with control mice immunized only with alum, but the RNAemia reduction of 0.5 Log10 for F108A groups was statistically similar to the control. Successful viral control by the lead VLP candidate following challenge supports further vaccine development for this candidate. Notably, nAb titer levels in the lead, but not F108A, VLP-immunized mice inversely correlated with RNAemia. Further evaluation of sera by an in vitro Ab-dependent enhancement assay demonstrated that the F108A VLP-induced immune sera had a significantly higher capacity to promote ZIKV infection in FcγR-expressing cells. These data indicate that a single amino acid change in the fusion loop resulted in increased VLP yields but that the immature F108A particles were significantly diminished in their capacity to induce nAbs and provide protection against ZIKV challenge. Author summary: Zika virus (ZIKV) is transmitted by mosquitoes and is a serious health threat due to potential epidemic spread. Infection in adults may lead to Guillain-Barré syndrome, a neurological disorder, or may cause harm to a developing fetus resulting in preterm birth, fetal death, or devastating congenital malformations. There are currently no approved vaccines against ZIKV. We previously developed a lead candidate vaccine based on a virus-like particle (VLP) that was generated in tissue culture. This ZIKV shell is devoid of any viral genetic material. In previous studies, this lead VLP candidate generated neutralizing antibodies (nAbs) that recognized wild-type ZIKV and prevented viral replication in both mice and non-human primates. To increase production of the lead VLP candidate and decrease cost-of-goods, we introduced a single amino acid change, phenylalanine to alanine, in the envelope glycoprotein. This change resulted in a modest increase in VLP yield. However, this single amino acid change resulted in reduced induction of nAbs following immunization and no significant reduction of RNAemia following challenge compared to the lead candidate. The results of this study suggest this investigational vaccine candidate is not suitable for further vaccine development and that ZIKV VLP maturation may have an important role in protection. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms are responsible for a wide range of biological phenomena in insects, controlling embryonic development, growth, aging and nutrition. Despite this, the role of epigenetics in shaping insect-pathogen interactions has received little attention. Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we explored the role of the Aedes aegypti histone acetyltransferase CBP (AaCBP) after infection with Zika virus (ZIKV), focusing on the two main immune tissues, the midgut and fat body. We showed that the expression and activity of AaCBP could be positively modulated by blood meal and ZIKV infection. Nevertheless, Zika-infected mosquitoes that were silenced for AaCBP revealed a significant reduction in the acetylation of H3K27 (CBP target marker), followed by downmodulation of the expression of immune genes, higher titers of ZIKV and lower survival rates. Importantly, in Zika-infected mosquitoes that were treated with sodium butyrate, a histone deacetylase inhibitor, their capacity to fight virus infection was rescued. Our data point to a direct correlation among histone hyperacetylation by AaCBP, upregulation of antimicrobial peptide genes and increased survival of Zika-infected-A. aegypti. Author summary: Pathogens have coevolved with mosquitoes to optimize transmission to hosts. As natural vectors, mosquitoes are permissive to and allow systemic and persistent arbovirus infection, which intriguingly does not result in dramatic pathological sequelae that affect their lifespan. In this regard, mosquitoes have evolved mechanisms to tolerate persistent infection and develop efficient antiviral strategies to restrict viral replication to nonpathogenic levels. There is a great deal of evidence supporting the implication of epigenetics in the modulation of the biological interaction between hosts and pathogens. This study reveals that Zika virus infection positively modulates the expression and activity of A. aegypti histone acetyltransferase CBP (AaCBP). This study shows that AaCBP plays a role in the activation of immune-responsive genes to limit Zika virus replication. This first description that Zika virus infection has epigenomic consequences in the regulation of A. aegypti immunity opens a new avenue for research on mosquito factors that can drive vector competence. [ABSTRACT FROM AUTHOR]

The Zika virus (ZIKV) epidemic, which was followed by an unprecedented outbreak of congenital microcephaly, emerged in Brazil unevenly, with apparent pockets of susceptibility. The present study aimed to detect high-risk areas for ZIKV infection and microcephaly in Goiania, a large city of 1.5 million inhabitants in Central-West Brazil. Using geocoded surveillance data from the Brazilian Information System for Notifiable Diseases (SINAN) and from the Public Health Event Registry (RESP-microcefalia), we analyzed the spatiotemporal distribution and socioeconomic indicators of laboratory confirmed (RT-PCR and/or anti-ZIKV IgM ELISA) symptomatic ZIKV infections among pregnant women and clinically confirmed microcephaly in neonates, from 2016 to 2020. We investigated temporal patterns by estimating the risk of symptomatic maternal ZIKV infections and microcephaly per 1000 live births per month. We examined the spatial distribution of maternal ZIKV infections and microcephaly cases across the 63 subdistricts of Goiania by manually plotting the geographical coordinates. We used spatial scan statistics estimated by discrete Poisson models to detect high clusters of maternal ZIKV infection and microcephaly and compared the distributions by socioeconomic indicators measured at the subdistrict level. In total, 382 lab-confirmed cases of maternal ZIKV infections, and 31 cases of microcephaly were registered in the city of Goiania. More than 90% of maternal cases were reported between 2016 and 2017. The highest incidence of ZIKV cases among pregnant women occurred between February and April 2016. A similar pattern was observed in the following year, although with a lower number of cases, indicating seasonality for ZIKV infection, during the local rainy season. Most congenital microcephaly cases occurred with a time-lag of 6 to 7 months after the peak of maternal ZIKV infection. The highest estimated incidence of maternal ZIKV infections and microcephaly were 39.3 and 2.5 cases per 1000 livebirths, respectively. Districts with better socioeconomic indicators and with higher proportions of self-identified white inhabitants were associated with lower risks of maternal ZIKV infection. Overall, the findings indicate heterogeneity in the spatiotemporal patterns of maternal ZIKV infections and microcephaly, which were correlated with seasonality and included a high-risk geographic cluster. Our findings identified geographically and socio-economically underprivileged groups that would benefit from targeted interventions to reduce exposure to vector-borne infections. Author summary: The first wave of Zika virus (ZIKV) epidemic and its Congenital Zika Syndrome, has vanished. However, the consequences have remained for the affected children and families ever since. In Brazil, the first cases of microcephaly, detected in the end of 2015 in the Northeast region, especially in coastal cities, quickly spread to other regions and cities in countryside of Brazil. Understanding the temporal and spatial dynamics of cases distribution is essential to identify areas of greater risk and enable preparedness for a future wave of cases. In this study, we analyzed the spatiotemporal distribution of cases of ZIKV infection in pregnant women and cases of microcephaly in newborns by district, over a five-year period, in a large city in Midwest Brazil. Additionally, cases of microcephaly were correlated with the socioeconomic and structural conditions at the local level. Our findings indicate heterogeneity in the spatiotemporal patterns of maternal ZIKV infections and microcephaly, which were correlated with seasonality and included a persistent high-risk geographic location (cluster) in the city of Goiania. We could identify geographically and socio-economically underprivileged groups, with higher risk for ZIKV infection, that would benefit from targeted interventions to reduce exposure to new vector borne infections. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) caused a large outbreak in Puerto Rico in 2014, followed by a Zika virus (ZIKV) outbreak in 2016. Communities Organized for the Prevention of Arboviruses (COPA) is a cohort study in southern Puerto Rico, initiated in 2018 to measure arboviral disease risk and provide a platform to evaluate interventions. To identify risk factors for infection, we assessed prevalence of previous CHIKV infection and recent ZIKV and DENV infection in a cross-sectional study among COPA participants. Participants aged 1–50 years (y) were recruited from randomly selected households in study clusters. Each participant completed an interview and provided a blood specimen, which was tested by anti-CHIKV IgG ELISA assay and anti-ZIKV and anti-DENV IgM MAC-ELISA assays. We assessed individual, household, and community factors associated with a positive result for CHIKV or ZIKV after adjusting for confounders. During 2018–2019, 4,090 participants were enrolled; 61% were female and median age was 28y (interquartile range [IQR]: 16–41). Among 4,035 participants tested for CHIKV, 1,268 (31.4%) had evidence of previous infection. CHIKV infection prevalence was lower among children 1–10 years old compared to people 11 and older (adjusted odds ratio [aOR] 2.30; 95% CI 1.71–3.08). Lower CHIKV infection prevalence was associated with home screens (aOR 0.51; 95% CI 0.42–0.61) and air conditioning (aOR 0.64; 95% CI 0.54–0.77). CHIKV infection prevalence also varied by study cluster of residence and insurance type. Few participants (16; 0.4%) had evidence of recent DENV infection by IgM. Among 4,035 participants tested for ZIKV, 651 (16%) had evidence of recent infection. Infection prevalence increased with older age, from 7% among 1–10y olds up to 19% among 41–50y olds (aOR 3.23; 95% CI 2.16–4.84). Males had an increased risk of Zika infection prevalence compared with females (aOR 1.31; 95% CI 1.09–1.57). ZIKV infection prevalence also decreased with the presence of home screens (aOR 0.66; 95% CI 0.54–0.82) and air conditioning (aOR 0.69; 95% CI 0.57–0.84). Similar infection patterns were observed for recent ZIKV infection prevalence and previous CHIKV infection prevalence by age, and the presence of screens and air conditioners in the home decreased infection risk from both viruses by as much as 50%. Author summary: We assessed baseline seroprevalence and risk factors for previous CHIKV or recent ZIKV infection among 4,035 participants in the Communities Organized to Prevent Arboviruses (COPA) study. Participants were recruited among people 1–50 years old living in 38 study clusters in the Ponce municipality of southern Puerto Rico. Among 4,035 participants, 31% had evidence of previous CHIKV infection, and 16% had evidence of recent ZIKV infection. For both viruses, older age was associated with greater risk for infection; male sex was also associated with higher rates of ZIKV infection. Seroprevalence varied by study cluster, with similar patterns between the two viruses. Home screens and air conditioning in the home were significant protective factors against infection in the multivariate analysis for both CHIKV and ZIKV. [ABSTRACT FROM AUTHOR]

Background: Transmission patterns and genetic diversity of dengue virus (DENV) circulating in Africa remains poorly understood. Circulation of the DENV serotype 1 (DENV1) in Angola was detected in 2013, while DENV serotype 2 (DENV2) was detected for the first time in 2018. Here, we report results from molecular and genomic investigations conducted at the Ministry of Health national reference laboratory (INIS) in Angola on suspected dengue cases detected between January 2017 and February 2019. Methods: A total of 401 serum samples from dengue suspected cases were collected in 13 of the 18 provinces in Angola. Of those, 351 samples had complete data for demographic and epidemiological analysis, including age, gender, province, type of residence, clinical symptoms, as well as dates of onset of symptoms and sample collection. RNA was extracted from samples and tested for DENV-RNA by two distinct real time RT-PCR protocols. On-site whole genome nanopore sequencing was performed for PCR+ cases. Bayesian coalescent models were used to estimate date and origin of outbreak emergence, as well as population growth rates. Results: Molecular diagnosis shows that 66 out of 351 (19%) suspected cases were DENV-RNA positive across 5 provinces in Angola. DENV PCR+ cases were diagnosed more frequently in urban sites compared to rural sites. Of the DENV positive samples, most were collected within 6 days of symptom onset. 93% of infections were confirmed by serotype-specific RT-PCR as DENV2 and 1 case (1.4%) was confirmed as DENV1. Six CHIKV RT-PCR positive cases were also detected during the study period, including 1 co-infection with DENV1. Most cases (87%) were detected in Luanda during the rainy season between April and October. Symptoms associated with severe dengue were observed in 11 patients, including 2 with a fatal outcome. On-site nanopore genome sequencing followed by genetic analysis revealed an introduction of DENV2 Cosmopolitan genotype (also known as DENV2-II genotype) possibly from India in or around October 2015, at least 1.5 years before its detection in the country. Coalescent models suggest relatively moderately rapid epidemic growth rates and doubling times, and a moderate expansion of DENV2 in Angola during the studied period. Conclusion: This study describes genomic, epidemiological and demographic characteristic of predominately urban transmission of DENV2 in Angola. We also find co-circulation of DENV2 with DENV1 and CHIKV and report several RT-PCR confirmed severe dengue cases in the country. Increasing dengue awareness in healthcare professional, expanding the monitorization of arboviral epidemics across the country, identifying most common mosquito breeding sites in urban settings, implementing innovative vector control interventions and dengue vaccination campaigns could help to reduce vector presence and DENV transmission in Angola. Author summary: Angola has experienced dengue, yellow fever and Zika virus outbreaks between 2013–2017. To improve the detection of arbovirus outbreaks in Angola, we set up the National Arbovirus Laboratory of Surveillance Programme at the National Institute of Health Research (INIS), Ministry of Health, Angola, which officially kick-started arbovirus surveillance activities in early 2017. In this study we tested samples collected from patients with suspected dengue cases January 2017 to February 2019. We report molecular and genomic findings on DENV2 transmission in Angola. On-site whole genome sequencing and subsequent phylogenetic analysis indicate a single introduction of the DENV2 Cosmopolitan genotype (also known as DENV2-II genotype) from Southern Asia, probably as a result of international travel, followed by moderate virus population growth. In Angola, most detections occurred in urban areas, although we detected DENV2 in 5 of the 18 provinces in Angola. This work emphasizes the importance of maintaining an active arbovirus surveillance programme throughout the country to investigate DENV transmission patterns and the risk of hyperendemicity in Angola. [ABSTRACT FROM AUTHOR]

During 2015–2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS). Notably, the scale and magnitude of outbreaks in Southeast Asia were significantly smaller compared to those in the Americas. Sequence comparison between epidemic-associated ZIKV strains from Southeast Asia with those from the Americas revealed a methionine to valine substitution at residue position 114 of the NS5 protein (NS5-M114V) in all the American isolates. Using an American isolate of ZIKV (Natal), we investigated the impact of NS5-M114V mutation on virus replication in cells, virulence in interferon (IFN) α/β receptor knockout (Ifnar-/-) mice, as well as replication and transmission potential in Aedes aegypti mosquitoes. We demonstrated that NS5-M114V mutation had insignificant effect on ZIKV replication efficiency in cells, its ability to degrade STAT2, and virulence in vivo, albeit viremia was slightly prolonged in mice. Furthermore, NS5-M114V mutation decreased mosquito infection and dissemination rates and had no effect on virus secretion into the saliva. Taken together, our findings support the notion that NS5-M114V mutation is unlikely to be a major determinant for virus replication and transmission potential. Author summary: Zika virus (ZIKV) emerged to cause outbreaks in Southeast Asia and the Americas during 2015–2016. However, the scale of outbreaks in Southeast Asia were significantly smaller compared to epidemic in the Americas. A methionine to valine amino acid mutation at residue position 114 of the NS5 protein (NS5-M114V) is hypothesized to influence the epidemic outcomes of ZIKV, which led to the large-scale epidemic that occurred in the Americas. By analyzing infection of mammalian and mosquito cells, IFNα/β receptor knockout (Ifnar-/-) mice and Aedes aegypti mosquitoes with engineered ZIKV isolates containing either methionine or valine at residue position 114 of the NS5 protein, we demonstrated that the NS5-M114V mutation did not affect virus replication efficiency and STAT2 degradation in cells, virulence in mice, or virus secretion into the mosquito saliva. The NS5-M114V mutation slightly prolonged viremia in Ifnar-/- mice and reduced mosquito infection rate. Collectively, our findings suggest that the NS5-M114V mutation is unlikely to have significantly influenced the ZIKV epidemic in the Americas. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne flavivirus and can cause neurodevelopmental disorders in fetus. As a neurotropic virus, ZIKV persistently infects neural tissues during pregnancy but the viral pathogenesis remains largely unknown. ZIKV has a positive-sense and single-stranded RNA genome, which encodes 7 non-structural (NS) proteins, participating in viral replication and dysregulation of host immunity. Like those in many other viruses, NS proteins are considered to be products evolutionarily beneficiary to viruses and some are virulence factors. However, we found that some NS proteins encoded by ZIKV genome appeared to function against the viral replication. In this report we showed that exogenously expressed ZIKV NS2A and NS4A inhibited ZIKV infection by inhibiting viral RNA replication in microglial cells and astrocytes. To understand how viral NS proteins suppressed viral replication, we analyzed the transcriptome of the microglial cells and astrocytes and found that expression of NS4A induced the upregulation of ISGs, including MX1/2, OAS1/2/3, IFITM1, IFIT1, IFI6, IFI27, ISG15 or BST2 through activating the ISGF3 signaling pathway. Upregulation of these ISGs seemed to be related to the inhibition of ZIKV replication, since the anti-ZIKV function of NS4A was partially attenuated when the cells were treated with Abrocitinib, an inhibitor of the ISGF3 signaling pathway, or were knocked down with STAT2. Aborting the protein expression of NS4A, but not its nucleic acid, eliminated the antiviral activity of NS4A effectively. Dynamic expression of viral NS proteins was examined in ZIKV-infected microglial cells and astrocytes, which showed comparatively NS4A occurred later than other NS proteins during the infection. We hypothesize that NS4A may possess intrinsic features to serve as a unique type of pathogen associated molecular pattern (PAMP), detectable by the cells to induce an innate immune response, or function with other mechanisms, to restrict the viral replication to a certain level as a negative feedback, which may help ZIKV maintain its persistent infection in fetal neural tissues. Author summary: The birth of microcephaly infants due to ZIKV infection in pregnant women is related to ZIKV persistent infection. However, it is unclear how ZIKV maintains its persistent infection. In this work, we observed the delayed appearance of ZIKV NS4A protein in neuroglia including microglia and astrocytes compared with other non-structural proteins. Subsequently, we revealed that ZIKV NS4A inhibited viral RNA replication by activating the ISGF3 signaling pathway and inducing the production of ISGs. Aborting NS4A protein expression totally rescued ZIKV viral replication. Our study, combined with the previous findings, suggests that viral non-structural proteins may regulate viral replication, thus perpetuating ZIKV infection. Our hypothesis provides a mechanism for ZIKV to maintain its status of a persistent infection during viral infection in fetus, which can shed lights on our further understanding of viral neuropathogenesis in ZIKV infection. [ABSTRACT FROM AUTHOR]

A Zika virus (ZIKV) infection during pregnancy can result in severe birth defects such as microcephaly. To date, it is incompletely understood how ZIKV can cross the human placenta. Furthermore, results from studies in pregnant mice and non-human primates are conflicting regarding the role of cross-reactive dengue virus (DENV) antibodies on transplacental ZIKV transmission. Elucidating how ZIKV can cross the placenta and which risk factors contribute to this is important for risk assessment and for potential intervention strategies for transplacental ZIKV transmission. In this study we use an ex vivo human placental perfusion model to study transplacental ZIKV transmission and the effect that cross-reactive DENV antibodies have on this transmission. By using this model, we demonstrate that DENV antibodies significantly increase ZIKV uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Furthermore, we show that cross-reactive DENV antibodies enhance ZIKV infection in term human placental explants and in primary fetal macrophages but not in primary trophoblasts. Our data supports the hypothesis that presence of cross-reactive DENV antibodies could be an important risk factor for transplacental ZIKV transmission. Furthermore, we demonstrate that the ex vivo placental perfusion model is a relevant and animal friendly model to study transplacental pathogen transmission. Author summary: Zika virus is a mosquito-transmitted virus that can cause severe birth defects such as microcephaly when the infection occurs during pregnancy. Understanding how Zika virus crosses the placenta during pregnancy is important for future prevention strategies for vertical Zika virus transmission. Despite significant efforts to study this, to date it remains incompletely understood how Zika virus can cross the placenta and which risk factors contribute to this form of transmission. In this study we use an ex vivo placental perfusion model to study transplacental Zika virus transmission. The ex vivo placental perfusion model is a highly physiological and animal friendly model that mimics the in vivo conditions during pregnancy. We found that antibodies against the closely related dengue virus can significantly enhance placental uptake of Zika virus and Zika virus infection of human placental explants and fetal macrophages. These findings indicate that presence of cross-reactive dengue virus antibodies could contribute to transplacental Zika virus transmission. [ABSTRACT FROM AUTHOR]

Background: While microcephaly is a significant adverse outcome of prenatal exposure to the Zika virus (ZIKV), subtle malformations of cortical development (MCD) have been observed in Zika-exposed children (ZEC), including delays in language, cognition, and motor domains, and visual acuity deficits. Interventions within the first 1,000 days of life can significantly improve developmental outcomes. This study examined a 12-week Responsive Caregiving Intervention on neurodevelopmental outcomes in 24-30-month-old ZEC. Methodology/Principal findings: A randomized controlled trial was implemented in Grenada, West Indies using an existing ZIKV cohort surveillance study. When children in that study turned 24 months, baseline child neurodevelopmental measures and caregiver interviews were administered. Caregivers who agreed to participate in the 12-week Responsive Caregiving Intervention, implemented when children were 24–30 months of age, were randomly assigned to the Intervention or Waitlist Control group. Children in both groups were re-assessed on the neurodevelopmental measures post-intervention. Conclusions/Significance: 233 children from the ZIKV surveillance study met inclusion criteria, of which n = 80 declined participation, n = 42 did not complete the Intervention, and n = 72 missed follow-up assessments given strict timelines in the study design. The final sample for analysis was N = 13 children in the Intervention group and N = 26 children in the Control group. A GEE model analysis showed significantly higher language (p = 0.021) and positive behaviour (p = 0.005) scores for children in the Intervention group compared to the Control group. The Intervention had a medium effect on child language (d = 0.66) and a large effect on positive behaviour (d = 0.83). A 12-week Responsive Caregiving Intervention Programme significantly improves language and positive behaviour scores in 30-month-old normocephalic children who were exposed to ZIKV in utero. The programme provides an option for mothers of ZIKV-exposed children who are seeking an evidence-based neurodevelopmental intervention regardless of known impact of the virus on cortical formation. Trial registration: The study was registered with clinicaltrials.gov (NCT04697147). Author summary: The relationship between Zika virus exposure during pregnancy and microcephaly (abnormally small head size) in infants is well known. Prenatal Zika virus exposure can also cause more subtle impacts in infant language, cognition, and motor function, and visual acuity, even in the absence of microcephaly. Interventions in the first 1,000 days of life can significantly improve these outcomes in at-risk children. Few studies have been carried out with the aim of improving neurodevelopment in Zika-exposed children. The present study utilized a randomized controlled trial to examine the impact of a 12-week Intervention on neurodevelopmental outcomes in 24-30-month-old Zika-exposed children. Analysis showed significantly higher language and positive behaviour scores among children in the Intervention group compared to children in the Control group. The Intervention used in this study is relatively low-cost and easy to implement. The application of interventions from the global early child development field has implications for governments, policy makers, public health officials, and parents of Zika-exposed children who are seeking effective interventions to mitigate the risk of neurodevelopment delays in those children. The fact that these much-needed interventions already exist and can be rolled out relatively quickly and effectively is encouraging. [ABSTRACT FROM AUTHOR]

The tropism of Zika virus (ZIKV) has been described in the nervous system, blood, placenta, thymus, and skeletal muscle. We investigated the mechanisms of skeletal muscle susceptibility to ZIKV using an in vitro model of human skeletal muscle myogenesis, in which myoblasts differentiate into myotubes. Myoblasts were permissive to ZIKV infection, generating productive viral particles, while myotubes controlled ZIKV replication. To investigate the underlying mechanisms, we used gene expression profiling. First, we assessed gene changes in myotubes compared with myoblasts in the model without infection. As expected, we observed an increase in genes and pathways related to the contractile muscle system in the myotubes, a reduction in processes linked to proliferation, migration and cytokine production, among others, confirming the myogenic capacity of our system in vitro. A comparison between non-infected and infected myoblasts revealed more than 500 differentially expressed genes (DEGs). In contrast, infected myotubes showed almost 2,000 DEGs, among which we detected genes and pathways highly or exclusively expressed in myotubes, including those related to antiviral and innate immune responses. Such gene modulation could explain our findings showing that ZIKV also invades myotubes but does not replicate in these differentiated cells. In conclusion, we showed that ZIKV largely (but differentially) disrupts gene expression in human myoblasts and myotubes. Identifying genes involved in myotube resistance can shed light on potential antiviral mechanisms against ZIKV infection. Author summary: The ZIKV was responsible for a large pandemic surge between 2015 and 2016; Brazil was the epicenter with very high numbers of congenital ZIKV syndrome that included microcephaly, myalgia, among other manifestations. We have observed in previous work that muscle progenitor cells (myoblasts) are infected by ZIKV, while differentiated cells (myotubes) do not present viral proteins. In the present study, we confirmed human myoblasts susceptibility to ZIKV infection and myotubes resistance. In fact, we show that myotubes are also susceptible to ZIKV entry but control the productive infection. We also elucidated the transcriptional mechanisms triggered by the ZIKV, which stimulated a more complex intrinsic antiviral response in myotubes than in myoblasts. Such particular gene modulation of the differentiated cells could offer clues to antiviral mechanisms. It would explain susceptibility differences between adults, where muscle progenitor cells are quiescent in the steady-state, and foetuses that have large amounts of these cells proliferating in order to grow muscles. [ABSTRACT FROM AUTHOR]

The tiger mosquito was introduced to the Eastern region of the Mediterranean basin more than twenty years ago. In Lebanon, it was first observed in 2002 in a limited number of locations mainly from the coastal area of the country. In the absence of national entomological control program, this invasive mosquito became an established species and is now considered in many localities, a source of nuisance because of its human biting behavior. Several entomological surveys were conducted to monitor the geographic spread and the seasonal dynamics of Aedes albopictus by collecting adult stages and by monitoring oviposition activity. Moreover, its susceptibility to the common groups of insecticides was assessed using WHO standard bioassays. Previous vector competence studies revealed that local strains were able to transmit Chikungunya and Dengue viruses. Due to the increased risk of Zika virus introduction in the country, we determined the competence of local populations to transmit this virus. Mapping results showed that Ae. albopictus is mainly spread in the relatively humid western versant of the Mount Lebanon chain reaching 1000m altitude, while it is absent from arid and semi-arid inland areas. Besides, this mosquito is active during 32 weeks from spring till the end of autumn. Local strains of the tiger mosquito are susceptible to pyrethroids and carbamates but resistant to organophosphates and organochlorines. They showed ability to transmit Zika virus; however, only 9% of females were capable to excrete the virus in their saliva at day 28 post infection. Current and previous observations highlight the need to establish a surveillance system in order to control this mosquito and monitor the potential introduction of related diseases. Author summary: Aedes albopictus, also called the tiger mosquito, is one the most invasive mosquito species worldwide. It is originated from South-East Asia and islands of the Pacific and Indian oceans and is known to be able to transmit to humans many viral diseases. During the last four decades it succeeded to invade many countries in all continents through an increasing international trade and travel. This mosquito was first observed in Lebanon, in the Middle East region, in 2002. Previous studies revealed that the introduced mosquito was able to transmit Chikungunya and Dengue viruses under experimental conditions. Therefore, it represents a health threat for the Lebanese population. In this study, the authors assessed the status of the tiger mosquito in the country two decades after its first record. They showed that it is widespread in the humid and sub-humid regions of Mount Lebanon chain and is active from spring through late autumn. The authors experimentally demonstrated the ability of this mosquito to transmit Zika virus, a virus that is highly likely to get introduced to Lebanon due to important population flow from South America to the country during the summer season. Finally, the authors found that local populations of tiger mosquito were susceptible to insecticides of the pyrethroids and carbamates groups but resistant to those of the organochlorines and organophosphates groups. The generated information should help national health authorities to establish a targeted surveillance and control strategies for this mosquito. [ABSTRACT FROM AUTHOR]

Understanding the burden and risk factors of dengue virus (DENV) infection in Puerto Rico is important for the prevention of dengue in local, traveler and military populations. Using sera from the Department of Defense Serum Repository, we estimated the prevalence and predictors of DENV seropositivity in those who had served in Puerto Rico, stratified by birth or prior residence ("birth/residence") in dengue-endemic versus non-endemic regions. We selected sera collected in early 2015 from 500 U.S. military members, a time-point also permitting detection of early cryptic Zika virus (ZIKV) circulation. 87.2% were born or resided in a DENV-endemic area before their military service in Puerto Rico. A high-throughput, flow-cytometry-based neutralization assay was employed to screen sera for ZIKV and DENV neutralizing antibodies, and confirmatory testing was done by plaque-reduction neutralization test (PRNT). We identified one Puerto Rico resident who seroconverted to ZIKV by June 2015, suggesting cryptic ZIKV circulation in Puerto Rico at least 4 months before the first reported cases. A further six PRNT-positive presumptive ZIKV infections which were resolved as DENV infections only by the use of paired sera. We noted 66.8% of the total study sample was DENV seropositive by early 2015. Logistic regression analysis indicated that birth/residence in a dengue non-endemic region (before military service in Puerto Rico) was associated with a lower odds of DENV exposure by January—June 2015 (aOR = 0.28, p = 0.001). Among those with birth/residence in a non-endemic country, we noted moderate evidence to support increase in odds of DENV exposure for each year of military service in Puerto Rico (aOR = 1.58, p = 0.06), but no association with age. In those with birth/residence in dengue-endemic regions (before military service in Puerto Rico), we noted that age (aOR = 1.04, p = 0.02), rather than duration of Puerto Rico service, was associated with dengue seropositivity, suggesting earlier lifetime DENV exposure. Our findings provide insights into the burden and predictors of DENV infection in local, traveler and military populations in Puerto Rico. Our study also highlights substantial PRNT ZIKV false-positivity when paired sera are not available, even during periods of very low ZIKV prevalence. Author summary: Understanding the risk and risk factors of dengue and Zika virus (DENV, ZIKV) infection improves their prevention. We leveraged a U.S. Department of Defense sera repository to identify sero-exposures to DENV and ZIKV in 500 adults performing military service in Puerto Rico through early 2015, the majority of who were born or previously resided in Puerto Rico. The mean duration of military service before sera were collected was 5.2 years. Serological results indicated ZIKV may have been circulating in Puerto Rico for at least 4 months before the first cases were detected in this territory. We noted ZIKV false-positivity to reference serological methods when paired sera are not available. 66.8% of the total study sample was DENV seropositive by 2015; at least 21.8% apparently had a prior single dengue infection. Among those with birth/residence in a non-dengue-endemic country, we noted an increase in the odds of DENV exposure for each year of military service in Puerto Rico. In those with birth/residence in dengue-endemic regions, we noted that age, rather than duration of Puerto Rico service, was associated with dengue seropositivity, suggesting earlier lifetime DENV exposure. Our findings provide insights into the risk and risk factors of these two important arboviruses. [ABSTRACT FROM AUTHOR]

The current outbreak of Zika virus poses a severe threat to human health. While the range of the virus has been cataloged growing slowly over the last 50 years, the recent explosive expansion in the Americas indicates that the full potential distribution of Zika remains uncertain. Moreover, many studies rely on its similarity to dengue fever, a phylogenetically closely related disease of unknown ecological comparability. Here we compile a comprehensive spatially-explicit occurrence dataset from Zika viral surveillance and serological surveys based in its native range, and construct ecological niche models to test basic hypotheses about its spread and potential establishment. The hypothesis that the outbreak of cases in Mexico and North America are anomalous and outside the native ecological niche of the disease, and may be linked to either genetic shifts between strains, or El Nino or similar climatic events, remains plausible at this time. Comparison of the Zika niche against the known distribution of dengue fever suggests that Zika is more constrained by the seasonality of precipitation and diurnal temperature fluctuations, likely confining autochthonous non-sexual transmission to the tropics without significant evolutionary change. Projecting the range of the diseases in conjunction with three major vector species (Aedes africanus, Ae. aegypti, and Ae. albopictus) that transmit the pathogens, under climate change, suggests that Zika has potential for northward expansion; but, based on current knowledge, our models indicate Zika is unlikely to fill the full range its vectors occupy, and public fear of a vector-borne Zika epidemic in the mainland United States is potentially informed by biased or limited scientific knowledge. With recent sexual transmission of the virus globally, we caution that our results only apply to the vector-borne transmission route of the pathogen, and while the threat of a mosquito-carried Zika pandemic may be overstated in the media, other transmission modes of the virus may emerge and facilitate naturalization worldwide.

BackgroundIn early 2015, a ZIKA Virus (ZIKV) infection outbreak was recognized in northeast Brazil, where concerns over its possible links with infant microcephaly have been discussed. Providing a causal link between ZIKV infection and birth defects is still a challenge. MicroRNAs (miRNAs) are small noncoding RNAs (sncRNAs) that regulate post-transcriptional gene expression by translational repression, and play important roles in viral pathogenesis and brain development. The potential for flavivirus-mediated miRNA signalling dysfunction in brain-tissue development provides a compelling hypothesis to test the perceived link between ZIKV and microcephaly.Methodology/principal findingsHere, we applied in silico analyses to provide novel insights to understand how Congenital ZIKA Syndrome symptoms may be related to an imbalance in miRNAs function. Moreover, following World Health Organization (WHO) recommendations, we have assembled a database to help target investigations of the possible relationship between ZIKV symptoms and miRNA-mediated human gene expression.Conclusions/significanceWe have computationally predicted both miRNAs encoded by ZIKV able to target genes in the human genome and cellular (human) miRNAs capable of interacting with ZIKV genomes. Our results represent a step forward in the ZIKV studies, providing new insights to support research in this field and identify potential targets for therapy.

Background: With the arrival of chikungunya (CHIKV) and zika (ZIKV) viruses in Mexico, there was a decrease in diagnosed dengue virus (DENV) cases. During the first years of cocirculation (2015–2017), the algorithms established by epidemiological surveillance systems and the installed capacity limited us to one diagnostic test per sample, so there was an underestimation of cases until September 2017, when a multiplex algorithm was implemented. Therefore, the objective of this study was determine the impact of the introduction of CHIKV and ZIKV on the incidence of diagnosed DENV in endemic areas of Mexico, when performing the rediagnosis, using the multiplex algorithm, in samples from the first three years of co-circulation of these arboviruses. Methodology and principal findings: For this, 1038 samples received by the Central Laboratory of Epidemiology between 2015 and 2017 were selected for this work. Viruses were identified by multiplex RT-qPCR, and the χ2 test was used to compare categorical variables. With the new multiplex algorithm, we identified 2.4 times the rate of arbovirosis as originally reported, evidencing an underestimation of the incidence of the three viruses. Even so, significantly less dengue was observed than in previous years. The high incidence rates of chikungunya and Zika coincided with periods of dengue decline. The endemic channel showed that the cases caused by DENV rose again after the circulation of CHIKV and ZIKV decreased. In addition, 23 cases of coinfection were identified, with combinations between all viruses. Conclusions and significance: The results obtained in this study show for the first time the real impact on the detected incidence of dengue after the introduction of CHIKV and ZIKV in Mexico, the degree of underestimation of these arboviruses in the country, as well as the co-infections between these viruses, whose importance clinical and epidemiological are still unknown. Author summary: Mexico is considered an endemic country for DENV as the number of cases each year is high. With the arrival of the CHIKV and ZIKV, this number decreased considerably. This led us to suspect that this decrease was not coincidental and could be attributed to two factors: the lack of expertise on the part of health teams to identify the clinical etiological agent and/or other complex ecological factors. Therefore, we decided to reanalyze 1038 suspected cases of dengue, chikungunya and Zika samples using a test that could identify any of the three viruses in a single reaction. We discovered that there was indeed an underestimation of cases, mostly of chikungunya and Zika but, contrary to what we imagined, a lower degree of dengue. We were also able to detect cases of coinfection, which had never been reported in Mexico. Thus, the present study lays the foundations for a better preparation for the arrival of other arboviruses, showing the need for the development and implementation of multiplex diagnostic techniques, in addition to providing more precise epidemiological information on this historical moment of co-circulation of these three arboviruses in our country. [ABSTRACT FROM AUTHOR]

Aedes aegypti is the primary vector of Zika virus (ZIKV), a flavivirus which typically presents itself as febrile-like symptoms in humans but can also cause neurological and pregnancy complications. The transmission cycle of mosquito-borne arboviruses such as ZIKV requires that various key tissues in the female mosquito including the salivary glands get productively infected with the virus before the mosquito can transmit the virus to another vertebrate host. Following ingestion of a viremic blood-meal from a vertebrate, ZIKV initially infects the midgut epithelium before exiting the midgut after blood-meal digestion to disseminate to secondary tissues including the salivary glands. Here we investigated whether smaller Ae. aegypti females resulting from food deprivation as larvae exhibited an altered vector competence for blood-meal acquired ZIKV relative to larger mosquitoes. Midguts from small 'Starve' and large 'Control' Ae. aegypti were dissected to visualize by transmission electron microscopy (TEM) the midgut basal lamina (BL) as physical evidence for the midgut escape barrier showing Starve mosquitoes with a significantly thinner midgut BL than Control mosquitoes at two timepoints. ZIKV replication was inhibited in Starve mosquitoes following intrathoracic injection of virus, however, Starve mosquitoes exhibited a significantly higher midgut escape and population dissemination rate at 9 days post-infection (dpi) via blood-meal, with more virus present in saliva and head tissue than Control by 10 dpi and 14 dpi, respectively. These results indicate that Ae. aegypti developing under stressful conditions potentially exhibit higher midgut infection and dissemination rates for ZIKV as adults, Thus, variation in food intake as larvae is potentially a source for variable vector competence levels of the emerged adults for the virus. Author summary: When mosquitoes are reared in a laboratory they are typically provided with ample nutrients as larvae so adults can grow to an optimal size; this ensures adults are robust for reproducible experiments. However, in the field not all larvae may have access to equal amounts of food. Studies including ours have shown that by restricting food as larvae, smaller adults can be produced, which can have an altered ability to be infected with and transmit arthropod-borne viruses. Zika virus is ingested into a female mosquito midgut when a blood-meal is acquired from an infected vertebrate host; the virus must infect midgut cells and escape this tissue to secondary tissues via the basal lamina, which surrounds the midgut. Viruses can then infect other organs including the salivary glands, for further transmission. In this study we focus on the impact limited nutrition as a larva has on the adult's transmission potential for Zika virus. [ABSTRACT FROM AUTHOR]

Background: Biological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC. Methods/principal findings: The NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately. Conclusions/significance: Our results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC. Author summary: Dengue represents a risk for almost half of the world's population, especially throughout the tropics. In New Caledonia, dengue outbreaks have become more frequent in the past decade along with the recent circulation of chikungunya and Zika viruses. The opportunity to use the biocontrol method involving the release of Wolbachia-infected Ae. aegypti mosquitoes has been investigated as an alternative solution to the traditional control methods, like elimination of larval habitats and pyrethroid insecticide application to kill adults, which are becoming insufficient. A local strain of Ae. aegypti carrying Wolbachia (NC-wMel) has been generated and tested to evaluate its pathogen blocking capacity for the four dengue virus serotypes as well as chikungunya and Zika viruses. The fitness of NC-wMel strain has also been assessed to estimate its ability to compete with the wild-type strain in the field. Noumea city, where a third of the population of New Caledonia resides, has been chosen as the first site to implement the method in New Caledonia. As Ae. aegypti is the only proven vector in New Caledonia, we expect a significant impact on dengue outbreaks occurring in Noumea as soon as a high frequency of NC-wMel is established in the population. [ABSTRACT FROM AUTHOR]