Development of a competition assay to assess the in vitro fitness of dengue virus serotypes using an optimized serotype-specific qRT-PCR.

Background: Comparing the in vitro fitness of dengue virus (DENV) isolates is a pivotal approach to assess the contribution of DENV strains' replicative fitness to epidemiological contexts, including serotype replacements. Competition assays are the gold standard to compare the in vitro replicative fitness of viral strains. Implementing competition assays between DENV serotypes requires an experimental setup and an appropriate read-out to quantify the viral progeny of strains belonging to different serotypes. Results: In the current study, we optimized an existing serotyping qRT-PCR by adapting primer/probe design and multiplexing the serotype-specific qRT-PCR reactions, allowing to accurately detect and quantify all four DENV serotypes. The qRT-PCR was specific, had a limit of detection of at least 5.08×101, 5.16×101, 7.14×101 and 1.36 ×101 genome copies/µL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R2) of 0.99975, 0.99975, 0.9985, 0.99965 for DENV-1, -2, -3 and -4 respectively. Challenge of this multiplex serotype-specific qRT-PCR on mixes of viral supernatants containing known concentrations of strains from two serotypes evidenced an accurate quantification of the amount of genome copies of each serotype. We next developed an in vitro assay to compare the replicative fitness of two DENV serotypes in the human hepatic cell line HuH7: quantification of the viral progeny of each serotype in the inoculum and the supernatant using the serotype-specific multiplex qRT-PCR unveiled an enrichment of the supernatant in DENV-1 genome copies, uncovering the enhanced replicative fitness of this DENV-1 isolate. Conclusions: This optimized qRT-PCR combined to a relevant cellular model allowed to accurately quantify the viral progeny of two DENV strains belonging to two different serotypes in a competition assay, allowing to determine which strain had a replicative advantage. This reliable experimental setup is adaptable to the comparative study of the replicative fitness of any DENV serotypes.

Levels and functionality of Pacific Islanders' hybrid humoral immune response to BNT162b2 vaccination and delta/omicron infection: A cohort study in New Caledonia.

BACKGROUND: Pacific Islanders are underrepresented in vaccine efficacy trials. Few studies describe their immune response to COVID-19 vaccination. Yet, this characterization is crucial to re-enforce vaccination strategies adapted to Pacific Islanders singularities. METHODS AND FINDINGS: We evaluated the humoral immune response of 585 adults, self-declaring as Melanesians, Europeans, Polynesians, or belonging to other communities, to the Pfizer BNT162b2 vaccine. Anti-spike and anti-nucleoprotein IgG levels, and their capacity to neutralize SARS-CoV-2 variants and to mediate antibody-dependent cellular cytotoxicity (ADCC) were assessed across communities at 1 and 3 months post-second dose or 1 and 6 months post-third dose. All sera tested contained anti-spike antibodies and 61.3% contained anti-nucleoprotein antibodies, evidencing mostly a hybrid immunity resulting from vaccination and SARS-CoV-2 infection. At 1-month post-immunization, the 4 ethnic communities exhibited no significant differences in their anti-spike IgG levels (p value = 0.17, in an univariate linear regression model), in their capacity to mediate omicron neutralization (p value = 0.59 and 0.60, in an univariate logistic regression model at 1-month after the second and third dose, respectively) and in their capacity to mediate ADCC (p value = 0.069 in a multivariate linear regression model), regardless of the infection status. Anti-spike IgG levels and functionalities of the hybrid humoral immune response remained equivalent across the 4 ethnic communities during follow-up and at 6 months post-third dose. CONCLUSIONS: Our study evidenced Pacific Islander's robust humoral immune response to Pfizer BNT162b2 vaccine, which is pivotal to re-enforce vaccination deployment in a population at risk for severe COVID-19. TRIAL REGISTRATION: This trial has been register in ClinicalTrials.gov (ID: NCT05135585).

Liver involvement in dengue: A systematic review.

Liver involvement is an unusual yet frequently overlooked dengue complication. Pivotal for an efficient clinical management, the early diagnosis of dengue-associated liver involvement relies on an accurate description of its clinical and biological characteristics, its prognosis factors, its association with severe dengue and its clinical management. We conducted a systematic review by searching PubMed and Web of Science databases for original case reports, cohort and cross-sectional studies reporting the clinical and/or biological features of dengue-associated liver involvement. The study was registered in PROSPERO (CRD42021262657). Of the 2552 articles identified, 167 were included. Dengue-associated liver involvement was characterised by clinical features including abdominal pain, hepatomegaly, jaundice, nausea/vomiting, and an echogenic liver exhibiting hepatocellular necrosis and minimal inflammation. Elevated Aspartate Aminotransferase and Alanine Aminotransferase but also elevated bilirubin, Alkaline Phosphatase, gamma-glutamyl transferase, increased International Normalised Ratio, creatinine and creatine kinase, lower albumin and prolonged prothrombin and activated partial thromboplastin time were prevalent in dengue-associated liver involvement. Cardiovascular and haematological systems were frequently affected, translating in a strong association with severe dengue. Liver involvement was more common in males and older adults. It was associated with dengue virus serotype-2 and secondary infections. Early paracetamol intake increased the risk of liver involvement, which clinical management was mostly conservative. In conclusion, this systematic review demonstrates that early monitoring of transaminases, clinical assessment, and ultrasound examination allow an efficient diagnosis of dengue-associated liver involvement, enabling the early identification and management of severe dengue.

A seroprevalence study of SARS-CoV-2 and seasonal coronaviruses after the first SARS-CoV-2 circulation in New Caledonia, Pacific region.

Objectives: This study aimed to determine the seroprevalence of immunoglobulin G antibodies targeting SARS-CoV-2 and other human coronaviruses after the first circulation of SARS-CoV-2 in New Caledonia, Pacific region. Methods: Blood samples were collected to detect the presence of SARS-CoV-2 immunoglobulin G antibodies. The sampling took place between July 2021 and July 2022 but was interrupted after the first circulation of SARS-CoV-2 (September 2021-March 2022) in New Caledonia. Data on ethnicity, age, gender, main residence, and anteriority of COVID-19 and vaccination were collected and analyzed. Results: A total of 747 participants, representative of New Caledonia's adult population, were included in the study. We found that 81% of the population had antibody responses to SARS-CoV-2 at the end of July 2022. The vaccination rate was 75%, whereas infections had affected 40% of the population. Individuals aged >45 years were significantly more vaccinated than those aged 18-44 years (80%, 95% confidence interval 74-84%). Oceanians were the most infected (50%, 95% confidence interval 42-57%). Conclusion: In New Caledonia, we show a high immunity rate (81%) after the first waves of SARS-CoV-2 circulation and the vaccination campaign. The analyses showed spatial heterogeneities in the infection rate across the territory and revealed that Oceanians were the most infected. Our study also highlighted high exposure of New Caledonia's population to other human coronaviruses.

Temperature and transmission of chikungunya, dengue, and Zika viruses: A systematic review of experimental studies on Aedes aegypti and Aedes albopictus.

Mosquito-borne viruses are leading causes of morbidity and mortality in many parts of the world. In recent years, modelling studies have shown that climate change strongly influences vector-borne disease transmission, particularly rising temperatures. As a result, the risk of epidemics has increased, posing a significant public health risk. This review aims to summarize all published laboratory experimental studies carried out over the years to determine the impact of temperature on the transmission of arboviruses by the mosquito vector. Given their high public health importance, we focus on dengue, chikungunya, and Zika viruses, which are transmitted by the mosquitoes Aedes aegypti and Aedes albopictus. Following PRISMA guidelines, 34 papers were included in this systematic review. Most studies found that increasing temperatures result in higher rates of infection, dissemination, and transmission of these viruses in mosquitoes, although several studies had differing findings. Overall, the studies reviewed here suggest that rising temperatures due to climate change would alter the vector competence of mosquitoes to increase epidemic risk, but that some critical research gaps remain.

Evaluating the strategies to control SARS-CoV-2 Delta variant spread in New Caledonia, a zero-COVID country until September 2021.

Objectives: New Caledonia, a former zero-COVID country, was confronted with a SARS-CoV-2 Delta variant outbreak in September 2021. We evaluate the relative contribution of vaccination, lockdown, and timing of interventions on healthcare burden. Methods: We developed an age-stratified mathematical model of SARS-CoV-2 transmission and vaccination calibrated for New Caledonia and evaluated three alternative scenarios. Results: High virus transmission early on was estimated, with R0 equal to 6.6 (95% confidence interval [6.4-6.7]). Lockdown reduced R0 by 73% (95% confidence interval [70-76%]). Easing the lockdown increased transmission (39% reduction of the initial R0); but we did not observe an epidemic rebound. This contrasts with the rebound in hospital admissions (+116% total hospital admissions) that would have been expected in the absence of an intensified vaccination campaign (76,220 people or 34% of the eligible population were first-dose vaccinated during 1 month of lockdown). A 15-day earlier lockdown would have led to a significant reduction in the magnitude of the epidemic (-53% total hospital admissions). Conclusion: The success of the response against the Delta variant epidemic in New Caledonia was due to an effective lockdown that provided additional time for people to vaccinate. Earlier lockdown would have greatly mitigated the magnitude of the epidemic.

Assessment of the Risk of Exotic Zika Virus Strain Transmission by Aedes aegypti and Culex quinquefasciatus from Senegal Compared to a Native Strain.

Zika virus (ZIKV) shows an enigmatic epidemiological profile in Africa. Despite its frequent detection in mosquitoes, few human cases have been reported. This could be due to the low infectious potential or low virulence of African ZIKV lineages. This study sought to assess the susceptibility of A. aegypti and C. quinquefasciatus to ZIKV strains from Senegal, Brazil, and New Caledonia. Vertical transmission was also investigated. Whole bodies, legs/wings and saliva samples were tested for ZIKV by real-time PCR to estimate infection, dissemination and transmission rates as well as the infection rate in the progeny of infected female A. aegypti. For A. aegypti, the Senegalese strain showed at 15 days post-exposure (dpe) a significantly higher infection rate (52.43%) than the Brazilian (10%) and New Caledonian (0%) strains. The Brazilian and Senegalese strains were disseminated but not detected in saliva. No A. aegypti offspring from females infected with Senegalese and Brazilian ZIKV strains tested positive. No infection was recorded for C. quinquefasciatus. We observed the incompetence of Senegalese A. aegypti to transmit ZIKV and the C. quinquefasciatus were completely refractory. The effect of freezing ZIKV had no significant impact on the vector competence of Aedes aegypti from Senegal, and vertical transmission was not reported in this study.

MALDI-TOF MS: An effective tool for a global surveillance of dengue vector species.

Dengue, Zika and chikungunya viruses cause significant human public health burdens in the world. These arboviruses are transmitted by vector mosquito species notably Aedes aegypti and Aedes albopictus. In the Pacific region, more vector species of arboviruses belonging to the Scutellaris Group are present. Due to the expansion of human travel and international trade, the threat of their dispersal in other world regions is on the rise. Strengthening of entomological surveillance ensuring rapid detection of introduced vector species is therefore required in order to avoid their establishment and the risk of arbovirus outbreaks. This surveillance relies on accurate species identification. The aim of this study was to assess the use of the Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) as a tool for an international identification and surveillance of these mosquito vectors of arboviruses. Field-mosquitoes belonging to 8 species (Ae. aegypti, Ae. albopictus, Aedes polynesiensis, Aedes scutellaris, Aedes pseudoscutellaris, Aedes malayensis, Aedes futunae and Culex quinquefasciatus) from 6 countries in the Pacific, Asian and Madagascar, were included in this study. Analysis provided evidence that a MALDI-TOF database created using mosquitoes from the Pacific region allowed suitable identification of mosquito species from the other regions. This technic was as efficient as the DNA sequencing method in identifying mosquito species. Indeed, with the exception of two Ae. pseudoscutellaris, an exact species identification was obtained for all individual mosquitoes. These findings highlight that the MALDI-TOF MS is a promising tool that could be used for a global comprehensive arbovirus vector surveillance.

Zika vector competence data reveals risks of outbreaks: the contribution of the European ZIKAlliance project.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika.

Modeling present and future climate risk of dengue outbreak, a case study in New Caledonia.

BACKGROUND: Dengue dynamics result from the complex interactions between the virus, the host and the vector, all being under the influence of the environment. Several studies explored the link between weather and dengue dynamics and some investigated the impact of climate change on these dynamics. Most attempted to predict incidence rate at a country scale or assess the environmental suitability at a global or regional scale. Here, we propose a new approach which consists in modeling the risk of dengue outbreak at a local scale according to climate conditions and study the evolution of this risk taking climate change into account. We apply this approach in New Caledonia, where high quality data are available. METHODS: We used a statistical estimation of the effective reproduction number (Rt) based on case counts to create a categorical target variable : epidemic week/non-epidemic week. A machine learning classifier has been trained using relevant climate indicators in order to estimate the probability for a week to be epidemic under current climate data and this probability was then estimated under climate change scenarios. RESULTS: Weekly probability of dengue outbreak was best predicted with the number of days when maximal temperature exceeded 30.8°C and the mean of daily precipitation over 80 and 60 days prior to the predicted week respectively. According to scenario RCP8.5, climate will allow dengue outbreak every year in New Caledonia if the epidemiological and entomological contexts remain the same. CONCLUSION: We identified locally relevant climatic factor driving dengue outbreaks in New Caledonia and assessed the inter-annual and seasonal risk of dengue outbreak under different climate change scenarios up to the year 2100. We introduced a new modeling approach to estimate the risk of dengue outbreak depending on climate conditions. This approach is easily reproducible in other countries provided that reliable epidemiological and climate data are available.

Wolbachia detection in Aedes aegypti using MALDI-TOF MS coupled to artificial intelligence.

The mosquito Aedes aegypti is the major vector of arboviruses like dengue, Zika and chikungunya viruses. Attempts to reduce arboviruses emergence focusing on Ae. aegypti control has proven challenging due to the increase of insecticide resistances. An emerging strategy which consists of releasing Ae. aegypti artificially infected with Wolbachia in natural mosquito populations is currently being developed. The monitoring of Wolbachia-positive Ae. aegypti in the field is performed in order to ensure the program effectiveness. Here, the reliability of the Matrix­Assisted Laser Desorption Ionization­Time Of Flight (MALDI­TOF) coupled with the machine learning methods like Convolutional Neural Network (CNN) to detect Wolbachia in field Ae. aegypti was assessed for the first time. For this purpose, laboratory reared and field Ae. aegypti were analyzed. The results showed that the CNN recognized Ae. aegypti spectral patterns associated with Wolbachia-infection. The MALDI-TOF coupled with the CNN (sensitivity = 93%, specificity = 99%, accuracy = 97%) was more efficient than the loop-mediated isothermal amplification (LAMP), and as efficient as qPCR for Wolbachia detection. It therefore represents an interesting method to evaluate the prevalence of Wolbachia in field Ae. aegypti mosquitoes.

Assessment of fitness and vector competence of a New Caledonia wMel Aedes aegypti strain before field-release.

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.

Non-Invasive versus Invasive Samples for Zika Virus Surveillance: A Comparative Study in New Caledonia and French Guiana in 2015-2016.

Zika virus, an arbovirus responsible for major outbreaks, can cause serious health issues, such as neurological diseases. In the present study, different types of samples (serum, saliva, and urine), collected in 2015-2016 in New Caledonia and French Guiana from 53 patients presenting symptoms and clinical signs triggered by arbovirus infections, were analyzed using a recently developed, and in-house validated, 4-plex RT-qPCR TaqMan method for simultaneous detection and discrimination of the Zika and Chikungunya viruses. Subsequently, statistical analyses were performed in order to potentially establish recommendations regarding the choice of samples type to use for an efficient and early stage Zika infection diagnosis. On this basis, the use of only urine samples presented the highest probability to detect viral RNA from Zika virus. Moreover, such a probability was improved using both urine and saliva samples. Consequently, the added value of non-invasive samples, associated with a higher acceptance level for collection among patients, instead of serum samples, for the detection of Zika infections was illustrated.

Replication Variance of African and Asian Lineage Zika Virus Strains in Different Cell Lines, Mosquitoes and Mice.

Since the epidemic in 2007, studies on vector competence for Zika virus (ZIKV) have intensified, showing that the transmission efficiency varies depending on the vector population, ZIKV strain, and dose of the infectious blood meal. In this study, we aimed to investigate the replication of African and Asian ZIKV strains in vitro and in vivo in order to reveal their phenotypic differences. In addition, we investigated the vector competence of Cambodian Aedes aegypti (Ae. aegypti) mosquitoes (urban and rural) for these ZIKV strains. We observed a significantly higher pathogenicity of the African ZIKV strain in vitro (in mosquito and mammalian cells), and in vivo in both Ae. aegypti and mice. Both mosquito populations were competent to transmit ZIKV as early as 7 days p.i., depending on the population and the ZIKV strain. Ae. aegypti from rural habitats showed significant higher transmission and survival rates than those from urban. We observed the highest transmission efficiency for the African ZIKV isolate (93.3% 14 days p.i.) and for the Cambodian ZIKV isolate (80% 14 days p.i.). Overall, our results highlight the phenotypic differences of the ZIKV lineages and the potential risk of ZIKV transmission by Ae. aegypti mosquitoes. Further investigations of Cambodian mosquito species and ZIKV specific surveillance in humans is necessary in order to improve the local risk assessment.

Potential role of vector-mediated natural selection in dengue virus genotype/lineage replacements in two epidemiologically contrasted settings.

Dengue virus (DENV) evolutionary dynamics are characterized by frequent DENV genotype/lineage replacements, potentially associated with changes in disease severity and human immunity. New Caledonia (NC) and Cambodia, two contrasted epidemiological settings, respectively experienced a DENV-1 genotype IV to I replacement in 2012 and a DENV-1 genotype I lineage 3-4 replacement in 2005-2007, both followed by a massive dengue outbreak. However, their underlying evolutionary drivers have not been elucidated. Here, we tested the hypothesis that these genotype/lineage switches reflected a higher transmission fitness of the replacing DENV genotype/lineage in the mosquito vector using in vivo competition experiments. For this purpose, field-derived Aedes aegypti from NC and Cambodia were orally challenged with epidemiologically relevant pairs of four DENV-1 genotype I and IV strains from NC or four DENV-1 genotype I lineage 3 and 4 strains from Cambodia, respectively. The relative transmission fitness of each DENV-1 genotype/lineage was measured by quantitative RT-PCR for infection, dissemination, and transmission rates. Results showed a clear transmission fitness advantage of the replacing DENV-1 genotype I from NC within the vector. A similar but more subtle pattern was observed for the DENV-1 lineage 4 replacement in Cambodia. Our results support the hypothesis that vector-driven selection contributed to the DENV-1 genotype/lineage replacements in these two contrasted epidemiological settings, and reinforce the idea that natural selection taking place within the mosquito vector plays an important role in DENV short-term evolutionary dynamics.

Development of a bedside score to predict dengue severity.

BACKGROUND: In 2017, New Caledonia experienced an outbreak of severe dengue causing high hospital burden (4379 cases, 416 hospital admissions, 15 deaths). We decided to build a local operational model predictive of dengue severity, which was needed to ease the healthcare circuit. METHODS: We retrospectively analyzed clinical and biological parameters associated with severe dengue in the cohort of patients hospitalized at the Territorial Hospital between January and July 2017 with confirmed dengue, in order to elaborate a comprehensive patient's score. Patients were compared in univariate and multivariate analyses. Predictive models for severity were built using a descending step-wise method. RESULTS: Out of 383 included patients, 130 (34%) developed severe dengue and 13 (3.4%) died. Major risk factors identified in univariate analysis were: age, comorbidities, presence of at least one alert sign, platelets count < 30 × 109/L, prothrombin time < 60%, AST and/or ALT > 10 N, and previous dengue infection. Severity was not influenced by the infecting dengue serotype nor by previous Zika infection. Two models to predict dengue severity were built according to sex. Best models for females and males had respectively a median Area Under the Curve = 0.80 and 0.88, a sensitivity = 84.5 and 84.5%, a specificity = 78.6 and 95.5%, a positive predictive value = 63.3 and 92.9%, a negative predictive value = 92.8 and 91.3%. Models were secondarily validated on 130 patients hospitalized for dengue in 2018. CONCLUSION: We built robust and efficient models to calculate a bedside score able to predict dengue severity in our setting. We propose the spreadsheet for dengue severity score calculations to health practitioners facing dengue outbreaks of enhanced severity in order to improve patients' medical management and hospitalization flow.

Viral evolution sustains a dengue outbreak of enhanced severity.

Compared to the previous 2013-2014 outbreak, dengue 2016-2017 outbreak in New Caledonia was characterized by an increased number of severe forms associated with hepatic presentations. In this study, we assessed the virological factors associated with this enhanced severity. Whole-genome sequences were retrieved from dengue virus (DENV)-1 strains collected in 2013-2014 and from severe and non-severe patients in 2016-2017. Fitness, hepatic tropism and cytopathogenicity of DENV 2016-2017 strains were compared to those of 2013-2014 strains using replication kinetics in the human hepatic cell line HuH7. Whole-genome sequencing identified four amino acid substitutions specific to 2016-2017 strains and absent from 2013-2014 strains. Three of these mutations occurred in predicted T cell epitopes, among which one was also a B cell epitope. Strains retrieved from severe forms did not exhibit specific genetic features. DENV strains from 2016-2017 exhibited a trend towards reduced replicative fitness and cytopathogenicity in vitro compared to strains from 2013-2014. Overall, the 2016-2017 dengue outbreak in New Caledonia was associated with a viral genetic evolution which had limited impact on DENV hepatic tropism and cytopathogenicity. These mutations, however, may have modified DENV strains antigenicity, altering the anti-DENV immune response in some patients, in turn favoring the development of severe forms.Trial registration: ClinicalTrials.gov identifier: NCT04615364.

First probable case of congenital Zika syndrome in Lao People's Democratic Republic.

Zika virus (ZIKV) is a Flavivirus transmitted by Aedes mosquitoes, and was responsible for a worldwide outbreak between 2013 and 2016. However, no ZIKV outbreak has been described in Southeast Asia since 2017. In this study, we report the first microcephaly case with probable ZIKV infection during pregnancy in Lao People's Democratic Republic.