In 2024-2025, Réunion Island experienced a large outbreak driven by East-Central-South African (ECSA)-2 lineage of chikungunya virus (CHIKV), leading to the implementation of a vaccination campaign using live-attenuated IXCHIQ (VLA1553). Currently, distinguishing vaccine-derived viremia from wild-type infection relies on genomic sequencing, which is resource-intensive and not routinely available in many diagnostic laboratories. To address this gap, we developed a duplex real-time RT-qPCR assay specifically designed to differentiate VLA1553 from wild-type ECSA strains circulating in La Réunion. The assay targets the non-structural protein 3 hypervariable region, including the 61-amino-acid deletion characteristic of the VLA1553 backbone. Analytical performance was assessed using a panel of CHIKV isolates representing different lineages and a VLA1553-like strain generated using an Infectious Subgenomic Amplicon system. Clinical performance was evaluated using samples from recently vaccinated individuals and patients infected with ECSA-2 strains, and compared with two RT-qPCR assays routinely used by the French National Reference Centers for arboviruses. The duplex assay proved to be specific, detecting wild-type ECSA strains exclusively in the FAM channel and the vaccine strain exclusively in the HEX channel, with no cross-reactivity. It demonstrated high sensitivity, with LOD95 values of 0.9 and 1.1 copies/µL for the vaccine-specific and wild-type-specific targets, respectively. All clinical samples were correctly classified, including one vaccinated individual simultaneously infected with a circulating wild-type strain, as confirmed by sequencing. This duplex assay provides a rapid and reliable method to distinguish vaccine-derived from naturally-acquired CHIKV viremia, supporting more accurate clinical and epidemiological investigations in settings where vaccination and viral circulation overlap.

Abstract Dengue virus (DENV) transmission in Bolivia has historically been restricted to tropical lowland regions, where the primary vector Ae. aegypti is endemic. In recent years, however, the vector has expanded into high-altitude areas. In 2024, Cochabamba, located at ~ 2,550 m above sea level, experienced its largest recorded dengue outbreak. We analyzed data from 9,576 suspected dengue cases reported between January and July 2024, of which 5,923 were laboratory-confirmed. A subset of DENV-positive samples underwent whole-genome sequencing and phylogenetic analysis. Dengue cases were detected in municipalities located at elevations up to 2,719 m, with the highest number reported in Cochabamba city (4,104/5,923, 69.3%; 2,558 m) and the highest incidence observed in Capinota (839 cases per 100,000 population; 2,386 m). Molecular analyses indicated predominant circulation of DENV-2 genotype II clade F.1.1.2. Phylogenetic reconstruction identified two distinct clades closely related to viruses previously detected in southern Brazil, suggesting multiple independent introductions into the department in late 2023. However, the limited availability of genomic data from Bolivia and neighboring countries limits precise identification of the outbreak’s origin. This study provides the first characterization of the 2024 dengue outbreak in Cochabamba, an exceptional event of substantial DENV circulation at high altitude, likely facilitated by the expanding geographic range of Ae. aegypti, potentially driven by climate change.

We report 2 cases of donor-derived West Nile virus infection in kidney transplant recipients in France. Both recipients had mild disease develop and recovered without sequelae. A more proactive screening strategy in France, particularly during periods of highest risk for West Nile virus circulation, would help reduce risk for donor-derived infections.

In 2025, chikungunya virus (CHIKV) re-emerged in Cuba after nine years without reported local transmission, with over 51 000 cases. We identified ten infected travellers returning to France from Cuba. Phylogenetic analysis showed that these viral genomes belong to the ECSA-2 genotype and cluster with recent Latin American sequences. These findings highlight the value of traveller-based genomic surveillance and underscore the risk of CHIKV expansion to other regions.

The circulation of arboviruses in sub-Saharan African countries remains poorly documented. The associated health burden may be underestimated and masked by the significance of malaria. Here, we have investigated acute undifferentiated fevers for arboviral infections in Mali (2016-2024). To estimate the proportion of patients with arboviral infection, and in particular dengue. A retrospective (2016-2022) and a prospective (2023-2024) studies were conducted in patients from health centers and hospitals of Mali (mainly in the Bamako region) selected by health professionals. Studies included patients with acute fever lasting less than 7 days; the prospective sub-study excluding pyogenic, urinary, tuberculosis, viral hepatitis, typhoid fever and post-traumatic infections. Blood samples were tested for arboviruses using molecular detection (including serotyping) and genomic sequencing. We collected demographic data and results of malaria testing for all patients and, in the prospective study, a set of clinical data. A total of 2,022 patients were included. Dengue virus (DENV) was the most frequently detected pathogen (retrospective study: 7.6%, 16/210 patients; prospective study: 29.5%, 535/1812 patients). We also detected chikungunya virus (n = 7), West Nile virus (n = 2) and Rift Valley fever virus (n = 1). Three serotypes of dengue were identified: DENV-2 (n = 185), DENV-1 (n = 113) and DENV-3 (n = 105); 148 DENV cases could not be typed. For each serotype, phylogenetic analyses identified a major lineage recently originating from the subregion (DENV-1-III; DENV-2-II; DENV-3-III). In contrast to malaria, the dengue detection rate was higher among patients over 18 years of age. The most frequently observed symptoms were headache, asthenia, arthralgia, myalgia and back pain. The mean number of those symptoms per patient was significantly higher in dengue patients. We recorded 6 cases of hemorrhagic dengue, but no deaths and no case requiring transfer to intensive care. Our findings confirm the threat posed by arbovirus infections in Mali, and more specifically the growing burden of dengue fever on public health. Monitoring dengue fever has become a major challenge in sub-Saharan countries in order to determine the conditions necessary for the future implementation of a dengue vaccination policy tailored to the public health objectives of these countries.

We report here the case of a blood donor who tested positive for chikungunya RNA by nucleic acid testing introduced in Guadeloupe (a French island in the Caribbean) to prevent the transmission of arboviral infections by transfusion. The strain was isolated, and further investigations revealed the presence of the IXCHIQ live attenuated vaccine strain in blood 1 month after vaccination.

Chikungunya virus (CHIKV) is a mosquito-borne arbovirus that causes acute febrile illness frequently associated with severe polyarthralgia and long-term disabling sequelae. In 2024–2025, La Réunion Island experienced a major resurgence of CHIKV transmission after more than a decade without documented autochthonous circulation. The live-attenuated chikungunya vaccine VLA1553 (IXCHIQ®, Valneva) was recently approved based primarily on immunogenicity data and a validated immune correlate of protection. However, real-world evidence regarding vaccine effectiveness, safety, and population-level impact during active outbreaks remains limited. The CHIK-RE-VAC study aims to evaluate the real-world effectiveness, safety, immunogenicity, and cost-effectiveness of the VLA1553 chikungunya vaccine during an ongoing epidemic on La Réunion Island. CHIK-RE-VAC is an ambispective, observational, multicenter, phase IV cohort study conducted across hospital and outpatient care clusters on La Réunion Island. Eligible adults are enrolled into vaccinated and unvaccinated groups according to their decision to receive vaccination in accordance with national recommendations. The study integrates both prospective recruitment and a retrospective cohort of individuals vaccinated prior to study initiation. Participants are followed for up to 12 months after vaccination or index date with active surveillance, including weekly symptom monitoring and triggered clinical visits. The primary outcome is vaccine effectiveness against laboratory-confirmed symptomatic chikungunya infection at 6 and 12 months. Secondary outcomes include severe disease, hospitalization, adverse events following vaccination, persistent symptoms, health-related quality of life, vaccine acceptability, and cost-effectiveness. A nested immunogenicity substudy evaluates neutralizing antibody responses and cellular immune responses over time. Statistical analyses will use propensity score–based weighting and generalized estimating equation models accounting for clustering. Recruitment began in April 2025 following the launch of a government-funded vaccination campaign targeting populations at high risk of severe chikungunya. An amendment introducing a retrospective cohort was approved in July 2025 to include individuals vaccinated prior to study initiation and to enhance recruitment and statistical power. As of March 2026, 260 participants have been enrolled across the prospective and retrospective components. Recruitment and follow-up are ongoing. The CHIK-RE-VAC study will provide the first comprehensive real-world evaluation of the effectiveness, safety, immunogenicity, and cost-effectiveness of the VLA1553 chikungunya vaccine during an active epidemic. The findings are expected to inform vaccination strategies, public health preparedness, and policy decisions regarding the deployment of chikungunya vaccines in outbreak settings. EU Clinical Trials Register: EU-CT 2025-521307-43-00; ClinicalTrials.gov: NCT06928753.

Importance:West Nile virus (WNV), a mosquito-borne orthoflavivirus, represents an increasing public health threat in Europe. In July 2025, WNV lineage 2 (WNV-L2) was associated with the first autochthonous cases ever detected in the metropolitan area around Paris, France; understanding the dispersal dynamics and geographic origin of these emergence events is critical for public health preparedness. Objectives:To characterize the spatial and temporal dynamics of WNV circulation in France from 2022 to 2025 and to determine the origin of the virus lineages responsible for the 2025 emergence in the Paris metropolitan area. Design, Setting, and Participants:This study was a genomic epidemiology assessment combining human, veterinary, and entomological monitoring, with viral genome sequencing and time-resolved, bayesian phylogenetic analysis. Surveillance was conducted across metropolitan France, with a focus on areas with documented WNV circulation (Eastern and Western Mediterranean, South Atlantic, and Paris). Surveillance included patients with confirmed WNV infection, avian and equine cases, and mosquito collections. Exposures:Natural exposure to WNV through mosquito vectors in affected areas. Main Outcomes and Measures:Phylogenetic relationships between emergence events across regions in France were inferred using a bayesian approach. Results:Genomic data from this epidemiological assessment encompassed 52 WNV-positive samples (6 human, 21 veterinary, and 25 entomological samples). WNV-L2 was the only lineage detected in France between 2022 and 2025. In 2024, Western Mediterranean strains were closely related to those from 2023 from the French South Atlantic area, suggesting west-to-south introduction, whereas Eastern Mediterranean strains represented genetically distinct clades associated with Northern Italy. The phylogenetic analysis of 2025 WNV sequences from the Paris area revealed that the outbreak virus strains all grouped together and originated from the L2 clade of sequences from the South Atlantic area in the 2023 to 2024 period. Conclusions and Relevance:In this study, genomic data from an epidemiologic assessment of 52 WNV-positive samples highlight the combined influence of local virus maintenance and long-distance dispersal in shaping WNV circulation in France. Strengthened genomic surveillance across ecological and administrative boundaries would be essential to anticipate further viral spread, inform response strategies, and protect populations at risk.

ABSTRACT Background Japanese encephalitis virus (JEV) is a mosquito-borne pathogen responsible for Japanese encephalitis (JE) clinical cases in Asia and the Western Pacific. Non-JE-vaccinated patients can develop potentially life-threatening neurologic forms. Case Summary Here, we present two severe, non-fatal JEV cases in returning travelers from Cambodia (Case 1) and Cambodia and Vietnam (Case 2), imported to France in 2023–2024. Neither patient was vaccinated, and both presented neurologic symptoms requiring hospitalization. Cases were confirmed by RT-qPCR, IgM, and IgG rise and seroneutralization. Interestingly, this is the second report to describe the detection of the JEV genome in urine by RT-qPCR. Conclusion This study highlights the critical importance of JE vaccination and the implementation of other personal protective measures to avoid mosquito bites when traveling to a JEV-endemic region.

Mosquito-borne viruses represent a large global health burden. With geographic expansion of competent vectors for chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV) in Europe, it is anticipated that the number of autochthonous cases of these tropical viruses in Europe will increase. Therefore, regular assessment of diagnostic capabilities in Europe is important. Our aim was to evaluate the mosquito-borne virus molecular detection capability of expert European laboratories by conducting an external quality assessment in October 2023. Molecular panels included 12 plasma samples: one alphavirus (CHIKV), four orthoflaviviruses (ZIKV, yellow fever virus [YFV], DENV, and Japanese encephalitis virus [JEV]), and two negative control samples. Mosquito-borne virus detection was assessed among 36 laboratories in 24 European countries. Adequate capabilities were lacking for YFV and JEV. Many laboratories relied on a mix of laboratory-developed tests (some of which were pan-orthoflavivirus or pan-alphavirus in combination with sequencing) and commercial assays. 47.2% of laboratories characterized all external quality assessment (EQA) samples correctly. Correct result rates were 100% for CHIKV and ZIKV and >99% for DENV, but laboratories lacked capacity, specificity, and sensitivity for JEV and YFV. Three of the viruses in this panel emerged and transiently circulated in Europe: CHIKV, ZIKV, and DENV. Molecular detection was excellent for those viruses, but <50% is accurate for the remainder of the panel. With the possibility or continuation of imported cases and a growing global concern about climate change and vector expansion, progress toward rapid, accurate mosquito-borne virus diagnostics in Europe is recommended, as well as regular EQAs to monitor it. IMPORTANCE The external quality assessment (EQA) focused on Aedes-borne viruses: chikungunya virus (CHIKV), dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). Japanese encephalitis virus, an orthoflavivirus that is spread by mosquito species belonging to the genus Culex, was included in the quality assessment as well. CHIKV, DENV, and ZIKV have proven potential for transient and limited circulation in Europe upon introduction of viremic travelers returning to Aedes albopictus-endemic regions. Results of this EQA were excellent for those viruses, but <50% is accurate for the remainder of the panel (YFV and Japanese encephalitis virus). Considering imported cases and the threat of climate change and competent vector expansion, progress toward rapid, accurate mosquito-borne virus diagnostics in Europe is recommended.

Mayaro virus (MAYV) is a mosquito-borne alphavirus that is widespread in the Amazon basin, where it co-circulates with the closely related chikungunya virus (CHIKV). Due to the limited surveillance and technical limitations of diagnostic assays (scarcity of commercial assays, serology cross-reactivity), the true burden of MAYV is uncertain. We designed a new RT-qPCR assay targeting the nsp1 gene for MAYV detection, which can be used in monoplex or duplex format. In the duplex format, the new MAYV assay is combined with a CHIKV assay and a second MAYV assay, both previously published. The lower limit of detection with a 95% positivity rate was determined to be <10 RNA copies/μL in monoplex and duplex formats for both MAYV and CHIKV. Monoplex and duplex assays proved to be linear within the tested range of approximately 108 to 102 RNA copies/μL and showed 100% specificity against a wide panel of arboviruses as well as several other pathogens in clinical samples. The testing of CHIKV-positive sera and MAYV-spiked plasma samples confirmed the suitability of the assays in a clinical setting. These assays offer a reliable tool for detection and differentiation of MAYV and CHIKV in endemic settings.IMPORTANCEMolecular diagnostic capabilities for detecting alphaviruses other than chikungunya virus (CHIKV) remain limited. Mayaro virus (MAYV), an emerging mosquito-borne alphavirus, co-circulates with CHIKV in the Americas, making clinical differentiation between the two viruses challenging. To address this, we developed a novel RT-qPCR assay specifically for the detection of MAYV, which can also be used in a duplex format to simultaneously detect and distinguish CHIKV. The assay was thoroughly evaluated in both monoplex and duplex formats and demonstrated high sensitivity and specificity. This new tool is particularly valuable for the detection of MAYV, especially in resource-limited settings, where its duplex format offers efficient and accurate differentiation of acute infections.

This study confirms the continued circulation of Toscana virus (TOSV) lineage B in France, with nine laboratory-confirmed autochthonous neuroinvasive cases identified between 2022 and 2024. All patients presented with central nervous system symptoms, predominantly during summer months, and most were confirmed by RT-qPCR (quantitative reverse transcription polymerase chain reaction) on cerebrospinal fluid. Phylogenetic analysis revealed close similarity to previously circulating French strains, supporting long-term local transmission. One case in eastern France raises concern for possible geographic expansion. These findings underscore the need for sustained surveillance and preventive measures in endemic regions, particularly for travellers and clinicians encountering summer-onset neurological syndromes.

Alphaviruses comprise over 30 identified species spread worldwide and carry a large global health burden. With vector expansion occurring in and around Europe, it is anticipated this burden will increase. Therefore, regular assessment of the diagnostic capabilities in Europe is important, e.g., by conducting external quality assessments (EQAs). Here we evaluated molecular detection of alphaviruses in expert European laboratories by conducting an EQA in March 2022. Molecular panels included 15 samples: nine alphaviruses, Barmah Forest virus (BFV), chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Mayaro virus (MAYV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Sindbis virus (SINV), Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV) and four negative control samples. Alphavirus detection was assessed among 23 laboratories in 16 European countries. Adequate capabilities were lacking for several viruses, and approximately half of the laboratories (11/23) relied on pan-alphavirus assays with varying sensitivity and specificity. Only 46% of laboratories characterized all EQA samples correctly. Correct result rates were > 90% for CHIKV, RRV and SINV, but laboratories lacked specificity for ONNV and MAYV and sensitivity for VEEV, BFV, and EEEV. Only two alphaviruses causing human disease circulate or have circulated in Europe, CHIKV and SINV. Molecular detection was satisfactory with both CHIKV and SINV, but < 50% correct for the entire alphaviruses panel. With continued imported cases, and a growing global concern about climate change and vector expansion, focus on progress toward rapid, accurate alphavirus diagnostics in Europe is recommended, as well as regular EQAs to monitor quality.

In August 2024, a case of Zika virus infection was identified in metropolitan France in a traveller returning from Seychelles. Genomic analysis confirmed that the strain belongs to the Asian lineage. Recent epidemiological findings provide the first evidence of ZIKV circulation in the western Indian Ocean region.

Current French guidelines on the diagnosis of dengue infection recommend both nucleic acid testing and serology as tools for laboratory confirmation. This study aimed to evaluate the performance of the fully automated Virclia IgM assay for the diagnosis of dengue infection. Samples from patients with a suspicion of dengue were prospectively tested using the Virclia Dengue IgM assay (Vircell) and subsequently underwent additional investigations (dengue RT-PCR and conventional dengue IgM EIA) at the French Reference Center for Arboviruses. A total of 104 patients were included with a median age of 34.3 years old and a median time since symptom (TSS) of 6 days. Dengue RT-PCR was positive in 57 patients (54.8%). The agreement was excellent (90.5%; κ = 0.81) between RT-PCR and Virclia Dengue IgM assay on samples collected from Day 5 postsymptom onset. On these samples, the sensitivity and specificity of the Virclia IgM assay were 95.7% (95% CI: 84.7%-96.9%) and 96.4% (95% CI: 80.8%-100%), respectively. In addition, the agreement was also excellent between the Virclia Dengue IgM assay and the Euroimmun plate-based Dengue IgM ELISA (92.7%; κ = 0.85). In conclusion, the Virclia Dengue IgM assay showed a good performance in the diagnosis of dengue infection and can be recommended in addition to nucleic acid testing to broaden the diagnostic window. The automation coupled with the monotest format is well-adapted for nonendemic areas.

Chikungunya virus (CHIKV) is known to circulate in Africa, but there is little evidence of CHIKV transmission in Côte d’Ivoire. Using genomic data, we linked CHIKV cases imported in France from Côte d’Ivoire to a lineage from the West African genotype, likely been circulating in the country since July 2021.

Chikungunya virus re-emerged on Réunion Island in August 2024, 18 years after a first major outbreak. Analysis of 173 genomes from the current epidemic reveals a monophyletic clade with mutations linked to adaptation to Aedes albopictus mosquitoes, including E1-A226V. Bayesian inference suggests only brief cryptic circulation before detection. The same lineage was also detected on Mayotte Island in March 2025. Continued spread and confirmed travel-related cases in mainland France and globally highlight the risk of wider regional and international dissemination.

With Aedes aegypti expanding to higher altitudes, dengue virus (DENV) cases soared to record levels in 2024 in Cochabamba, Bolivia (2,558 meters above sea level). Using genome sequencing, we identified at least two distinct clades of DENV-2 genotype II circulating during the outbreak. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was financially supported by the National Research Council of Thailand (NRCT): (NRCT5-RGJ63012-125), Grant No. RGNS 64-172 from the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (MHESI); and the international postdoctoral fellowship 2022 provided by Mahidol University. This work was also supported by the ARBOGEN project, funded by the MSDAVENIR Foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Ethics Committee of the Sindicato Medico y Ramas Afines de la Caja Nacional de Salud, Cochabamba, Bolivia (No. 01/2024). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript