Yuki Takamatsu

Neutralizing antibodies provide critical protection against dengue virus (DENV) infections. However, under certain conditions, antibodies can also enhance infection and contribute to severe disease through antibody-dependent enhancement (ADE). ADE can occur through extrinsic mechanisms, in which antibody-opsonized virions engage with Fcγ receptors (FcγRs) on target cells to facilitate increased viral entry, while intrinsic mechanisms have been proposed in which FcγR-mediated uptake triggers intracellular pathways that promote viral replication. The extensive genetic diversity of DENV further complicates the evaluation of antibody functionality, as antibodies may exhibit distinct neutralizing and enhancing activities against different viral genotypes. Here, we developed a single-round infectious particle (SRIP) panel representing 17 DENV genotypes across all four DENV serotypes. This genotype-inclusive platform enables assessment of antibody-mediated neutralization and FcγR-dependent enhancement of infection through luciferase reporter quantification. Using this panel, we evaluated antibody responses from DENV-infected individuals, healthy volunteers, and flavivirus-cross-reactive or DENV-specific monoclonal antibodies. Our findings reveal genotype-specific variations in antibody-mediated neutralization and enhancement, emphasizing the complex interplay between antibody responses and DENV genetic diversity. This SRIP panel provides a robust approach for profiling functional antibodies against genetically diverse DENV strains. While the current platform primarily quantifies extrinsic ADE, its single-round infection format and intracellular replicon-based readout provide a foundation for future investigations of intrinsic ADE mechanisms. Overall, this platform offers broad applications for studying dengue immunity, evaluating vaccine-induced antibody responses, and advancing our understanding of antibody-mediated protection and disease enhancement. IMPORTANCE: Dengue virus (DENV) comprises four serotypes and substantial genetic diversity, which can influence the ability of antibodies to neutralize or enhance infection. However, comparative assessment of antibody functionality across diverse DENV genotypes remains challenging because differences in viral genetic backgrounds can confound functional measurements. We developed a single-round infectious particle (SRIP) panel representing 17 DENV genotypes in which the prM and E structural proteins are varied on a common replicon backbone. This design provides a standardized platform for directly comparing antibody-mediated neutralization and Fcγ receptor-dependent enhancement across genetically diverse DENV strains. By characterizing antibody responses against a broad range of genotypes, the panel can reveal differences in functional antibody activity that may be overlooked when using a limited number of representative strains. The platform is therefore useful for investigating how DENV genetic diversity influences antibody-mediated protection and extrinsic antibody-dependent enhancement (ADE), as well as for evaluating the breadth of antibody responses induced by infection or vaccination. Although the current platform primarily quantifies extrinsic ADE, its single-round infection format and quantitative intracellular reporter readout provide a foundation for future studies investigating intrinsic ADE in more physiologically relevant immune cell models. This genotype-inclusive panel offers a practical approach for standardized functional profiling of dengue antibodies and may facilitate improved evaluation of vaccine-induced immunity and antibody responses across the genetic diversity of DENV.

All four dengue virus serotypes (DENV-1 to DENV-4) can cause severe and potentially fatal disease. Understanding the interplay between host immune responses and viral genetic diversity is particularly crucial in hyperendemic regions, where serotype shifts frequently occur during outbreaks. In this cross-sectional, surveillance-based study, we analyzed 350 patients with confirmed secondary dengue infection in Lao PDR, with samples collected between 2022 and 2023. Of these, 80 were successfully serotyped by RT-PCR and included in the serotype-specific analyses. Viral genotyping was performed using envelope (E) gene sequencing, and neutralizing antibody (nAb) titers and antibody-dependent enhancement (ADE) activities against all four DENV serotypes were assessed using single-round infectious particles in both FcγRIIA-negative and FcγRIIA-positive BHK-21 cells. The nAb titers were highest against DENV-2, followed by DENV-1, DENV-4, and DENV-3. Nevertheless, DENV-2 exhibited the highest ADE activity, suggesting that high nAb titers may not necessarily prevent ADE. We also documented the co-circulation of DENV-2 Cosmopolitan and Asian I genotypes. Our findings highlight the complex interplay between neutralizing and enhancing antibody responses during secondary infection, particularly in the context of DENV-2 infection. Our results emphasize the need to understand the dynamics of DENV transmission in hyperendemic settings and may inform future vaccine development and evaluation strategies.

The Zika virus (ZIKV) remains an important public health concern owing to its association with neurological disorders and congenital abnormalities. Genetically, ZIKV is classified into two major lineages—African and Asian—and continues to evolve, with evidence of genetic diversification following its introduction into the Americas. The study of the envelope (E) gene of ZIKV is a key tool for understanding viral biology, phylogenetic analysis, and developing antiviral drugs and vaccine candidates. However, few studies have focused on designing primers capable of efficiently detecting and amplifying this essential gene. In this study, we developed universal primer sets capable of amplifying and sequencing the full-length ZIKV E gene across genetically diverse lineages and strains. Primers were designed based on representative ZIKV sequences available in public databases and validated using viral isolates and in vitro-spiked plasma samples. The assay demonstrated high specificity and analytical sensitivity, with the analytical detection limit established using serially diluted DNA amplicons from the target region. Furthermore, ZIKV was successfully detected in plasma samples spiked at 1 × 10³ PFU/mL, which enabled complete sequencing of the E gene. These findings highlight the utility of our primers for molecular epidemiological studies, strengthening arbovirus surveillance and improving diagnostic capacity in regions where multiple arboviruses co-circulate, using a simple and reliable assay.

Dengue has remained a public health challenge in the Lao People's Democratic Republic (Lao PDR) for several decades, with all four dengue serotypes circulating. This study aimed to investigate the epidemiology, clinical characteristics, and role of co-circulation with factors associated with severe dengue in this setting. We conducted a cross-sectional study to retrieve all serum samples from patients suspected of having dengue collected through the National Dengue Surveillance System between 2022 and 2023 in Lao PDR, as well as their demographic and clinical information. All samples were tested for DENV detection and serotyping by reverse-transcription PCR. In-house DENV IgM/IgG ELISAs confirmed the infections and classified them as primary or secondary infections. Multinomial logistic regression was used to analyze the factors associated with disease severity. Of the 1098 serum samples from suspected dengue patients, 615 (56%) tested DENV-positive. The median age of the DENV-positive patients was 22 years (IQR 15-33), and 339 (55.1%) were female. Overall, 178 (29%) infections were classified as primary and 437 (71%) as secondary infections. DENV-positive patients were common in the northern region (37.4%) compared to other regions, with significant geographic differences in serotype distribution (p < 0.0001): DENV-2 was identified in the south (62.5%), while DENV-1 was detected in the north (45.9%) (p < 0.0001). In the southern region, secondary infections due to DENV-2 (66.7%) were more common, whereas primary infections due to DENV-1 were more frequent in the northern region (p = 0.0002). Greater disease severity was observed in DENV-2 infection (83.3%) (p = 0.002). Severe dengue was more likely in secondary infection (OR = 2.38, 95% CI: 0.84-6.74) compared to primary infection and was significantly higher in the southern (OR = 11.16, 95% CI: 3.05-40.77) (p < 0.001) and central regions (OR = 10.34, 95% CI: 2.81-38.09) (p < 0.001). Collectively, our findings identified the key factors that contributed to the dengue epidemic in 2022-2023. DENV-2, secondary infection, and geographic variation were associated with severe outcomes. Enhanced genomic and epidemiological surveillance is critical for addressing the dengue burden in the Lao PDR.

Dengue cases are reported annually in Vung Tau province, southern Vietnam; however, data on viral and host factors associated with disease severity are limited, particularly during epidemics driven by specific serotypes. We investigated the prevalence of dengue virus (DENV), its molecular epidemiology, and clinical-laboratory correlates of severity in patients with suspected dengue at Vung Tau General Hospital between September 2020 and May 2021. Among 1435 suspected cases, 639 (44.5%) were classified as DENV infection-negative, 60 (4.2%) presented with primary infection and 736 (51.3%) with secondary infection. DENV serotyping indicated that DENV-4 was the most prevalent serotype (50 patients, 37.6%), followed by DENV-2 (41, 30.8%), DENV-1 (39, 29.3%), and DENV-3 (3, 2.3%). Phylogenetic analysis revealed DENV-1 genotype I, DENV-2 genotype II (cosmopolitan) and genotype V (Asian I), and DENV-4 genotype I. Notably, DENV-4 exhibited two coexisting clades, suggesting that variations in clade distribution may have contributed to the epidemic. Although parameters commonly associated with dengue severity, such as white blood cell counts, platelet counts, and liver enzyme levels (AST and ALT), were comparable across serotypes, hemoglobin and hematocrit levels were elevated in DENV-4 infections compared with other serotypes. This elevation may indicate a higher risk of plasma leakage, a key feature of severe dengue, underscoring the potential clinical significance of DENV-4 during this epidemic. These findings highlight the epidemiological and clinical importance of DENV-4 and provide evidence for refining dengue control strategies in endemic areas.

Background: Chikungunya virus (CHIKV) and dengue virus (DENV) frequently co-occur in Myanmar and present with overlapping symptoms, complicating diagnosis. During the 2019 dengue outbreak in Yangon, Myanmar, molecular data on CHIKV were limited among dengue-suspected patients and there were no publicly available CHIKV genome sequences from Yangon in international databases. To address this gap and potential diagnostic overlap, we investigated the prevalence of CHIKV infection and described the genomic characteristics of detected strains. Methods: Serum samples from 267 dengue-suspected patients collected in 2019 were screened for anti-CHIKV IgM and IgG by in-house ELISA and 211 samples with sufficient remaining volume were further analyzed by RT-qPCR, isolation of the virus, and whole-genome sequencing for mutation analysis. Results: CHIKV antibodies were found in 24.7% (66/267) of samples (IgM 3.4%, IgG 21.3%), and viral RNA was detected in 10.9% (23/211) of samples. Fifteen viral isolates were successfully obtained (7.1% of those tested), including two co-detections with DENV-2 by RT-PCR. All isolates belonged to the East/Central/South African genotype, Indian Ocean Lineage (ECSA-IOL), and clustered with strains from Thailand, China, and Mandalay, Myanmar. Whole-genome analysis identified 33 non-synonymous mutations across nonstructural and structural proteins, including mutations previously reported in regional ECSA-IOL strains such as E1:K211E and E2:V264A, with 11 amino acid changes not previously reported in available Myanmar reference sequences. Discussion: Serological and molecular findings indicate CHIKV circulation during the 2019 dengue outbreak in Yangon and highlight the limitations of single-target testing. Serological evidence indicate the presence of anti-CHIKV IgM and IgG antibodies, reflecting CHIKV exposure within the study population. Notably, all RNA-positive cases were seronegative for both IgM and IgG, a pattern consistent with the temporal dynamics of infection and the inherent constraints of serological detection in co-endemic settings. Molecular co-detection with DENV-2 and genomic findings highlight the potential value of multiplex diagnostic approaches in co-endemic settings. Conclusion: This study documents CHIKV detection and genomic characterization in dengue-suspected patients in Yangon and highlights the potential value of multiplex diagnostic approaches and continued genomic surveillance as broader public health considerations for arboviral detection in Myanmar.

Background: The Lao People's Democratic Republic (Lao PDR) is hyperendemic for dengue, with all four dengue virus (DENV) serotypes circulating since 1979, posing substantial health risks. Understanding serotype co-circulation, phylogenetic diversity, and disease severity determinants is crucial for effective surveillance and control.Methods: Serum samples from suspected dengue patients in Lao PDR (2022–23) were analyzed through reverse-transcription PCR for DENV detection and serotyping. Genotyping was performed via envelope (E) gene sequencing. In-house DENV IgM/IgG ELISAs confirmed infections and classified primary versus secondary infections. Neutralizing antibody (nAb) titers and antibody-dependent enhancement (ADE) activity were assessed against all serotypes in BHK21 cells.Findings: Of 1098 samples, 615 (56%) were DENV-positive. Median age was 22 years, with 55·1% female. Primary infections were 29% and secondary 71%. Significant regional serotype differences were observed (p < 0·0001), with DENV-2 predominating in the south and DENV-1 in the north. Greater severity was linked to DENV-2 (p = 0·002), secondary infections (p = 0·005), and central/southern residency (p < 0.0001). DENV-2 showed highest ADE and nAb titers. A novel amino acid substitution was identified in Lao DENV-2 strains.Interpretation: This study illuminates the regional transmission patterns and virological factors influencing dengue epidemiology in Lao PDR. Enhanced genomic and epidemiological surveillance is critical for addressing the dengue burden. Results emphasize the role of ADE and highlight considerations for vaccine strategies in hyperendemic zones.

On 9–10 December 2025, the Coalition for Epidemic Preparedness Innovations (CEPI) and the International Vaccine Institute (IVI) convened a workshop in Seoul under CEPI’s Disease X Program. The primary objective was to identify existing gaps needing to be filled and streamline vaccine development and preparedness for Severe Fever with Thrombocytopenia Syndrome (SFTS). CEPI’s partners and experts discussed a multifaceted agenda, ranging from understanding the evolving epidemiology to the refinement of animal models and immunological assay harmonization. Key outcomes included the refinement of Target Product Profiles (TPPs) specifying use cases for both peacetime and outbreak contexts, alongside a recommendation for a core immunoassay panel aimed at harmonizing evaluation frameworks and mitigating the challenges posed by low SFTS prevalence. Integration of the One Health approach emerged as a critical strategy for SFTS prevention, complemented by proactive regulatory engagement to compress vaccine development timelines. This report summarizes these key insights from the workshop, delineating a strategic framework for delivering safe, effective, and accessible vaccines for SFTS and broader Disease X threats.