Frontiers in Virology

In an infection, Hepatitis B Virus (HBV) core protein (HBc) normally assembles into icosahedral capsids. Capsid Assembly Modulators (CAMs) are direct acting antivirals that induce HBc mis-assembly and are the subject of active research and development. Two versions of HBc are used in structural studies of CAM-HBc complexes: Cp150 and Cp149-Y132A. Cp150 forms empty icosahedral capsids that are structurally indistinguishable from those found in virions. The Y132A mutation of Cp149 leads to an assembly defective soluble protein that crystalizes as flat hexagonal sheets, where the hexagons resemble icosahedral quasi-sixfold vertices. In this study, we compare structures of CAM-bound Cp150 to CAM-bound Cp149-Y132A. In capsids, the residues forming the CAM site shift to match the structure of bound CAMs, an induced fit. In Cp149-Y132A crystals, CAM sites show little structural adjustment in response to different CAMs binding. In turn, the array of residues that interact with CAMs varies from CAM to CAM in capsid structures but remains nearly constant in Cp149-Y132A crystals. These results illustrate important differences between CAM binding in Cp149-Y132A and Cp150 structures that will contribute to future CAM design.

RNA helicases encoded by positive-strand RNA viruses are essential for genome replication, yet the specific biological functions and mechanochemical basis underlying these functions remain poorly defined. Progress has been limited by the difficulty of resolving individual catalytic steps under single-turnover conditions, which are often experimentally inaccessible for viral enzymes. Alphaviruses replicate within membrane-bound spherules that may alter local metabolite concentrations, raising the possibility that the enzymatic properties of alphaviral proteins differ from those of viruses with greater cytosolic exposure. Here, we present a kinetic and binding analysis of full-length non-structural protein 2 (nsP2) from Chikungunya virus, a multifunctional superfamily 1B NTPase and RNA helicase. Purified nsP2 binds nucleoside triphosphates with high affinity, exhibiting equilibrium dissociation constants in the single digit micromolar range. This property enabled single-turnover, pre-steady-state, and isotope-trapping experiments that are rarely feasible for viral helicases. These analyses identified two sequential conformational-change steps required for nucleotide hydrolysis. Molecular dynamics simulations suggest tightening of the RecA1 and RecA2 domains upon ATP binding followed by compaction of the enzyme mediated by interactions between the 1B subdomain and RecA2 domain. Product inhibition patterns support random release of ADP and inorganic phosphate, with relative binding affinities indicating that ADP dissociates first. The reaction is irreversible. Although nsP2 binds RNA tightly, strand separation under single-turnover conditions is too slow to represent ATP-driven unwinding, instead likely reflecting formation of an unwinding-competent nsP2-RNA complex. Together, these findings establish a quantitative framework for nsP2 function and provide a roadmap for mechanistic studies of alphaviral helicases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/723793v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@13899a1org.highwire.dtl.DTLVardef@ee1aadorg.highwire.dtl.DTLVardef@1991e1org.highwire.dtl.DTLVardef@b877f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tomato brown rugose fruit virus (Tobamovirus fructirugosum) is an emerging virus that affects tomatoes, capsicum, and chili. Since its first detection in Jordan in 2015, the virus was reported in more than 40 countries across all the continents. In Morocco, the virus was reported for the first time in October 2021. However, its genetic diversity remains unexplored. In this work, we used a collection of tomato fruits from local markets to investigate the variability of the virus in the country. We explored the different pressures acting on the N-terminus of the RNA-dependent RNA polymerase, the movement protein, and the coat protein genes. Then, we used haplotype network analyses to reveal the population structure within the Moroccan isolates and studied their relationships with the ones from the world. We found that genetic diversity is low, which is consistent with the global situation. No signatures of diversifying selection were detected across the analyzed genes. However, the virus sequences from Morocco showed a clear geographic structure, suggesting that geographic factors probably combined with agricultural practices may contribute to shaping the population structure of ToBRFV in Morocco.

Since the initial distribution of the SARS-CoV-19 vaccine, its widespread use has been hypothesized to act as a selective pressure that drives the COVID-19 virus to mutate. This study aims to investigate the correlation between global vaccination rates and the mutation rate of the SARS-CoV-2 Beta variant (B.1.351). From January to July 2021, nucleotide diversity increased in tandem with vaccination rates, demonstrating that the virus evolved more rapidly in response to selective pressure from mass vaccination. Statistical analysis revealed statistically significant positive correlations between both vaccination rates and vaccine doses administered with nucleotide diversity. Thus, our findings indicate a positive correlation between rising vaccination rates and nucleotide diversity, suggesting that increased vaccination coverage acted as a selective pressure that accelerated viral evolution of SARS CoV2.

Temperate regions of the world, such as southern Australia, often experience increased health burden from respiratory pathogens during winter. The ability to forecast short-term trends in cases of these pathogens is of significant interest to public health. Across the 2024 southern hemisphere winter period, the Australia--Aotearoa Consortium for Epidemic Forecasting and Analytics (ACEFA) ran a pilot respiratory virus forecasting initiative in collaboration with the Victorian Department of Health. Each week from the 9th of May 2024 through to 12th September 2024, the consortium solicited 28-day forecasts of daily case incidence for influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV) from multiple research groups. Four component model forecasts were contributed by three different research groups, with a fourth group utilising the component forecasts to generate ensemble forecasts (making a total of six models, four component models and two ensembles). Here we statistically evaluated the performance of each forecast and a baseline model against the observed case data. The two ensemble models were found to be frequently the top performing models. All models performed worse than the baseline model around the epidemic peaks for each pathogen.

Numerous studies have shown that the abundance of antibiotic-resistant bacteria (ARBs) or antibiotic-resistance genes (ARGs) in soil increases after irrigation with wastewater. However, it is unclear whether this increase is due to the selection effects of pharmaceutical residues in the irrigation water or the continuous introduction of ARBs and ARGs with the wastewater. Further, it is unclear how the binding of antibiotics to natural colloids (1-1000 nm) affects their biological effects compared to truly dissolved substances (< 1 nm). We conducted competition experiments with resistant and susceptible Acinetobacter baylyi BD413 strains in wastewater, as well as in colloidal and truly dissolved extracts of soils irrigated with wastewater. Although the concentrations of our six target antibiotics were far below the measured minimum selective concentrations of the tested strains, we demonstrate that the resistant strain was favored in the wastewater and the colloidal extracts. In contrast, the truly dissolved fractions exhibited weaker and more variable selective effects. A non-targeted analysis revealed the presence of 82 additional substances in our extracts, including further antibiotics, pesticides, and different non-antibiotic drugs that may influence the selection of our resistant A. baylyi BD413 strain. Our findings suggest that antibiotic resistance is selected for in wastewater and wastewater-irrigated soils. This cannot be explained by antibiotic concentrations alone, but may also arise from the effects of complex mixtures of co-occurring contaminants, particularly those associated with colloidal particles.

We prepared siRNA libraries against the H5N2 virus NP gene, and the PA, PB1 and PB2 genes that express the proteins that form the virus polymerase complex. The antiviral activity of the siRNA libraries in H5N2 virus infected cells was initially assessed by using qPCR to measure the corresponding mRNA levels in the siRNA-treated cells. In this way siRNA molecules within each library were identified that exhibited to a greater than 70% reduction in levels of each target mRNA. A selection of these siRNA molecules was further evaluated for their antiviral activity in a multi-cycle H5N2 MDCK cell model. The siRNA molecules identified were successful in blocking virus transmission and lead to a reduction in influenza virus progeny virus production. This antiviral activity correlated with both the inhibition of nuclear export of the newly formed RNP complexs that arise from the transcriptional activity of the input virus, and the inhibition of the polymerase activity of the newly formed virus polymerase complexes. This study highlights the potential use of siRNA as a strategy to block virus transmission by targeting the avian influenza virus polymerase complex.

Citrus yellow vein clearing virus (CYVCV) is the causal agent of an emerging disease representing a potentially high-impact threat for citrus production. Despite remaining outside Europe for decades, CYVCV has now expanded towards two important European citrus producers, Italy and, more recently, Spain. The presence of this virus in the EPPO region represents a current threat with unpredictable and potentially devastating consequences for European citriculture. Therefore, urgent protective measures need to be taken to prevent CYVCV spread and minimize its impact. Diagnostics is a key measure in the management of viral diseases, highlighting the need for harmonized methods suitable for reliable routine detection of the currently known CYVCV diversity. In this study, an inclusive, efficient and highly sensitive real-time RT-qPCR for the detection of CYVCV in plant material and transmission vectors has been developed and validated according to EPPO standards. Moreover, the validated method has been successfully adapted to both PCR digital platforms, that allow high-sensitive absolute quantitative detection, essential in the diagnostics at low viral concentrations; and PCR portable tools, that can be applied in a real diagnostic context for on-site detection. This versatility combines standard validated performance, absolute sensitive quantitation and real on-site detection. The study has also addressed sampling strategies to support reliable molecular diagnostic performance. Our results represent an improvement in the detection of CYVCV to be applied in epidemiological studies and different real diagnostic contexts for the containment of this important citrus pathogen.

Belemia belongs to Nyctaginaceae and comprises two species of delicate vines. Both species are endemic to Brazil. Belemia fucsioides, the type species, described in 1981, occurs in a restricted area of the Atlantic Forest in southeastern Brazil. Belemia cordata, described in 2020, is known from only two records from the same area in the Cerrado of northern Brazil. Here, we describe the taxonomic history of Belemia and provide the first synopsis for the genus. We include species description, distribution map, identification key, and anatomical data. We used field observations over the past decade and modeled anthropogenic changes in the species range to conduct a conservation assessment in accordance with the IUCN Red List criteria. Conservation assessments indicate significant concerns for Belemia, classified as either endangered (B. fucsioides) or critically endangered (B. cordata). The species are threatened primarily by habitat loss to land used for agriculture, forestry, and livestock production. This study contributes to ongoing initiatives exploring plant diversity in the Neotropics and supports efforts to identify threats to biodiversity.

In recent years, detection of local dengue cases in Florida have increased in both frequency and geographical extent. From 2022 to 2024, consecutive outbreaks in Miami-Dade County were mainly caused by a single lineage of dengue virus (DENV) serotype 3, prompting questions about changing epidemiology and a transition towards endemicity. In this study, we used mathematical modeling and genomic epidemiology to reveal the spatiotemporal dynamics and drivers of local dengue cases in Florida. We found that annual clusters and outbreaks were caused by frequent short-lived DENV introductions, primarily from the Caribbean, and did not find evidence for local trans-seasonal DENV lineage persistence. Further, we show that the climate-driven increases in local suitability for Aedes aegypti transmission and travel-associated cases were the greatest risk factors for outbreaks in Miami-Dade and the geographic expansion of dengue in Florida. Overall, while we do not yet find evidence for endemicity, we demonstrate how climatic trends are enhancing the local public health risk caused by dengue in Florida.

Currently circulating swine influenza viruses (SIVs) mainly include H1N1, H1N2, and H3N2 subtypes. In this study, two G4 genotype Eurasian avian-like (EA) H1N1 SIVs were isolated from 556 samples collected between 2023 and 2026. A systematic analysis was conducted on the two EA H1N1 isolates (FYD30 and YZF69) to assess their pandemic potential. The hemagglutinin (HA) proteins of both H1N1 viruses possessed residues 225E and 228S, indicating enhanced affinity for human-like -2,6-linked sialic acid receptors, which was confirmed by receptor-binding assays. Polymerase activity tests demonstrated that the two SIVs exhibited significantly higher activity in mammalian cells, relative to avian cells, which is consistent with the efficient replication in mammalian cells. Challenge experiments revealed that both H1N1 caused significant pathogenicity in mice and pigs, with YZF69 exhibited higher virulence than FYD30. The higher virulence of YZF69 may be attributed to its molecular features, including the NP Q357K mutation, and an additional glycosylation site in HA. In conclusion, currently circulating EA H1N1 SIVs have acquired key molecular signatures of mammalian adaptation, exhibit enhanced virulence in mammals, and continue to undergo extensive reassortment driven by international swine trade. These findings highlight the potential pandemic risk of SIVs and underscore the urgent need for strengthened surveillance.

The automated classification of animals from photos is important in ecology and conservation biology for organizing and understanding the immense diversity of species, as well as facilitating effective conservation and management practices. It is equally important for disease surveillance systems, allowing prompt detection of anomalies in species distributions and boosting citizen-scientist platforms by making user-reported data more accurate and convenient. Image classification uses photos and can also rely on the geographical locations of animals to improve performance. While image classification models have difficulties in classifying low-quality images, unbalanced datasets, and with a small number of images, species distribution models have difficulty in classifying species that coexist in a given region. We propose here strategies for combining image classification models based on deep neural networks with species distribution models using genetic algorithms. The proposal is applied to a real-world dataset comprising fifteen classes of animals from the Brazilian fauna obtained from Fiocruzs citizen-scientist Wildlife Health Information System (SISS-Geo). The SISS-Geo photos portray the reality of animals in their environments, with varying quality, and pose numerous difficulties for classification. Experimental results demonstrate that the proposed integration consistently outperforms standalone models. While individual SDMs achieve Top-1 accuracies of 27.79% (MaxEnt) and 31.76% (Bioclim), and CNN-based classifiers reach 58.17% with ResNet50 and 64.13% with ResNet-152, the hybrid strategies yield substantial improvements. The genetic algorithm-based integration with a single global weight achieves up to 67.96% Top-1 accuracy, whereas the class-specific integration using fifteen parameters attains the best overall performance, reaching 69.03%.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

Tomato fruit blotch virus (ToFBV) is an emerging multipartite positive-sense RNA virus associated with blotchy symptoms on tomato fruits and classified within the genus Blunervirus (family Kitaviridae). Despite its increasing agricultural relevance, the study of ToFBV has been hindered by the lack of mechanical transmissibility and the difficulty in reproducing infections under controlled conditions. In this work, we report a preliminary step toward the development of the first infectious agroclone system for ToFBV, based on full-length cDNA copies of its four genomic RNAs. We demonstrate that the cloned viral genome is capable of initiating cell autonomous replication in Nicotiana benthamiana, as indicated by the accumulation of negative-sense RNA intermediates in infiltrated tissues. To further validate the system, RNA3 was engineered to express GFP, enabling visualization of infection foci and confirming active viral replication in both N. benthamiana and tomato. Functional assays of RNA4-encoded proteins demonstrated that it encodes a movement protein capable of complementing movement-deficient viral vectors and a putative suppressor of post-transcriptional gene silencing (PTGS). Together, these results establish a versatile reverse genetics platform for ToFBV, providing new insights into the replication and functional organization of blunerviruses and enabling future studies on virus-host interactions, pathogenicity, and control strategies.

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReEDs mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.

Avian influenza viruses (AIVs) are endemic in the Americas and responsible for outbreaks in both domestic and wild birds that occasionally spill over into humans. We report the first known outbreak of AIV H9N2 in lesser rhea (Rhea pennata), also known as Darwins rhea, in the region of Puno-Peru. The animals in this study lived in an isolated conservation center located in remote highlands above 4,000 m.a.s.l. Between June and July 2025, a total of 46/92 animals were recorded sick, with symptoms including greenish diarrhea (100%), hyporexia (24%), dyspnea (76%), nasal discharge (42%), drowsiness (18%) and isolation from the flock (73%), and 94% later died. Gross pathology exams revealed septicemia characterized by severe hepatitis, pneumonia, tracheitis, enteritis, and encephalitis. Swab and necropsy samples tested positive for Influenza A by PCR and were later identified as H9N2 through whole genome sequencing. We generated complete H9N2 genomes for two individuals. No additional pathogens were found. Phylogenetic analysis across all eight segments revealed that the viruses were low pathogenicity H9N2 AIV strains of North American origin, which indicated this outbreak was a new introduction of the virus into South America. We also performed a comparative mutational analysis and identified multiple mutations previously associated with mammalian host adaptation, increased virulence, increased pathogenicity, and increased virus binding to 2-6 receptors, which may explain the high mortality rates observed despite the supposedly low pathogenicity of the strain. We also identified novel mutations specific to rhea viruses that will need to be experimentally validated. This is the first report of a natural H9N2 systemic infection in an avian host, highlighting a need for increased surveillance efforts for zoonotic influenza viruses with pandemic potential. Author SummaryAvian influenza viruses (AIVs) are endemic in the Americas and cause more than 7,600 infections annually in domestic and wild birds worldwide each year. We report detection of AIV H9N2 in lesser rhea during an outbreak that occurred in June-July 2025 in the Andean highlands of Puno in Peru. Multiple sick animals were reported with symptoms of respiratory and gastrointestinal disease and 94% of them later died. Samples collected tested positive for Influenza A and they were subtyped as H9N2 of low pathogenic origin from North America. This is the third time H9N2 enters South America from North America, presumably through wild birds, some of which migrate along the Pacific Flyway. Comparison with other H9N2 sequences revealed a total of 44 mutations of interest that may explain the elevated death rates observed. Surveillance in wild birds remains patchy at best and needs to be strengthened in order to prevent spillover events into other animals, including humans.

The fully mycoheterotrophic, non-photosynthetic Afrothismia fonensis Cheek & G.Delhaye sp. nov. (Afrothismiaceae), is described and illustrated from two sites in submontane forest in or adjacent to the Pic de Fon Foret Classee, Simandou Range, Republic of Guinea. This is the first record of the genus and family in West Africa west of Nigeria. The new species is remarkable for its small size, and for being unique in the genus in the entirely connate intertepaline lobes (in other species of the genus they are free or only partly united) and the longitudinal ridges on the outer perianth tube (unknown in other species). The provisional extinction risk assessment for Afrothismia fonensis is Critically Endangered (CR B1ab (iii)+2ab(iii)+D1) using the IUCN 2012 categories and criteria, due to less than 50 individuals being recorded, and due to the both the very small range and the immediate threats from foraging by red river hogs, trampling by cattle and from de-watering of the adjacent Oueleba iron-ore body where mining began in 2025. It should be noted that mitigation actions are expected to adequately address the risks associated with mining activities, and direct impacts to both areas of Afrothismia fonensis habitat have been fully avoided through relocation of planned infrastructure. We review the importance of the Boyboyba forest, Simandou range, as the West African centre of diversity for non-photosynthetic heteromycotrophs. This new discovery is examined in the context of other recently discovered range extensions to Guinea of Central African genera and families.

Two threatened new species of Podostemaceae belonging to the genus Inversodicraea, I. joulei and I. lebbiei, both from the Republic of Sierra Leone, are described and illustrated. A first record in Sierra Leone of the genus Lestestuella is also reported. Inversodicraea is the most species-rich genus of Podostemaceae in Africa and now comprises 38 species. Inversodicraea joulei is easily recognised because it has a persistent spine distally on the median rib of each fruit valve, and scattered, membranous scale-leaves with broadly rounded apices, while Inversodicraea lebbiei is distinct in having narrowly triangular robust scale-leaves which are inrolled, spreading distally, and completely covering the stem, arranged in five ranks. Inversodicraea joulei is known from a single location with three sites while I. lebbiei is known from two locations each with one site. Using the latest IUCN Red List guidance, Inversodicraea joulei is assessed as Critically Endangered and I. lebbiei is assessed as Endangered, due to threats from dam construction projects, agricultural practices and mining activities, resulting in high levels of siltation on rocks in the fast-flowing rivers where these species grow.

Successive dengue virus (DENV) outbreaks can progressively reshape population immunity influencing disease expression and diagnostic performance. Objectives The aim was to evaluate the impact of secondary infections across sequential outbreaks on clinical severity, serotype dynamics and diagnostic concordance. Methods This retrospective study analyzed 976 febrile-stage samples from three sequential outbreaks in Misiones, Argentina. For serotyping and clinical analyses, 869 viremic samples confirmed by at least one direct method were included (2016: n=512; 2019: n=148; 2024: n=209). Additionally, 318 samples, including 107 non-viremic cases, were used to compare NS1 rapid diagnostic tests (NS1 Ag) and RT-PCR. Viral serotyping and clinical and laboratory markers of disease severity were evaluated. Results Secondary infections increased from 31.05% (2016) to 43.24% (2019) and 53.87% (2024) (p<0.0010). Serotype distribution shifted from DENV-1 predominance in 2016 (95.12%), DENV-1/DENV-4 co-circulation in 2019 (60.71%/39.29%), and DENV-2 predominance in 2024 (97.60%). Secondary infections were associated with more severe disease manifestations, particularly in 2024, with higher hematocrit (p=0.0120) and hemoglobin (p=0.0080), lower white blood cells (p=0.020) and platelet counts (p=0.0030), and elevated AST (p=0.0007) and ALT (p=0.0130). Concordance between NS1 Ag and RT-PCR was lower in secondary infections (k=0.457 vs k=0.759, p=0.0013). Conclusions The rising frequency of secondary infections may affect both clinical severity and diagnostic performance during outbreaks. The clinical impact was more evident in 2024, likely associated with the introduction of a new serotype. These findings highlight the need for optimized surveillance and diagnostic strategies to improve case detection and patient management during epidemics.