Infection, Genetics and Evolution

Carbapenem-resistant (CR) bacteria have emerged and been spreading beyond healthcare-associated facilities into the environment. It is recognized that toilet bowl water in patient rooms of healthcare-associated facilities can be one of internal reservoirs of CR bacteria. In accordance with this idea, toilet bowl water samples were collected from patient rooms in a tertiary healthcare-associated facility in North Macedonia, and meropenem (MEM)-resistant bacterial isolates were obtained from the toilet bowl water. In this study, because a MEM-resistant C. braakii isolate, that was one of MEM-resistant opportunistic pathogens, was obtained from the toilet water, whole-genome sequencing (WGS) of this isolate was performed to obtain genetic characteristics of the blaNDM-1-positive C. braakii isolate. By the WGS, four contigs were constructed, the longest contig, contig 1 (5,189,681 bp), contained blaCTX-M with some additional antimicrobial-resistance genes (ARGs). Interestingly, blaNDM-1 was detected in contig 2 (177,260 bp) and contig 3 (64,168 bp). Plasmid replicon of contig 2 was IncA/C2 but plasmid replicon of contig 3 was IncN and different from one of contig 2. Genetic structures surrounding blaNDM-1 were different between these two blaNDM-1-positive plasmids implying transfer or insertion of blaNDM-1 had occurred by IS or other mechanism. Further molecular epidemiology will be needed to explain the mechanism that allowed the C. braakii isolate to possess two structurally different blaNDM-1 plasmids.

In the past decade, dengue fever has emerged as a major public health on Reunion Island in the Southwest Indian Ocean. During the 2018-2022 outbreak, an unusual increase in ocular complications was reported in some patients. To investigate a potential viral cause, we analysed 447 blood samples from hospitalized patients with and without ophthalmic symptoms. Genetic sequencing revealed the co-circulation of two strains of dengue virus serotype 1, both genetically linked to strains previously identified in Asia. Notably, all patients with ophthalmic symptoms were infected with viruses from a single cluster within genotype I, which harbored several unique mutations. These findings suggest that the rare ocular complications observed during this outbreak may be associated with specific viral cluster. Further laboratory studies are required to confirm this potential link.

The Entamoeba histolytica/dispar/moshkovskii complex comprises morphologically indistinguishable species, making accurate diagnosis difficult and leading to overestimations in amebiasis prevalence. This study aimed to establish a sensitive and specific molecular method to differentiate these species in human fecal samples from Coyaima, Tolima, Colombia. A polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA (18S rRNA) gene was developed and optimized under various amplification conditions. The nested PCR showed higher sensitivity and specificity than the multiplex approach, achieving a detection limit of 160 picograms of E. histolytica DNA. Cross-reactivity tests confirmed the methods specificity, as no amplification was observed with other intestinal protozoa or helminths. Epidemiological analysis revealed a prevalence of 24.54% for E. dispar, 1.04% for E. histolytica, and 0.78% for mixed E. histolytica/dispar infections, while E. moshkovskii was not detected in the study area. These findings demonstrate the effectiveness of the optimized protocol as a reliable molecular tool for differential diagnosis and epidemiological surveillance of the E. histolytica/dispar/moshkovskii complex, contributing to improved understanding of its distribution and transmission dynamics in endemic Colombian populations.

Age related macular degeneration is known to be one of the major causes of irreversible blindness among the older generation. We present a mathematical model of partial differential equations for the therapy of this disease, which is based on the intravitreal injection of a drug into the vitreous body. For the treatment to work, the drug has to travel past the inner-limiting membrane into the retina and reduce the free vascular endothelial growth factor (VEGF) concentration by binding to at least one of the two binding sites of the VEGF molecule. Therefore, our model consists of two compartments, the vitreous and the retina. In the vitreous we employ four coupled convection-diffusion-reaction equations with an additional coupling to the underlying aqueous humor flow and four coupled diffusion-reaction equations in the retina. The resulting PDE system is solved numerically in a realistic 3D eye geometry. Temporal discretization is based on one-step theta schemes and spatial discretization is done using the Finite Element method. The numerical results are used to demonstrate the therapy concept and to analyze the drug efficacy of aflibercept and ranibizumab. The results show, among other things, that only about 20 % of the drug reaches the retina through the inner-limiting membrane and that 50 % of the VEGF concentration has been rebuilt in the retina after 38.19 days for a single ranibizumab injection.

We genotyped 1189 multidrug-resistant Mycobacterium tuberculosis isolates identified during 2013-2022 in Argentina, through a mixed strategy using PCR-based methods and whole-genome sequencing. Epidemiological, geographic distribution and microbiological data were integrated. Most cases belonged to a cluster (75.7%). The proportion of orphan and clustered cases varied across regions. The Euro-American lineage4 was virtually predominant. The most important clusters, M, Ra, Rb and Callao2 strains, comprised 45.9% of the newly diagnosed cases, and their relative importance varied over time. A preliminary genomic analysis showed that several local transmission chains due to the Callao2 strain, imported from Peru, were active, including a superspreading event that occurred circa 2020. A good performance of the current second-line regimes can be expected for most of the cases. Heightening suspicion of drug-resistance and enhancing timely and active surveillance in specific risk groups could contribute to the tuberculosis management in Argentina, tackling transmission and resistance amplification. BiosketchBiochemist Federico Lorenzo is a professional of the Servicio de Micobacterias, Departamento de Bacteriologia, INEI, ANLIS "C. G. Malbran" and is specialized in the microbiological diagnosis of mycobacterial diseases using next-generation sequencing technologies. His research interests are drug-resistant tuberculosis, non-tuberculous mycobacteria and bioinformatic analysis applied to diagnostics. One-sentence summaryWe evaluated the genotypes associated with multidrug-resistant tuberculosis in Argentina, 2013-2022.

Since 2014, Cubas National Tuberculosis Control and Elimination Program (PNCET, acronym in Spanish) has been using the Xpert(R) MTB/RIF technique (Cepheid, Sunnyvale, CA, USA) for rapid molecular diagnosis of tuberculosis (TB). Due to financial limitations and difficult access to kits and cartridges (both of which are of U.S. origin), this test is only intended for prioritized vulnerable groups. Thus, only one third of the cases reported annually are diagnosed by rapid tests, as an initial technique. The scope of Xpert diagnosis can be increased by using the pooling sputum method, where several sputum samples from different persons are pooled and applied in a single Xpert(R) MTB/RIF Ultra (or simply Xpert Ultra) cartridge. This method of pooling sputum with the Xpert Ultra has been used with samples from symptomatic respiratory patients from countries with a high burden of disease, and its use could be extended to household contacts without expectoration, over 8 years of age. Sputum may not always be produced spontaneously in people with incipient, asymptomatic disease, and children often have problems expectorating. The use of a positive expiratory pressure (PEP) device, Lung Flute (LF) ECO (Acoustic Innovation, Tokyo, Japan), helps to produce sputum more easily and its result could be very similar to the use of a hypertonic solution. To increase rapid molecular diagnosis in people with suspected TB and at an early stage of the disease, this project aims to: 1. evaluate the pooled sputum method and the Xpert Ultra molecular assay in the rapid diagnosis of TB in household contacts of cases with bacteriologically confirmed pulmonary TB. 2. Estimate the proportion of household contacts that are able to produce a sputum sample suitable for Xpert Ultra using the LF ECO device. In Latin America, Xpert(R) MTB/RIF, and more recently Xpert Ultra, is used in almost all countries as the first TB diagnostic technique, but the percentages are still low, mainly due to the scarce economic resources available in the region. Therefore, the use of novel strategies, such as the use of sample mixtures pooled in a single Xpert Ultra cartridge, will increase the coverage of testing for TB cases, reduce the sources of infection in the community and reduce the costs of cases diagnosed by rapid tests. This study will provide important information on the combination of a powerful molecular tool (Xpert(R) MTB/RIF Ultra) for TB diagnosis, the application of a pooling sputum method and the use of a PEP device, Lung Flute ECO, in household contacts with and without respiratory symptoms. The synergistic methods in this study could serve as a reference for other countries in the process of TB elimination. These countries need to vastly increase their testing to find a rapidly shrinking number of cases, and our efficient strategy represents a potentially sustainable way to do this. Low income countries face challenging hurdles to acquire sufficient imported proprietary consumables for high volume testing. Pooling is increasingly viewed as a way to promote equity and access in settings without preferential pricing or favorable trade relations.

Drug-resistant tuberculosis (DR-TB) remains a major health concern, especially in high-burden areas like Gabons Haut-Ogooue region. This study investigates the prevalence of drug-resistant tuberculosis (TB) and associated factors in the Haut-Ogooue region. Drug resistance poses a significant threat to global TB control efforts. We conducted a retrospective and prospective study at Centre Hospitalier Universitaire de Franceville from August 2022 to August 2024. A total of 1,792 participants were included. Sputum samples were collected and analyzed using GeneXpert MTB/RIF ULTRA and MTB/XDR assays. Data on demographic characteristics, HIV status, and previous TB treatment history were collected and analyzed using R software. The majority of participants were male (62.1%). Multidrug-resistant TB (MDR-TB) was the predominant resistance profile (94.44% of resistant isolates). A significant association was found between previous TB treatment history and drug-resistant TB (p < 0.001). No significant associations were observed between drug-resistant TB and sex or age. A high proportion of participants had unknown HIV status (47.8%), and most resided in urban areas (85.5%). The high prevalence of MDR-TB and its association with previous TB treatment highlight the urgent need to strengthen TB control programs, improve treatment practices, and address the social determinants of TB in Gabon.

Campylobacter jejuni is a zoonotic bacterium causing foodborne gastroenteritis in humans worldwide, whose main symptom is diarrhea. The infection is severe mostly in children and in immunocompromised individuals. Currently, the bacterium has become increasingly resistant to antibiotics, especially those first-choice drugs used to treat campylobacteriosis posing a significant health threat towards the treatment outcomes. The burden of campylobacteriosis and Antimicrobial Resistance (AMR) remains significant with limited genomic surveillance. This study aimed to characterize the resistome (ARGs), virulence factors as well as population structure across Homo sapiens, Milk (from dairy cattle), goat, Bos indicus, Ovis aries, and Gallus gallus in three countries Ethiopia, Kenya and Tanzania through the use of One Health Whole Genome Sequencing (WGS) approach. A total of 161 C. jejuni publicly available WGS were retrieved from NCBI database and analyzed by using established WGS bioinformatics pipelines from genome assembly and annotation, AMR gene identification via ResFinder -ABRIcate, virulence genes were detected via ABRicate/ VFDB. Visualization of gene distribution and population structure were done using heatmap, Venn diagrams, principal component analysis and minimum spanning tree for comparative analysis. Out of 161 C. jejuni WGS, 130 (80.75%) sequences were positive to one or more than one ARGs. Among detected ARGs, the resistome was dominated by {beta}-lactam (blaOXA 193, blaOXA-61, blaOXA-184 and blaOXA-489) genes. Two genes linked to tetracycline resistance (tet(O/32/O), and tet(O) were found in Ethiopia and Tanzania while resistance to aminoglycoside ant (6)-Ia was the least detected. The Gallus gallus-Homo sapiens transmission (zoonotic transmission) was portrayed by the overlap of ARGs (blaOXA-193 and tet(O) and PCA clustering. The conserved virulence gene profiles were shared by all isolated (cadF, jlpA, cdtA, cdtB, cdtC and flagellar genes). The present study adds to the current knowledge on molecular epidemiology and AMR development in C. jejuni species in Eastern African countries and globally. The findings underscore the need for sustained region-specific genome surveillance under One Health framework to inform AMR stewardship and public health interventions.

IntroductionDengue has been prevalent in a regular fashion in Bangladesh and Chattogram for the last 6-7 years and is showing some serotype twisting. So, the objectives of the present study were to explore the burden of dengue serotypes in Chattogram. MethodsIn this study, 223 Dengue RT-PCR positive patients were evaluated for serotyping. Gender and age group, along with cycle threshold (CT) values, were also collected. Data after collection were compiled, analyzed, and plotted in Microsoft Excel and GraphPad Prism 10.4. Ethical clearance was taken to conduct the study. ResultsAmong 223 patients analyzed, males and females were found near equal (113 and 110). Middle-aged patients were more than the extremes of age. The mean {+/-} SD of age was 33.55 {+/-} 13.67 years. Regarding serotype distributions, isolated Den 1, Den 2 and Den 3 were found 1.3%, 73.1% and 6.7%, respectively. Concurrent infections with multiple serotypes were observed in several patients, most notably the Den 2 and Den 3 combination, which accounted for 14.3% (n=32) of the cases. Other co-infections were less frequent: the Den 1 and Den 2 pairing appeared in 3.6% (n=8) of the cohort, while triple-serotype infections (Den 1, 2, and 3) and Den 3/Den 4 pairings were rare, each occurring in only 0.4% of patients. Statistical analysis of CT values revealed no significant sex-based differences for Den 2 and Den 3. However, significant variations in CT values were observed when comparing Den 1 against both Den 2 and Den 3 (p < 0.05). In contrast, the difference between Den 2 and Den 3 Ct values remained statistically insignificant. ConclusionIn the year 2025, Dengue serotypes 2 and 3 were found to be the most prevalent, both in isolated or in combinations and Den 1 and Den 4 were found minimum. Exposure to multiple serotypes and twisting from one serotype to another might influence the dengue outcome in future, which needs further exploration.

IntroductionHemophagocytic lymphohistiocytosis (HLH) is a serious condition induced by Dengue virus which becomes fatal if not detected early and treated appropriately. So objectives of the present study are to observe the different patterns of presentations, clinical features and outcome of HLH induced by Dengue. MethodsIn this observational study, 14 patients admitted and diagnosed HLH as per diagnostic criteria, were included after informed written consent. Study conducted in a period of six months from 01/07/2025 to 31/12/2025. All patients were followed up till discharge. After collection, all data were analyzed by Microsoft Excel 2010. Ethical clearance was taken from Ethical Review Board of the Medical College. ResultsAmong 14 cases, male were more affected then the female (78.6% VS 21.4%) and majority were in between 20 to 50 years age groups. Clinical data showed, all 14 cases had fever for >7 days, joint pain 3(21.4%), headache 11(78.6%), skin rashes 10(71.4%), retro-orbital pain 2(14.3%), vomiting 11(78.6%),bleeding 10(71.4%), cough 4(28.6%), loose motion 9(64.3%), abdominal pain 7(50.0%), anorexia 2(14.3%), Melaena 2(14.3%), jaundice 4(28.6%) and spleenomegaly 9(64.3%). One(7.1%) case had history of Hypertension. Laboratory data showed different level of Bi or Pancytopenia, high ferritin, high TG, low fibrinogen, raised liver enzymes and low sodium. Dengue RT PCR and serology results showed 8(42.9%) cases were both IG M and Ig G dengue antibody positive, 6 cases were RT PCR positive, 2 cases were IgM and another 4 cases were IgG positive. Outcome of patients revealed, among all 14 cases12(85.8%) patients improved uneventfully and 2 were shifted to ICU where one improved and one died. ConclusionDengue is prevailing for long time and different complications are evolving and HLH is a relatively newer incident among the dengue patients. Infection by different serotypes at different time or multiple dengue serotype infection may be related with HLH and it might be a future subject to explore and to evaluate.

BackgroundChildren who are HIV-exposed uninfected (HEU) show greater morbidity and mortality than HIV-unexposed children (HUU). In this study we investigate sex differences in growth, infection rates and antibody response among HEU and HUU infants. MethodsThe study enrolled 107 pregnant women with HIV and 103 pregnant women without HIV with follow-up of their infants from birth to 12 months of age. Study measures assessed included growth parameters, the prevalence of children with overt disease symptoms as reported by the mother, PCR-based assessment of infections (cytomegalovirus (CMV), respiratory syncytial virus (RSV), rhinovirus, influenza A & B, rotavirus and malaria) as well as antibody profile to CMV, RSV and enterovirus infections. ResultsCompared to male HUU, male HEU infants had lower Height-for-age-z-scores ({beta} -0.75; P=0.047) in mixed-effect model accounting for age. Additionally, they showed transiently lower Weight-for-age-z-scores at 3 months (1.07 vs 0.05, P=0.04), with higher risk of rhinorrhea (RR=2.29, P=0.02) and lower enterovirus titers at birth (P=0.0066). Female HEU showed transiently higher stunting at 6 months (0% vs 21%; P=0.01) and lower CMV viremia at 6 months, with elevated CMV antibody titers at 3 months (P=0.04) compared to female HUU. With prevalence ranging from 25%-61%, CMV and Rhinovirus infections were dominant in all groups. HEU and HUU exhibited similar antibody decay and acquisition patterns for CMV, RSV, and Enterovirus across both sexes. ConclusionHEU infants show transient sex-based differences in growth, infection and immune profiles raising the relevance for considering sex as a key parameter to assess infant health.

This paper looked at the exploration of Lassa fever transmission dynamics through stochastic models which yielded valuable insights into the interplay of factors influencing the probability of extinction and persistence of the virus within a population. By embracing the inherent randomness and variability in the system, the model provided a more realistic representation of the complex ecological and epidemiological dynamics of Lassa fever. We developed the deterministic model using a system of ordinary differential equations and the stochastic model using the Continuous Time Markov Chain. The probability of extinction and persistence underscored the need for a proactive and flexible approach to public health management. Our study revealed that introducing Lassa virus at the onset of an epidemic through various routes affects the likelihood of pathogen extinction. The presence of multiple infection routes increased the probability of pathogen persistence, highlighting complex transmission dynamics. Variations in contact rates, particularly between susceptible rodents and the environment community pathogen load, play a crucial role in influencing pathogen dynamics. This interconnected nature of transmission pathways underscores the factors governing Lassa virus persistence or extinction in a population, providing valuable insights for targeted management and control strategies for Lassa fever.

Microbial keratitis is a sight-threatening corneal infection with varying etiological agents, primarily bacteria and fungi. Assessing and contrasting the virulence factors of microorganisms isolated from a non-contact lens-associated keratitis (NCLAK) and contact lens-associated keratitis (CLAK) is the goal of the current investigation. Samples were collected from over 60 patients and analysed using standard microbiological techniques, including culture, Gram staining, KOH mount, biochemical tests, antimicrobial susceptibility testing, and biofilm assays. The results demonstrated that CLAK isolates were predominantly bacterial, especially Pseudomonas aeruginosa, known for strong biofilm production and high multidrug resistance. In contrast, NCLAK showed a higher incidence of fungal infections, particularly Candida albicans. The results highlight the significance of early diagnosis, tailored and improved awareness regarding contact lens hygiene to prevent complications associated with keratitis.

Influenza A(H3N2) subclade K virus was detected in Canada early in the 2025/26 influenza season, bearing an antigenic transition in the hemagglutinin (HA) glycoprotein. Analysis of 396 HA sequences from Canada showed antigenic divergence from 2025/26 influenza vaccine strains, consistent with partial mismatch. Phylodynamic analysis revealed sustained pre-vaccine transmission without clear post-vaccine expansion. Phylogenetic and phylogeographic analyses indicated interprovincial mixing within a highly connected metapopulation, highlighting the value of genomic surveillance for real-time epidemiologic inference and public health decision-making.

The measles outbreak in Jalisco, Mexico (January-February 2026) experienced vigorous sustained transmission with an exponential growth rate = 0.10 (95% CI: 0.10-0.11) per day, doubling time = 6.3 days (95% CI: 6.3-6.9), yielding the effective reproduction number at 3.34 (95% CI: 3.16-3.54), with elevated incidence among infants and young adults.

BackgroundMore than 6.2 million people have died already from COVID-19. Drug resistance and relapse cases from first generation therapeutics calls for development of new drugs in alternative medicine. Complementary and Alternative Medicines (CAM) that include herbal remedies and phytochemicals are usually not fully integrated into mainstream healthcare systems. The study proposes a CAM remedy, a new polyherbal formulation NAOQ19 against the SARS-CoV-2. MethodsThe present study consists of invitro and invivo evaluation of NAOQ19 against SARS-CoV-2 infection. First, invitro testing of NAOQ19 anti-viral activity was carried out on three relevant cell lines: Vero E6, A549ACE2 and Huh 7.5.1 ACE2TMPRSS2. Next, animal model testing of NAOQ19 was performed in Syrian golden hamsters along with positive control Remdesivir and infection control for 3 days to determine the efficacy and safety of the formulation. Finally, a double blind randomized clinical trial with mild to moderate COVID-19 infected patients were evaluated to test the efficacy of NAOQ19 in human settings. ResultsThis study demonstrated a strong anti-viral (low EC50) activity in cell culture with live virus and exhibits reduced plaque forming units (high antiviral activity) in the Syrian golden hamster model. Moreover, in the clinical trials, NAOQ19 shows high efficacy demonstrating early recovery and reduced levels of inflammatory biomarkers among COVID-19 infected patients. ConclusionThis novel polyherbal formulation NAOQ19, demonstrates strong anti-viral activity in preclinical and clinical study; thereby proving its candidacy as a low-cost alternative medicine with minimal adverse effects.

A new H3N2 variant (named subclade K) possesses several key hemagglutinin substitutions and is circulating widely during the 2025-2026 influenza season. In this report, we completed experiments to determine if the 2025-2026 seasonal influenza vaccine elicits antibodies in humans that recognize this variant. We find that H3N2 subclade K viruses are antigenically advanced; however, the 2025-2026 seasonal influenza vaccine elicited antibodies in many individuals that efficiently recognized these viruses. Thus, the current seasonal influenza vaccine will likely be somewhat effective at preventing H3N2 subclade K virus infections.

The emergence of vaccine covered serotypes causing invasive pneumococcal disease (IPD) is a serious concern worldwide. We investigated the unexpected rise of serotype 4 causing IPD primarily in non-vaccinated young adults after the COVID-19 pandemic that further spread to adults [&ge;] 65 years in recent years. For this purpose, we conducted a retrospective study of serotype 4 IPD cases (n=827) reported in Spain between 2009 and 2024. Whole-genome sequencing was performed to assess clonal lineages and phylogenetic relationships. Clinical and epidemiological data were compared between serotype 4 and all other serotypes causing IPD. Epidemiological and genomic analysis confirmed that the rise started as an abrupt cluster of IPD cases in Seville (Andalusia) in the year 2022 due to the ST15063 within GPSC12 lineage. This outbreak initially caused pneumonia episodes that required hospitalization in young individuals associated with high rates of tobacco smoking, alcohol, and inhaled drugs such as cannabis and cocaine, followed by a general distribution pattern throughout the country in the following years, affecting the elderly population. Experimental studies to evaluate potential underlying mechanisms confirmed that ST15063 serotype 4 strains displayed enhanced infection rates of human lung cells that significantly increased in the presence of cigarette smoke exposure and by influenza H3N2 virus coinfection, but not with H1N1. These findings highlight the need for targeted vaccination strategies not only against pneumococcus but also against respiratory viruses such as influenza, RSV and COVID-19 and demonstrate the importance of molecular surveillance to establish effective interventions in high-risk populations.

Malaria remains a major public health concern in Cameroon, with Plasmodium falciparum responsible for most morbidity and mortality, particularly among children under five. In response to rising cases, Cameroon began implementing the RTS,S/AS01 malaria vaccine in early 2024. Given the vaccines strain-specific efficacy, understanding antigenic diversity and complexity of infection (COI) is critical for evaluating long-term impact. We analyzed 100 P. falciparum-positive dried blood spots collected in Mapoussere, Kaele Health District (2022-2023). Using the 4CAST amplicon sequencing assay, we targeted four genes: csp (circumsporozoite protein), ama (apical membrane antigen 1), sera2 (serine repeat antigen 2), and trap (thrombospondin-related anonymous protein). Haplotypes were identified using SeekDeep, and diversity metrics including heterozygosity (He), nucleotide diversity ({pi}), and selection statistics (Tajimas D, Fu and Lis D*, F*) were computed. We successfully genotyped csp in 35% of samples, identifying 22 haplotypes (He = 0.908; {pi} = 0.021). The vaccine-matched haplotype was present in 20% of genotyped infections. The T cell TH2 and TH3 epitopes of csp showed signs of balancing selection. Both ama and sera2 exhibited higher genotyping success and diversity, with ama showing significant Tajimas D values. COI was highest for ama (mean COI = 2.8), followed by sera2 (2.1), csp (1.3), and trap (1.2). This study provides a baseline of P. falciparum antigenic diversity and COI in a vaccine-targeted region. The presence of vaccine-matched strains and high diversity in TH2/TH3 epitopes in csp may influence vaccine efficacy. Continued molecular surveillance is essential to monitor antigenic shifts and guide future strategies.

ObjectiveWest Nile virus lineage 2 (WNV-2) is a growing public health concern in Europe causing West Nile fever or West Nile neuroinvasive disease (WNND) with substantial morbidity and mortality; however, genomic data from Southern Italy are limited despite recent expansion of autochthonous transmission. The aim of the study was to characterise the phylogenetic and molecular features of the WNV-2 strain responsible for the first autochthonous human infection reported in Calabria (2023), and two more additional WNND cases detected in 2024. MethodsFull WNV-2 genomes were generated from the three cases. Phylogenetic analysis was performed using all publicly available WNV sequences up to September 2025. Amino-acid changes in the polyprotein were compared with known WNV-2 lineage and sub-lineage signatures. ResultsThe three sequences formed a monophyletic group within sub-lineage WNV-2a, clustering with strains circulating in Central-South-Eastern Europe and showing closest affinity to Hungarian sequences. Non-synonymous substitutions characteristic of the Hungary 578/10 ancestor (NS2B-119I, NS4B-14G, NS4B-49A and NS5-298A), were identified and absent from Central-Northern-Western European and previously reported Italian sequences. Additional substitutions (E-159T, E-399R and NS3-249P) corresponded to signatures from a fatal WNV-2 infection in a Great Grey owl in Slovakia. ConclusionsOur study provides the first report of Central-South-Eastern European WNV-2 circulation outside Eastern Europe supporting its likely spread through the Balkans into Italy by 2022. These findings underscore the rapid spread of WNV-2 in newly affected areas and highlight the critical need for sustained molecular surveillance.