Enterovirus D68 circulation in Portugal, 2024–2025

Enterovirus D68 circulation in Portugal, 2024–2025 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enterovirus D68 circulation in Portugal, 2024–2025 Sérgio Santos-Silva, Guilherme Moreira, André Palma, Soraia Rodrigues, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9161762/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Enterovirus D68 (EV-D68) is a reemerging respiratory pathogen that is increasingly associated with severe respiratory disease and occasional neurological complications, yet its circulation in Portugal remains poorly documented. In the present study, we conducted molecular surveillance in southern Portugal (Algarve region) from October 2024 to February 2025. Screening of 150 respiratory samples from patients (115 from children and 35 from adults), all positive for rhinovirus/enterovirus, revealed eight EV-D68–positive cases, corresponding to a detection rate of 5.44%, spanning a broad age range from infancy to the elderly. Clinical manifestations ranged from mild respiratory illness to severe conditions, including pneumonia and acute respiratory distress syndrome. In addition, neurological involvement such as complex febrile seizures and encephalomyelitis was observed. Some cases involved coinfections, while others presented with EV-D68 as the sole detected pathogen. Genomic characterization from one isolate revealed a VP1 fragment belonging to clade B3, and a complete genome from another isolate clustered within clade A2. Phylogenetic analysis confirmed clustering with contemporary European isolates. These findings highlight the circulation of multiple EV-D68 clades in Portugal, underscore the clinical diversity of infections, and reinforce the importance of genomic surveillance to monitor viral evolution and potential public health impact. Non-polio enterovirus EV-D68 (sub)clades A2 and B3 epidemiology surveillance Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Enterovirus D68 (EV-D68) is a reemerging non-polio enterovirus in the family Picornaviridae , first isolated in 1962, that primarily causes respiratory illness yet has increasingly been associated with more severe lower respiratory tract disease and, in rare cases, neurologic complications such as acute flaccid myelitis [ 1 ]. Historically, EV-D68 caused sporadic cases or small clusters of acute respiratory illness but from around 2014 onward, several large outbreaks of acute respiratory illness have been well documented in North America [ 2 – 4 ] and Europe [ 5 – 7 ]. These outbreaks revealed not only elevated numbers of respiratory disease, often among children, but also occasional neurological manifestations, raising concern about viral evolution, diversity, and surveillance gaps [ 8 , 9 ]. Molecular studies have improved the understanding of EV-D68 genotype and clade structure, identifying four main clades (A, B, C, and D), some of which further subdivide into subclades (A1, A2, B1, B2, B3, D1, D2), with subclades such as B3 being especially prominent in recent years [ 10 ]. Complete genome sequencing is still relatively rare in many settings, limiting resolution of viral evolution, transmission dynamics, and potential recombination or mutation events outside highly variable regions like VP1 [ 11 ]. Recent work by the European Non-Polio Enterovirus Network (ENPEN) has shown a marked upsurge of EV-D68 in Europe during the fall–winter 2021–2022 season, with severe acute respiratory distress commonly observed, often presenting with fever and, in some cases, neurological complications, including acute flaccid myelitis diagnosed in young children, alongside the emergence of B3-derived lineages [ 10 , 12 ]. Moreover, sustained circulation of EV-D68 and evolution of these B3-derived lineages (including novel amino acid substitutions) demonstrate the rapid adaptive capacity of EV-D68, even during periods of presumed low viral activity, and have once again been documented across Europe [ 10 ], in the United States of America [ 13 ] and parts of Asia [ 14 – 16 ]. These findings point to both ongoing genetic diversification and the need for improved, harmonized detection and surveillance [ 10 ]. In Portugal, the circulation or genetic diversity of EV-D68 remains largely uncharacterized. To address this gap, we conducted a study in southern Portugal (Algarve region) from October 2024 to February 2025, screening nasopharyngeal samples from children and adults who presented to the emergency department or required hospitalization with acute and severe respiratory infections and tested positive for rhinovirus/enterovirus (RV/EV), in order to detect the presence of EV-D68. We also aimed to characterize the EV-D68 isolates identified at the genomic level by performing amplification of the VP1 region and, when possible, attempting whole genome sequencing. This study will contribute to the limited data available on EV-D68 in Portugal by evaluating its presence, molecular diversity, and phylogenetic relationships with circulating lineages. 2. Materials and Methods 2.1. Sample Collection A total of 150 nasopharyngeal samples were collected from 115 children and 35 adults who attended the emergency department or were hospitalized at Unidade Hospitalar de Faro (Algarve, Portugal) with acute and severe respiratory infections between October 2024 and February 2025. All samples tested positive for Rhinovirus/Enterovirus (RV/EV) using the BioFire® FilmArray® Respiratory 2.1 Panel (BioMérieux). All the nasopharyngeal swab samples collected in viral transport medium were stored at − 80°C until further processing. 2.2. Nucleic Acid Extraction for EV-D68 detection Nucleic acids were extracted from 200 µL of each of the 150 original nasopharyngeal swab samples using the QIAamp Viral Mini Kit (Qiagen, Hilden, Germany) on the QIAcube® automated platform (Qiagen), following the instructions from the manufacturer. The resulting DNA and RNA were eluted in RNase-free water and stored at − 80°C until further processing. 2.3. Detection of EV-D68 and genomic characterization EV-D68 RNA screening was performed in all 150 RV/EV-positive samples using a real-time RT-PCR (RT-qPCR) assay targeting a ~ 94 bp fragment in the VP1 region (NU assay) [ 17 ]. RT-qPCR reactions were performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the Xpert OneStep Fast Probe kit (GRiSP®, Porto, Portugal), according to the manufacturer’s instructions. Thermal cycling included an initial reverse transcription step at 50°C for 15 min, followed by reverse transcriptase inactivation and cDNA denaturation at 95°C for 5 min. Amplification was then carried out over 40 cycles, with denaturation at 95°C for 5 s and annealing/extension at 55°C for 20 s. Data were analyzed using CFX Maestro software version 4.0.2325.0418 (Bio-Rad, Hercules, CA, USA). Further EV-D68 genomic characterization was performed by nested PCR amplification of the VP1 region (~ 590 bp) in all EV-D68–positive samples using the set of primers AN1019, AN1014 and AN1021, AN1022 previously described [ 17 ]. All PCR reactions were performed on a T100 thermocycler (Bio-Rad, Hercules, CA, USA). The first PCR round was carried out using the Xpert One-Step RT-PCR kit (GriSP®, Porto, Portugal), followed by the second round with the Xpert Fast Hotstart Mastermix 2× with dye (GriSP®, Porto, Portugal). Thermal cycling for the first round included cDNA synthesis at 45°C for 15 min, initial denaturation at 95°C for 3 min, then 40 cycles of 95°C (denaturation) for 10 s, 60°C for 10 s (annealing), and 72°C for 15 s (extension), with a final extension at 72°C for 10 min. The second round consisted of an initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C (denaturation) for 15 s, 52°C for 15 s (annealing), and 72°C for 2 s (extension), concluding with a final extension at 72°C for 10 min. The PCR products were visualized by electrophoresis on a 1% agarose gel stained with Xpert Green Safe DNA gel dye (GriSP®, Porto, Portugal) and run at 120 V for 30 minutes. The results were confirmed using a UV transilluminator. Amplicons of the expected size were purified using the GRS PCR & Gel Band Purification Kit (GriSP®, Porto, Portugal). Purified products were then subjected to bidirectional Sanger sequencing using the appropriate specific internal primers for the target gene. The resulting sequences were edited and aligned using BioEdit v7.1.9 and compared with sequences available in the NCBI GenBank nucleotide database, retrieved on 1 September 2025 ( http://blast.ncbi.nlm.nih.gov/Blast ). 2.4. Whole-genome sequencing Sequence-independent single-primer amplification (SISPA) was performed on positive RT-qPCR samples with a previously described protocol [ 18 ]. The cDNA generated through SISPA was sequenced on an Oxford Nanopore Technologies (ONT) PromethION 24 platform using an R10.4.1 flow cell. Libraries were prepared with the Native Barcoding Kit 96 V14 (SQK-NBD114.96). 2.5. Bioinformatic analysis Raw FASTQ reads were basecalled in super-accurate mode using ont-dorado-for-promethion v.7.4.12, with a minimum Q-score threshold of 10, and adapters and barcodes trimmed via MinKNOW. Initial read quality was assessed with NanoPlot v.1.43.0 [ 19 ]. Sequencing adapters and barcodes were further removed using Porechop v.0.2.4 [ 20 ], and reads were filtered with NanoFilt v.2.8.0 [ 19 ] to retain only those with an average quality score of at least 10. Filtered sequencing reads were first screened to remove host- and contaminant-derived sequences. This was done by aligning the reads to reference FASTA files representing the host genome and potential contaminants using Minimap2 [ 21 ]. Reads that failed to align (i.e., unmapped reads) were kept for downstream analyses. These unmapped reads were taxonomically classified with Kraken2 [ 22 ] against the RefSeq viral database retrieved on 1 September 2025 [ 23 ], to identify candidate viral sequences. Reads classified as EV-D68 were then extracted using the extract_kraken_reads.py script [ 22 ] which retrieves reads based on Kraken2 classification results. The extracted EV-D68 reads were subsequently mapped to a reference EV-D68 genome. The reference genome was indexed with Minimap2 (v2.30) [ 21 ], which is optimized for long-read sequencing data such as Oxford Nanopore. Reads were aligned with the map-ont preset, generating a SAM file that was converted to a sorted and indexed BAM file using Samtools (v1.22) [ 24 ], for efficient handling. Genome-wide coverage was then calculated from the BAM file with Pysam (v0.23.3) [ 25 ]. Per-base coverage was computed by iterating through pileup columns and the resulting coverage profiles were generated using an in-house Python script [ 26 ], using Matplotlib [ 27 ]. A consensus sequence was assembled from the aligned reads in regions with sufficient depth and overlap. This consensus sequence was queried against the NCBI nucleotide database using BLASTn [ 28 ] to validate viral identity and assess sequence similarity. 2.6. Phylogenetic analysis The sequences obtained in this study were compared with representative EV-D68 sequences retrieved from GenBank for phylogenetic analysis. Multiple sequence alignment was conducted using MAFFT software version 7.407 [ 29 ]. Maximum-likelihood phylogenetic trees were then inferred with IQ-TREE version 3.0.1 [ 30 ], employing automatic model selection and 1,000 bootstrap replicates to assess node support. Trees were visualized and annotated using the Interactive Tree Of Life (iTOL) platform. 3. Results 3.1. Demographic and clinical characteristics of EV/RV-positive patients From the 150 patients studied, all shared a common finding: they were all positive for RV/EV. The study population consisted of 115 children and 35 adults. The children’s ages ranged from 1 week to 17 years, with a median age of 12 months and a mean age of approximately 2 years and 7 months. The group was predominantly male ( n = 75). The adult group age ranged from 20 to 100 years with a median age of 58 years, and a mean of approximately 60 years and 2 months. From this group 19 were males and 16 females. The most frequent clinical signs in children at hospital admission included fever ( n = 39), cough ( n = 14), bronchiolitis ( n = 7), broncospasm ( n = 5) and asthma ( n = 5). In addition, more severe clinical manifestations were documented, including acute respiratory distress syndrome (ARDS) ( n = 1), complex febrile seizures ( n = 1), and other serious conditions such as autoimmune encephalitis ( n = 1). For adults the most frequent clinical signs at hospital admission included pneumonia ( n = 5), fever ( n = 3), bronchiolitis ( n = 1), cough ( n = 1), difficulty breathing ( n = 2), and asthma or chronic lung disease ( n = 2 ). In addition, more severe manifestations were documented, including hypocalcemia ( n = 1), diabetic ketoacidosis ( n = 1), neutropenia with fever ( n = 1), and immunosuppression ( n = 2). In addition to testing positive for RV/EV, co-infections with other respiratory viruses were identified in some patients, including adenovirus ( n = 22), respiratory syncytial virus (RSV) ( n = 8), parainfluenza viruses types 1, 2, and 3 ( n = 8), influenza A viruses (H1 and H3) ( n = 5), influenza B virus ( n = 4), coronaviruses NL63 ( n = 3) and OC43 ( n = 3), human metapneumovirus ( n = 3), and SARS-CoV-2 ( n = 1). 3.2. Demographic and clinical characteristics of EV-D68 positive patients Among the 150 RV/EV-positive samples analyzed for EV-D68, eight tested positive, five were from children and three from adults (Table 1 ). Table 1 Clinical and virological characteristics of the eight EV-D68–positive patients, including age, sex, clinical presentation, coinfections, sample collection date, and viral clade/subclade when available. Sample ID Age Sex Clinical presentation Coinfections Collection date Clade/subclade detected (Accession number) 1 8M Male ARDS - November 2024 - 2 1Y Male Tachypnea, difficulty breathing and cough Adenovirus December 2025 B/B3 (PX277134) 3 2Y Male Global polypnea, bronchospasm, persistent cough Adenovirus December 2024 - 4 2Y Female Complex febrile seizure Adenovirus and RSV January 2025 - 5 8Y Male Encephalomyelitis, persistent fever, global respiratory failure requiring invasive ventilation - December 2024 - 6 68Y Male Pneumonia, global respiratory failure - January 2025 A/A2 (PV418226) 7 80Y Female Severe fatigue and loss of strength in the lower limbs - November 2024 - 8 95Y Female Tracheobronchitis, hypoxemic respiratory failure - November 2024 - ARDS – Acute respiratory distress syndrome; RSV – Respiratory syncytial virus; M – Months; Y – Years. The EV-D68 positive children were aged 8 months, 1 year, 2 years, and 8 years old, comprising four males and one female. Co-infections of EV-D68 and adenovirus were observed in three cases, one of which was also infected with RSV and presented with complex febrile seizures. Noteworthy, the two children with the most severe clinical conditions, one with ARDS and the other with global respiratory failure requiring invasive ventilation, periods of drowsiness, and encephalomyelitis, did not present any co-infections, as such infections associated only with EV-D48. The three adults who tested positive for EV-D68 were 68, 80, and 95 years old, and included two males and one female. No co-infections were identified in this group, and the clinical conditions included tracheobronchitis, pneumonia, global respiratory failure, with one case reporting weakness of the lower limbs. 3.3. Analysis of EV-D68 Strains In order to characterize EV-D68 at the genomic level, nested PCR amplification of the VP1 region (~ 590 bp) was performed on all the eight EV-D68–positive samples. Only one sample, obtained from a 2-year-old child yielded a VP1 gene fragment suitable for analysis. The sequence has been deposited in GenBank under the accession number PX277134 (Table 1 ). Using the SISPA protocol, we successfully recovered a complete EV-D68 genome out of the eight EV-D68 positive samples. The genome obtained from a 68-year-old patient has been deposited in the GenBank database under accession number PV418226 (Table 1 ). Depth-of-coverage analysis of the assembled genome indicated high and relatively uniform coverage across most of the genome (Fig. 1 ). A graphical representation of the coverage confirmed sufficient read depth to ensure high confidence in consensus sequence generation. Three phylogenetic trees were performed to determine the evolutionary placement of the obtained sequences. First, a tree based on the complete VP1 gene was constructed using all available reference sequences from Nextstrain [ 31 ], allowing comparison with a broad set of isolates (Fig. 2 ). In this analysis, the VP1 gene fragment obtained from the child (PX277134) clustered within clade B, subclade B3, while the VP1 gene from the full genome obtained from the 68-year-old patient (PV418226) clustered within clade A, subclade A2. Second, a smaller VP1-based tree was generated using a subset of representative sequences, including five sequences from each clade and subclade available in Nextstrain [ 31 ], to provide a simplified overview of lineage relationships (Fig. 3 ). This analysis confirmed that our sequences belong to the same lineage as shown in the Fig. 2 phylogenetic tree. Finally, a full-genome phylogenic tree was constructed incorporating all available complete EV-D68 genomes from Nextstrain [ 31 ], alongside the full genome recovered in this study PV418226 (Fig. 4 ). The full genome clustered within clade A, subclade A2, along with other European isolates, such was the case with the VP1-only analyses. BLAST analysis of this full genome indicated that it shared 99.57% identity with an EV-D68 full genome respiratory sample isolated from a human in France (PQ612569), and 99.33–99.39% identity with EV-D68 full genome respiratory samples isolated from humans in the USA (PV624835, PV178657), respectively. 4. Discussion EV-D68, a reemerging respiratory pathogen increasingly linked to severe respiratory disease and neurological complications [ 1 , 32 ], has shown genetic diversification and widespread circulation across Europe [ 10 , 12 ], yet its presence in Portugal remains poorly documented. The present study contributes to the limited data on EV-D68 in Portugal by evaluating its presence, molecular diversity, and phylogenetic relationships with circulating European lineages, providing new insights into viral diversity in this region of the Iberian Peninsula, highlighting ongoing surveillance gaps, and underscoring the importance of molecular epidemiology in understanding EV-D68 circulation. Of the 150 patients studied, all of whom were RV/EV-positive, eight tested positive for EV-D68, corresponding to a detection rate of 5.44% in nasopharyngeal samples. The age of patients infected with EV-D68 ranged from 8 months to 95 years, with both sexes represented. Clinical presentations were heterogeneous for both pediatric and adult patients. Among children infected with EV-D68, clinical manifestations included acute respiratory distress syndrome-ARDS (8-month-old, male), tachypnea with difficulty breathing and cough (1-year-old, male), global polypnea with bronchospasm and persistent cough (2-year-old, male), complex febrile seizure (2-year-old, female), and encephalomyelitis with persistent fever and global respiratory failure requiring invasive ventilation (8-year-old, male). Among adults, presentations included pneumonia with global respiratory failure (68-year-old, male), severe fatigue and loss of strength in the lower limbs (80-year-old, female), and tracheobronchitis with hypoxemic respiratory failure (95-year-old, female). This is in line with what has been described in other studies where several international reports confirming that EV-D68 can cause a spectrum of disease ranging from mild illness to severe respiratory failure, in both children and adults, especially in the presence of respiratory comorbidities, such as asthma or chronic lung disease [ 12 , 33 – 36 ]. In children, infection is often associated with respiratory symptoms such as cough, wheezing, shortness of breath, and fever, and it may progress to the need for intensive support, including mechanical ventilation, especially in patients with preexisting asthma or underlying lung disease [ 12 , 34 , 37 – 39 ]. Neurological complications, such as AFM, although rare, have been documented, reinforcing the neurotropic potential of the virus [ 40 – 42 ]. In adults, the clinical presentation is also predominantly respiratory, ranging from mild symptoms to severe respiratory failure, with greater severity observed in older adults and in those with comorbidities such as heart disease, asthma, and chronic obstructive pulmonary disease [ 33 , 36 ]. Symptoms such as cough, shortness of breath, wheezing, chest pain, and fever are common, and hospitalization may be necessary, especially in patients with risk factors. Cases of neurological involvement in adults are rare, but have been reported [ 36 , 41 ]. Importantly, coinfections with adenovirus and/or respiratory syncytial virus were detected only among pediatric patients, whereas in adults EV-D68 was the sole pathogen identified. Molecular characterization was performed on two EV-D68 isolates, one from a 2-year-old patient and the other from a 68-year-old patient, revealing distinct genetic profiles. The VP1 gene fragment isolated from the sampled child patient clustered with subclade B3, while the full genome isolated from the adult was classified as subclade A2. According to Nextstrain representation of isolates over the past six years, the A2 subclade has been predominantly detected in Spain, France, and Italy, with the latter being the closest related sequences. These results provide valuable phylogenetic insight into the circulation of EV-D68 in Portugal, showing consistency with previous studies indicating that, since 2017, only genotypes B3 and A2/D2 have continued to circulate in Europe, as well as in Asia and the United States [ 1 , 10 , 43 ]. Moreover, the Algarve region in southern Portugal [ 44 ], as an international tourist destination, may contribute to the introduction and spread of diverse EV-D68 strains, highlighting the importance of continuous surveillance in areas with high population mobility. The identification of a full genome clustering in clade A2, together with a VP1 fragment from subclade B3 aligns with recent European trends where B3 has been the dominant lineage whilst A2 continues to be observed in circulation [ 32 ]. In 2018, European EV-D68 isolates showed distinct clusters in both B3 and A2, with different phylogenetic origins. Subclade B3 emerged mainly from U.S. and European lineages, whereas A2 was traced back to strains from East Asia [ 32 ]. Interestingly, subclade B3 has been primarily associated with pediatric infections (median age: 5 years), while A2 has been more common in adults (median age: 42 years) [ 45 ], which is in accordance with our results. Complete genome and VP1 fragment analyses confirm the co-circulation of these lineages, reflecting EV-D68 diversity and evolutionary dynamics that have been previously described in Europe [ 32 ]. Although our dataset is too limited to analyze age or clinical severity associations, these findings highlight the need for closer examination of the clinical impact of EV-D68 classical and emerging clades in Portugal. When compared with other European studies, the 5.44% detection rate observed in our study falls within similar ranges, likely due to the use of respiratory samples, including remnant specimens that had previously tested positive for RV/EV. It should be noted that for the diagnosis of EV-D68, respiratory samples are considered the gold standard, however, in cases of neurological involvement, cerebrospinal fluid (CSF) analysis can also provide useful information, although viral detection in the CSF is rare [ 46 , 47 ]. Moreover, the lack of standardized inclusion of EV-D68 in routine respiratory virus panels may contribute to the limited national-level data. For instance, large-scale surveillance during the 2021–2022 upsurge in Europe documented detection rates in respiratory samples ranging from below 5% in some hospital cohorts to over 10% [ 12 ]. Other surveys have also reported detection rates in respiratory or nasopharyngeal samples ranging from 2% to 5% in hospitalized children and exceeding 10% in pediatric outbreak settings [ 48 , 49 ]. These data confirm that the 5.44% rate detected in the present study is consistent with the range reported in various European surveillance settings, some of which also included samples previously positive for RV/EV, as was the case in our study. Furthermore, other surveillance studies have noted substantial variability across countries and seasons, often influenced by differences in sampling strategy, case mix, and testing intensity [ 32 ]. Although our dataset is limited in size and geographic scope, the measurable detection of EV-D68 in Portugal indicates ongoing viral circulation. The antigenic evolution of EV-D68 remains a concern, particularly with amino acid substitutions in the VP1 loops and other surface-exposed protein regions [ 32 ]. European surveillance has demonstrated that these mutations can accumulate rapidly under immune pressure, potentially altering neutralization profiles and population susceptibility [ 50 ]. Future studies incorporating broader sequencing, epitope mapping, and serological assays will be essential to assess whether strains retrieved in Portugal show signs of immune escape or altered pathogenicity. From a public health perspective, our findings carry several implications. First, EV-D68 is confirmed to be circulating in Portugal, and continued surveillance will be required to monitor its prevalence, genetic diversity, and potential clinical impact. Second, sequence-based monitoring must become a priority, since PCR detection alone does not provide the resolution needed to track introductions, clade shifts, or evolutionary dynamics. Finally, integration of molecular data with clinical and epidemiological information will be necessary to determine whether subclades A2 and B3 infections in Portugal show different severity or age distribution, as suggested in other European studies. Also, continuous collaborative data sharing with European partners is essential, given that EV-D68 shows rapid cross-border mixing, with migration rates between countries estimated at multiple introductions per year. This is particularly relevant for southern Portugal, including the Algarve region, which receives migratory flows from Africa and for which clinical and epidemiological surveillance data remain largely lacking. In spite of these results, some limitations should be acknowledged. The present study was season restricted and geographically limited to the Algarve region, preventing assessment of national-level trends or longer-term evolutionary dynamics. In conclusion, this study provides evidence of EV-D68 circulation in Portugal, including detection of clade A2, which has been circulating across Europe. These findings underscore the need to strengthen national surveillance capacity, expand genomic sequencing efforts, and integrate clinical and epidemiological data to better assess the potential burden and public health significance of EV-D68 in Portugal. Declarations Acknowledgments Guilherme Moreira thanks Fundação para a Ciência e a Tecnologia (FCT) for the financial support of their Ph.D work under the scholarships and 2025.05151.BD. Author contributions Sérgio Santos-Silva: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Guilherme Moreira : Writing – review & editing, Methodology, Investigation, Data curation. André Palma : Writing – review & editing, Methodology, Formal analysis. Soraia Rodrigues: Writing – review & editing, Methodology. Margarida Simões : Writing – review & editing. Raquel Guerreiro : Writing – review & editing, Methodology. Maria S.J. Nascimento : Writing – review & editing, Supervision, Investigation, Formal analysis, Conceptualization. João R. Mesquita: Writing – review & editing, Supervision, Investigation, Funding acquisition, Formal analysis, Conceptualization. Funding This research was funded by Fundação para Ciência e Tecnologia (FCT), grant number 2025.05151.BD. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interest The authors declare no competing interests. Conflict of interest All authors declare that they have no conflict of interest. Ethical statement This study was approved by an Ethics Commission (Comissão de Ética para a Saúde da Unidade Local de Saúde do Algarve; reference number: 084/2025), and all participants provided an informed consent. References Grizer CS, Messacar K, Mattapallil JJ (2024) Enterovirus-D68 - A Reemerging Non-Polio Enterovirus that Causes Severe Respiratory and Neurological Disease in Children. Front Virol (Lausanne Switzerland). ;4 Midgley CM, Watson JT, Nix WA, Curns AT, Rogers SL, Brown BA et al (2015) Severe respiratory illness associated with a nationwide outbreak of enterovirus D68 in the USA (2014): a descriptive epidemiological investigation. Lancet Respir Med 3:879–887 Messacar K, Abzug MJ, Dominguez SR (2016) 2014 outbreak of enterovirus D68 in North America. J Med Virol 88:739–745 Drews SJ, Simmonds K, Usman HR, Yee K, Fathima S, Tipples G et al (2015) Characterization of enterovirus activity, including that of enterovirus D68, in pediatric patients in Alberta, Canada, in 2014. J Clin Microbiol 53:1042–1045 Midgley SE, Christiansen CB, Poulsen MW, Hansen CH, Fischer TK (2015) Emergence of enterovirus D68 in Denmark, June 2014 to February 2015. Euro Surveill Bull Eur sur les Mal Transm = Eur. Commun Dis Bull. ;20 Bragstad K, Jakobsen K, Rojahn AE, Skram MK, Vainio K, Holberg-Petersen M et al (2015) High frequency of enterovirus D68 in children hospitalised with respiratory illness in Norway, autumn 2014. Influenza Other Respi Viruses 9:59–63 Poelman R, Schölvinck EH, Borger R, Niesters HGM, van Leer-Buter C (2015) The emergence of enterovirus D68 in a Dutch University Medical Center and the necessity for routinely screening for respiratory viruses. J Clin Virol Off Publ Pan Am Soc Clin Virol 62:1–5 Messacar K, Asturias EJ, Hixon AM, Van Leer-Buter C, Niesters HGM, Tyler KL et al (2018) Enterovirus D68 and acute flaccid myelitis-evaluating the evidence for causality. Lancet Infect Dis 18:e239–e247 Helfferich J, Knoester M, Van Leer-Buter CC, Neuteboom RF, Meiners LC, Niesters HG et al (2019) Acute flaccid myelitis and enterovirus D68: lessons from the past and present. Eur J Pediatr 178:1305–1315 Hirvonen A, Johannesen CK, Simmonds P, Fischer TK, Harvala H, Benschop KSM (2025) Sustained circulation of enterovirus D68 in Europe in 2023 and the continued evolution of enterovirus D68 B3-lineages associated with distinct amino acid substitutions in VP1 protein. J Clin Virol. ;178 Giardina FA, Romano G, Ferrari G, Pellegrinelli L, Seiti A, Galli C et al (2025) A newly developed whole genome sequencing protocol enables early tracking of enterovirus D68 molecular evolution. J Virol Methods 335:115131 Simoes MP, Hodcroft EB, Simmonds P, Albert J, Alidjinou EK, Ambert-Balay K et al (2024) Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021–2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study. J Infect Dis 230:e917–e928 Gummersheimer S, Hayes A, Harrison C, Lee B, Schuster J, Dhar M et al (2023) Prevalence and clinical presentation of EV-D68 infections in Kansas City children during the 2022 season. Diagn Microbiol Infect Dis 107:115992 Xu S, Tang Y, Li M, Zhang L, Su Y, Wang Y et al (2024) Clinical characteristics of Chinese children with EV-D68-associated pneumonia: A single-center retrospective analysis. Pediatr Pulmonol 59:3660–3666 Li Q, Chen X, Ai J, Li L, Li C, Zhu Y et al (2023) Clinical and molecular epidemiologic features of enterovirus D68 infection in children with acute lower respiratory tract infection in China. Arch Virol 168:206 Yun KW, Ahn B, Choi SH, Kang DY, Kim TS, Lee MK et al (2023) First Detection of Enterovirus D68 in Korean Children, September 2022. Infect Chemother 55:422–430 Ikuse T, Aizawa Y, Takihara H, Okuda S, Watanabe K, Saitoh A (2021) Development of Novel PCR Assays for Improved Detection of Enterovirus D68. J Clin Microbiol. ;59 Moreno G, Gage Moreno DO, Sequence-Independent (2020) Single-Primer Amplification of RNA viruses. protocols.io De Coster W, Rademakers R (2023) NanoPack2: population-scale evaluation of long-read sequencing data. Bioinformatics. ;39 Wick RR (2018) Porechop [Internet]. Available from: https://github.com/rrwick/Porechop Li H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094–3100 Lu J, Rincon N, Wood DE, Breitwieser FP, Pockrandt C, Langmead B et al (2022) Metagenome analysis using the Kraken software suite. Nat Protoc 17:2815–2839 Langmead B AWS Public Datasets: Kraken2 Standard and Viral Databases [Internet]. 2025 [cited 2025 Sep 9]. Available from: https://benlangmead.github.io/aws-indexes/k2 Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO et al (2021) Twelve years of SAMtools and BCFtools. Gigascience 10:1–4 Pysam Developers Pysam [Internet]. 2025 [cited 2025 Jun 1]. Available from: https://github.com/pysam-developers/pysam Moreira G PUPI-Plot: Phylogenetically Unique and Popular Identifier Plot [Internet]. 2025 [cited 2025 Sep 1]. Available from: https://github.com/GmoreiraVet/PUPI-Plot/tree/main Hunter JD, Matplotlib (2007) A 2D Graphics Environment. Comput Sci Eng 9:90–95 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K et al (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421 Rozewicki J, Li S, Amada KM, Standley DM, Katoh K (2019) MAFFT-DASH: Integrated protein sequence and structural alignment. Nucleic Acids Res 47:W5–10 Nguyen L-T, von Haeseler A, Minh BQ (2018) Complex Models of Sequence Evolution Require Accurate Estimators as Exemplified with the Invariable Site Plus Gamma Model. Syst Biol 67:552–558 Nextstrain (2025) Enterovirus D68 / genome [Internet]. [cited 2025 Sep 1]. Available from: https://nextstrain.org/enterovirus/d68/genome Hodcroft EB, Dyrdak R, Andrés C, Egli A, Reist J, de Artola DGM et al (2022) Evolution, geographic spreading, and demographic distribution of Enterovirus D68. PLoS Pathog 18:1–18 Esposito S, Bosis S, Niesters H, Principi N (2015) Enterovirus D68 Infection. Viruses [Internet]. ;7:6043–50. Available from: https://consensus.app/papers/enterovirus-d68-infection-esposito-bosis/00999a827fad5aebb15743be3d020c39 / Andrés C, Vila J, Creus-Costa A, Piñana M, González-Sánchez A, Esperalba J et al (2022) Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014–2021. Emerg Infect Dis 28:1327–1331 Ott C, Dutilh G, Reist J, Bingisser R, Egli A, Heininger U (2024) Clinical Presentation of Enterovirus D68 in a Swiss Pediatric University Center. Pediatr Infect Dis J 43:1135–1140 Imamura T, Oshitani H (2015) Global reemergence of enterovirus D68 as an important pathogen for acute respiratory infections. Rev Med Virol 25:102–114 Vermillion MS, Dearing J, Zhang Y, Adney DR, Scheuermann RH, Pekosz A et al (2022) Animal Models of Enterovirus D68 Infection and Disease. J Virol 96:e0083322 Lugo D, Krogstad P (2016) Enteroviruses in the early 21st century: new manifestations and challenges. Curr Opin Pediatr 28:107–113 Waghmare A, Pergam SA, Jerome KR, Englund JA, Boeckh M, Kuypers J (2015) Clinical disease due to enterovirus D68 in adult hematologic malignancy patients and hematopoietic cell transplant recipients. Blood 125:1724–1729 Sooksawasdi Na Ayudhya S, Laksono BM, van Riel D (2021) The pathogenesis and virulence of enterovirus-D68 infection. Virulence 12:2060–2072 Holm-Hansen CC, Midgley SE, Fischer TK (2016) Global emergence of enterovirus D68: a systematic review. Lancet Infect Dis 16:e64–75 Howson-Wells HC, Tsoleridis T, Zainuddin I, Tarr AW, Irving WL, Ball JK et al (2022) Enterovirus D68 epidemic, UK, 2018, was caused by subclades B3 and D1, predominantly in children and adults, respectively, with both subclades exhibiting extensive genetic diversity. Microb genomics. ;8 Midgley SE, Benschop K, Dyrdak R, Mirand A, Bailly J-L, Bierbaum S et al (2020) Co-circulation of multiple enterovirus D68 subclades, including a novel B3 cluster, across Europe in a season of expected low prevalence, 2019/20. Euro Surveill Bull Eur sur les Mal Transm = Eur. Commun Dis Bull. ;25 Maia C, Dionísio L, Afonso MO, Neto L, Cristóvão JM, Campino L (2013) Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal. Mem Inst Oswaldo Cruz 108:481–487 Fall A, Norton JM, Abdullah O, Pekosz A, Klein E, Mostafa HH et al (2024) Enhanced genomic surveillance of enteroviruses reveals a surge in enterovirus D68 cases, the Johns Hopkins health system, Maryland, 2025;63 Knoester M, Helfferich J, Poelman R, Van Leer-Buter C, Brouwer O, Niesters H (2018) Twenty-nine Cases of Enterovirus-D68–associated Acute Flaccid Myelitis in Europe 2016. Pediatr Infect Dis J [Internet]. ;38:16–21. Available from: https://consensus.app/papers/twentynine-cases-of-enterovirusd68–associated-acute-knoester-helfferich/46258e47a046597c993667c2b84420a7 / Harvala H, Broberg E, Benschop K, Berginc N, Ladhani S, Susi P et al (2018) Recommendations for enterovirus diagnostics and characterisation within and beyond Europe. J Clin Virol Off Publ Pan Am Soc Clin Virol 101:11–17 Schuffenecker I, Mirand A, Josset L, Henquell C, Hecquet D, Pilorgé L et al (2016) Epidemiological and clinical characteristics of patients infected with enterovirus D68, France, July to December 2014. Eurosurveillance 21:1–12 Fall A, Kenmoe S, Ebogo-Belobo JT, Mbaga DS, Bowo-Ngandji A, Foe-Essomba JR et al (2022) Global prevalence and case fatality rate of Enterovirus D68 infections, a systematic review and meta-analysis. PLoS Negl Trop Dis 16:1–24 Fang Y, Chen Q, Wang H, Wang L, Rong H, Liao Q et al (2021) The role of conformational epitopes in the evolutionary divergence of enterovirus D68 clades: A bioinformatics-based study. Infect Genet Evol [Internet]. ;93:104992. Available from: https://doi.org/10.1016/j.meegid.2021.104992 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Reviews received at journal 29 Mar, 2026 Reviews received at journal 24 Mar, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers agreed at journal 20 Mar, 2026 Reviewers agreed at journal 19 Mar, 2026 Reviewers invited by journal 19 Mar, 2026 Editor assigned by journal 19 Mar, 2026 Submission checks completed at journal 19 Mar, 2026 First submitted to journal 18 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9161762","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":609482842,"identity":"29d692bb-9b5a-4194-a8f2-dab0c7b29f72","order_by":0,"name":"Sérgio Santos-Silva","email":"","orcid":"","institution":"LAQV, REQUIMTE, University of Porto","correspondingAuthor":false,"prefix":"","firstName":"Sérgio","middleName":"","lastName":"Santos-Silva","suffix":""},{"id":609482843,"identity":"839602d7-f1d9-415f-9ac1-a8c86c4d6d67","order_by":1,"name":"Guilherme Moreira","email":"","orcid":"","institution":"University of Porto","correspondingAuthor":false,"prefix":"","firstName":"Guilherme","middleName":"","lastName":"Moreira","suffix":""},{"id":609482844,"identity":"d0671ff3-7bff-4965-9914-04cc9e609e67","order_by":2,"name":"André Palma","email":"","orcid":"","institution":"Serviço de Patologia Clínica, Unidade Local de Saúde do Algarve – Unidade Hospitalar de Faro","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"","lastName":"Palma","suffix":""},{"id":609482845,"identity":"a5396537-f7ed-4bcd-a0e0-9d4a467495d7","order_by":3,"name":"Soraia Rodrigues","email":"","orcid":"","institution":"University of Porto","correspondingAuthor":false,"prefix":"","firstName":"Soraia","middleName":"","lastName":"Rodrigues","suffix":""},{"id":609482846,"identity":"c85a7055-607b-488b-bad7-c3c2942761f3","order_by":4,"name":"Margarida Simões","email":"","orcid":"","institution":"University of Évora, School of Science and Technology, Dept. Veterinary Medicine, Comprehensive Health Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Margarida","middleName":"","lastName":"Simões","suffix":""},{"id":609482847,"identity":"fb86e2ae-2c6d-496e-8d29-16c4c87a0d34","order_by":5,"name":"Raquel Guerreiro","email":"","orcid":"","institution":"Serviço de Patologia Clínica, Unidade Local de Saúde do Algarve – Unidade Hospitalar de Faro","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Guerreiro","suffix":""},{"id":609482848,"identity":"a81fe0e0-b7d5-41cc-8e1a-da5f3b46431b","order_by":6,"name":"Maria S.J. Nascimento","email":"","orcid":"","institution":"University of Porto (FFUP)","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"S.J.","lastName":"Nascimento","suffix":""},{"id":609482849,"identity":"95f8bcae-9873-49ac-bd11-c052c7badb80","order_by":7,"name":"João R. Mesquita","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACxgYIzcPAwHzgAIxHnBYeBrYEJC0JROjlYeAxYCBKC/Ps04kPf5QdlrFnP/PxwM8ddvYMErkHmAt/4HFYX+5mY55zh3l4eHI3HOw9k5zYIJGXwDwDjy2MPbzbpBnbgFoYcjccZmxjTmCQyDFg5sGvZfvPnyAt/G8eALXU2xOjZRsDL0iLRA4DUMthxgYitGyW5jmXzsNz45kB0C/HE9t43iUcnpGGW4thD+/Gjz/KrO3Z+5Mff/i5o9qenz334OMCGzxaGkAkG5IIGzCKDuPWwMAgz4CuBRSrzPi0jIJRMApGwYgDACOrTfsEMjfqAAAAAElFTkSuQmCC","orcid":"","institution":"LAQV, REQUIMTE, University of Porto","correspondingAuthor":true,"prefix":"","firstName":"João","middleName":"R.","lastName":"Mesquita","suffix":""}],"badges":[],"createdAt":"2026-03-18 16:54:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9161762/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9161762/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105352111,"identity":"8aa2cfbc-7563-460b-b815-ef82d9ec5ce8","added_by":"auto","created_at":"2026-03-25 06:04:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102229,"visible":true,"origin":"","legend":"\u003cp\u003eCoverage depth across the full assembled genome of PV418226 obtained by SISPA amplification and Oxford Nanopore sequencing. The depth of coverage is shown on the y-axis, and the genome position is shown on the x-axis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9161762/v1/621eaadc8909d4ba31e09a76.png"},{"id":105352112,"identity":"93ac4a8c-bad2-471c-a54e-d44185a8e9ee","added_by":"auto","created_at":"2026-03-25 06:04:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":386628,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood phylogenetic tree of EV-D68 based on the full VP1 gene. The tree was inferred using the TIM+F+R4 substitution model (Transition model with empirical base frequencies and a FreeRate model of rate heterogeneity using four discrete rate categories), selected as the best-fitting model according to the Bayesian Information Criterion (BIC). All available Nextstrain VP1 sequences were included to provide a comprehensive overview. The full VP1 sequences obtained in this study (PX277134 and PV418226) are highlighted by red arrows.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9161762/v1/c59c9e62f7c8540ea48ea17b.png"},{"id":105564817,"identity":"fb618e85-e233-4cbf-b88f-e944ccec11b0","added_by":"auto","created_at":"2026-03-27 12:50:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131672,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood phylogenetic tree of EV-D68 based on the full VP1 gene. The tree was inferred using the TN+F+G4 substitution model (Tamura-Nei model with empirical base frequencies and a Gamma model of rate heterogeneity using four discrete rate categories), selected as the best-fitting model according to the Bayesian Information Criterion (BIC). To simplify interpretation, the tree includes only five reference VP1 sequences per clade/subclade. Accession sequence numbers are shown together with clade/subclade designations, and the VP1 sequences obtained in this study (PX277134 and PV418226) are highlighted in green bold.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9161762/v1/9adf98978dcfe56d716107d0.png"},{"id":105352113,"identity":"3a04ecf8-a538-4511-9f9f-9f0af90a8a3c","added_by":"auto","created_at":"2026-03-25 06:04:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":261323,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood phylogenetic tree of EV-D68 based on the full genome. The tree was inferred using theTIM+F+I+R6 substitution model (Transition model with empirical base frequencies and an Invar+FreeRate model of rate heterogeneity using six discrete rate categories), selected as the best-fitting model according to the Bayesian Information Criterion (BIC). The full genome PV418226 obtained in this study is highlighted by a red arrow.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9161762/v1/ee2233e45e7e67541d558601.png"},{"id":105569608,"identity":"d899990a-25ac-4549-ad5e-c80cdaee6842","added_by":"auto","created_at":"2026-03-27 13:12:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1584277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9161762/v1/009424c9-35c1-447a-82eb-c60821e22ac6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enterovirus D68 circulation in Portugal, 2024–2025","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEnterovirus D68 (EV-D68) is a reemerging non-polio enterovirus in the family \u003cem\u003ePicornaviridae\u003c/em\u003e, first isolated in 1962, that primarily causes respiratory illness yet has increasingly been associated with more severe lower respiratory tract disease and, in rare cases, neurologic complications such as acute flaccid myelitis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHistorically, EV-D68 caused sporadic cases or small clusters of acute respiratory illness but from around 2014 onward, several large outbreaks of acute respiratory illness have been well documented in North America [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and Europe [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These outbreaks revealed not only elevated numbers of respiratory disease, often among children, but also occasional neurological manifestations, raising concern about viral evolution, diversity, and surveillance gaps [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMolecular studies have improved the understanding of EV-D68 genotype and clade structure, identifying four main clades (A, B, C, and D), some of which further subdivide into subclades (A1, A2, B1, B2, B3, D1, D2), with subclades such as B3 being especially prominent in recent years [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Complete genome sequencing is still relatively rare in many settings, limiting resolution of viral evolution, transmission dynamics, and potential recombination or mutation events outside highly variable regions like VP1 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent work by the European Non-Polio Enterovirus Network (ENPEN) has shown a marked upsurge of EV-D68 in Europe during the fall\u0026ndash;winter 2021\u0026ndash;2022 season, with severe acute respiratory distress commonly observed, often presenting with fever and, in some cases, neurological complications, including acute flaccid myelitis diagnosed in young children, alongside the emergence of B3-derived lineages [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, sustained circulation of EV-D68 and evolution of these B3-derived lineages (including novel amino acid substitutions) demonstrate the rapid adaptive capacity of EV-D68, even during periods of presumed low viral activity, and have once again been documented across Europe [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], in the United States of America [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and parts of Asia [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These findings point to both ongoing genetic diversification and the need for improved, harmonized detection and surveillance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Portugal, the circulation or genetic diversity of EV-D68 remains largely uncharacterized. To address this gap, we conducted a study in southern Portugal (Algarve region) from October 2024 to February 2025, screening nasopharyngeal samples from children and adults who presented to the emergency department or required hospitalization with acute and severe respiratory infections and tested positive for rhinovirus/enterovirus (RV/EV), in order to detect the presence of EV-D68. We also aimed to characterize the EV-D68 isolates identified at the genomic level by performing amplification of the VP1 region and, when possible, attempting whole genome sequencing.\u003c/p\u003e \u003cp\u003eThis study will contribute to the limited data available on EV-D68 in Portugal by evaluating its presence, molecular diversity, and phylogenetic relationships with circulating lineages.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sample Collection\u003c/h2\u003e \u003cp\u003eA total of 150 nasopharyngeal samples were collected from 115 children and 35 adults who attended the emergency department or were hospitalized at Unidade Hospitalar de Faro (Algarve, Portugal) with acute and severe respiratory infections between October 2024 and February 2025. All samples tested positive for Rhinovirus/Enterovirus (RV/EV) using the BioFire\u0026reg; FilmArray\u0026reg; Respiratory 2.1 Panel (BioM\u0026eacute;rieux). All the nasopharyngeal swab samples collected in viral transport medium were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Nucleic Acid Extraction for EV-D68 detection\u003c/h2\u003e \u003cp\u003eNucleic acids were extracted from 200 \u0026micro;L of each of the 150 original nasopharyngeal swab samples using the QIAamp Viral Mini Kit (Qiagen, Hilden, Germany) on the QIAcube\u0026reg; automated platform (Qiagen), following the instructions from the manufacturer. The resulting DNA and RNA were eluted in RNase-free water and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Detection of EV-D68 and genomic characterization\u003c/h2\u003e \u003cp\u003eEV-D68 RNA screening was performed in all 150 RV/EV-positive samples using a real-time RT-PCR (RT-qPCR) assay targeting a\u0026thinsp;~\u0026thinsp;94 bp fragment in the VP1 region (NU assay) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. RT-qPCR reactions were performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the Xpert OneStep Fast Probe kit (GRiSP\u0026reg;, Porto, Portugal), according to the manufacturer\u0026rsquo;s instructions. Thermal cycling included an initial reverse transcription step at 50\u0026deg;C for 15 min, followed by reverse transcriptase inactivation and cDNA denaturation at 95\u0026deg;C for 5 min. Amplification was then carried out over 40 cycles, with denaturation at 95\u0026deg;C for 5 s and annealing/extension at 55\u0026deg;C for 20 s. Data were analyzed using CFX Maestro software version 4.0.2325.0418 (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003cp\u003eFurther EV-D68 genomic characterization was performed by nested PCR amplification of the VP1 region (~\u0026thinsp;590 bp) in all EV-D68\u0026ndash;positive samples using the set of primers AN1019, AN1014 and AN1021, AN1022 previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. All PCR reactions were performed on a T100 thermocycler (Bio-Rad, Hercules, CA, USA). The first PCR round was carried out using the Xpert One-Step RT-PCR kit (GriSP\u0026reg;, Porto, Portugal), followed by the second round with the Xpert Fast Hotstart Mastermix 2\u0026times; with dye (GriSP\u0026reg;, Porto, Portugal). Thermal cycling for the first round included cDNA synthesis at 45\u0026deg;C for 15 min, initial denaturation at 95\u0026deg;C for 3 min, then 40 cycles of 95\u0026deg;C (denaturation) for 10 s, 60\u0026deg;C for 10 s (annealing), and 72\u0026deg;C for 15 s (extension), with a final extension at 72\u0026deg;C for 10 min. The second round consisted of an initial denaturation at 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C (denaturation) for 15 s, 52\u0026deg;C for 15 s (annealing), and 72\u0026deg;C for 2 s (extension), concluding with a final extension at 72\u0026deg;C for 10 min.\u003c/p\u003e \u003cp\u003eThe PCR products were visualized by electrophoresis on a 1% agarose gel stained with Xpert Green Safe DNA gel dye (GriSP\u0026reg;, Porto, Portugal) and run at 120 V for 30 minutes. The results were confirmed using a UV transilluminator.\u003c/p\u003e \u003cp\u003eAmplicons of the expected size were purified using the GRS PCR \u0026amp; Gel Band Purification Kit (GriSP\u0026reg;, Porto, Portugal). Purified products were then subjected to bidirectional Sanger sequencing using the appropriate specific internal primers for the target gene. The resulting sequences were edited and aligned using BioEdit v7.1.9 and compared with sequences available in the NCBI GenBank nucleotide database, retrieved on 1 September 2025 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih.gov/Blast\u003c/span\u003e\u003cspan address=\"http://blast.ncbi.nlm.nih.gov/Blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Whole-genome sequencing\u003c/h2\u003e \u003cp\u003eSequence-independent single-primer amplification (SISPA) was performed on positive RT-qPCR samples with a previously described protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The cDNA generated through SISPA was sequenced on an Oxford Nanopore Technologies (ONT) PromethION 24 platform using an R10.4.1 flow cell. Libraries were prepared with the Native Barcoding Kit 96 V14 (SQK-NBD114.96).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Bioinformatic analysis\u003c/h2\u003e \u003cp\u003eRaw FASTQ reads were basecalled in super-accurate mode using ont-dorado-for-promethion v.7.4.12, with a minimum Q-score threshold of 10, and adapters and barcodes trimmed via MinKNOW. Initial read quality was assessed with NanoPlot v.1.43.0 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Sequencing adapters and barcodes were further removed using Porechop v.0.2.4 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and reads were filtered with NanoFilt v.2.8.0 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] to retain only those with an average quality score of at least 10.\u003c/p\u003e \u003cp\u003eFiltered sequencing reads were first screened to remove host- and contaminant-derived sequences. This was done by aligning the reads to reference FASTA files representing the host genome and potential contaminants using Minimap2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Reads that failed to align (i.e., unmapped reads) were kept for downstream analyses. These unmapped reads were taxonomically classified with Kraken2 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] against the RefSeq viral database retrieved on 1 September 2025 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], to identify candidate viral sequences. Reads classified as EV-D68 were then extracted using the extract_kraken_reads.py script [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] which retrieves reads based on Kraken2 classification results.\u003c/p\u003e \u003cp\u003eThe extracted EV-D68 reads were subsequently mapped to a reference EV-D68 genome. The reference genome was indexed with Minimap2 (v2.30) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which is optimized for long-read sequencing data such as Oxford Nanopore. Reads were aligned with the map-ont preset, generating a SAM file that was converted to a sorted and indexed BAM file using Samtools (v1.22) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], for efficient handling. Genome-wide coverage was then calculated from the BAM file with Pysam (v0.23.3) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Per-base coverage was computed by iterating through pileup columns and the resulting coverage profiles were generated using an in-house Python script [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], using Matplotlib [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA consensus sequence was assembled from the aligned reads in regions with sufficient depth and overlap. This consensus sequence was queried against the NCBI nucleotide database using BLASTn [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to validate viral identity and assess sequence similarity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe sequences obtained in this study were compared with representative EV-D68 sequences retrieved from GenBank for phylogenetic analysis. Multiple sequence alignment was conducted using MAFFT software version 7.407 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Maximum-likelihood phylogenetic trees were then inferred with IQ-TREE version 3.0.1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], employing automatic model selection and 1,000 bootstrap replicates to assess node support. Trees were visualized and annotated using the Interactive Tree Of Life (iTOL) platform.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Demographic and clinical characteristics of EV/RV-positive patients\u003c/h2\u003e \u003cp\u003eFrom the 150 patients studied, all shared a common finding: they were all positive for RV/EV. The study population consisted of 115 children and 35 adults. The children\u0026rsquo;s ages ranged from 1 week to 17 years, with a median age of 12 months and a mean age of approximately 2 years and 7 months. The group was predominantly male (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;75). The adult group age ranged from 20 to 100 years with a median age of 58 years, and a mean of approximately 60 years and 2 months. From this group 19 were males and 16 females.\u003c/p\u003e \u003cp\u003eThe most frequent clinical signs in children at hospital admission included fever (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;39), cough (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14), bronchiolitis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7), broncospasm (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) and asthma (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5). In addition, more severe clinical manifestations were documented, including acute respiratory distress syndrome (ARDS) (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), complex febrile seizures (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), and other serious conditions such as autoimmune encephalitis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1). For adults the most frequent clinical signs at hospital admission included pneumonia (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5), fever (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3), bronchiolitis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), cough (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), difficulty breathing (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2), and asthma or chronic lung disease (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003e2\u003c/em\u003e). In addition, more severe manifestations were documented, including hypocalcemia (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), diabetic ketoacidosis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), neutropenia with fever (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), and immunosuppression (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2).\u003c/p\u003e \u003cp\u003eIn addition to testing positive for RV/EV, co-infections with other respiratory viruses were identified in some patients, including adenovirus (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22), respiratory syncytial virus (RSV) (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), parainfluenza viruses types 1, 2, and 3 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), influenza A viruses (H1 and H3) (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5), influenza B virus (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4), coronaviruses NL63 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) and OC43 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3), human metapneumovirus (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3), and SARS-CoV-2 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Demographic and clinical characteristics of EV-D68 positive patients\u003c/h2\u003e \u003cp\u003eAmong the 150 RV/EV-positive samples analyzed for EV-D68, eight tested positive, five were from children and three from adults (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical and virological characteristics of the eight EV-D68\u0026ndash;positive patients, including age, sex, clinical presentation, coinfections, sample collection date, and viral clade/subclade when available.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClinical presentation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCoinfections\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCollection date\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eClade/subclade detected (Accession number)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eARDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNovember 2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTachypnea, difficulty breathing and cough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdenovirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDecember 2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eB/B3 (PX277134)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlobal polypnea, bronchospasm, persistent cough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdenovirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDecember 2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eComplex febrile seizure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdenovirus and RSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJanuary 2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEncephalomyelitis, persistent fever, global respiratory failure requiring invasive ventilation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDecember 2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePneumonia, global respiratory failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJanuary 2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA/A2 (PV418226)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSevere fatigue and loss of strength in the lower limbs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNovember 2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTracheobronchitis, hypoxemic respiratory failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNovember 2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eARDS \u0026ndash; Acute respiratory distress syndrome; RSV \u0026ndash; Respiratory syncytial virus; M \u0026ndash; Months; Y \u0026ndash; Years.\u003c/p\u003e \u003cp\u003eThe EV-D68 positive children were aged 8 months, 1 year, 2 years, and 8 years old, comprising four males and one female. Co-infections of EV-D68 and adenovirus were observed in three cases, one of which was also infected with RSV and presented with complex febrile seizures. Noteworthy, the two children with the most severe clinical conditions, one with ARDS and the other with global respiratory failure requiring invasive ventilation, periods of drowsiness, and encephalomyelitis, did not present any co-infections, as such infections associated only with EV-D48.\u003c/p\u003e \u003cp\u003eThe three adults who tested positive for EV-D68 were 68, 80, and 95 years old, and included two males and one female. No co-infections were identified in this group, and the clinical conditions included tracheobronchitis, pneumonia, global respiratory failure, with one case reporting weakness of the lower limbs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Analysis of EV-D68 Strains\u003c/h2\u003e \u003cp\u003eIn order to characterize EV-D68 at the genomic level, nested PCR amplification of the VP1 region (~\u0026thinsp;590 bp) was performed on all the eight EV-D68\u0026ndash;positive samples.\u003c/p\u003e \u003cp\u003eOnly one sample, obtained from a 2-year-old child yielded a VP1 gene fragment suitable for analysis. The sequence has been deposited in GenBank under the accession number PX277134 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUsing the SISPA protocol, we successfully recovered a complete EV-D68 genome out of the eight EV-D68 positive samples. The genome obtained from a 68-year-old patient has been deposited in the GenBank database under accession number PV418226 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDepth-of-coverage analysis of the assembled genome indicated high and relatively uniform coverage across most of the genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A graphical representation of the coverage confirmed sufficient read depth to ensure high confidence in consensus sequence generation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThree phylogenetic trees were performed to determine the evolutionary placement of the obtained sequences. First, a tree based on the complete VP1 gene was constructed using all available reference sequences from Nextstrain [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], allowing comparison with a broad set of isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In this analysis, the VP1 gene fragment obtained from the child (PX277134) clustered within clade B, subclade B3, while the VP1 gene from the full genome obtained from the 68-year-old patient (PV418226) clustered within clade A, subclade A2. Second, a smaller VP1-based tree was generated using a subset of representative sequences, including five sequences from each clade and subclade available in Nextstrain [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], to provide a simplified overview of lineage relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This analysis confirmed that our sequences belong to the same lineage as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e phylogenetic tree. Finally, a full-genome phylogenic tree was constructed incorporating all available complete EV-D68 genomes from Nextstrain [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], alongside the full genome recovered in this study PV418226 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The full genome clustered within clade A, subclade A2, along with other European isolates, such was the case with the VP1-only analyses. BLAST analysis of this full genome indicated that it shared 99.57% identity with an EV-D68 full genome respiratory sample isolated from a human in France (PQ612569), and 99.33\u0026ndash;99.39% identity with EV-D68 full genome respiratory samples isolated from humans in the USA (PV624835, PV178657), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eEV-D68, a reemerging respiratory pathogen increasingly linked to severe respiratory disease and neurological complications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], has shown genetic diversification and widespread circulation across Europe [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], yet its presence in Portugal remains poorly documented. The present study contributes to the limited data on EV-D68 in Portugal by evaluating its presence, molecular diversity, and phylogenetic relationships with circulating European lineages, providing new insights into viral diversity in this region of the Iberian Peninsula, highlighting ongoing surveillance gaps, and underscoring the importance of molecular epidemiology in understanding EV-D68 circulation.\u003c/p\u003e \u003cp\u003eOf the 150 patients studied, all of whom were RV/EV-positive, eight tested positive for EV-D68, corresponding to a detection rate of 5.44% in nasopharyngeal samples. The age of patients infected with EV-D68 ranged from 8 months to 95 years, with both sexes represented. Clinical presentations were heterogeneous for both pediatric and adult patients.\u003c/p\u003e \u003cp\u003eAmong children infected with EV-D68, clinical manifestations included acute respiratory distress syndrome-ARDS (8-month-old, male), tachypnea with difficulty breathing and cough (1-year-old, male), global polypnea with bronchospasm and persistent cough (2-year-old, male), complex febrile seizure (2-year-old, female), and encephalomyelitis with persistent fever and global respiratory failure requiring invasive ventilation (8-year-old, male). Among adults, presentations included pneumonia with global respiratory failure (68-year-old, male), severe fatigue and loss of strength in the lower limbs (80-year-old, female), and tracheobronchitis with hypoxemic respiratory failure (95-year-old, female). This is in line with what has been described in other studies where several international reports confirming that EV-D68 can cause a spectrum of disease ranging from mild illness to severe respiratory failure, in both children and adults, especially in the presence of respiratory comorbidities, such as asthma or chronic lung disease [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In children, infection is often associated with respiratory symptoms such as cough, wheezing, shortness of breath, and fever, and it may progress to the need for intensive support, including mechanical ventilation, especially in patients with preexisting asthma or underlying lung disease [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Neurological complications, such as AFM, although rare, have been documented, reinforcing the neurotropic potential of the virus [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In adults, the clinical presentation is also predominantly respiratory, ranging from mild symptoms to severe respiratory failure, with greater severity observed in older adults and in those with comorbidities such as heart disease, asthma, and chronic obstructive pulmonary disease [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Symptoms such as cough, shortness of breath, wheezing, chest pain, and fever are common, and hospitalization may be necessary, especially in patients with risk factors. Cases of neurological involvement in adults are rare, but have been reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImportantly, coinfections with adenovirus and/or respiratory syncytial virus were detected only among pediatric patients, whereas in adults EV-D68 was the sole pathogen identified.\u003c/p\u003e \u003cp\u003eMolecular characterization was performed on two EV-D68 isolates, one from a 2-year-old patient and the other from a 68-year-old patient, revealing distinct genetic profiles. The VP1 gene fragment isolated from the sampled child patient clustered with subclade B3, while the full genome isolated from the adult was classified as subclade A2. According to Nextstrain representation of isolates over the past six years, the A2 subclade has been predominantly detected in Spain, France, and Italy, with the latter being the closest related sequences.\u003c/p\u003e \u003cp\u003eThese results provide valuable phylogenetic insight into the circulation of EV-D68 in Portugal, showing consistency with previous studies indicating that, since 2017, only genotypes B3 and A2/D2 have continued to circulate in Europe, as well as in Asia and the United States [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Moreover, the Algarve region in southern Portugal [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], as an international tourist destination, may contribute to the introduction and spread of diverse EV-D68 strains, highlighting the importance of continuous surveillance in areas with high population mobility. The identification of a full genome clustering in clade A2, together with a VP1 fragment from subclade B3 aligns with recent European trends where B3 has been the dominant lineage whilst A2 continues to be observed in circulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In 2018, European EV-D68 isolates showed distinct clusters in both B3 and A2, with different phylogenetic origins. Subclade B3 emerged mainly from U.S. and European lineages, whereas A2 was traced back to strains from East Asia [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Interestingly, subclade B3 has been primarily associated with pediatric infections (median age: 5 years), while A2 has been more common in adults (median age: 42 years) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], which is in accordance with our results. Complete genome and VP1 fragment analyses confirm the co-circulation of these lineages, reflecting EV-D68 diversity and evolutionary dynamics that have been previously described in Europe [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough our dataset is too limited to analyze age or clinical severity associations, these findings highlight the need for closer examination of the clinical impact of EV-D68 classical and emerging clades in Portugal.\u003c/p\u003e \u003cp\u003eWhen compared with other European studies, the 5.44% detection rate observed in our study falls within similar ranges, likely due to the use of respiratory samples, including remnant specimens that had previously tested positive for RV/EV. It should be noted that for the diagnosis of EV-D68, respiratory samples are considered the gold standard, however, in cases of neurological involvement, cerebrospinal fluid (CSF) analysis can also provide useful information, although viral detection in the CSF is rare [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, the lack of standardized inclusion of EV-D68 in routine respiratory virus panels may contribute to the limited national-level data. For instance, large-scale surveillance during the 2021\u0026ndash;2022 upsurge in Europe documented detection rates in respiratory samples ranging from below 5% in some hospital cohorts to over 10% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Other surveys have also reported detection rates in respiratory or nasopharyngeal samples ranging from 2% to 5% in hospitalized children and exceeding 10% in pediatric outbreak settings [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These data confirm that the 5.44% rate detected in the present study is consistent with the range reported in various European surveillance settings, some of which also included samples previously positive for RV/EV, as was the case in our study. Furthermore, other surveillance studies have noted substantial variability across countries and seasons, often influenced by differences in sampling strategy, case mix, and testing intensity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although our dataset is limited in size and geographic scope, the measurable detection of EV-D68 in Portugal indicates ongoing viral circulation.\u003c/p\u003e \u003cp\u003eThe antigenic evolution of EV-D68 remains a concern, particularly with amino acid substitutions in the VP1 loops and other surface-exposed protein regions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. European surveillance has demonstrated that these mutations can accumulate rapidly under immune pressure, potentially altering neutralization profiles and population susceptibility [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Future studies incorporating broader sequencing, epitope mapping, and serological assays will be essential to assess whether strains retrieved in Portugal show signs of immune escape or altered pathogenicity.\u003c/p\u003e \u003cp\u003eFrom a public health perspective, our findings carry several implications. First, EV-D68 is confirmed to be circulating in Portugal, and continued surveillance will be required to monitor its prevalence, genetic diversity, and potential clinical impact. Second, sequence-based monitoring must become a priority, since PCR detection alone does not provide the resolution needed to track introductions, clade shifts, or evolutionary dynamics. Finally, integration of molecular data with clinical and epidemiological information will be necessary to determine whether subclades A2 and B3 infections in Portugal show different severity or age distribution, as suggested in other European studies. Also, continuous collaborative data sharing with European partners is essential, given that EV-D68 shows rapid cross-border mixing, with migration rates between countries estimated at multiple introductions per year. This is particularly relevant for southern Portugal, including the Algarve region, which receives migratory flows from Africa and for which clinical and epidemiological surveillance data remain largely lacking.\u003c/p\u003e \u003cp\u003eIn spite of these results, some limitations should be acknowledged. The present study was season restricted and geographically limited to the Algarve region, preventing assessment of national-level trends or longer-term evolutionary dynamics.\u003c/p\u003e \u003cp\u003eIn conclusion, this study provides evidence of EV-D68 circulation in Portugal, including detection of clade A2, which has been circulating across Europe. These findings underscore the need to strengthen national surveillance capacity, expand genomic sequencing efforts, and integrate clinical and epidemiological data to better assess the potential burden and public health significance of EV-D68 in Portugal.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuilherme Moreira thanks Fundação para a Ciência e a Tecnologia (FCT) for the financial support of their Ph.D work under the scholarships and 2025.05151.BD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSérgio Santos-Silva:\u003c/strong\u003e Writing – review \u0026amp; editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. \u003cstrong\u003eGuilherme Moreira\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing – review \u0026amp; editing, Methodology, Investigation, Data curation. \u003cstrong\u003eAndré Palma\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing – review \u0026amp; editing, Methodology, Formal analysis. \u003cstrong\u003eSoraia Rodrigues:\u003c/strong\u003e Writing – review \u0026amp; editing, Methodology. \u003cstrong\u003eMargarida Simões\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing – review \u0026amp; editing. \u003cstrong\u003eRaquel Guerreiro\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing – review \u0026amp; editing, Methodology. \u003cstrong\u003eMaria S.J. Nascimento\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Writing – review \u0026amp; editing, Supervision, Investigation, Formal analysis, Conceptualization. \u003cstrong\u003eJoão R. Mesquita:\u003c/strong\u003e Writing – review \u0026amp; editing, Supervision, Investigation, Funding acquisition, Formal analysis, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Fundação para Ciência e Tecnologia (FCT), grant number 2025.05151.BD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by an Ethics Commission (Comissão de Ética para a Saúde da Unidade Local de Saúde do Algarve; reference number: 084/2025), and all participants provided an informed consent.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGrizer CS, Messacar K, Mattapallil JJ (2024) Enterovirus-D68 - A Reemerging Non-Polio Enterovirus that Causes Severe Respiratory and Neurological Disease in Children. Front Virol (Lausanne Switzerland). ;4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMidgley CM, Watson JT, Nix WA, Curns AT, Rogers SL, Brown BA et al (2015) Severe respiratory illness associated with a nationwide outbreak of enterovirus D68 in the USA (2014): a descriptive epidemiological investigation. Lancet Respir Med 3:879\u0026ndash;887\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMessacar K, Abzug MJ, Dominguez SR (2016) 2014 outbreak of enterovirus D68 in North America. J Med Virol 88:739\u0026ndash;745\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrews SJ, Simmonds K, Usman HR, Yee K, Fathima S, Tipples G et al (2015) Characterization of enterovirus activity, including that of enterovirus D68, in pediatric patients in Alberta, Canada, in 2014. J Clin Microbiol 53:1042\u0026ndash;1045\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMidgley SE, Christiansen CB, Poulsen MW, Hansen CH, Fischer TK (2015) Emergence of enterovirus D68 in Denmark, June 2014 to February 2015. Euro Surveill Bull Eur sur les Mal Transm\u0026thinsp;=\u0026thinsp;Eur. Commun Dis Bull. ;20\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBragstad K, Jakobsen K, Rojahn AE, Skram MK, Vainio K, Holberg-Petersen M et al (2015) High frequency of enterovirus D68 in children hospitalised with respiratory illness in Norway, autumn 2014. Influenza Other Respi Viruses 9:59\u0026ndash;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoelman R, Sch\u0026ouml;lvinck EH, Borger R, Niesters HGM, van Leer-Buter C (2015) The emergence of enterovirus D68 in a Dutch University Medical Center and the necessity for routinely screening for respiratory viruses. J Clin Virol Off Publ Pan Am Soc Clin Virol 62:1\u0026ndash;5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMessacar K, Asturias EJ, Hixon AM, Van Leer-Buter C, Niesters HGM, Tyler KL et al (2018) Enterovirus D68 and acute flaccid myelitis-evaluating the evidence for causality. Lancet Infect Dis 18:e239\u0026ndash;e247\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHelfferich J, Knoester M, Van Leer-Buter CC, Neuteboom RF, Meiners LC, Niesters HG et al (2019) Acute flaccid myelitis and enterovirus D68: lessons from the past and present. Eur J Pediatr 178:1305\u0026ndash;1315\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirvonen A, Johannesen CK, Simmonds P, Fischer TK, Harvala H, Benschop KSM (2025) Sustained circulation of enterovirus D68 in Europe in 2023 and the continued evolution of enterovirus D68 B3-lineages associated with distinct amino acid substitutions in VP1 protein. J Clin Virol. ;178\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiardina FA, Romano G, Ferrari G, Pellegrinelli L, Seiti A, Galli C et al (2025) A newly developed whole genome sequencing protocol enables early tracking of enterovirus D68 molecular evolution. J Virol Methods 335:115131\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimoes MP, Hodcroft EB, Simmonds P, Albert J, Alidjinou EK, Ambert-Balay K et al (2024) Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021\u0026ndash;2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study. J Infect Dis 230:e917\u0026ndash;e928\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGummersheimer S, Hayes A, Harrison C, Lee B, Schuster J, Dhar M et al (2023) Prevalence and clinical presentation of EV-D68 infections in Kansas City children during the 2022 season. Diagn Microbiol Infect Dis 107:115992\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu S, Tang Y, Li M, Zhang L, Su Y, Wang Y et al (2024) Clinical characteristics of Chinese children with EV-D68-associated pneumonia: A single-center retrospective analysis. Pediatr Pulmonol 59:3660\u0026ndash;3666\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Chen X, Ai J, Li L, Li C, Zhu Y et al (2023) Clinical and molecular epidemiologic features of enterovirus D68 infection in children with acute lower respiratory tract infection in China. Arch Virol 168:206\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYun KW, Ahn B, Choi SH, Kang DY, Kim TS, Lee MK et al (2023) First Detection of Enterovirus D68 in Korean Children, September 2022. Infect Chemother 55:422\u0026ndash;430\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkuse T, Aizawa Y, Takihara H, Okuda S, Watanabe K, Saitoh A (2021) Development of Novel PCR Assays for Improved Detection of Enterovirus D68. J Clin Microbiol. ;59\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreno G, Gage Moreno DO, Sequence-Independent (2020) Single-Primer Amplification of RNA viruses. protocols.io\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Coster W, Rademakers R (2023) NanoPack2: population-scale evaluation of long-read sequencing data. Bioinformatics. ;39\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWick RR (2018) Porechop [Internet]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rrwick/Porechop\u003c/span\u003e\u003cspan address=\"https://github.com/rrwick/Porechop\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094\u0026ndash;3100\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J, Rincon N, Wood DE, Breitwieser FP, Pockrandt C, Langmead B et al (2022) Metagenome analysis using the Kraken software suite. Nat Protoc 17:2815\u0026ndash;2839\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangmead B AWS Public Datasets: Kraken2 Standard and Viral Databases [Internet]. 2025 [cited 2025 Sep 9]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://benlangmead.github.io/aws-indexes/k2\u003c/span\u003e\u003cspan address=\"https://benlangmead.github.io/aws-indexes/k2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO et al (2021) Twelve years of SAMtools and BCFtools. Gigascience 10:1\u0026ndash;4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePysam Developers Pysam [Internet]. 2025 [cited 2025 Jun 1]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/pysam-developers/pysam\u003c/span\u003e\u003cspan address=\"https://github.com/pysam-developers/pysam\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira G PUPI-Plot: Phylogenetically Unique and Popular Identifier Plot [Internet]. 2025 [cited 2025 Sep 1]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/GmoreiraVet/PUPI-Plot/tree/main\u003c/span\u003e\u003cspan address=\"https://github.com/GmoreiraVet/PUPI-Plot/tree/main\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunter JD, Matplotlib (2007) A 2D Graphics Environment. Comput Sci Eng 9:90\u0026ndash;95\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K et al (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRozewicki J, Li S, Amada KM, Standley DM, Katoh K (2019) MAFFT-DASH: Integrated protein sequence and structural alignment. Nucleic Acids Res 47:W5\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen L-T, von Haeseler A, Minh BQ (2018) Complex Models of Sequence Evolution Require Accurate Estimators as Exemplified with the Invariable Site Plus Gamma Model. Syst Biol 67:552\u0026ndash;558\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNextstrain (2025) Enterovirus D68 / genome [Internet]. [cited 2025 Sep 1]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nextstrain.org/enterovirus/d68/genome\u003c/span\u003e\u003cspan address=\"https://nextstrain.org/enterovirus/d68/genome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHodcroft EB, Dyrdak R, Andr\u0026eacute;s C, Egli A, Reist J, de Artola DGM et al (2022) Evolution, geographic spreading, and demographic distribution of Enterovirus D68. PLoS Pathog 18:1\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsposito S, Bosis S, Niesters H, Principi N (2015) Enterovirus D68 Infection. Viruses [Internet]. ;7:6043\u0026ndash;50. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://consensus.app/papers/enterovirus-d68-infection-esposito-bosis/00999a827fad5aebb15743be3d020c39\u003c/span\u003e\u003cspan address=\"https://consensus.app/papers/enterovirus-d68-infection-esposito-bosis/00999a827fad5aebb15743be3d020c39\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndr\u0026eacute;s C, Vila J, Creus-Costa A, Pi\u0026ntilde;ana M, Gonz\u0026aacute;lez-S\u0026aacute;nchez A, Esperalba J et al (2022) Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014\u0026ndash;2021. Emerg Infect Dis 28:1327\u0026ndash;1331\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtt C, Dutilh G, Reist J, Bingisser R, Egli A, Heininger U (2024) Clinical Presentation of Enterovirus D68 in a Swiss Pediatric University Center. Pediatr Infect Dis J 43:1135\u0026ndash;1140\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImamura T, Oshitani H (2015) Global reemergence of enterovirus D68 as an important pathogen for acute respiratory infections. Rev Med Virol 25:102\u0026ndash;114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVermillion MS, Dearing J, Zhang Y, Adney DR, Scheuermann RH, Pekosz A et al (2022) Animal Models of Enterovirus D68 Infection and Disease. J Virol 96:e0083322\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLugo D, Krogstad P (2016) Enteroviruses in the early 21st century: new manifestations and challenges. Curr Opin Pediatr 28:107\u0026ndash;113\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaghmare A, Pergam SA, Jerome KR, Englund JA, Boeckh M, Kuypers J (2015) Clinical disease due to enterovirus D68 in adult hematologic malignancy patients and hematopoietic cell transplant recipients. Blood 125:1724\u0026ndash;1729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSooksawasdi Na Ayudhya S, Laksono BM, van Riel D (2021) The pathogenesis and virulence of enterovirus-D68 infection. Virulence 12:2060\u0026ndash;2072\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolm-Hansen CC, Midgley SE, Fischer TK (2016) Global emergence of enterovirus D68: a systematic review. Lancet Infect Dis 16:e64\u0026ndash;75\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHowson-Wells HC, Tsoleridis T, Zainuddin I, Tarr AW, Irving WL, Ball JK et al (2022) Enterovirus D68 epidemic, UK, 2018, was caused by subclades B3 and D1, predominantly in children and adults, respectively, with both subclades exhibiting extensive genetic diversity. Microb genomics. ;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMidgley SE, Benschop K, Dyrdak R, Mirand A, Bailly J-L, Bierbaum S et al (2020) Co-circulation of multiple enterovirus D68 subclades, including a novel B3 cluster, across Europe in a season of expected low prevalence, 2019/20. Euro Surveill Bull Eur sur les Mal Transm\u0026thinsp;=\u0026thinsp;Eur. Commun Dis Bull. ;25\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaia C, Dion\u0026iacute;sio L, Afonso MO, Neto L, Crist\u0026oacute;v\u0026atilde;o JM, Campino L (2013) Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal. Mem Inst Oswaldo Cruz 108:481\u0026ndash;487\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFall A, Norton JM, Abdullah O, Pekosz A, Klein E, Mostafa HH et al (2024) Enhanced genomic surveillance of enteroviruses reveals a surge in enterovirus D68 cases, the Johns Hopkins health system, Maryland, 2025;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnoester M, Helfferich J, Poelman R, Van Leer-Buter C, Brouwer O, Niesters H (2018) Twenty-nine Cases of Enterovirus-D68\u0026ndash;associated Acute Flaccid Myelitis in Europe 2016. Pediatr Infect Dis J [Internet]. ;38:16\u0026ndash;21. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://consensus.app/papers/twentynine-cases-of-enterovirusd68\u0026ndash;associated-acute-knoester-helfferich/46258e47a046597c993667c2b84420a7\u003c/span\u003e\u003cspan address=\"https://consensus.app/papers/twentynine-cases-of-enterovirusd68\u0026ndash;associated-acute-knoester-helfferich/46258e47a046597c993667c2b84420a7\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarvala H, Broberg E, Benschop K, Berginc N, Ladhani S, Susi P et al (2018) Recommendations for enterovirus diagnostics and characterisation within and beyond Europe. J Clin Virol Off Publ Pan Am Soc Clin Virol 101:11\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuffenecker I, Mirand A, Josset L, Henquell C, Hecquet D, Pilorg\u0026eacute; L et al (2016) Epidemiological and clinical characteristics of patients infected with enterovirus D68, France, July to December 2014. Eurosurveillance 21:1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFall A, Kenmoe S, Ebogo-Belobo JT, Mbaga DS, Bowo-Ngandji A, Foe-Essomba JR et al (2022) Global prevalence and case fatality rate of Enterovirus D68 infections, a systematic review and meta-analysis. PLoS Negl Trop Dis 16:1\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang Y, Chen Q, Wang H, Wang L, Rong H, Liao Q et al (2021) The role of conformational epitopes in the evolutionary divergence of enterovirus D68 clades: A bioinformatics-based study. Infect Genet Evol [Internet]. ;93:104992. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.meegid.2021.104992\u003c/span\u003e\u003cspan address=\"10.1016/j.meegid.2021.104992\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Non-polio enterovirus, EV-D68, (sub)clades A2 and B3, epidemiology, surveillance","lastPublishedDoi":"10.21203/rs.3.rs-9161762/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9161762/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnterovirus D68 (EV-D68) is a reemerging respiratory pathogen that is increasingly associated with severe respiratory disease and occasional neurological complications, yet its circulation in Portugal remains poorly documented. In the present study, we conducted molecular surveillance in southern Portugal (Algarve region) from October 2024 to February 2025. Screening of 150 respiratory samples from patients (115 from children and 35 from adults), all positive for rhinovirus/enterovirus, revealed eight EV-D68\u0026ndash;positive cases, corresponding to a detection rate of 5.44%, spanning a broad age range from infancy to the elderly. Clinical manifestations ranged from mild respiratory illness to severe conditions, including pneumonia and acute respiratory distress syndrome. In addition, neurological involvement such as complex febrile seizures and encephalomyelitis was observed. Some cases involved coinfections, while others presented with EV-D68 as the sole detected pathogen. Genomic characterization from one isolate revealed a VP1 fragment belonging to clade B3, and a complete genome from another isolate clustered within clade A2. Phylogenetic analysis confirmed clustering with contemporary European isolates. These findings highlight the circulation of multiple EV-D68 clades in Portugal, underscore the clinical diversity of infections, and reinforce the importance of genomic surveillance to monitor viral evolution and potential public health impact.\u003c/p\u003e","manuscriptTitle":"Enterovirus D68 circulation in Portugal, 2024–2025","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 06:03:56","doi":"10.21203/rs.3.rs-9161762/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-31T11:14:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T00:22:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-24T18:34:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207835934312450390862301757647086977798","date":"2026-03-20T06:51:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51963515433342867511733135638539604629","date":"2026-03-20T04:42:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170595923740882443222131873779458483144","date":"2026-03-19T21:48:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-19T21:33:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-19T10:51:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-19T10:51:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2026-03-18T16:41:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"88756fd0-c59a-432c-9c12-b9442aeaa8c2","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T21:24:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 06:03:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9161762","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9161762","identity":"rs-9161762","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}