Oropouche virus (OROV; Orthobunyavirus) is an emerging arbovirus endemic to South America and the Caribbean, with imported cases in European countries, including Spain. Although primarily transmitted by biting midges (Culicoides spp.), OROV has been detected in several mosquito species, raising concerns about potential establishment in nonendemic regions. European populations of Aedes albopictus, and Culex pipiens, as well as the invasive Aedes aegypti, represent relevant models for assessing vector competence. Here, we evaluated the vector competence of Spanish Cx. pipiens biotype molestus, Spanish Ae. albopictus, and Ae. aegypti (Liverpool strain), for the 2024 OROV outbreak strain. Female mosquitoes were orally exposed to infectious blood meals and maintained under controlled insectary conditions at 27 °C. In addition, an additional group received a second noninfectious blood meal. The survival of the mosquitoes was monitored, and infection, dissemination, and transmission rates were assessed at 7, 14, and 21 days post infection. Vertical transmission of the virus to the progenies was also analyzed. Overall, Ae. albopictus exhibited low infection rates, with occasional dissemination and transmission events. Aedes aegypti and Cx. pipiens biotype molestus showed infection and occasional dissemination, but no evidence of transmission. A second noninfectious blood meal did not significantly affect infection, dissemination, or transmission rates in any species. Viral loads in bodies and legs were low and did not differ significantly between species, time points, or feeding regimens. Survival was not affected by infection or blood-feeding regime. We did not find vertical transmission of OROV to the progenies. Regardless of virus dissemination in mosquitoes, our study indicates a poor vector competence of Spanish Ae. albopictus and no evidence of transmission under the conditions tested in Ae. aegypti and Cx. pipiens biotype molestus for the circulating OROV strain. These findings suggest a low risk for local OROV establishment in Spain, although continuous surveillance and research are warranted to monitor potential vector–virus adaptation.

OBJECTIVES:To describe the first documented case of Andes virus (ANDV) hantavirus cardiopulmonary syndrome (HCPS) in Spain and the first worldwide use of a therapeutic regime including two antivirals (favipiravir and ribavirin) and two host-directed drugs (baricitinib and icatibant). METHODS:A 69-year-old Spanish man, repatriated after a multinational ANDV outbreak aboard a cruise ship, was managed in a high-level isolation unit. Diagnosis was established by RT-PCR and serology while he was still asymptomatic as part of protocol-driven screening. Under compassionate-use authorization and written informed consent, the patient received ribavirin (initially intravenous, then switched to oral on day +4), oral favipiravir, subcutaneous icatibant, and oral baricitinib, with serial clinical, laboratory, and radiological monitoring. RESULTS:Hypoxaemia, bilateral B-lines, thrombocytopaenia, lymphopaenia, and hyponatraemia developed within 24 hours after diagnosis. The combination regimen was initiated on day 0, and baricitinib was added on day +1, coinciding with the need for high-flow nasal oxygen. Sustained clinical, laboratory, and radiological recovery occurred from day +2 onwards, without progression to invasive ventilation or vasopressors. Mild diarrhoea attributed to ribavirin led to its discontinuation on day +5, shortly after the intravenous-to-oral switch. Severe recurrent diarrhoea on day +8, attributed to favipiravir, prompted its withdrawal before completion of the planned 10-day course; baricitinib was completed on day +10. CONCLUSIONS:This sentinel case of imported HCPS in nonendemic Europe was managed with, to our knowledge, the first reported combined antiviral and host-directed regimen for this syndrome and the first reported use of favipiravir in a patient with hantavirus infection. The favourable outcome supports prospective evaluation of antiviral combinations and adjunctive immunomodulation within international preparedness protocols.

Leishmaniasis is a well-known vector borne disease with a high incidence in southern European countries. In Spain, Leishmania infantum infections typically occur as sporadic and infrequent outbreaks. However, in recent years, several outbreaks have been reported in different regions, accompanied by increasing hospitalization rates. In 2023, 31 human cases of Leishmania infection were reported in hospitals across 15 municipalities in Córdoba (southern Spain). Visceral leishmaniasis was the predominant form (77.4%), followed by cutaneous (19.4%), and mucocutaneous (3.2%) forms. A retrospective analysis of Phlebotomus captures from the mosquito monitoring network was conducted to characterize the phenology, abundance, and feeding behaviour of sand flies in the main outbreak area. A total of 22,220 sand fly specimens were recorded, with male identification revealing five species: Phlebotomus perniciosus (94.3%), Sergentomyia minuta (4.3%), Phlebotomus ariasi (0.08%), Phlebotomus sergenti (0.03%), Phlebotomus papatasi (0.005%) and 1.3% undetermined. Sand fly captures peaked in September, preceding the major peak in human case notifications. Most of the identified blood meals (75.0%, n = 163) came from Iberian rabbits (Oryctolagus cuniculus algirus), although blood from six other mammal species, including two samples of human origin, and one bird species was also detected. A total of 5855 female sand flies analysed, grouped into 145 pools, showed a Leishmania infection prevalence of 3.8%, peaking in October and November. Our findings suggest that the Iberian rabbit may play a key role in the local epidemiology of L. infantum, given its high frequency as a blood source for Ph. perniciosus and the elevated vector densities and infection rates observed in the outbreak area. These results emphasize the need for continuous entomological surveillance and proactive vector management, integrating the monitoring and control of potential reservoir hosts to effectively reduce transmission risk in the affected areas.

West Nile virus (WNV) is an emerging mosquito-borne pathogen in Europe, responsible for recurrent outbreaks affecting humans, horses, and wildlife. Although WNV circulation has been documented in Portugal, genomic data from mosquito populations remain scarce, limiting our understanding of viral diversity and transmission dynamics in the western Iberian Peninsula. Here, we investigated the presence and genetic diversity of WNV in mosquitoes collected from wetland ecosystems in southern Portugal in 2025. Mosquitoes were collected and grouped into pools by collection date, location, and species. Pools were screened for WNV RNA by RT-qPCR, and positive samples were subjected to whole-genome sequencing using a hybrid-capture target enrichment method. Phylogenetic analyses were conducted to determine the evolutionary relationships of the detected viruses with previously reported WNV strains circulating worldwide. WNV RNA was detected in several mosquito pools, predominantly in Culex univittatus , from which seven near-complete viral genomes were recovered. All sequences belonged to WNV lineage 1A but segregated into two distinct genetic clusters. One cluster grouped with strains previously reported from the Iberian Peninsula, whereas the second formed a divergent variant previously undetected. These findings indicate the co-circulation of genetically distinct WNV strains in Portuguese wetlands. Our results provide the phylogenetic characterization of WNV from mosquito populations in Portugal and reveal previously undocumented viral diversity in the region. This study highlights the importance of integrating entomological surveillance with genomic approaches to better understand the mechanisms underlying WNV introduction, maintenance, and spread in southern Europe.

Viral central nervous system (CNS) infections in adults frequently remain unresolved after routine diagnostic testing. We applied probe-based viral metagenomic next-generation sequencing (vmNGS) to cerebrospinal fluid samples from adults with suspected CNS infection and negative conventional diagnostics in a retrospective multicentre study conducted in Spain between 2022 and 2023. Among 40 idiopathic cases, vmNGS detected viral sequences in 6 patients without evidence of coinfection: human pegivirus (HPgV, n=3), Toscana virus (TOSV, n=1), herpes simplex virus type 1 (HSV-1, n=1) and varicella-zoster virus (VZV, n=1). Two HPgV-positive patients were transplant recipients, with neurological disease occurring more than 2 years after transplantation, compatible with possible long-term viral persistence in immunocompromised hosts. TOSV genotype B was identified in a patient residing in central Spain, supporting consideration of TOSV in selected cases of unexplained aseptic meningitis during the vector season, including outside traditionally recognized Mediterranean coastal regions. Furthermore, the failure of syndromic panel testing to detect HSV-1 and VZV highlights the need for complementary diagnostic strategies when clinical suspicion remains high. Overall, the detection of unexpected viral sequences, together with missed clinically actionable infections, supports the use of complementary molecular testing in selected cases of unexplained CNS syndromes when routine diagnostics are negative. These findings highlight the added diagnostic value of vmNGS and provide sequence-level data for future studies of viral diversity and molecular epidemiology in neurological disease.

INTRODUCTION:Dengue is the most significant vector-borne viral disease in global morbidity and mortality, and it is considered a re-emerging disease spreading into new regions. The risk of autochthonous dengue virus transmission in Spain remains high due to the increasing number of travellers returning from endemic areas and the presence of Aedes albopictus within our territory. Conducting epidemiological and molecular studies on returning travellers from endemic areas may be crucial to discern transmission patterns and track the global spread of the virus. This study focuses on the molecular characterization of suspected imported dengue cases from 2022 to 2024. METHODS:We analysed 600 samples from 539 suspected dengue-infected travellers between 2022 and 2024. All samples were tested by a quantitative RT-PCR, and PCR-positive cases were confirmed by performing a non-overlapping nested RT-PCR, going under subsequent sequencing to identify viral diversity. RESULTS:Of the 539 suspected cases, 183 were confirmed as DENV-positive, with Cuba as the most common travel origin associated with infections. Molecular analysis of positive samples identified all four DENV serotypes and ten genotype groups, with DENV-3 genotype III as the most predominant. Additionally, among confirmed cases reported from regions with Aedes albopictus presence, 73% (94/129) occurred during the vector's active season (May-November), increasing the risk of local transmission. CONCLUSION:Our results highlight the importance of returning travellers as sentinels for ongoing dengue outbreaks and epidemiology in endemic regions. Additionally, these emphasize the importance of early case identification through sentinel surveillance to prevent potential autochthonous transmission.

Viruses represent a major component of global biodiversity and are integral to ecological and evolutionary processes shaping host populations and communities. Bats, with high species richness and ecological diversity, provide a system to examine how host traits and environmental context structure viral communities. Here, we characterize viral diversity from three bat species (Pipistrellus kuhlii, Cnephaeus isabellinus, and Nyctalus lasiopterus) using samples across two environments in southern Spain: a well-preserved Mediterranean forest (Sierras de Cazorla, Segura, and Las Villas Natural Park, CSVNP) and a human-modified wetland-agrosystem mosaic (Doñana National Park, DNP). Metagenomics detected 72 eukaryotic virus species, including viruses reported in mammals, insects, arachnids, and plants. Viral richness and composition varied among samples, bat species, and environments. Samples of P. kuhlii exhibited the highest richness, driven by insect-associated viruses. C. isabellinus showed a higher contribution of vertebrate-related viruses, whereas N. lasiopterus exhibited the lowest richness. CSVNP samples showed higher viral richness and more exclusive taxa, whereas DNP samples exhibited lower richness, with a greater contribution of arthropod-associated viruses, potentially reflecting prey communities. Despite this, diversity metrics were similar between environments, indicating structurally comparable communities composed of distinct taxa. Bat viromes appear to be associated with host ecology, trophic behavior, and environmental context.

[This corrects the article DOI: 10.1016/j.onehlt.2024.100947.].

BACKGROUND:Mpox, formerly known as monkeypox, is an infectious disease caused by the mpox virus. Systemic involvement is rare and mpox-associated meningoencephalitis is an uncommon clinical presentation. Because mpox-associated meningoencephalitis is characterized by nonspecific clinical and incidental imaging findings, the disease is difficult to diagnose. CASE SUMMARY:We present a case of fatal mpox-associated meningoencephalitis with negative polymerase chain reaction in cerebrospinal fluid (CSF) diagnosed at autopsy. A young immunocompetent patient with no significant medical history initially presented with genital mpox infection. Within 48 hours, he showed neurologic involvement requiring orotracheal intubation. His condition deteriorated rapidly, progressing to cerebral edema and brain death consistent with meningoencephalitis. Despite negative testing for mpox virus in CSF, the diagnosis was confirmed posthumously by autopsy, where histological examination revealed the presence of mpox virus in brain tissue. CONCLUSIONS:Mpox-associated meningoencephalitis should be considered as a potential diagnosis even in the absence of the mpox virus genome in CSF.

Flaviviruses are emerging pathogens, mostly transmitted by arthropod vectors, responsible for human, animal, and zoonotic diseases. The emergence of flaviviruses has been favored in recent decades by factors related to climate change and globalization, which contribute to the arrival and establishment of their vectors in new geographic areas, thus promoting epidemic outbreaks and facilitating these viruses to become endemic in these areas. This is the case of the West Nile virus (WNV) in Europe, which affects its natural bird host populations and also accidental hosts such as humans and horses. Flaviviruses are antigenically related, which induces cross-reactivity, making their serological diagnosis difficult, especially in areas where several flaviviruses co-circulate. Here, we have developed WNV biosensors in which the enzymatic activity of the viral protease, expressed during infection, allows its detection using fluorescence-based techniques. These biosensors carry WNV-specific protease cleavage sites and show high specificity for the detection of both lineages 1 and 2, with limits of detection (LODs) ranging from 0.001-0.0001 MOI at 48 hours post-infection (h.p.i.). These LODs are even lower at 72 h.p.i. reaching as low as an MOI of 2.5 × 10-6. They are also capable of detecting, although with lower sensitivity, other flaviviruses such as dengue, Zika, and Usutu viruses, without showing reactivity against unrelated viruses. These biosensors have been validated in viral neutralization assays with sera from infected mice and humans, as well as in antiviral screening, with results comparable to those of currently used systems, showing significant potential as clinical and laboratory tools.

This review examines the emerging public health threat posed by the Oropouche virus (OROV), an arbovirus circulating in the Latin Americas. It focuses on its genomic organization, transmission dynamics, clinical manifestations, and vector competence while evaluating environmental and anthropogenic factors influencing its spread. Recent studies reveal that climate change, deforestation, and urbanization have expanded OROV’s geographic range. Outbreaks have been reported in Brazil, Panama, Cuba, and Guyana, between others, with imported cases detected in Europe and the United States. Reassortment events contribute to the virus's genetic diversity and complicate its diagnosis along with co-circulation with other arboviruses with similar symptoms. OROV is an underestimated pathogen requiring enhanced surveillance and multidisciplinary control measures. Strengthening diagnostic capabilities and implementing coordinated public health strategies are essential to mitigate its spread and address severe complications, including neurological and maternal-fetal outcomes.

BACKGROUND:Considering the increase of West Nile virus (WNV) circulation in Europe, blood banks perform WNV Nucleic Acid Test testing to ensure transfusion safety during the WNV transmission season. Usutu virus (USUV), an arbovirus related to WNV, has relevant molecular and serological cross-reactivity with WNV. STUDY DESIGN AND METHODS:During the 2024 WNV season in Europe, 15,957 blood donations from the Balearic Islands Blood Bank and 79,400 from the Catalonia Blood Bank were tested for WNV using the Cobas WNV real-time PCR (Roche Diagnostics, USA) and the Procleix WNV/Procleix ArboPlex transcription-mediated assays (Grifols Diagnostic Solutions Inc., USA), respectively. Serological tests for flavivirus, PCR using USUV- and WNV-specific primers, sequencing of viral RNA, and neutralization tests (NT) were performed to confirm positive results. RESULTS:We identified three donors with USUV infection. In July, the first donor was detected in Majorca (Balearic Islands), whose infection was confirmed by NT. In September, two donors with USUV Africa 3 lineage were identified by RT-PCR and sequencing in Catalonia. There were three USUV-positive cases in 95,357 donations (1 in 31,786; 95% CI: 1 in 10,877 to 1 in 154,083) during the 2024 WNV season in Spain. DISCUSSION:We report three cases of autochthonous USUV infection in blood donors from the Balearic Islands and Catalonia, two of which were infected by the USUV Africa 3 lineage. Health authorities should be aware that positive WNV screening may be due to USUV, an emerging zoonotic virus that can be underreported.

AimOral antibiotic prophylaxis (OAP) lowers rates of surgical site infection (SSI) and may aid anastomotic healing in colorectal surgery. The aim of this study was to analyse the understudied impact of OAP on SSI microbiology after colorectal surgery.MethodA post hoc analysis was performed on a previous prospective, multicentre study of elective colorectal surgery. For 1000 patients with SSI, this study compared the microbiology of SSIs in procedures without OAP (SSI/OAP-) and with OAP (SSI/OAP+).ResultsThere were 340 patients in the SSI/OAP- group and 660 in the SSI/OAP+ group. The use of OAP increased the presence of Gram-positive cocci (GPC) (OR 1.542, 95% CI 1.153-2.062) and fungi (OR 2.037, 95% CI 1.206-3.440), but reduced rates of Gram-negative bacteria (GNB) (OR 1.461, 95% CI 1.022-2.088) and anaerobe isolation (OR 0.331, 95% CI 0.158-0.696). Specifically, it led to increases in the isolation of Enterococcus faecium (OR 1.450, 95% CI 0.812-2.591), methicillin-resistant Staphylococcus aureus (OR 2.000, 95% CI 1.043-3.834) and Candida spp. (OR 2.037, 95% CI 1.206-3.440). In colon surgery with OAP, GPC infections were more likely (OR 1.461, 95% CI 1.022-2.088). In rectal surgery, organ/space SSIs had a higher risk of harbouring GPC (OR 1.860, 95% CI 1.153-2.999) and a lower risk of GNB (OR 0.321, 95% CI 0.200-0.515).ConclusionOAP reduced the presence of anaerobes and GNB in SSIs, but increased the isolation of GPCs and fungi, with E. faecium and Candida being of particular concern. This information should guide empirical antibiotic therapy for postoperative colorectal SSIs in patients who have received preoperative OAP.

Oropouche fever is a vector-borne disease endemic in Central and South America. Infection with Oropouche virus (OROV) was confirmed in June and July 2024 in 13 patients travelling from Cuba to Spain. These patients presented fever, headache, myalgia and arthralgia, and three patients exhibited a biphasic course, with two developing transient neurological symptoms. Oropouche virus infection was diagnosed using reverse transcription quantitative PCR (RT-qPCR) and neutralisation tests. Viral RNA was detected in specimens from serum, urine, plasma and whole blood; from four urine samples up to 24 days post-symptom onset. Phylogenetic analysis of one OROV strain (OROV SP2024) isolated from one patient, demonstrated it clustered closely with reassortant strains circulating in Brazil and imported cases in Italy. These findings underscore the importance of including OROV in the differential diagnosis of febrile illnesses in returning travellers and demonstrate the diagnostic value of analysing multiple sample types. Enhanced clinical awareness and diagnostic capacity are essential to improve detection and surveillance of OROV among international travellers from affected regions.

West Nile virus (WNV), mainly transmitted by Culex mosquitoes, poses significant health risks to humans and horses, particularly in endemic regions. The first detection of WNV lineage 2 in Spain was in 2017 in Catalonia (northeastern Spain). In 2023, WNV was confirmed in a young yellow-legged gull and a probable human case was notified within the urban settings. We aimed to define the zone of WNV circulation in the Barcelona Metropolitan Area where these infections occurred and the effectiveness of the One Health approach for early WNV detection. The Catalan WNV surveillance and control programs includes the testing of horses, birds, mosquitoes and humans following molecular and serological methods. Phylogenetic analyses were performed to determine the origin of the circulating virus. IgM-positive data from both active and passive surveillance in horses identified the area of WNV circulation and suggested that WNV circulation happened either before or concurrently with human and bird infections in the agricultural and peri-urban areas. Furthermore, a new WNV introduction was discarded by phylogenetic studies, demonstrating that WNV lineage 2 has been established in Catalonia, albeit at a low level of circulation since the virus was not detected in blood donors. Our findings underscore the importance of integrating active and passive surveillance strategies to early assess WNV circulation and activate public health responses. The study highlights the role of wildlife in the WNV transmission and emphasizes the need for ongoing monitoring in animals and also mosquito control measures to mitigate the risk of animal and human infections.

Introduction: Recently, as the effects of anthropogenic climate change (ACC) become evident, vector-borne diseases (VBD) are spreading and establishing in temperate regions of the world. We investigated whether these two phenomena are related. We hypothesized that ACC has turned many areas, previously considered “unsuitable” for sustaining VBD cycles, into “suitable” ones. The hypothesis assumes that VBD pathogens have historically been able to reach temperate areas, but only started to thrive there under the influence of ACC, hence their current spread. To support this hypothesis, which we tentatively named “The frying pan theory”, we have chosen the recent spread of West Nile virus (WNV) in Spain as a model of study. Methods & Materials: The study area was Southern Spain, now endemic for WNV. We analyzed eighteen years (2003 to 2020) of data on WNV seroprevalence in common coots and horses and compared them statistically with annual series of climate variables (temperature, pluviometry, etc). We also performed phylogenetic analyses using WNV sequences from samples of mosquitoes, birds and horses, collected in the area up to 2022, and analyzed the pathogenicity of representative Spanish WNV isolates from this area and period in a mouse model. Results: Coots’ and horses’ seroprevalences exhibited a biphasic curve indicating at least two epizootic waves, one before and another after 2010. Seroprevalence in coots correlated with maximum winter temperature and average spring temperature. Horses’ seroprevalence correlated positively with average minimum annual temperature and the number of rain days per year and negatively with total annual precipitations. Phylogenetic analyses revealed at least six independent introductions of WNV into Southern Spain during the study period. Some introductions succeeded in establishing in the area and spreading to neighboring regions, while others did not. No significant changes in the pathogenicity of the strains studied were observed. Discussion: Long-term comparisons between annual variation of pathogen exposure and climate variables allow forecasting VBD trends. In this regard, higher minimum temperatures are among the ACC trends that may favor vector biology and hence VBD. Our data support that VBD pathogens like WNV can spread to new territories, but they will only establish their cycles successfully if they find suitable conditions. In Spain, competent WNV hosts (birds) and vectors (Culex mosquitoes), have always been present, and the virus has likely been introduced occasionally since long, e.g. by bird migrations. However, local WNV cycles would only be established if these necessary ingredients are “heated” by ACC much like well-cooked bacon and eggs may result only if heated in a frying pan. Conclusion: The data obtained support our hypothesis, which may also apply to most of Europe and other temperate areas of the world, not only for WNV but also for many other VBD.

Mosquito-borne pathogens produce relevant diseases causing human fatalities worldwide. In addition, mosquitoes transmit a variety of pathogens to livestock and wildlife, negatively affecting local economies and causing ecological impacts. Mosquitoes collected in a highly protected wetland from southern Spain were molecularly screened for the presence of three major pathogens, including the zoonotic flavivirus West Nile virus (WNV), avian Plasmodium, and filarioid nematodes. Overall, 95 mosquito pools including 1376 females corresponding to 4 Culex species were molecularly analyzed, including 40 mosquito pools containing 390 Culex pipiens, 42 mosquito pools containing 880 Culex perexiguus, 10 mosquito pools containing 102 Culex modestus, and 3 mosquito pools containing 4 Culex laticinctus. WNV was detected in 5 Cx. perexiguus pools. Avian Plasmodium was found in 28 mosquito pools tested, including 17 pools of Cx. perexiguus and 11 pools of Cx. pipiens. Three different Plasmodium spp. lineages were found in mosquitoes, corresponding to the morphospecies: P. vaughani (SYAT05; n = 22), P. matutinum (LINN1; n = 4), and the Plasmodium sp. (SGS2; n = 1). One positive sample was not identified at the lineage level. Plasmodium prevalence was significantly associated with mosquito species and sampling session and marginally related with mosquito pool size. None of the pools tested were positive for the presence of Dirofilaria spp. These results represent the most taxon extensive survey of pathogens in mosquitoes in Doñana. This study expands the knowledge of the diversity of pathogens naturally circulating in this protected wetland in southern Spain. Recommendations for the population are considered.

Lymphocytic choriomeningitis virus (LCMV) is a neglected rodent-borne virus, with a worldwide distribution. The common mouse Mus musculus acts as reservoir and vector in the biological cycle of the virus. Surveillance of LCMV infection in mice is of importance as they are a widely used animal model in research and, through contact with them or their fluids, humans can be infected. Although most human cases are asymptomatic, LCMV infection can cause mild to severe, even fatal, and new diagnostic tools need to be developed to improve its detection. In the present work we report the development of a new method for the detection of LCMV RNA by quantitative reverse transcription polymerase chain reaction (RT-qPCR), able to detect all LCMV strains described to date. RT-qPCR targeting the S segment was developed and evaluated. Specificity and sensitivity were determined, and its limit of detection (LOD) was defined. The method designed is able to detect all 5 LCMV lineages described to date, with a LOD of 5.6 genome copies/μL. Its design with a built-in internal amplification control allows the detection of false negative results. Other arenaviruses were found not to cross-react with the method designed. In conclusion, a new diagnostic RT-qPCR for the detection of LCMV have successfully designed and validated. Improved detection techniques allow to reduce the turnaround time in the diagnosis of infections and to improve epidemiological surveillance in humans and animals.