All methods were carried out in accordance with relevant guidelines and regulations, and experimental protocols were approved by the National Institute of Agricultural and Veterinary Research (INIAV I.P.).

Samples

A total of 138 serum samples from cattle, sheep, goats, and red deer (Cervus elaphus) were analyzed. In the case of domestic ruminants, blood was collected from the jugular or the tail vein, while in hunted wild animals it was obtained by cardiac puncture performed post-mortem. Blood was drawn into plain tubes and, after coagulation at room temperature, samples were centrifuged for 10 min at 1500 g at 4 °C. The serum was preserved at − 20 °C until analysis.

These samples originated from 35 farms and 10 hunting areas located in various regions of northern and eastern Portugal (Fig. 1). The sera, mainly from adult female cattle, were collected between July and October 2024 as part of the national surveillance programs for Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) and were submitted to the National Institute of Agrarian and Veterinary Research (INIAV, I.P.) for virological diagnosis. This opportunistic sampling focused on the Bragança region, where the human case was identified, as well as on nearby northeastern areas along the Spanish border, where locally acquired human cases have previously been reported.

Map of sampling areas in northern and eastern mainland Portugal comprising Bragança, Vila Real, Viseu, Guarda and Castelo Branco districts. Icons indicate the sampled animal species. Under each species the number of samples considered is indicated along with the number of farms from which they belong to.

Ticks (n = 9) were collected from the surrounding vegetation of a CCHFV seropositive cattle farm located in the municipality of Sabugal, within the Guarda district on September 3rd using the blanket dragging method, where white fabric strips were systematically dragged across vegetation in animal resting areas to attract and capture ticks¹². Additionally, ticks were also collected from cattle (n = 6) in the same farm in mid-October. All specimens were transferred to microtubes containing 70% ethanol (v/v).

Tick classification

Ticks were identified to the genus level based on morphological characteristics using a stereomicroscope and standard taxonomic keys^13,14. Identification focused on key distinguishing traits specific to Hyalomma species, the primary vectors of CCHF.

To enhance accuracy of the morphological classification, molecular confirmation was performed using 16 S ribosomal DNA sequence analysis, as previously described¹⁵.

Antibody ELISA

Antibody detection in sera was performed using the ID Screen CCHF Double Antigen Multispecies ELISA kit, following the manufacturer’s instructions (IDvet, Grabels, France). ​The Sample-to-Positive (S/P) ratio was calculated by dividing the optical density (OD) of each sample by the OD of the positive control, and then multiplying the result by 100 to express it as a percentage (%S/P) (Supplementary Table 1) to provide a quantitative measure of the sample’s antibody concentration relative to the control, with higher values indicating stronger antibody presence and potential exposure to the target pathogen. Samples with a %S/P > 30 were considered positive. Each ELISA plate validation was performed as recommended by the manufacturer.

Indirect Immunofluorescence assay

A subset of 9 ELISA-positive and 3 ELISA-negative bovine sera and 4 ELISA-positive red deer sera were tested by a commercial immunofluorescence biochip (Euroimmun, Lübeck, Germany).

The selected sera were tested under different dilution conditions to optimize the signal-to-noise ratio in the immunofluorescence assay. The serum samples were diluted to 1:50, 1:100, and 1:200, using both PBS-Tween and the sample buffer provided with the commercial kit. Concurrently, the FITC-conjugated secondary antibody was tested at dilutions of 1:100, 1:200, and 1:500 in PBS containing 1% BSA. All conditions were tested in quadruplicate to ensure the reproducibility and consistency of the results. The combination of conditions that yielded the strongest and most specific fluorescence signal with minimal background was subsequently adopted for final image acquisition.

Each serum was added to wells containing CCHFV-GPC (glycoprotein precursor), CCHFV-N (nucleocapsid protein) and EU 90 (control-transfected cells) antigens in three different locations. All the sera were diluted 1:50 in sample buffer from the kit and incubated for 45 min at 25 ºC in a humid chamber.

After two washes (1 min and 5 min) with shaking in PBS-Tween, the secondary antibody diluted 1:100 in PBS with 1% BSA (Thermo Fisher Scientific, MA, USA) was incubated for 30 min at 25ºC in a humid chamber followed by two washes (1 min and 5 min) with shaking using PBS-Tween. A Rabbit anti-Bovine IgG (H + L) Secondary Antibody, FITC (Invitrogen, MA, USA) and a Rabbit anti-Deer IgG (H + L) Secondary Antibody, FITC (Seracare, MA, USA) were used for bovine and deer serum samples, respectively.

Slides were mounted using antifade mounting medium with DAPI (Vectashield, CA, USA) and observed on a Nikon Eclipse TI-FL inverted microscope.

Nucleic-acid extraction and viral RNA detection

Individual ticks were homogenized at 20% (w/v) with phosphate-buffered saline (PBS) under mechanical homogenization with 0.5 mm zirconium beads (Sigma-Aldrich, St. Louis, Missouri, EUA) using four cycles of 15 s at 3000 rpm with an interval of 10 s (Precellys^® Evolution, Belgic) and then clarified at 3,000 g for 5 min.

Total nucleic acid was extracted from 200 µL of the clarified tick supernatant or blood samples, using the IndiMag^® Pathogen Kit (Indical, Leipzig, Germany) in a KingFisher Flex extractor (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Nucleic acids were preserved at −20 °C until use. The extraction process was validated using an 18 S rDNA qPCR¹⁶ and an RT-qPCR for the detection of spiked synthetic RNA (VLP-RNA EXTRACTION, Meridian Life Science, Memphis, TN, USA), which was added to the sample (4 µL/sample) prior to extraction.

Detection of the CCHFV genome in blood samples and in ticks was performed using RT-qPCR targeting the nucleoprotein gene (S segment), as described by Koehler et al.¹, with the One-step NZYSpeedy RT-qPCR Probe kit (NZYtech, Portugal).