Equine sera: 260 equine blood samples (251 horses, 7 donkeys, and 2 mules; mean age = 10.9 years, range = 1–32 years) were collected from seven regions of Kosovo between January and September 2018: Prishtina n = 32, Mitrovica n = 28, Peja n = 25, Prizren n = 47, Gjakova n = 12, Ferizaj n = 50, and Gjilan, n = 66. The samples originated from 177 different animal owners. There are 2,353 equines in Kosovo . Blood samples were collected randomly from healthy horses with no history of WN diseases or symptoms of neurological disorders. Sera were kept at -20 °C until use.
Bird sampling: 50 sera of domestic birds (backyard chickens) were collected. The birds, aged from 1 to 3 years old, were purchased from free-range farms. After slaughtering, 5 ml blood was collected from each animal while 5 g of viscera tissues from the brain, liver, heart, kidney, and lungs were pooled and stored in -80 °C freezer. In addition, 51 wild bird samples from the Corvidae family were collected randomly, as found dead by hunters and stored in -20 °C freezer. The birds’ viscera (brain, liver, heart, kidney, and lungs) were pooled and stored at -80 °C.
Mosquito sampling: Most of the mosquito samples were collected from the same farms as the equine samples. Before RT-PCR screening, all mosquitoes were pooled from 5 to 30 individuals, depending on species and collection sites. Collections were made using an Insect Monitoring Trap (IMT) with dry ice. Collected specimens were stored in a -20° C freezer. The 626 mosquitoes (60 pools) were grouped in five genera, including Culex spp. (226 individuals; 26 pools), Aedes spp. (136 individuals; 16 pools), Anopheles spp. (184 individuals; 7 pools), Culiseta spp. (34 individuals; 4 pools), and Phlebotomus spp. (46 individuals; 7 pools). The sampling period coincided with the peak activity period for these mosquito species. Adult specimens were morphologically identified to species level using interactive identification keys for mosquitoes of the Euro-Mediterranean Region .
ELISA (Enzyme-linked immunosorbent assay): Sera were tested for WNV-specific antibodies using a commercially available ELISA-blocking enzyme immunoassay, which allowed recognition of WNV-IgG-antibodies (INGEZIM west Nile COMPAC, Madrid, Spain). ELISA was performed according to the manufacturer’s instructions.
PRNT (Plaque Reduction Neutralization Test): All ELISA-positive serum samples were then subjected to PRNT to confirm the presence of virus specific antibodies. The test was performed on two-fold dilutions of the serum samples (starting from 1:20) against a 100 pfu/100 ∝L diluted NY99 (lineage 1) strain of the WNV. Serum samples blocking 90% of plaque occurrence were considered positive for WNV-specific antibodies.
VNT (Virus Neutralization Test): eight ELISA and PRNT positive horse sera were then confirmed by virus neutralization test (VNT), with neutralizing antibody titers from 1:320 to 1:1,280, using WNV strain NY99 and USUV strain 1477 on Vero E6 cells .
RNA extraction from mosquito and bird organs: the mosquito pools (5–30 individuals) and 0.5 g of bird organ (0.1 g of brain, lung, heart, kidney, and lungs) were placed in a 2 ml Eppendorf tube containing three to five steel beads (7 mm, Qiagen, Hilden, Germany), frozen on liquid nitrogen and directly pulverized in a Tissue-Lyser LT (Qiagen, Hilden, Germany) with a − 20 °C pre-cooled rotor at 50 Hz for 2–5 min. Repeated freezing and lysis was necessary in some cases. The samples were then re-suspended in 300–500 µl PBS (depending on tissue size) containing 500 IU/ml penicillin, 10% fetal calf serum, and 500 µg/ml amphotericin, and centrifuged at 2,000 × g. Viral RNA extraction was done with QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
RT-PCR: 25 µl RNA of each sample extractions were tested using RealStar® WNV RT-PCR Kit 1.0, (Altona Diagnostics, Hamburg, Germany) for the detection of West Nile virus (WNV) RNA, according to the manufacturer’s instructions.