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 West Nile neuroinvasive diseases (WNND) in animals. . Sera were kept at -20°C until use.
Bird sampling: 50 sera of domestic birds (backyard chickens) were collected between June-October 2019. The chickens aged from 1 to 3 years old, were purchased from free-range farms and kept outdoors. 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, such a Hooded Crown (Corvus corone) 2 samples, Jackdaw (Corvus monedula) 9 samples and Raven (Corvus corax) 40 samples, as they 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: Mosquito samples were collected in the period from June-October 2019, from the same farms of the equine samples. Collections were made using an Insect Monitoring Trap (IMT “Genicco SRL”, Italy) with dry ice. The traps were in place overnight. After the morphological identification of mosquitoes on the same day of collection, specimens were stored immediately in a -20° C for one week and after that in a -80° C. Before RT-PCR screening, all mosquitoes were pooled from 5 to 30 individuals, depending on its genera and collection sites. The 580 mosquitoes, adult females (53 pools) were grouped in for 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). The sampling period coincided with the peak activity period for these mosquito species. Adult specimens were morphologically identified under binocular stereomicroscope (Motic, SMZ-161, Hong Kong) to genus level based on main morphological characteristics using interactive identification keys for mosquitoes of the Euro-Mediterranean Region .
ELISA (Enzyme-linked immunosorbent assay): Sera were tested for WNV-reactive 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 mL diluted NY99 (lineage 1) strain of the WNV on Vero cells (ATCC CCL81). 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-qPCR: 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.