**3. Discussion**

West Nile fever is now the most important mosquito-borne viral disease in Europe. The incidence of WNV peaked in 2018 with a total number of 2083 confirmed human cases (a 7.2-fold increase over the previous year) in Europe [18]. The massive outbreaks affected mainly Southern (Italy, Greece, Spain, France), Eastern (Croatia, Serbia, Bulgaria, Romania) and Central (Austria, Hungary, the Czech Republic) Europe, and expansion into previously virus-free regions (Slovenia, Kosovo). In a broader context, we should also consider the role of genetic, ecological, environmental and possible socio-economic aspects that may have played a role in increased WNV activity during the 2018 transmission season, most importantly suitable environmental factors for mosquito vectors, particularly increased day temperature [18] as well as more specific environmental factors such as a large number of vessels with stagnant water (barrels, watering cans and containers), which are constantly found in urban areas in the summer months (applies mainly to the Czech Republic). These objects represent ideal places for mass breeding of WNV vectors.

The Czech Republic and Germany are countries with the northernmost spread of WNV in Europe [3,19]. Increased surveillance, including large-scale surveys of mosquitoes, horses and birds, carried out during two large-scale EC-funded cooperation projects (EDEN and EDENext) between 2008 and 2015, has long indicated that WNV cases may occur in the country. As for the supervision of birds, Hubálek et al. [20] examined 54 domestic birds (geese and ducks) and 391 wild birds representing 28 migratory and resident species, using VNT in the South Moravian fishpond ecosystem. Antibodies to WNV were not detected in domestic waterfowl, but 23 (5.9%) wild birds of 10 species showed a positive response. Straková et al. [21] examined antibodies against WNV and USUV in 146 common coots (*Fulica atra*) on ponds in Moravia. Our results show that both WNV and USUV infections occur in common coots, and this species of bird can serve as an "indicator" of the presence of these viruses in fishpond and wetlands in Central Europe. In addition, two goshawks (*Accipiter gentilis*) held captive by falconers in Moravia died of WNV encephalitis in 2017 [22] and among predators, especially goshawks (several of them wild), WNV encephalitis broke out in the Czech Republic in 2018 [23].

According to recent data from several European countries [24,25], goshawks can serve as suitable indicators for active WNV circulation during the summer season in Europe. As far as horse surveillance is concerned, no case of West Nile fever has been reported in horses so far. The State Veterinary Institute in cooperation with the reference laboratory for arboviruses, regularly examines horse sera from all districts in the Czech Republic. Blood sera from 163 horses were examined from various parts of the Czech Republic in a plaque reduction neutralization test (VNT), but no specific WNV antibodies were detected [26]. A similar examination of a much larger sample of horses (2349 animals) revealed 11 horses (0.47%) with specific antibodies to WNV [27]. Regarding mosquito monitoring, WNV-2 was detected (RT-PCR) in *Culex modestus* mosquitoes collected in ponds in South Moravia during August 2013 and also isolated (newborn mice). Phylogenetic analysis has shown that these Czech WNV strains are closely related to the Austrian, Italian and Serbian strains reported in 2008, 2011 and 2012, respectively [3]. A total of 61,770 female *Cx. modestus* were collected in South Moravian ponds in the years 2010 to 2014, and 1243 samples were examined for the presence of flaviviruses by RT-PCR. Nine strains of WNV lineage 2 were detected in *Cx. modestus* collected in the same reed ecosystem. USUV and WNV co-circulate in the same wetland ecosystem, characterized by the presence of waterfowl and *Cx. modestus* mosquitoes, serving as hosts and vectors, respectively, for both viruses [8]. In addition, ornithophilic *Cx. pipiens* was demonstrated as a vector in 2015 [4]. A total of 28,287 hibernating mosquitoes caught in February or March from 2011 to 2017 in a WNVendemic area of South Moravia were screened for the presence of WNV RNA. No WNV

positive pools were found from 2011 to 2016, while lineage 2 WNV RNA was detected in 3 pools of *Cx. pipiens* mosquitoes collected in 2017 at 2 study sites. The data support the hypothesis of possible WNV persistence in mosquitoes throughout the winter season in Europe [28]. Interestingly, antibodies to WNV (overall 5.9% prevalence) were documented by VNT in the blood sera of wild artiodactyls including roe deer, red deer, fallow deer, mouflons and wild boars, sampled in the South Moravian district of Bˇreclav [29].

Blood safety testing started after the first WNV human cases were confirmed in the affected area (from September until November 2018). The Transfusion and Tissue Department of the University Hospital Brno started WNV testing of blood donors by PCR in September 2018 and finished at the end of November 2018. This solution was based on an epidemiological situation in South Moravia published on the websites of the European Centre for Disease Prevention and Control (ECDC). During this period, 4400 blood donors were tested with negative results.
