Additionally, volunteer bird ringers collected 31 samples from 30 chickens at seven locations during the study period, four of which were within the 15 km radius in Utrecht (seeFigure 1). not detected by other existing surveillance schemes in mosquitoes, wild birds, horses and humans. In addition, the results show rapid decay of USUV antibodies in approximately 20 weeks. Our findings support the power and the added value of petting zoo chickens as sentinels for monitoring USUV and WNV circulation compared to other available methods. Seroconversions observed in petting zoos and backyard chickens living in or near densely populated urban areas further highlighted potential public health risks that went undetected. KEYWORDS:West Nile computer virus, Usutu computer virus, Orthoflavivirus, chicken, sentinel, serology, surveillance == Introduction == West Nile computer virus (WNV) and Usutu computer virus (USUV) are both zoonotic mosquito-borne viruses in the Japanese encephalitis serogroup and belong to theFlaviviridaefamily and genusOrthoflavivirus[1]. UNC1079 Both viruses are maintained in an enzootic transmission cycle between birds and mosquitoes (primarilyCulexmosquitoes) and are known to co-circulate in parts of Europe [24].Culex pipiensis recognized as the primary vector for WNV and USUV in Europe. In the Netherlands,Cx. pipiensis a ubiquitous and highly abundant mosquito species [57]. Culex pipiensare known to feed both on avian and mammalian hosts including humans, which may facilitate the spread and spillover of USUV and WNV [8]. The Usutu computer virus has been circulating in continental Europe for more than two decades [9]. In the Netherlands, USUV was detected in 2016 for the first time and has caused UNC1079 significant outbreaks in birds, with associated mortality specifically in wild blackbirds (Turdus merula) and captive owls (Strigiformes) from 2016 to 2018 [10,11]. In 2018, a study on Dutch blood donors revealed multiple (asymptomatic) human USUV infections, which occurred concurrently with an observed increase in bird mortality in the study area [12]. Surveillance of live and lifeless wild birds and mosquitoes has shown ongoing circulation of USUV in the years after [13]. Over the last decades, WNV has become one of the most widespread UNC1079 arboviruses in MGC33570 the world [14]. Outbreaks of disease caused by WNV have been extensively described in southern Europe. In 2018 a major outbreak resulted in 1311 confirmed human cases across Europe [15]. In the same 12 months, the computer virus also was detected northwards as the first WNV cases in Germany were observed in birds and horses [16]. In August 2020, the computer virus was detected for the first time in the Netherlands in a common whitethroat (Curruca communis) andCulexmosquitoes [17]. UNC1079 In October of the same 12 months, the first autochthonous case of human WNV neuroinvasive disease was identified in the Netherlands. Retrospective analysis revealed six additional clinical cases [18]. All WNV detections within the Netherlands were restricted to two specific areas; in the municipality of Utrecht where most WNV positive birds and all positive mosquitoes were found; and near the municipality of Arnhem where a seventh human case was identified in October [19,20]. According to the World Organisation for Animal Health (WOAH), sentinels are defined as susceptible animals of known health or immune status that are regularly tested in specific (outbreak-prone) geographical locations to detect the occurrence of diseases or infections, often through serological testing [21]. Captive sentinel birds, such as chickens (Gallus gallus domesticus) and pigeons (Columbia livia) have been routinely utilized for arbovirus monitoring and surveillance in various settings and across different continents [2224]. Chickens do not show clinical signs following infection but do develop neutralizing antibodies. Experimental studies have shown that chickens do not contribute to the vector-host transmission cycle [25,26]. In UNC1079 addition, sentinels such as chickens can be repeatedly sampled at the same desired locations, while also leveraging on their historical data on origin and movement patterns. Hence, chickens can serve as.