**1. Introduction**

West Nile virus (WNV) is an enveloped spherical, single-stranded positive-sense RNA virus belonging to the *Flaviviridae* family [1]. It is maintained in nature in an enzootic cycle involving ornithophilic mosquitoes (mainly *Culex*) as transmission vectors and certain birds as reservoir hosts. Spill-over from this cycle occasionally results in severe outbreaks in horses and humans that are considered dead-end hosts, which means that they cannot transmit the virus to feeding mosquitoes due to their low and transient viremia [2].

Although most human infections are asymptomatic, in some instances (~20%) the virus can cause a febrile syndrome (WNV fever). Around 1% of the cases progress to severe neuro-invasive disease with a fatality rate of around 10% [3]. In horses, the infection is generally asymptomatic, but approximately 10% of the infected animals develop neurological symptoms such as ataxia, limb paralysis, skin fasciculation and muscle tremors [4]. In birds, the pathogenic potential greatly di ffers among species and also depends on the viral strain. Passerine birds (especially corvids) and some raptor species are particularly susceptible to WNV infection [5].

Phylogenetic studies have revealed the existence of at least seven genetic lineages, of which lineages 1 (L1) and 2 (L2) are the most widespread and relevant for human and animal health [6]. Circulation of the virus has been regularly reported in wide areas of the Euro-Mediterranean area since 1998 [7]. However, in recent years, the virus has dramatically expanded, with an upsurge in the number and incidence of outbreaks in humans and animals caused by both L1 and L2 strains. In fact, in 2018 the transmission season started earlier and the number of human autochthonous cases reported in EU and neighboring countries (2083 cases) exceeded the global number from the previous seven years. In horses, 285 cases were notified, which represents an increase of 30% in comparison with 2017 [8]. However, epidemiological data on WNV from Black Sea countries is scarce and therefore surveillance e fforts in this region needs to be harmonized with those implemented in other countries.

The epidemiological situation in the Mediterranean basin is complex because, apart from WNV L1 and L2, other flaviviruses circulate in overlapping areas [6,9]. This is the case with Usutu virus (USUV), a zoonotic arbovirus closely related to WNV that has spread throughout Europe since 2001, when it was first detected in Austria [10]. Nowadays many countries have reported the presence of both viruses in mosquitos, birds and humans [9,11]. Of the countries participating in this EQA, in at least three (Serbia, Tunisia and Morocco) the co-circulation of both viruses has been demonstrated [12–14].

WNV diagnostic methods include virus isolation, reverse-transcription polymerase chain reaction (RT-PCR) and serological tests. Isolation procedures are laborious and require biosecurity level 3 (BSL-3) facilities. By contrast, molecular methods, such as RT-PCR, and particularly real-time RT-PCR (RRT-PCR), can be easily applied in basic laboratories, are fast and sensitive, enabling timely detection and early outbreak response [6]. In the current context, RRT-PCRs that allow for simultaneous detection of WNV L1, L2 and other flaviviruses such as USUV are extremely useful for outbreak investigations and epidemiological studies, maximizing the information obtained from each sample [15].

Among the antibody detection tools, ELISA tests are the most widely used with several commercial kits available. However, a relevant limitation of WNV ELISA tests is the cross-reactivity of antibodies raised against di fferent flaviviruses that can lead to diagnostic misinterpretations [6,16]. To confirm WNV infection, the gold standard method is virus neutralization (VNT) that enables di fferential diagnosis by titration of neutralizing antibodies in parallel against di fferent flaviviruses that could cross-react in serological tests. Nevertheless, VNT is time-consuming and has to be performed in BSL-3 labs.

Prevention and control e fforts substantially rely on e ffective surveillance of the infection in animals and vectors that can act as early warning triggers [17]. The implementation of locally adapted surveillance systems in birds, horses and mosquitos and the upgrade of the diagnostic capacities of veterinary laboratories is crucial to fight the disease.

MediLabSecure is an EU-funded project whose main objective is to create a framework for collaboration to promote arbovirus surveillance under a One Health approach in 19 countries of the Mediterranean and Black Sea regions [18,19]. Since the beginning of the project in 2014, the MediLabSecure animal virology network has implemented numerous actions to enhance capacity building of veterinary labs to face health threats caused by emerging arboviruses.

In a specific questionnaire delivered to identify the priorities of the beneficiary countries in the field of arboviral diseases, all the labs recognized WNV as a common health priority in the region. A training curriculum was implemented to improve diagnostic performance of the veterinary labs for this pathogen, including two diagnostic workshops (molecular and serological diagnosis) that were organized in 2015 and 2016 at Centro de Investigación en Sanidad Animal (INIA-CISA) (Madrid, Spain). After these training sessions, an external quality assessment (EQA) was organized between October 2016 and March 2017 to evaluate the degree of learning and the capacity of the labs to incorporate the molecular and serological techniques into their routine diagnostic activities. In this study we report the results of this inter-laboratory trial and provide relevant information about the current WNV (and USUV) diagnostic capacities of veterinary labs in the Mediterranean and Black Sea regions. This exercise also enabled an extensive reproducibility assessment of the recommended tests for WNV and USUV diagnostics.
