*4.3. EQA Details*

For the molecular detection, two assays were proposed. First, for generic detection of flaviviruses we recommended the hemi-nested conventional RT-PCR described by Scaramozzino et al. [35] with some modifications as described in the previous section. This RT-PCR targets the highly conserved NS5 region and it is a useful first-line molecular screening test for an unknown flavivirus. However, definitive flavivirus determination requires post-amplification identification techniques (e.g., genome sequencing) or the application of RT-PCR techniques for species-specific detection of flaviviruses. For this reason, the second recommended assay was a triplex RRT-PCR that enables simultaneous

detection and differentiation of WNV L1, L2 and USUV [15]. This is based on different sets of primers and fluorogenic probes specific to each virus that are labelled with selective, non-overlapping fluorogen-quencher pairs (FAM for WNV L1, VIC for WNV L2 and Cy5 for USUV). This multiplex RRT-PCR is very sensitive and specific and has been widely validated with experimental and field samples [15,39]. Taking into account the epidemiological situation of the concerned region, where different WNV lineages co-circulate with other related flaviviruses, and especially USUV, this diagnostic approach was considered highly beneficial for surveillance and diagnostic studies.

The labs reported the use of eight different real-time thermocyclers: Rotor-Gene 3000 (5 labs), Applied Biosystems 7500 (5 labs), Applied Biosystems 7300 (2 labs), Aria Mx Agilent (1 lab), Bioer Gene Max (1 lab), Abi QuantStudio (1 lab), StepOnePlus (1 lab) and Stratagene Mx3005 (1 lab).

For serological diagnosis of WNV infection, two commercially available ELISA tests were recommended. The first was the INgezim West Nile Compac ELISA kit (Ingenasa) that allows the detection of anti-E domain III antibodies [40]. It is a multispecies ELISA that only requires 10 μL of sample which is very advantageous for the analysis of sera from small birds. Several studies have proved that this assay is more specific than other commercial ELISAs that, although designed to identify WNV antibodies, also detect cross-reacting antibodies directed against other flaviviruses and especially USUV [16,29]. Moreover, the INgezim West Nile Compac ELISA detects both IgG and IgM antibodies and, based on the results of previous EQAs, it seems that this kit enables more efficient detection of recently-infected animals than other commercial kits [29]. To specifically identify recent (acute) infection in horses, we selected an IgM antibody capture ELISA (MAC-ELISA) from the same company, the Ingezim WNV IgM ELISA. This assay was recommended based on our own comparative studies and on the results of an EQA organized in 2013 where this kit demonstrated the highest analytical sensitivity in horses experimentally infected with WNV L1 and L2 [29].

Detailed standard operating procedures for the recommended assays were distributed and all the reagents and kits were provided to each laboratory, including the mentioned ELISA kits, the extraction kit (QIAamp® Cador Pathogen Mini Kit, QIAGEN), the RT-PCR kit (SuperScript® III One-Step RT-PCR System, Invitrogen) and primers for generic flavivirus detection, the RRT-PCR kit (QuantiTect Probe RT-PCR kit, QIAGEN), and primers and probes for the triplex RRT-PCR. As explained earlier, extraction and reaction positive controls were also delivered to all the labs.

For the molecular panel, specific instructions were provided to reconstitute the lyophilized samples as well as to prepare the positive extraction control. For the serological panel, the laboratories were asked to analyze the panel samples using the recommended kits following manufacturer's instructions. Additionally, the labs were encouraged to analyze both panels using alternative methods (other protocols that may be established in the labs) and report the results together with those derived from the recommended methods.

The extraction kit, the lyophilized samples, the positive extraction control and the ELISA kits were shipped at room temperature. The panel of sera, the RT-PCR kits, the positive reaction control and the primers and probes were shipped in dry ice. A number code was assigned to each laboratory to ensure a blind analysis of the results.

**Author Contributions:** Conceptualization: J.F.-P., M.Á.J.-C., E.P.-R., C.C.-G., F.L.; Formal analysis: E.P.-R., C.C.-G., F.L.; Funding acquisition: M.Á.J.-C.; Investigation: E.P.-R., C.C.-G., F.L., J.F.-P., M.Á.J.-C., A.V., L.V., N.T., K.S., S.S., A.O., A.K., K.K., I.K.-H., N.M.H., J.E.H., H.D., M.S.B., B.A., N.A. Methodology: E.P.-R., C.C.-G., F.L., J.F.-P., M.A.J.-C. Project administration: M.Á.J.-C., E.P.-R.; Supervision: J.F.-P., M.Á.J.-C.; Writing—original draft: E.P.-R.; Writing—review & editing: E.P.-R., C.C.-G., F.L., J.F.-P., M.Á.J.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** MediLabSecure project is supported by the European Commission (EU DG DEVCO: IFS/21010/23/-194 and 2018/402-247). www.medilabsecure.com.

*Pathogens* **2020**, *9*, 1038

**Acknowledgments:** We thank María del Carmen Barbero, Pilar Aguilera, Ana María Robles and Amalia Villalba for excellent technical assistance during preparation and testing of the panels. We are grateful to Ana Moreno (IZSLER, Brescia), Norbert Nowotny (University of Vienna) and Ana Vázquez (ISCIII, Spain) for kindly providing the USUV, WNV L2 and Nakayama JEV strains that were used to prepare the molecular panel. We thank Jordi Figuerola and Ramón Soriguer (EBD-CSIC, Spain) for providing the sera from naturally infected horses and to Sylvie Lecollinet (ANSES, France) for providing the reference serum. We are also grateful to Lyudmila Maruschak and Elena Coada for participating in the analysis of the panels in Ukraine and Moldova.

**Conflicts of Interest:** The authors declare no conflict of interest. The contents of this article are the sole responsibility of the authors and do not necessarily reflect the views of the European Union.

**Ethical Statement:** The tissue samples and sera used for the preparation of the EQA panels were selected from the biobank of INIA-CISA (Madrid, Spain). None of them were specifically collected for this study. All the samples used to prepare the molecular panel originated from animal studies that were authorized by INIA Animal Experimentation Ethics Committee according to European Directive 2010/63/EU (Spanish Royal Decree 53/2013). The sera used in the serological panel were obtained from naturally infected horses in Doñana National park (Southern Spain) that were sampled by expert veterinarians in the framework of a WNV sero-surveillance research project. All procedures to obtain these samples were approved by CSIC Ethics Committee.
