*4.3. Serology*

Blood samples were collected in dry tubes, allowed to clot, and centrifuged at 1500 rpm for 10 min and stored at +4 ◦C during 1 month at most or at −20 ◦C for long term archiving.

For equine suspected cases reported to the French NRL, sera were first screened for anti-WNV antibodies by competition ELISA (ID Screen West Nile competition kit, IDVet Company, Montpellier, France) in local veterinary laboratories. Then IgG positive sera were further analyzed by M-antibody capture ELISA for IgM detection (ID screen West Nile IgM capture, IDVet company, Montpellier, France) in local veterinary laboratories and confirmed at the NRL. Analysis and interpretation of ELISAs were performed according to the manufacturer's instructions. In the event of IgM positive screening, the first samples collected during WNV outbreaks were confirmed by microneutralization test (MNT) as described in Beck et al. [66]. A confirmed case was therefore defined as a clinical suspected horse with at least a positive IgM ELISA test.

For human WNV diagnosis, sera and cerebrospinal fluid (CSF) were tested by in-house ELISAs (indirect IgG and MAC-ELISAs) using precipitated and inactivated virus. A case of WNV infection is confirmed with the presence of IgM in CSF and/or IgM and IgG in sera and anti-WNV neutralizing antibodies [67].
