Test panels were composed of 8–10 coded samples. A single panel was sent each year to test the different techniques routinely used in the participating laboratory as part of its animal rabies post-mortem diagnostic process. All the participants received the same panel but with samples coded differently. Samples contained 1 mL of lyophilised brain homogenate of mouse, dog, pig or raccoon dog origin experimentally infected by various fixed (CVS-27) or field strains of lyssavirus such as RABV (Rabies lyssavirus), EBLV-1 (European bat lyssavirus 1), EBLV-2 (European bat lyssavirus 2), DUVV (Duvenhage lyssavirus), BBLV (Bokeloh bat lyssavirus) or ABLV (Australian bat lyssavirus). At least one RABV and one EBLV-1 sample were included each year in the test (Table 2). Negative samples (NEG) originated from confirmed negative red fox or pig brain samples from France (a country free from infection with RABV). Some batches named “RABV dil” were RABV samples (from a French RABV GS7 strain isolated in 1990) diluted with a rabies-negative brain sample. All the ABLV, BBLV and DUVV batches used during the study were produced from a single unique virus strain (strain 127900 for BBLV, strain 96132 for DUVV and strain 96/0648 for ABLV). EBLV-2 was from strain RV1332 from 2009 to 2012, and then from RV1787 from 2013 to 2019. EBLV-1 was from EBLV-1a (strain 122936) or EBLV-1b (strain 121411 from 2009 to 2012 and strain 123008 from 2013 to 2019) (see S1 Table). RABV batches were from different rabies viruses isolated in various parts of the Maghreb or Europe (See S2 Table). Various additional samples from previous batches were also included in panel tests (from 2016 to 2019) in order to vary the composition of panels from one participant to another and thus avoid collusion between laboratories. These samples were not considered for the evaluation.

Virus batches were produced in vivo according to Robardet et al. (2011) [18]. Briefly, mice, foxes, dogs or raccoon dogs were inoculated intracerebrally. Animals having shown signs suggestive of rabies (experimentation continued up to stage 4/5 of the disease [19]) validated a posteriori by FAT diagnosis [20,21] were humanely euthanised. The first animal of a batch production with suggestive signs was diagnosed by the FAT to confirm rabies infection. For each batch of virus, the brain samples from euthanised animals were excised then mixed, homogenised and aliquoted into 1 mL tubes before being freeze-dried. Whenever possible, brain samples of foxes, dogs and raccoon dogs were collected from other scientific research being carried out in the laboratory as long as the virus inoculation protocol was the same as that used for virus production dedicated to rabies diagnosis proficiency test studies. Panel samples were the lyophilised homogenates of fresh infected brains, each sample representing different viral strains and all blindly coded.

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