Evolution of avian encephalomyelitis virus during embryo-adaptation

•AE outbreaks after vaccination were not caused by embryo-adaption of the vaccines.•Two vaccines and one field isolate were embryo-adapted after only seven passages.•Two SNPs distinguishing wild-type and embryo-adapted AEV strains were found.•Embryo-adaption was not correlated with higher viral load. Wild-type avian encephalomyelitis virus (AEV) causes neurological signs in young chicks but no disease in pullets after oral or intracutaneous infection. However, if the virus gets embryo-adapted by serial passaging in chicken embryos, it will cause AE after intracutaneous infection in chickens of all ages. Recently, several cases of AE in layer pullets occurring shortly after intracutaneous vaccination were described. The present investigation was initiated to determine if vaccines that had inadvertently been embryo-adapted were responsible for these outbreaks. Virus isolation was done from two vaccines and one field sample. One of the vaccines had been used in one of the flocks before the outbreak. After the first passage, regardless of the inoculum, no embryo was paralyzed, indicating that the vaccines and the field isolate were not embryo-adapted. After seven passages all three strains were fully embryo-adapted causing typical lesions in the embryos. Viral load as determined by RT-qPCR remained constant during the passages. Partial sequences of the VP2 gene of vaccines, the field sample and four other field isolates were nearly identical and highly similar to published sequences from all over the world; only sequences originating from non-vaccinated birds were clearly set apart. Analysis of whole genomes identified two single nucleotide polymorphisms (SNPs) that distinguished wild-type and embryo-adapted strains. Sanger sequencing brains and nerves of the five field isolates and of the first, third and fifth passages of the isolates showed that the mutations indicating embryo-adaptation were first observed in the fifth passage.