The recent outbreaks of avian influenza (AI) worldwide have highlighted the difficulties in controlling this disease. Vaccination has become a recommended tool to support the eradication efforts and to limit the economic losses due to AI. A vaccination system based on the use of a vaccine containing a heterologous neuraminidase to the field virus has been shown to be efficacious in reducing the viral shedding and clinical symptoms and in differentiating vaccinated from infected animals. To further develop this so called differentiating infected from vaccinated animal vaccination system, two reassortant avian influenza viruses of the H7N5 subtype have been generated. The aim of this study was to generate a prototype strain with a rare N subtype to avoid interference with the anti-N discriminatory test.