Influenza viruses are highly genetically variable and escape from immunogenic pressure by antigenic changes in their surface proteins referred to as antigenic drift and antigenic shift. To assess the potential genetic plasticity under strong selection pressure, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was passaged in embryonated chicken eggs in the presence of a neutralizing, polyclonal chicken serum for 50 times. The resulting mutant had acquired major alterations in the neuraminidase (NA)-encoding segment. Extensive deletions and rearrangements were detected in contrast to only 12 amino acid substitutions within all other segments. Interestingly, this new neuraminidase segment resulted from complex sequence shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to the wild-type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA-segment could be generated, however, required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent despite the still present HPAIV polybasic hemagglutinin cleavage site. Overall, the variants reported here are the first HPAIV H5N1-viruses without functional neuraminidase which grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the neuraminidase in virus replication and pathogenicity.