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Avian influenza viruses (AIV) are classified as either low pathogenic (LP) or highly pathogenic (HP) due to their virulence in
chickens. Highly pathogenic avian influenza viruses (HPAIV) exhibit a polybasic cleavage site (PCS) within the hemagglutinin
(HA) protein and therefore the HA can be cleaved and activated by ubiquitous proteases causing severe systemic disease with high
lethality. Naturally occurring HPAIV have always been of subtype H5 or H7 with very rare exceptions. Recently we showed that
HPAIV can be created with other HA subtypes exhibiting an artificial PCS in a H5 HPAIV background and the introduction of a
PCS within the HA in the parental background was not sufficient. Therefore, the objective of the study was to investigate the minimal
requirement for exhibiting a highly pathogenic phenotype of non-H5/H7 LPAI viruses. Reverse genetics systems were established for
LPAIV strains H4N6 and H8N4. Reassortants of LPAIV HA with artificial PCS and gene segments of a H5N1 HPAIV were generated
and the virulence was ascertained in SPF chickens. In summary, the HPAIV H5N1 nucleoprotein (NP), neuraminidase (NA) and
the matrix protein (M) segments conferred increased virulence. Whereas the impact on virulence of the NA and M gene segments
differed, the NP gene of H5 HPAIV increased virulence in both H4 and H8 backgrounds. Furthermore, the impact of single NP
amino acids was assessed.