Virulence of chimeric avian influenza viruses of subtypes H4 and H8 with polybasic cleavage site

Avian influenza viruses (AIV) are classified according to their pathogenicity in chickens into low pathogenic (LP), exhibiting hemagglutinins (HA) of all subtypes (H1-H16), or highly pathogenic (HP), restricted to the subtypes H5 and H7. In the latter subtypes, existence of polybasic cleavage site (PCS) within the HA is mostly the main virulence determinant. To investigate if the HP phenotype is restricted to these two subtypes, we cloned the HA genes of nearly all HA subtypes, introduced an artificial PCS and generated the respective reassortants in an HPAIV H5N1 background. The generated mutants carrying the H8, H2 or H4 HA were lethal for infected chickens. Thereafter, the role of PCS, HA and/or other segments in virulence of the LPAIV H4 and H8 in chickens were studied using reassortants of H4/H5, H8/H5 and H4/H8 exhibiting PCS motifs. The introduction of a PCS into these avirulent LPAIV caused only mild disease. Within the H4 background the introduction of the HPAIV H5 HA showed the highest increase in virulence followed by the nucleoprotein (NP). In contrast, in the H8 background a recombinant virus containing the H5 NP was more virulent than that carrying the H5 HA. Interestingly, a reassortant of H8 HA with PCS in the H4 background was also capable of causing lethal disease. Thus, our results indicated the possible emergence of non-H5/H7 HPAIV and posed the question about the role of NP in contribution to virulence. Therefore, LPAIV infections in poultry should not be underestimated.