Avian Influenza Virus Hemagglutinins H2, H4 and H8 support a highly pathogenic phenotype in chicken

Introduction Highly pathogenic avian influenza viruses (HPAIV) evolve from low-pathogenic precursors specifying the hemagglutinin (HA) serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. Since the reason for that HA serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site to the HA protein or unique predisposition for insertion mutations. Methods Using reverse genetics, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10 or H15 and rescued HA reassortants after co-transfection with the seven remaining gene segments from either a lowpathogenic H9N2 or high-pathogenic H5N1 strain. To investigate the virulence of the HA reassortants, we subjected them to oculonasal infection in chicken. For assessment of pathogenicity according to the OIE criteria, we then determined the intravenous pathogenicity index (IVPI). The most virulent reassortants were studied in contact transmission experiments. Results Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4 or H8 in the HPAIV background were lethal for all animals and exhibited IVPI of 2.79, 2.37 or 2.85, displaying values equivalent to conventional H5 or H7 HPAIV. Furthermore, all three reassortants were shed via oral and cloacal routes at lower titers compared with the homologous H5N1 HPAIV and were transmitted to some contact animals. Once infected, those contact animals succumbed to death whereas no surviving chickens developed any clinical symptoms, displayed viral antigen in several organs or sero-converted. Conclusion Taken together, with a polybasic HA cleavage site present, nonH5/H7 viruses can exhibit a highly pathogenic phenotype yet requiring further adaptation for establishment in the field. Therefore, the well-known restriction of authentic HPAIV to the serotypes H5 and H7 is likely due to their unique predisposition for acquisition of a polybasic HA cleavage site.