Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

Abstract

High-pathogenic avian influenza viruses (HPAIVs) evolve from low-pathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.

Fig. 1.

Course of disease in chickens infected with the Hp-HA_poly reassortants. Daily clinical scores after oculonasal inoculation with 10⁶ pfu of Hp-Wt, Hp-H8_poly, Hp-H2_poly, Hp-H4_poly, Hp-H5_It98, Hp-H6_poly, Hp-H14_poly, Hp-H1_poly, or Hp-H15_poly. Scores from birds infected with Hp-H3_poly, Hp-H10_poly, or Hp-H11_poly and from mock-infected animals were 0 or close to 0 throughout the whole observation period and are therefore shown in Table 3 only. For each group, three animals were infected except the Hp-H4_poly, Hp-H6_poly, and Hp-H8_poly groups, in which four animals were included. Animals were scored as healthy (score 0), ill (score 1), severely ill (score 2), or dead (score 3); the arithmetic mean of individual values gives the daily clinical score.

Fig. 2.

Transmission among chickens. Daily clinical scores of animals inoculated oculonasally with 10⁶ pfu of Hp-Wt, Hp-H2_poly, Hp-H4_poly, or Hp-H8_poly and of contact chickens included on the following day. The six primarily inoculated chickens were divided into two subgroups of three individuals to which two contact animals were added. All birds were observed daily for clinical signs and scored as healthy (score 0), ill (score 1), severely ill (score 2), or dead (score 3).