The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken

Abstract

Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV. Here, we aimed to unravel such virulence determinants in addition to the HA gene. Stepwise reduction of HPAIV genes revealed that the HPAIV HA and NA form a minimum set of virulence determinants, sufficient for a lethal phenotype in chicken. Abolishing the NA stalk deletion considerably reduced lethality and prevented transmission. Conversely, the analogous stalk deletion reconstructed in the NA of an LPAIV reassortant carrying only the HPAIV HA resulted in 100% lethality both after primary and contact infection. Remarkably, the unmodified LPAIV NA with its long stalk, when exclusively introduced into the H5N1 HPAIV, still enabled high virulence and efficient transmission. Therefore, irrespective of an NA stalk deletion, minor virulence determinants in addition to the essential polybasic HACS contribute to high virulence, whereas the NA stalk deletion alone may serve as major virulence determinant.

Figure 1. Simultaneous presence of TG05 and R65 genes determined by Sanger sequencing of supernatants following plasmid co-transfection to obtain mixtures of random reassortants.

(A) The TG05 polymerase gene plasmids (PB2, PB1, PA, NP) and the R65 HA plasmid were co-transfected together with mixtures of the NA, M, and NS plasmids originating both from TG05 and R65. (B) The TG05 plasmids except the HA plasmid were co-transfected together with all eight R65 plasmids.

Figure 2. High lethality following oculonasal infection of chickens with 600 μl co-transfection supernatants.

Daily clinical score: 0: healthy, 1: ill, 2: severely ill or 3: dead. (A) Birds infected with supernatant from co-transfection of the TG05 polymerase gene plasmids (PB2, PB1, PA, NP), the R65 HA plasmid and mixtures of the NA, M, and NS plasmids originating both from TG05 and R65. (B) Birds infected with supernatant from co-transfection of the TG05 plasmids except the HA plasmid and all eight R65 plasmids.

Figure 3. Minimal R65 gene constellations sufficient for a lethal phenotype and transmission in chicken.

We infected the birds oculonasally with 10⁵ pfu virus and added bystander animals on day 1 p. i. Daily clinical score: 0: healthy, 1: ill, 2: severely ill or 3: dead.

Figure 4. NA stalk deletion is crucial for transmission among chickens.

We infected chickens oculonasally with 10⁵ pfu virus and added bystander animals on day 1 p. i. Daily clinical score: 0: healthy, 1: ill, 2: severely ill or 3: dead.