Two independent evolutionary pathways of HPAIV

HPAIV originate from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS). Beside this prime virulence determinant, additional adaptive changes might accumulate already in those precursors during their circulation in gallinaceous poultry prior to the emergence of an HPAIV. Since this evolutionary process is not well understood, we aimed to unravel the genetic determinants which, beyond the polybasic HACS, are required for transformation of LPAIV into HPAIV. To select a minimal gene constellation sufficient for high virulence, we co-transfected plasmids coding for all eight genes from an H5N1 HPAIV and seven, except HA, from an H5N1 LPAIV, and used the supernatant to infect chickens. Shed reassortants carried the HPAIV PB2, NP, HA, NA, and M genes; a reconstituted virus was highly pathogenic and transmissible like the wild-type. A tailored H5N1 reassortant, carrying the LPAIV NA, exhibited 100% lethality both in inoculated and contact chickens. The same lethal phenotype was exhibited by an LPAIV reassortant carrying only the HPAIV HA and NA. However, abolishing the NA stalk deletion led to considerably reduced lethality and no transmission. Conversely, an LPAIV reassortant carrying only the HPAIV HA but the LPAIV NA with engineered stalk deletion displayed 100% lethality both after primary or contact infection. Such virulent phenotypes require a polybasic HACS and either the adapted polymerase, M and NS genes together, or an NA with stalk deletion, indicating the existence of at least two different sets of virulence determinants and, therefore, several independent evolutionary pathways for HPAIV.