Two independent evolutionary pathways of HPAIV

Background HPAIV develop from low-pathogenic precursors of hemagglutinin (HA) serotypes H5 or H7, requiring the acquisition of a polybasic HA cleavage site (HACS). However, introduction of a polybasic HACS into LPAIV does not necessarily result in high virulence for chicken indicating that, beside the essential polybasic HACS, the existence of additional virulence determinants. Those additional adaptive changes might accumulate in the low-pathogenic precursors during their circulation in gallinaceous poultry prior to emergence of an HPAIV. Since this evolutionary process is not well understood, we aimed to unravel the genetic determinants which, beside the polybasic HACS, are required for transformation of LPAIV into HPAIV. Results To select a minimal gene constellation sufficient for a highly pathogenic virus, 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 virus reconstituted by reverse genetics, was highly pathogenic and transmissible like the wild-type. Furthermore, by stepwise exchange of the HPAIV genes, we eventually found that an LPAIV reassortant carrying only the HPAIV HA and NA contains the minimum set of HPAIV genes enabling high virulence, albeit at a prolonged mean death time. Furthermore, we abolished the NA stalk deletion leading to considerably reduced lethality and no transmission in chicken. 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. Therefore, the NA stalk deletion is an essential virulence determinant beside the polybasic HACS. Remarkably, the LPAIV NA which contains no stalk deletion, introduced into the H5N1 HPAIV exclusively, did not reduce virulence and transmission compared to the HPAIV parent virus. Taken together, the HPAIV HA and NA are sufficient for high virulence in an LPAIV background. Moreover, the NA stalk deletion along with the polybasic HACS form a minimum set of virulence determinants. However, our selection experiment in chickens, infected with transfection supernatants, indicates that beside the HPAIV HA and NA, the PB2, NP, and M enable maximal virulence. For that reason, we surveyed all public PB2, NP, and M1 protein sequences from Genbank and GISAID. Data mining revealed that there are natural HPAIV strains corresponding to the here described HA/NA reassortant, confirming the HA with polybasic HACS and the NA with stalk deletion as a minimum set of virulence determinants. However, natural HPAI strains without stalk deletion but with several NP mutations from the HPAIV studied, corresponding to the NA/ HPAIV reassortant, indicate an alternative set of virulence determinants. Conclusions Therefore, those two sets of virulence determinants indicate two independent pathways of evolution of LPAIV into HPAIV.

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