Hemagglutinin Subtype Specificity and Mechanisms of Highly Pathogenic Avian Influenza Virus Genesis

Abstract

Highly Pathogenic Avian Influenza Viruses (HPAIVs) arise from low pathogenic precursors following spillover from wild waterfowl into poultry populations. The main virulence determinant of HPAIVs is the presence of a multi-basic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein. The MBCS allows for HA cleavage and, consequently, activation by ubiquitous proteases, which results in systemic dissemination in terrestrial poultry. Since 1959, 51 independent MBCS acquisition events have been documented, virtually all in HA from the H5 and H7 subtypes. In the present article, data from natural LPAIV to HPAIV conversions and experimental in vitro and in vivo studies were reviewed in order to compile recent advances in understanding HA cleavage efficiency, protease usage, and MBCS acquisition mechanisms. Finally, recent hypotheses that might explain the unique predisposition of the H5 and H7 HA sequences to obtain an MBCS in nature are discussed.

In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis

“…Sequences were aligned using MUSCLE [ 41 ], and phylogenetic HA ORF trees were constructed using FastTree2 [ 42 ] to ensure HA sequences were sorted correctly by subtype ( Figure 1 ). Since the mutations leading to HPAIV genesis have always occurred between the P5 and P1′ codons of the cleavage site (P1/P1′ corresponding to the R*G cleavage site) [ 4 ], the P6 to P1′ region was extracted from the ORFs, and only cleavage sites containing no insertions were kept to focus on LPAIV sequences. As tetrabasic cleavage sites, i.e., MBCSs acquired via substitution, have been observed in some HPAIV HAs [ 4 ], 51/1220 H5 HA sequences with a tetrabasic MBCS were also excluded.…”

“…However, AIVs from the H5 and H7 subtypes can evolve into highly pathogenic avian influenza viruses (HPAIVs) [ 2 ]. Virtually all documented HPAIVs have emerged in poultry, and in these species, they cause severe systemic disease leading to mortality rates as high as 100% [ 4 ]. The main virulence determinant of HPAIVs in poultry is the HA proteolytic cleavage site, which is changed from a monobasic cleavage site in LPAIV HAs (arginine (R)/lysine (K)) into a multi-basic cleavage site (MBCS, typically (R n /K n -)R-R/K-R/K-R) through substitutions or, more commonly, via insertion of additional nucleotides by the influenza virus RNA-dependent RNA polymerase (RdRp) [ 4 ].…”

“…HAs with an MBCS can be cleaved by ubiquitous furin-like proteases rather than tissue-restricted LPAIV-activating trypsin-like proteases, supporting the systemic spread of HPAIVs in poultry. Since 1959, there have been 51 independent recorded emergences of AIVs with an MBCS, all of them of the H5 and H7 subtypes [ 4 ], with the exception of an H4 virus which remained trypsin-dependent [ 6 ].…”

“…In some H5 HPAIVs, the MBCS is acquired via nucleotide substitutions, leading to the replacement of non-basic amino acids by basic ones. More commonly, MBCS acquisition results from an insertion of at least six nucleotides coding for two basic amino acids [ 4 ]. Substitutions can be explained by the low fidelity of the influenza RdRp [ 12 , 13 ], but the exact mechanisms underlying insertions at the HA cleavage site remain to be elucidated.…”

“…Secondly, a subset of MBCS acquisitions, so far only documented in H7, are the result of non-homologous recombination (NHR) leading to the insertion of 9 to 30 nucleotides of other viral or host RNAs at the HA cleavage site [ 18 , 19 , 20 , 21 , 22 ]. We focused here on MBCS acquisition via stuttering or backtracking, which has occurred in both H5 and H7 LPAIVs [ 4 ].…”