Investigating the Detection and Acquisition of Multibasic Cleavage Sites in H7 Avian Influenza Virus

Abstract

The recent rise in avian influenza virus (AIV), particularly the H5 and H7 subtypes, highlights the need for improved understanding and detection of pathogenic variants. These subtypes exist in two pathotypes: low-pathogenic (LP) and high-pathogenic (HP). LPAIV is characterized by a monobasic cleavage site in the hemagglutinin protein, whereas HPAIV contains a multibasic cleavage site (MBCS) composed of at least four basic amino acids. This structural difference in HPAIV enables cleavage by ubiquitous furin proteases, increasing viral tropism and contributing to enhanced pathogenicity. Despite the critical role of MBCSs in virulence, efficient diagnostic tools for their detection remain limited, and the mechanisms underlying their acquisition are not fully understood or investigated, particularly in H7 AIV. This study investigates both the detection and potential formation mechanisms of MBCSs by evaluating Cas13-based assays to distinguish H7 LP and HP variants across European, North American, and Asian lineages, alongside computational modeling and circular dichroism (CD) spectroscopy to assess transient RNA secondary structures near cleavage sites. Cas13 demonstrated sequence-dependent effectiveness in differentiating pathogenic variants. Computational predictions provided no evidence for G-quadruplex (G4) formation, while CD offered less conclusive evidence of their absence in the analyzed regions. Computational analyses, however, suggested a propensity for T-loop structures to form near the cleavage site. These findings highlight both the promise and limitations of current diagnostic strategies and suggest structural mechanisms that may contribute to MBCS acquisition in H7 AIV, which warrants further investigation to validate these observations and their biological significance.