Influenza A H5N1 hemagglutinin (HA) plays a crucial role in viral pathogenesis and changes in the HA receptor binding domain (RBD) have been attributed to alterations in viral pathogenesis. Mutations often occur within the HA which in-turn results in HA structural changes that consequently contribute to protein evolution. However, the possible occurrence of mutations that results to reversion of the HA protein (going back to an ancestral protein conformation) which in-turn creates distinct HA structural patterns across the 1959-2023 H5N1 viral evolution has never been investigated. Here, we generated and verified the quality of the HA models, identified similar HA structural patterns, and elucidated the possible variations in HA RBD structural dynamics. Our results show that there are 7 distinct structural patterns occurring among the 1959-2023 H5N1 HA models which suggests that reversion of the HA protein putatively occurs during viral evolution. Similarly, we found that the HA RBD structural dynamics vary among the 7 distinct structural patterns possibly affecting viral pathogenesis.

Introduction
Among the known influenza A strains, the H5N1 strain is generally a flu strain that has been infecting various avian species since 1959 (Lycett et al., 2019) with occasional human and mammal infections (Gambotto et al., 2008). Moreover, the receptor binding domain (RBD) plays an important role in the H5N1 hemagglutinin (HA) protein structure as it functions as a major determinant of host range restriction (Imai et al., 2012; Wu et al., 2018; Yang et al., 2015). Additionally, the HA RBD is responsible for viral binding to host receptor cells by interacting with membrane receptor molecules which contain sialic acid (SA) (Rogers et al., 1983). Previous studies demonstrated that the HA from influenza A virus interacts with SA receptors on the host cell surface and mediates the entry of viral particles in the two wild-type SAs [α(2,3) or α(2,6)], and, moreover, the H5N1 HA preferentially binds to the α(2,3) SA receptors (Lu et al., 2013; Rogers et al., 1983). Strains binding to host SA is the key proponent of viral infectivity which in-turn serves as a major determinant of host infection (Cao et al., 2011). Furthermore, since H5N1 pathogenecity relies heavily on HA RBD binding to SA, this emphasizes the structural importance of HA RBD with regards to viral infection (Cao et al., 2011; Hensley et al., 2009; Kongchanagul et al., 2008).
Influenza A H5N1 HA protein has had multiple mutations that can either be advantageous or deleterious to the HA protein (Ayora-Talavera et al., 2009), which in-turn contributes to HA protein evolution since the mutations can generate diversity (advantageous) and/or loss through selection (deleterious) (Popova et al., 2019; Wilker et al., 2013). This emphasizes the importance of studying the function of HA protein mutations (Castonguay et al., 2021). Interestingly, earlier works have shown that viral proteins sometimes revert back to an ancestral conformation (Rokas and Carroll, 2008; Shah et al., 2015) which can potentially result to viral proteins having the same structural pattern and conformation during protein evolution. Surprisingly, at present, there is no study that establishes and evaluates the possible occurrence of HA structural similarities (ascribable to reversion) that may have existed during the 1959-2023 H5N1 HA protein evolution. A better understanding of the occurrence of HA structural similarities during the course of HA protein evolution may contribute to our understanding of the avian H5N1 HA protein evolution which in-turn could result to antiviral therapeutic strategies as well as predictions for future viral evolution and outbreaks.