Exploration of genes and identification of evolutionary evidence in adeno-associated viruses

Adeno-associated virus (AAV) commonly infects humans and non-human primates, generally inducing mild or even asymptomatic outcomes. AAVs have been shaped and diversified by evolutionary pressures, resulting in the identification of 13 serotypes thus far. Each serotype of AAV exhibits distinct tissue tropisms, targeting various organs, including the lung, central nervous system (CNS), liver, and skeletal muscle, thereby establishing AAVs as widely utilized vectors for therapeutic gene delivery. Bioinformatics analysis of specific viruses enables the inference of evolutionary patterns and offers valuable insights for predicting the emergence of novel viruses. While DNA sequence-based analysis has effectively facilitated the observation of mutation patterns accumulating within specific genes, it often provides limited insight into the actual impact of these mutations on proteins, the fundamental functional units. Utilizing proteotyping, an amino acid sequence-based comparative analysis, we identified hypervariable regions (HVR) within the AAV Cap gene and revealed concentrated evolutionary pressures in serotypes 4, 5, 11, and 12. Furthermore, we found that AAV-5 proteins exhibited considerable amino acid sequence divergence compared to those of other serotypes. Despite divergence, all AAV-5 proteins maintained a noticeable structural similarity to their counterparts in other serotypes. Our findings provide sequence-based insights into the evolutionary processes of AAV, facilitating the efficient identification of novel viruses.