Robust AAV Genotyping Based on Genetic Distances in Rep Gene That Are Maintained by Ubiquitous Recombination

Adeno-associated viruses (AAVs) are a convenient tool for gene therapy delivery. According to the current classification, they are divided into the species AAV A and AAV B within the genus Dependoparvovirus. Historically AAVs were also subdivided on the intraspecies level into 13 serotypes, which differ in tissue tropism and targeted gene delivery capacity. Serotype, however, is not a universal taxonomic category, and their assignment is not always robust. Cross-reactivity has been shown, indicating that classification could not rely on the results of serological tests alone. Moreover, since the isolation of AAV4, all subsequent AAVs were subdivided into serotypes based primarily on genetic differences and phylogenetic reconstructions. An increased interest in the use of AAV as a gene delivery tool justifies the need to improve the existing classification. Here, we suggest genotype-based AAV classification below the species level based on the rep gene. A robust threshold was established as 10% nt differences within the 1248 nt genome fragment, with 4 distinct AAV genotypes identified. This distinct sub-species structure is maintained by ubiquitous recombination within, but not between, rep genes of the suggested genotypes.