Characterizing Complex Populations of Endogenous Adeno-Associated Viruses by Single-Genome Amplification

The isolation of adeno-associated virus (AAV) genomes from biomaterials at the molecular level has traditionally relied on polymerase chain reaction-based and cloning-based techniques. However, when applied to samples containing multiple species, traditional techniques for isolating viral genomes can amplify artificial recombinants and introduce polymerase misincorporation errors. In this study, we describe AAV single-genome amplification (AAV-SGA): a powerful technique to isolate, amplify, and sequence single AAV genomes from mammalian genomic DNA, which can then be used to construct vectors for gene therapy. We used AAV-SGA to precisely isolate 15 novel AAV genomes belonging to AAV clades A, D, and E and the Fringe outgroup. This technique also enables investigations of AAV population dynamics and recombination events to provide insights into virus-host interactions and virus biology. Using AAV-SGA, we identified regional heterogeneity within AAV populations from different lobes of the liver of a rhesus macaque and found evidence of frequent genomic recombination between AAV populations. This study highlights the strengths of AAV-SGA and demonstrates its capability to provide valuable insights into the biology and diversity of AAVs.

Automated summary

Traditional techniques for isolating adeno-associated virus (AAV) genomes from complex samples are not effective, while AAV-SGA technique can isolate, amplify, and sequence AAV genomes individually, providing important tools for constructing gene therapy vectors. In addition, AAV-SGA technique can also study AAV population dynamics and recombination events, enhancing the understanding of virus-host interactions and virus biology.