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

Area of Science:

• Molecular biology
• Virology
• Genomics

Background:

• Traditional methods for isolating adeno-associated virus (AAV) genomes, such as PCR and cloning, can introduce errors and amplify artificial recombinants when analyzing complex samples.
• Accurate isolation of intact viral genomes is crucial for understanding viral diversity and developing gene therapy vectors.

Purpose of the Study:

• To introduce and validate a novel technique, adeno-associated virus single-genome amplification (AAV-SGA), for the precise isolation, amplification, and sequencing of single AAV genomes.
• To explore AAV diversity, population dynamics, and recombination events using the AAV-SGA method.

Main Methods:

• Development and application of the AAV single-genome amplification (AAV-SGA) technique.
• Isolation and sequencing of single AAV genomes from mammalian genomic DNA.
• Analysis of AAV populations from rhesus macaque liver samples.

Main Results:

• Successfully isolated and sequenced 15 novel adeno-associated virus (AAV) genomes across multiple clades (A, D, E) and an outgroup.
• Identified regional heterogeneity within AAV populations in different liver lobes.
• Provided evidence of frequent genomic recombination between distinct AAV populations within the host.

Conclusions:

• AAV-SGA is a powerful tool for accurately isolating and sequencing single AAV genomes, overcoming limitations of traditional methods.
• The technique facilitates the discovery of novel AAV diversity and provides insights into AAV biology, population dynamics, and recombination.
• AAV-SGA enables the construction of precise AAV vectors for gene therapy applications and aids in studying virus-host interactions.