The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors. Adeno-associated virus (AAV) is a member of the genusDependovirus, which lies within the Parvoviridae family (17). An interest in this family of viruses has been stimulated because of their potential use as gene transfer vectors (14).Little is known about the biology of AAV infections, although a significant proportion of humans and nonhuman primates have antibodies in their blood that react to some of the six existing serotypes of AAV (5, 7). This suggests that primates are hosts for infection with AAV, although the clinical sequelae of these infections have yet to be identified.The study of AAV has been limited to the previously described six serotypes, of which five were isolated as contaminants in laboratory preparations of adenoviruses (1,3,16). Our lack of understanding of AAV clinical infections has complicated the search for clinical isolates of the virus. Members of our laboratory recently described a strategy for evaluating latent or persistent AAV genomes from tissues of asymptomatic nonhuman primates through the use of PCR. These studies led to the discovery of two novel AAV serotypes, called AAV7 and AAV8, that have improved properties as vectors for gene therapy (10). In nonhuman primates, AAV sequences were quite prevalent and heterogenous (9).The goal of this study was to determine if latent AAVs exist in humans, and if so, to characterize their structural, serologic, and functional properties. MATERIALS AND METHODSCollection of primate tissues. Our sources of nonhuman primate tissues were described previously (9). Human tissues were collected under two independent IRB protocols approved by the Institutional Review Board of the University of Pennsylvania from either surgical procedures, postmortem examinations, or organ donors through two major national human tissue providers, the Cooperative Human Tissue Network and the National Disease Research Interchange. The human tissues used for this study were comprised of 18 different tissue types that included the colon, liver, lung, spl...

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Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.A deno-associated viruses (AAVs) belong to the Parvoviridae family, which is characterized as small animal viruses with linear single-stranded DNA genomes that replicate in the presence of helper virus such as adenovirus (1). AAVs are being evaluated as vectors for human gene therapy (2). The initial characterization of this group of viruses was based on serologic crossreactivity by using complement fixation and neutralizing assays (3). Six distinct serotypes of AAV have been described, of which five were initially isolated as contaminants of adenovirus preparations (4-6). Sequence analysis of selected AAV isolates revealed divergence throughout the genome that is most concentrated in hypervariable regions (HVRs) of the capsid proteins (7-10). Epidemiological data indicate that all known serotypes are endemic to primates, although isolation of clinical isolates has been restricted to AAV2 and AAV3 from anal and throat swabs of human infants and AAV5 from a human condylomatous wart (11)(12)(13)(14). No known clinical sequelae have been associated with AAV infection. Vectors based on replication-defective forms of AAV have been evaluated in preclinical and clinical models of gene therapy (2). Detection and Recovery of AAV Sequences. DNA was extracted and analyzed for the presence of AAV DNA by using a PCR strategy to amplify a 255-bp (15) fragment called the ''signature region'' by using conserved oligonucleotides. To directly amplify a 3.1-kb full-length Cap fragment from NHP tissue and blood DNAs, two other highly conserved regions were identified in AAV genomes for use in PCR amplification of large fragments. A primer within a conserved region located in the middle of the Rep gene was selected (AV1ns, 5Ј-GCTGCGTCA ACTGGACCA AT-GAGAAC-3Ј) in combination with the 3Ј primer located in another conserved region downstream of the Cap gene (AV2cas, 5Ј-CGCAGAGACCAAAGTTCAACTGAAACGA-3Ј) for amplification of full-length cap fragments. The PCR products were Topo-cloned (Invitrogen), and sequence analysis was performed by Qiagengenomics (Qiagengenomics, Seattle) with an accuracy of Ն99.9%. A total of 50 capsid clones were i...