What Diseases Do Adeno-Associated Viruses Cause?

The party line about adeno-associated viruses is that they are "nonpathogenic." However, this claim is based on very limited investigation. Lack of acute symptoms of infection doesn't mean that they are harmless. It has been known since the early 1970s that AAVs cause latent infections by integrating into the cellular DNA, from which they can be reactivated by helper viruses including adenoviruses and herpesviruses. (Also, exposure to adenoviruses and herpesviruses is more common among the lower socioeconomic classes.) Although up to 70% of adults are seropositive for AAV, there was virtually no interest in the effects of AAV integration and viral proteins until after AAVs began to be developed as potential gene therapy vectors.

EXAMPLE: "Despite its spectacular achievements, the first successful form of gene therapy will for now be used only as a last resort due to the risk of cancer. And that's not the only bad news. The latest studies suggest that some other gene therapies using different viruses might also trigger cancer. The Necker hospital in Paris pioneered the use of gene therapy to cure the severe inherited immune disease called X-SCID. But two of the 11 boys treated have developed leukemia. Both these cases appear to be due to the corrective gene being inserted near another gene called Lmo2, which helps control cell growth and can contribute to cancer if turned on at the wrong time (New Scientist, 25 January, p 12). Calculations by Christof von Kalle of the University of Cincinnati College of Medicine in Ohio suggest that rather than being an extraordinary coincidence the engineered mouse retrovirus used to deliver the gene in the French study may insert it near Lmo2 about once per 100,000 insertions. Since millions of bone marrow cells are modified and returned to the boys, such insertions may crop up in most if not all of the patients." (Cancer risk clouds gene cures. New Scientist March 15, 2003; Gene therapy 'caused T-cell leukemia." By Jo Lyford. The Scientist, Oct. 20, 2003; a third child later developed leukemia: January 2005, third adverse event in Paris! By Sandro Rusconi. Swiss Gene Therapy Web Page.) Although AAV was not used in this therapy, the principle is the same. And, what they do not mention in these articles is that the same thing may happen in natural infections - but, despite foreboding evidence, there has been little or no investigation before the establishment rushed into gene therapy.

Cancer risk clouds gene therapy / EurekAlert
Gene therapy 'caused T-cell leukemia' / Biomed Central
January 2005, third adverse event in Paris! / Swiss Gene Therapy Web Page

LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1. S Hacein-Bey-Abina, C Von Kalle, M. Schmidt, MP McCormack, N Wulffraat, P Leboulch, A Lim, C S Osborne, R Pawliuk, E Morillon, R Sorensen, A Forster, P Fraser, J I Cohen, G de Saint Basile, I Alexander, U Wintergerst, T Frebourg, A. Aurias, D Stoppa-Lyonnet, S Romana, I Radford-Weiss, F Gross, F Valensi, E Delabesse, E Macintyre, F. Sigaux, J Soulier, LE Leiva, M Wissler, C Prinz, TH Rabbitts, F Le Deist, A Fischer, M Cavazzana-Calvo. Science 2003 October 17;302(5644):415-419.

Hacein-Bey-Abina et al / Science 2003 abstract

The Rep68 protein of adeno-associated virus type 2 stimulates expression of the platelet-derived growth factor B c-sis proto-oncogene. RS Wonderling, RA Owens. J Virol 1996 Jul;70(7):4783-4786. "The inappropriate expression of PDGF genes and their mitogenic products has been linked to several proliferative disorders such as fibrosis, atherosclerosis, and neoplasia. The ability of Rep68 to upregulate the c-sis proto-oncogene may imply a role for wild-type AAV in these diseases." Expression of several other genes to which Rep68 bound was not seen; however, some disorders are caused by reduced expression rather than increased expression of genes.

Wonderling / J Virol 1996 full article (pdf)
Platelet-derived growth factor c-sis proto-oncogene, 22q12.3-q13.1 / OMIM
Carcinoma marker GA733-1, 1p32-q12 / OMIM
Alpha-A-crystallin gene, 21q22.3 / OMIM

In a later study, Wonderling and Owens found 18 additional binding sites for AAV Rep68 protein (Binding sites for adeno-associated virus Rep proteins within the human genome. RS Wonderling, RA Owens. J Virol 1997 Mar;71(3):2528-2534).

Wonderling / J Virol 1997 full article (pdf)
Colony stimulating factor-1 (CSF-1), 5q33.2-q33.3 / OMIM
Insulin-like growth factor binding protein 2 (IGFBP-2), 2q33-q34 / OMIM
Histone H2B.1, 1q21-q23 / OMIM
Basement membrane heparin sulfate proteoglycan (HSPG2); perlecan, 1p36.1 / OMIM
Inhibin beta-B subunit, 2cen-q13 / OMIM
Interleukin-2 enhancer binding factor (ILF), 17q25 / OMIM
p53-related protein p63, 3q27 / OMIM
Global transcription activator hSNF2L, Xq25 / OMIM
Midkine, 11p11.2 / OMIM
Growth-arrest- and DNA-damage inducible protein GADD45, 1p34-p12 / OMIM
Cyclin-dependent kinase inhibitor KIP2, 11p15.5 / OMIM
Hereditary breast and ovarian cancer gene BRCA1, 17q21 / OMIM
Beta-actin repair domain, 7p22-p12 / OMIM
ERCC1 gene, 19q13.2-q13.3 / OMIM

Sites of recombinant adeno-associated virus integration. ED Rivadeneira, NC Popescu, DB Zimonjic, GS Cheng, PJ Nelson, MD Ross, JA DiPaolo, ME Klotman. Int J Oncol 1998 Apr;12(4):805-810. "Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost... Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors."

Rivadeneira - Int J Oncol 1998 abstract / PubMed

Adeno-associated virus site-specific integration and AAVS1 disruption. H Hamilton, J Gomos, KI Berns, E Falck-Pedersen. J Virol 2004 Aug;78(15):7874-7882. "Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 ( approximately 100% of infected cells)."

Hamilton / J Virol 2004 full article
Hamilton - J Virol 2004 full article / PubMed Central

Large-scale analysis of adeno-associated virus vector integration sites in normal human cells. DG Miller, GD Trobridge, LM Petek, MA Jacobs, R Kaul, DW Russell. J Virology 2005 Sep;79(17):11434-11442. "[A]n extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials." The authors have identified 977 integration junctions. "AAV vectors had a 4.8-fold preference for integration in CpG islands, which did not extend into surrounding regions... There was also a significant preference for integration in CpG islands (4.03% versus 0.84% for calculated random integrants), which typically act as transcriptional control elements for nearby genes. Surprisingly, some of our results were similar to those of murine leukemia virus vectors, where integrations also occurred preferentially near transcription start sites and CpG islands, with only a slight overall preference for genes. Thus, despite the distinct life cycles of these viruses, they may share aspects of integration site selection."

Miller / J Virology 2005 full article

Characterization of adeno-associated virus genomes isolated from human tissues. BC Schnepp, RL Jensen, CL Chen, PR Johnson, KR Clark. J Virol 2005 Dec;79(23):14793-14803. Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events... Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues."

Schnepp / J Virol 2005 full article
Schnepp - J Virol 2005 full article / PubMed Central

Increased complexity of wild-type adeno-associated virus-chromosomal junctions as determined by analysis of unselected cellular genomes. HR Drew, LJ Lockett, GW Both. J Gen Virol 2007 Jun;88(Pt 6):1722-1732. "For the plasmid system, we found, as expected, that viral p5-ITR elements may confer substantial Rep-mediated AAVS1 integration specificity on a selectable gene (Philpott et al., 2002). However, for wtAAV in unselected cells, we found that the virus (or parts thereof) can integrate specifically into chromosome 19 AAVS1 or join to other chromosomes via a few preferred ‘GGTC’ sequences that lie mainly within the 138 nt AAV p5 promoter. The overall complexity of wtAAV integration in unselected cells thus appears far greater than when only certain parts of that virus (e.g. Rep, p5 and ITR) are studied in a typical selectable cell system."

Drew / J Gen Virol 2007 full article

Site-specific integration of adeno-associated virus involves partial duplication of the target locus. E Henckaerts, N Dutheil, N Zeltner, S Kattman, E Kohlbrenner, P Ward, N Clément, P Rebollo, M Kennedy, GM Keller, RM Linden. Proc Natl Acad Sci USA 2009 May 5;106(18):7571-7576. AAV integration causes an extensive but partial duplication of the target gene, MBS85 (myosin-binding subunit 85), "thought to be involved in the regulation of actin–myosin fiber assembly." In latently infected mouse ES cells, expression levels of Tnnt1 and Tnni3 genes were not significantly different from those observed in the parental cell line, and "expression levels of Mbs85 or any of the Mbs85-linked genes were not influenced by the integration event." The cells were able to differentiate into contracting cardiomyocytes and neurons, and formed normal embryoid bodies.

Henckaerts - PNAS 2009 full article / PubMed Central
Henckaerts / PNAS 2009 full article

Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. S Boutin, V Monteilhet, P Veron, C Leborgne, O Benveniste, MF Montus, C Masurier. Hum Gene Ther 2010 Jun;21(6):704-712. "Natural exposure to AAV types 1, 2, 5, 6, 8, and 9 can result in the production of antibodies from all four IgG subclasses, with a predominant IgG1 response and low IgG2, IgG3, and IgG4 responses. Prevalences of anti-AAV1 and -AAV2 total IgG determined by enzyme-linked immunosorbent assay were higher (67 and 72%) than those of anti-AAV5 (40%), anti-AAV6 (46%), anti-AAV8 (38%), and anti-AAV9 (47%). Furthermore, data showed that cross-reactions are important. The two highest neutralizing factor seroprevalences were observed for AAV2 (59%) and AAV1 (50.5%) and the lowest were observed for AAV8 (19%) and AAV5 (3.2%)... Furthermore, among individuals seropositive for AAV5, AAV8, and AAV9, about 70-100% present low titers."

Boutin - Hum Gene Ther 2010 abstract

Integration preferences of wildtype AAV-2 for consensus rep-binding sites at numerous loci in the human genome. D Hüser, A Gogol-Döring, T Lutter, S Weger, K Winter, EM Hammer, T Cathomen, K Reinert, R Heilbronn. PLoS Pathog 2010 Jul 8;6(7):e1000985. "Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome." AAVS1 is at 19q13.42. "The junctions identified on chr. 19 span the region of the transcribed gene for protein phosphatase 1, regulatory subunit 12C (PPP1R12C). The 8 kb AAVS2 sequence identified on chr. 5p13.3 represents an intergenic region to the best of current knowledge... Apparently, PPP1R12C is essential, since the majority of latently infected cell lines display gene duplications [57] and simultaneous AAV integrations in both alleles have never been reported."

Hüser / PLoS Pathog 2010 full article

High-throughput sequencing reveals principles of Adeno-Associated Virus Serotype 2 integration. T Janovitz, IA Klein, T Oliveira, P Mukherjee, MC Nussenzweig, M Sadelain, E Falck-Pedersen. J Virol 2013 Aug;87(15):8559-8568. "Using a novel high-throughput approach, Integrant-Capture Sequencing, nearly twelve million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes."

Janovitz - J Virol 2013 abstract / PubMed

Highly divergent integration profile of Adeno-Associated Virus Serotype 5 revealed by high-throughput sequencing. T Janovitz, T Oliveira, M Sadelain, E Falck-Pedersen. J Virol 2014 Mar;88(5):2481-2488. "The integration activity of AAV-5 was 99.7% distinct from AAV-2 and favored intronic sequences. Genome-wide integration was highly correlated with viral replication protein binding and endonuclease sites, and a 39 base pair consensus integration motif was revealed that included these features. Algorithmic scanning identified 126 AAV-5 hotspots, the largest of which encompassed 3.3% of all integration events."

Janovitz - J Virol 2014 full article / PubMed Central
Janovitz / J Virol 2014 full article

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing. D Hüser, A Gogol-Döring, W Chen, R Heilbronn. J Virol 2014 Oct 1;88(19):11253-11263. "Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats."

Hüser - J Virol 2014 abstract / PubMed

High prevalence of infectious adeno-associated virus (AAV) in human peripheral blood mononuclear cells indicative of T lymphocytes as sites of AAV persistence. D Hüser, D Khalid, T Lutter, EM Hammer, S Weger, M Heßler, U Kalus, Y Tauchmann, K Hensel-Wiegel, D Lassner, R Heilbronn. J Virol. 2017 Jan 31;91(4). pii: e02137-16. "In genomic DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV-positive, predominantly AAV2 (77%), AAV5 (19%), and additional serotypes. Roughly 11% of blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, where 76% were AAV-positive... Leukocyte separation revealed that AAV resided in CD3+ T-lymphocytes, perceived as putative in vivo site of AAV persistence. Moreover, infectious AAV of various serotypes could be rescued and propagated from numerous samples."

Hüser - J Virol. 2017 abstract / PubMed

AAV2 Found in Liver Cancer

Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas. JC Nault, S Datta, S Imbeaud, A Franconi, M Mallet, G Couchy, E Letouzé, C Pilati, B Verret, JF Blanc, C Balabaud, J Calderaro, A Laurent, M Letexier, P Bioulac-Sage, F Calvo, J Zucman-Rossi. Nat Genet 2015 Oct;47(10):1187-1193. "We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC." Of the 11 patients, one had HCV, one HBV plus alcohol, and three tumors were alcohol-related. Three patients were female (Supplementary Table 2).

Nault - Nat Genet 2015 abstract / PubMed
Nault / Nat Genet 2015 abstract
Nault / Nat Genet 2015 suppl (pdf, 15 pp)

Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma. JC Nault, S Datta, S Imbeaud, A Franconi, J Zucman-Rossi. Mol Cell Oncol 2016 Jan 19;3(2):e1095271. "We identified somatic AAV2 integration in a subset of 11 hepatocellular carcinomas"

Nault - Mol Cell Oncol 2016 abstract / PubMed

Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients. KJ Park, J Lee, JH Park, JW Joh, CH Kwon, JW Kim. Ann Lab Med 2016 Sep;36(5):469-474. 289 HCC cases (159 Hepatitis-B-related, 16 Hepatitis-C-related, and 114 viral serology-negative cases). AAV2 was found in two. "Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis."

Park / Ann Lab Med 2016 abstract/landing

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome. GJ Logan, AP Dane, CV Hallwirth, CM Smyth, EE Wilkie, AK Amaya, E Zhu, N Khandekar, SL Ginn, SHY Liao, SC Cunningham, N Sasaki, M Cabanes-Creus, PPL Tam, DW Russell, L Lisowski, IE Alexander. Nat Genet 2017 Aug;49(8):1267-1273. "Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC."

Logan - Nat Genet 2017 abstract / PubMed

Adeno-associated virus in the liver: natural history and consequences in tumour development. T La Bella, S Imbeaud, C Peneau, I Mami, S Datta, Q Bayard, S Caruso, TZ Hirsch, J Calderaro, G Morcrette, C Guettier, V Paradis, G Amaddeo, A Laurent, L Possenti, L Chiche, P Bioulac-Sage, JF Blanc, E Letouze, JC Nault, J Zucman-Rossi. Gut 2019 Aug 2 [Epub ahead of print]. Tumour and non-tumour liver tissues of 1461 patients. "AAV DNA was detected in 21% of the patients, including 8% of the tumour tissues, equally distributed in two major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumour tissues, frequently associated with viral RNA expression and human herpesvirus type 6, the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B and GLI1/INHBE. AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localisation of the integration site."

La Bella - Gut 2019 abstract / PubMed

AAV and skeletal muscle gene TNNT1

Adeno-associated virus site-specifically integrates into a muscle-specific DNA region. N Dutheil, F Shi, T Dupressoir, RM Linden. Proc Natl Acad Sci USA 2000 Apr;97(9):4862-4866. "These data indicate that integration of AAV into the AAVS1 locus can lead to rearrangements of the TNNT1 gene." TNNT1 is the slow skeletal troponin T gene.

Dutheil / PNAS 2000 full article
Dutheil - PNAS 2000 full article / PubMed Central
Slow skeletal troponin T gene TNNT1, 19q13.4 / OMIM

AAV and skin

Ubiquitous human adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. C Meyers, M Mane, N Kokorina, S Alam, PL Hermonat. Virology 2000 Jul 5;272(2):338-346. "Since its discovery in 1966, adeno-associated virus type 2 (AAV) has been described as a helper-dependent parvovirus. However, in this study we demonstrate that AAV undergoes its complete life cycle, devoid of helper viruses or genotoxic agents, in the organotypic epithelial raft culture system, a model of normal skin... Additionally, dosage dependent histologic changes, some of which might be interpreted as cytopathology, were induced in the AAV-infected epithelial tissues."

Meyers - Virology 2000 abstract / PubMed

Adeno-associated virus expresses transgenes in hair follicles and epidermis. UR Hengge, A Mirmohammadsadegh. Mol Ther 2000 Sep;2(3):188-194. "Interestingly, expression was present not only in dividing and postmitotic epidermal keratinocytes but also in hair follicle epithelial cells and eccrine sweat glands."

Hengge - Mol Ther 2000 abstract / PubMed

AAV in bone marrow progenitor cells

Detection of adeno-associated virus type 2 in sorted human bone marrow progenitor cells. RJ Anderson, G Galatowicz, ID Macdonald, MW Lowdell, TJ Corbett, HG Prentice. Exp Hematol 1997 Mar;25(3):256-262. Integrated virus was found in 18/106 bone marrow samples; in 0/3 CD34+ cells, but 3/3 CD3+ cells from PCR-positive individuals.

Anderson - Exp Hematol 1997 abstract / PubMed

AAV is implicated in male sterility

DNA of adeno-associated virus (AAV) in testicular tissue and in abnormal semen samples. K Erles, V Rohde, M Thaele, S Roth, L Edler, JR Schlehofer. Hum Reprod 2001 Nov;16(11):2333-2337. And: Adeno-associated virus infection linked to male infertility. (News) Medscape - Reuters Health 2001 Oct 30.

Erles - Hum Reprod 2001 abstract / PubMed
Erles / Medscape - Reuters Health News 2001

Evidence of chromosomal integration of AAV DNA in human testis tissue. S Mehrle, V Rohde, JR Schlehofer. Virus Genes 2004 Jan;28(1):61-69. "These data demonstrate that AAV DNA can integrate also after natural infection, and that integration occurs within the AAVS1 region, at least in some cases."

Mehrle - Virus Genes 2004 abstract / PubMed

Presence of DNA of adeno-associated virus in subfertile couples, but no association with fertility factors. JR Schlehofer, C Boeke, M Reuland, W Eggert-Kruse. Hum Reprod 2012 Mar;27(3):770-778. 146 male and 134 female partners of asymptomatic subfertile couples. "The presence of AAV DNA in semen was not significantly related to semen quality, including sperm functional capacity or local ASA, nor was it coupled to the presence of AAV in the endocervical material of female partners. The presence of AAV DNA was not associated with the presence of other micro-organisms of the lower genital tract or with seminal WBC in men. AAV DNA in endocervical material was not related to a reduced quality of cervical mucus or to other female infertility factors."

Schlehofer - Hum Reprod 2012 abstract / PubMed

AAV Infection During Pregnancy Causes Spontaneous Abortions and Trophoblastic Disease

Detection of adeno-associated virus DNA in human genital tissue and in material from spontaneous abortion. E Tobiasch, M Rabreau, K Geletneky, S Laruë-Charlus, F Severin, N Becker, JR Schlehofer. J Med Virol 1994 Oct;44(2):215-222. "Using the polymerase chain reaction (PCR) AAV type 2 DNA was amplified in histological sections of 19 of 30 biopsies of the uterine mucosa. In addition, AAV-2 DNA was detected in abortion material during the first trimester of pregnancy (12/30 cases were positive) but not in material of abortion from the second or third trimester (9 cases). Whereas in tissues from the uterus AAV DNA was found only by PCR, large amounts of viral DNA were detectable by Southern blot analysis in abortion material. In situ hybridization revealed DNA of AAV to be present in the villous moiety (trophoblast) of the placenta but not in the embryo or decidua. in the same cells, AAV proteins (including the replication-associated rep proteins) were detected by immunofluorescence analysis. These results suggest (1) that AAV infects the uterine mucosa (possibly persistently) and (2) that it can replicate in trophoblast cells. This might disturb placenta development and may play a role in early miscarriage."

Tobiasch - J Med Virol 1994 abstract / PubMed

Presence of integrated DNA sequences of adeno-associated virus type 2 in four cell lines of human embryonic origin. M Dutheil, O Malhomme, N Provost, P Becquart, T Burguette, JR Schlehofer, T Dupressoir. J Gen Virol 1997 Nov;78 ( Pt 11):3039-3043. In material from first trimester miscarriage, "AAV type 2 (AAV-2) DNA and viral proteins were detected mainly in the trophoblast cell layer of placenta. In this report, we present evidence that AAV DNA is also present in established human trophoblast cell lines (JEG-3, JAr, BeWo) and in the human amnion cell line FL. In cells of these lines, AAV-2 DNA could be detected both by PCR and Southern blot analysis. Restriction enzyme analysis indicated that AAV DNA was integrated into the host cell genome."

Dutheil / J Gen Virol 1997 full article (pdf, 5pp)

Evidence for infection of the human embryo with adeno-associated virus in pregnancy. T Burguete, M Rabreau, M Fontanges-Darriet, E Roset, HD Hager, A Koppel, P Bischof, JR Schlehofer. Hum Reprod 1999 Sep;14(9):2396-2401. "In mothers with AAV DNA-positive amnion fluids, premature amniorrhexis and premature labour occurred significantly more frequently (P<0.001)."

Burguete - Hum Reprod 1999 abstract / PubMed

Adeno-associated virus DNA in human gestational trophoblastic disease. K Kiehl, JR Schlehofer, R Schultz, M Zugaib, E Armbruster-Moraes. Placenta 2002 May;23(5):410-415. "AAV DNA was found in 43 samples (28/49 hydatiform moles, 4/14 choriocarcinomas, 11/15 miscarriage material). These findings confirm AAV infection of embryo-derived tissue in humans and further suggest a role of AAV in miscarriage and trophoblastic disease."

Kiehl - Placenta 2002 abstract / PubMed

Adeno-associated virus-2 (AAV-2) causes trophoblast dysfunction, and placental AAV-2 infection is associated with preeclampsia. F Arechavaleta-Velasco, Y Ma, J Zhang, CM McGrath, S Parry. Am J Pathol 2006 Jun;168(6):1951-1959. "We determined that transformed extravillous trophoblast (HTR-8/SVneo) cells were susceptible to AAV-2 infection in the presence or absence of adenovirus, which provides helper function for AAV-2 replication, and that AAV-2 infection reduced invasion of HTR-8/SVneo cells through an extracellular matrix before cytopathic effects were detected. In a case-control study, AAV-2 DNA was found more frequently in trophoblast cells from cases of severe preeclampsia (22/40) than from normal term deliveries (5/27, P = 0.002)."

Arechavaleta-Velasco / Am J Pathol 2006 full article
Arechavaleta-Velasco - Am J Pathol 2006 full article / PubMed Central

Adverse reproductive outcomes in urban women with adeno-associated virus-2 infections in early pregnancy. F Arechavaleta-Velasco, L Gomez, Y Ma, J Zhao, CM McGrath, MD Sammel, DB Nelson, S Parry. Hum Reprod 2008 Jan;23(1):29-36. "First trimester maternal IgM seropositivity was 5.6 times more prevalent among pre-eclampsia/IUGR/stillbirth cases (P = 0.0004) and 7.6 times more prevalent among preterm deliveries (P < 0.0001) than among controls. CMV and adenovirus IgM antibodies and chronic AAV-2 infections (IgG seropositivity) were not associated with adverse pregnancy outcomes. CONCLUSIONS Primary or reactivated AAV-2 infection (maternal IgM seropositivity) early in pregnancy was associated with adverse reproductive outcomes associated with placental dysfunction, including pre-eclampsia, stillbirth and spontaneous preterm delivery."

Arechavaleta-Velasco - Hum Reprod 2008 abstract / PubMed

Molecular detection of adeno-associated virus in cases of spontaneous and intentional human abortion. CC Pereira, LB de Freitas, PR de Vargas, ML de Azevedo, JP do Nascimento, LC Spano. J Med Virol 2010 Oct;82(10):1689-1693. Nucleic acid of AAV-2, 3, and 5 types from 118 decidual and chorionic tissues. "AAV was observed in 28.4% (23/81) of the cases, of which, 78.3% (18/23) were in the decidua and 21.7% (5/23) in the extravillous trophoblast, the chorionic plate, or chorionic villi fragments. AAV-2, the only type detected, occurred in 32.3% (22/68) and in 7.7% (1/13) of the spontaneous and intentional abortions, respectively. ISH revealed AAV in the decidua, chorionic tissue or chorionic plate and extravillous trophoblast. The detection of only AAV-2 type indicates that it is the most frequent in the population studied and/or shows tissue tropism. The presence of AAV in decidual or trophoblastic cells in cases of abortion, as observed by ISH, implies that the virus could jeopardize the pregnancy. The significant predominance in spontaneous cases suggests possibly a causal association between AAV and abortion."

Pereira - J Med Virol 2010 abstract / PubMed


There were earlier reports that AAV infection might protect against cervical cancer by interfering with human papillomavirus infection. However, epidemiological studies have not confirmed this. (Adeno-associated virus and development of cervical neoplasia. HD Strickler, R Viscidi, C Escoffery, C Rattray, KL Kotloff, J Goldberg, A Manns, C Rabkin, R Daniel, B Hanchard, C Brown, M Hutchinson, D Zanizer, J Palefsky, RD Burk, B Cranston, B Clayman, KV Shah. J Med Virol 1999 Sep;59(1):60-65; and: Evaluation of the possible protective role of adeno-associated virus type 2 infection in HPV-associated premalignant disease of the cervix. KO Odunsi, CC van Ee, TS Ganesan, AN Shelling. Gynecol Oncol 2000 Sep;78(3 Pt 1):342-345.)

Strickler - J Med Virol 1999 abstract / PubMed
Odunsi - Gynecol Oncol 2000 abstract / PubMed

Detection of cervical infections in colposcopy clinic patients. S Lanham, A Herbert, A Basarab, P Watt. J Clin Microbiol 2001 Aug;39(8):2946-2950. "The frequency of AAV remained at 15 to 20% in all age ranges. In the study presented here, 31 samples contained AAV DNA; of these, 9 (29.0%) were CIN 3. In comparison, 200 samples did not contain detectable AAV DNA, of which 59 (29.5%) were CIN 3 (P value, 0.96). Hence, AAV showed no protective effect on the colposcopy patients studied." "It is known that primary infection with AAV usually occurs in childhood, but an unstable AAV antibody response may allow lifelong reinfection or reactivation of persisting virus (6). In the study presented here, AAV occurred in similar proportions of all age groups studied, indicating that reactivation of latent infection rather than reinfection would be the most likely source of AAV. This suggests that many women will have latent AAV infection from an early age and hence possible susceptibility to HPV transformation if cells in the endocervix are infected."

Lanham - J Clin Microbiol 2001 full article / PubMed Central

Altered biology of adeno-associated virus type 2 and human papillomavirus during dual infection of natural host tissue. C Meyers, S Alam, M Mane, PL Hermonat. Virology 2001 Aug 15;287(1):30-39. In cultured cells, "Raft tissues that were actively replicating HPV were superinfected with AAV type 2 (AAV-2). We observed a multiplicity of infection (m.o.i.)-dependent enhancement and inhibition of HPV DNA replication, concomitant with AAV-2 replication. The data suggest that at low m.o.i. of AAV-2 infection, HPV DNA replication was slightly increased compared to controls and AAV-2 replicated poorly. At high AAV-2 m.o.i., HPV DNA replication was reduced and AAV-2 replicated to high levels. AAV-2 replication was increased in the presence of HPV compared to primary human keratinocyte, squamous cell carcinoma, and HaCat raft cultures infected with AAV-2 alone. These data suggest that HPV may provide types of "enhancer/helper" functions for AAV-2 replication and progeny formation. Infection with AAV-2 had significant effects on epithelial morphology. During infection with low m.o.i. of AAV-2 the epithelium stratified to a greater extent than in controls. With high m.o.i. of AAV-2 infections, tissue cytopathic effects were observed, indicating an additional factor responsible for the effect of AAV-2 on HPV replication and infection."

Meyers - Virology 2001 abstract / PubMed

Temporal acceleration of the human papillomavirus life cycle by adeno-associated virus (AAV) type 2 superinfection in natural host tissue. N Agrawal, M Mane, M Chiriva-Internati, JJ Roman, PL Hermonat. Virology 2002 Jun 5;297(2):203-210. "Here it is shown that the presence of AAV-2 at a variety of multiplicities of infection (m.o.i.) resulted in early onset HPV-31b DNA replication. Using plasmids which each expressed only one of the four rep proteins, an enhancement affect was seen for all four rep proteins of AAV, with Rep40 having the highest activity. Furthermore, AAV (m.o.i. of 5) also resulted in a temporally accelerated production of HPV infectious units, seen as early as Day 4, with high levels of viral progeny being produced by Day 6.5. Like earlier studies at Day 12, histological differences were seen at Day 6.5 between AAV-infected and mock-infected HPV/rafts."

Agrawal - Virology 2002 abstract / PubMed

Correlation of human papillomavirus and adeno-associated virus 2 infection in cytology with Korean women. J Hyun-Sun, C Heung-Jae, K Byung-Hun, C Young-Lae, N Gye-Hyun, C Ho-Sun, K Chong-Kook, H Byoung-Don, B Su-Mi, A Woong-Shick. Int J Gynecol Cancer 2006 Mar-Apr;16(2):604-609. "The correlation between AAV 2 and HPV 16 was statistically significant in normal and CIN I/II group only, and AAV 2 and HPV 16 and/or HPV 18 showed no correlation. Therefore, the correlation between AAV and HPV were not statistically significant. These data support the previous reports that AAV might not be associated with cervical tumorigenesis."

Hyun-Sun - Int J Gynecol Cancer 2006 abstract / PubMed

Adeno-associated virus infection and cervical neoplasia: is there a protective role against human papillomavirus-related carcinogenesis? T Agorastos, S Chrisafi, AF Lambropoulos, T Mikos, TC Constandinides, JR Schlehofer, B Schlehofer, A Kotsis, JN Bontis. Eur J Cancer Prev 2008 Aug;17(4):364-368. 373 women aged 19-65. "AAV detection was not statistically different between HPV (-) and HPV (+) controls (P=0.06). In the disease groups, however, the prevalence of AAV was statistically significantly lower in the HPV (+) relative to the HPV (-) patients (P=0.0009, P=0.00001, and P=0.0225, for women with low-grade cervical lesions, for women with high-grade cervical lesions, and for women with cervical cancer, respectively). No difference in the prevalence of AAV DNA between HPV-positive and HPV-negative unaffected (control) women is observed."

Agorastos - Eur J Cancer Prev 2008 abstract / PubMed

Adeno-associated virus type 2 induces apoptosis in human papillomavirus-infected cell lines but not in normal keratinocytes. S Alam, C Meyers. J Virol 2009 Oct;83(19):10286-10292. In cell line CIN-612 9E which maintains episomal genomes of HPV type 31b. "The induction of apoptosis coincided with AAV2 Rep protein expression; increased S-phase progression; upregulated pRb displaying both hyper- and hypophosphorylated forms; increased levels of p21(WAF1), p16(INK4), and p27(KIP1) proteins; and diminished levels of E7 oncoprotein. In contrast, normal keratinocytes that were infected with AAV2 or transfected with the cloned full-length AAV2 genome failed to express Rep proteins or undergo apoptosis."

Alam / J Virol 2009 full article

Adeno-associated virus may play a protective role against human papillomavirus-induced cervical lesions independent of HIV serostatus. LB de Freitas, AT de Mattos, BM Lima, AE Miranda, LC Spano. Int J STD AIDS 2012 Apr;23(4):258-261. "AAV prevalence was 19.7% (56/284), with 18.7% (21/112) and 20.3% (35/172) in HIV-positive and -negative women, respectively. AAV type 2 was the single virus type detected. AAV was detected with higher frequency in HPV-infected women (P < 0.05) as was HPV in HIV-positive women (P < 0.05). The AAV-HPV co-infected women showed a lower rate of atypical squamous cells of undetermined significance or cervical intraepithelial neoplasia development compared with those infected only with HPV."

de Freitas - Int J STD AIDS 2012 abstract / PubMed

Prevalence of adeno-associated virus and human papillomavirus DNA in Iranian women with and without cervical cancer. NZ Shafiei-Jandaghi, J Yavarian, E Faghihloo, N Ghavami, Z Yousefi Ghalejoogh, SJ Kiani, S Shatizadeh Malekshahi, R Shahsiah, E Jahanzad, M Hosseini, T Mokhtari Azad. Pathol Res Pract 2017 May;213(5):457-460. 61 cases, 50 controls. "AAV DNA was detected in 7 cases (14%) of HCs and 9 specimens (14.8%) of case group."

Shafiei-Jandaghi - Pathol Res Pract 2017 abstract / PubMed

AAV Vector Epidemic Possible?

Reducing the risk of adeno-associated virus (AAV) vector mobilization with AAV type 5 vectors. FC Hewitt, C Li, SJ Gray, S Cockrell, M Washburn, RJ Samulski. J Virol 2009 Apr;83(8):3919-3929. "Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates."

Hewitt / J Virol 2009 full article
Hewitt - J Virol 2009 full article / PubMed Central

Doubletalk about AAV

Virus-mediated killing of cells that lack p53 activity. K Raj, P Ogston, P Beard. Nature 2001 Aug 30;412(6850):914-917; and: Virus selectively kills p53-deficient cancer cells. Medscape - Reuters Health 2001 Aug 29. The study indicates that AAV DNA "elicits in cells a DNA damage response," which should not be surprising considering that AAV integrates into cellular DNA. However, in the news report the authors state: "Whereas further work will elucidate what kind of DNA damage AAV DNA is mimicking [sic!] and details of which pathway is being triggered, the immediate importance of these findings is the introduction of the principle of using viruses to efficiently deliver DNA with unusual or modified structures into cells to eliminate those cells that lack p53 activity." "Mimicking" DNA damage?? That is a peculiar choice of words. It suggests not only a preference for discovering a new use for AAV in cancer therapy over their role in the causation of disease, but also a desire to overlook those kind of implications. And no doubt it is because a new cancer therapy is more likely to win its discoverer(s) one of those much-sought trips to Stockholm than is a contribution to the prevention of cancer.

Raj - Nature 2001 abstract / PubMed
Raj / Medscape - Reuters Health 2001

It is possible that this simply won't work. Offner et al. found that "malignant cells may leave the primary carcinoma at an early stage of its development when p53 is not mutated yet, and targeting mutated p53 is unlikely to be a successful strategy to detect and to eliminate these potential precursors of subsequent overt metastasis." (P53 gene mutations are not required for early dissemination of cancer cells. S Offner, W Schnaus, K Witter, GB Baretton, G Schlimok, B Passlick, G Riethmuller, K Pantel. PNAS 1999 Jun;96:6942-6946.) It has also been found that "Isolated tumor cells in bone marrow predict reduced survival in node-negative non-small cell lung cancer" (B Passlick, B Kubuschok, JR Izbicki, O Thetter, K Pantel. Ann Thoracic Surg 1999 Dec;68(6):2053-2058). So once again, energies are being misdirected on a wild goose chase, while the implications for etiology and prevention are ignored.

Offner / PNAS 1999 full article
Passlick - Ann Thorac Surg 1999 abstract / PubMed

Inhibition of S-phase progession by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb. P Saudan, J Vlach, P Beard. EMBO J 2000;19(16):4351-4361. In cells infected with an AAV vector which lacks the Rep gene, the adenovirus E4orf6 protein also arrests cells in S-phase, with reversibility by cyclin A. "Hence, in a wild-type AAV infection, Rep78, which we have shown to affect the cells similarly, could promote the establishment of a latent infection."

Saudan / EMBO J 2000 full article

[Note that the adenovirus E4orf6 protein has since been implicated in "hit-and-run" transformation!] "Hit-and-run" transformation by adenovirus oncogenes. M Nevels, B Tauber, T Spruss, H Wolf, T Dobner. J Virol 2001 Apr;75(7):3089-3094.) Is AAV rep78 protein similar to adenovirus E4orf6 protein in this respect as well?

Nevels - J Virol 2001 abstract / PubMed

Adeno-associated virus and adenovirus coinfection induces a cellular DNA damage and repair response via redundant phosphatidylinositol 3-like kinase pathways. RF Collaco, JM Bevington, V Bhrigu, V Kalman-Maltese, JP Trempe. Virology 2009 Sep 15;392(1):24-33. "AAV/Ad coinfections induce a robust DNA damage response (DDR) that is distinct from that induced by Ad infection alone. Using chemical inhibitors, deficient cell lines and siRNA knockdowns of the DDR kinases, ATM, ATR and DNA-PK, we determined that DNA-PK and ATM kinases are the initial transducers of this response. AAV/Ad coinfection induces ATM-and DNA-PK mediated phosphorylation of RPA2, NBS1, H2AX and the checkpoint kinases CHK1/2. Inhibition of one or more of the DDR kinases reduces the level of phosphorylation of downstream targets but does not dramatically reduce Ad or AAV protein expression. However, AAV DNA levels are moderately affected by kinase inhibition."

Collaco - Virology 2009 author manuscript / PubMed Central

The DNA damage response to non-replicating adeno-associated virus: Centriole overduplication and mitotic catastrophe independent of the spindle checkpoint. C Ingemarsdotter, D Keller, P Beard. Virology 2010 May 10;400(2):271-286. "We found that the virus uncouples centriole duplication from the cell cycle, inducing centrosome overamplification that is dependent on Chk1, ATR and CDK kinases, and on G2 arrest. Interference with spindle checkpoint components Mad2 and BubR1 revealed unexpectedly that mitotic catastrophe occurs independently of spindle checkpoint function. We conclude that, in the p53-deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis."

Ingemarsdotter - Virology 2010 abstract / PubMed

See Also:

Parvovirus B19 Causes Fetal Loss


cast 08-21-19