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TT virus (TTV) is a novel single-strand (ss) DNA viras originally detected in a patient with transfusion-transmitted hepatitis. However, little is known about this viras, including its role in disease, and the link with hepatitis is especially controversial. We previously reported detection of TTV DNA in the blood of - 80% of American blood donors, and the detection of viral RNA, an indicator of active TT viral transcription, in hematopoietic cells, particularly B lymphocytes and CD34 cells. We developed an assay to detect double stranded (ds) replicating TTV DNA based on the replication pattern of chicken anemia virus of the same Circoviridae family, which was used in a novel in vitro TTV culture system. Replicating TTV was confirmed to be present in bone marrow cells (mainly CD34 cells), hematopoietic progenitors, and peripheral blood mononuclear cells (PBMNCs; mainly B cells). Replicating virus was detected in serum or urine of an infected volunteer, and these samples were used to test cell lines for permissivity for TTV. Following inoculation, ds TTV DNA, TTV RNA and TTV protein could all be detected in Epstein-Barr virus (EBV)-transformed B cell lines (B95-8, Raji, Daudi, Skw6.4, ARH77), but not EBV-negative lines (CA46 and ST486). Coinfection with EBV and TTV resulted in EBNA-2 expression and TTV expression and replication in ST486 cells. In contrast, CA46 did not express EBNA-2 and remained non-permissive for TTV. We could not detect replicating TTV DNA in myeloid (HL-60, U937, HEL, KG1, KGla, K562 and UT-7/Epo) and T cell lines (Jurkat, CEM, CESS, and Dl.l). Electron microscopy and cesium chloride gradient density studies demonstrated that TTV is a 13-15nm nonenveloped icosahedral virus with a density of 1.32-1.33 g/ml. Due to the dependence of EBV infection on cell line permissivity we determined the correlation of EBV seropositivity and TTV DNA detection in healthy blood donors. TTV DNA was detected in PBMNCs of 13 of 18 healthy bone marrow donors, and all TTV positive samples also showed evidence of EBV infection. In serum samples (n=100), TTV DNA was only found in patients with anti-EBV antibody, while in anti-EBV antibody negative samples (15/100), TTV DNA was not detected. Our results show that TTV replicates in hematopoietic cells, particularly B cells, and our data from both cell lines and patient studies suggest that TTV may require a helper viras, such as EBV, for productive infection.

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.

Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration.