Productive replication of TT virus in hematopoietic cells: Development of an in vitro culture system and evidence that TTV requires a helper virus
Handa A., Agbandje-Mckenna M., Brown KE.
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.