The diversity of torque teno viruses: in vitro replication leads to the formation of additional replication-competent subviral molecules

The family Anelloviridae comprises torque teno viruses (TTVs) diverse in genome structure and organization. The isolation of a large number of TTV genomes (TTV Heidelberg [TTV-HD]) of 26 TTV types is reported. Several isolates from the same type indicate sequence variation within open reading frame 1 (ORF1), resulting in considerably modified open reading frames. We demonstrate in vitro replication of 12 full-length genomes of TTV-HD in 293TT cells. Propagation of virus was achieved by several rounds of infections using supernatant and frozen whole cells of initially infected cells. Replication of virus was measured by PCR amplification and transcription analyses. Subgenomic molecules (TTV), arising early during propagation and ranging in size from 401 to 913 bases, were cloned and characterized. Propagation of these TTV in in vitro cultures was demonstrated in the absence of full-length genomes. The family Anelloviridae includes torque teno viruses (TTVs), TT-midiviruses (TTMDV), and TT-miniviruses (TTMV), the majority originating from samples of human or-igin (2, 37, 39, 41, 56). The plurality of this family of single-stranded DNA (ssDNA) viruses is reflected not only in DNA sequence, but also in genome size and organization. Infections occur within the first days of life, with close to 100 % of infants being infected at 1 year of age. The primary route of infection, however, remains unclear (23, 38, 48). The ubiquitous nature of TTV infections has hampered efforts to associate it with the pathogenesis of disease (9, 15, 25, 41). A possible etiological association with diseases of the liver (re-viewed in reference 36) and respiratory tract (3, 30, 31, 49)