Molecular and biophysical characterization of TT virus: evidence for a new virus family infecting humans

Abstract

The recent isolation of a novel DNA virus from the serum of a Japanese patient (T.T.) has provided the latest possible candidate virus associated with cryptogenic hepatitis. In the present study, we report the complete nucleotide sequence of this virus (TTV) isolated from the serum of a West African. Based on PCR studies designed to amplify overlapping regions of the viral genome and sensitivity to digestion with mung bean nuclease, the viral genome is circular and negative stranded, and comprises 3,852 nt, which is 113 nt longer than the prototype isolate from Japan. Cesium chloride density gradient centrifugation demonstrated banding of the virus at 1.31-1.34 g/ml; filtration studies indicated that TTV had a particle size of 30-50 nm. These results suggest that the virus is similar to the Circoviridae, viruses known to infect plants and vertebrates (e. g., birds and swine); however, sequence similarity searches of available databases did not reveal identity between TTV and other viruses. Phylogenetic analyses of a 260-nt region from 151 globally distributed isolates demonstrated the existence of three major TTV genotypes. Several individuals at high risk for infection with parenterally transmitted viruses were infected with more than one genotype. There was no correlation between genotype and geographic origin. Finally, intravenous inoculation of TTV-positive human serum into chimpanzees demonstrated that TTV can be transmitted to primates; no biochemical or histological evidence for hepatitis was obtained. The distinct biophysical and molecular characteristics of TTV suggest that it is a member of a new family of viruses, which we have tentatively named the Circinoviridae.

Figure 1

TTV circular genome (thin line) with ORFs 1 and 2 indicated. Anchored PCR extension clones extending upstream and downstream from the N22 clone sequence (gray box) described by Nishizawa et al. (1) are indicated by the arrows inside the circular genome. The inverse PCR product (ud) that overlaps the anchored PCR products is also shown. The 113-nt sequence identified in the GH1 isolate is indicated by the hatched box. Position 1 is indicated by the arrow.

Figure 2

Distribution of the pairwise genetic distances observed between 157 TTV nucleotide sequences. Genetic distances are on the x axis; the frequency of occurrence of discrete distances is on the y axis.

Figure 3

Consensus phylogenetic tree (unrooted) of 260 nt (amplification primers removed) from 163 TTV isolates. Branch lengths are proportional to the genetic distance (scale shown). Isolates representing the subtypes (1a, 1b, 2a, and 2b) proposed by Okamoto et al. (2) are labeled. GenBank accession numbers are given where appropriate. Geographical designations: AR, Argentina; EG, Egypt; GE, Greece; GH, Ghana; JA, Japan; NL, The Netherlands; NZ, New Zealand; US, United States. Sequences isolated from a single individual are designated with the isolate number followed by the lower case letters a, b, or c. Bootstrap values are shown at the nodes for 1,000 resamplings of the data. Genetic groups are indicated as genotypes 1, 2, and 3 and subtypes 2.1 and 2.2.