Detection of TT virus DNA in liver biopsies by in situ hybridization

Abstract

A novel hepatitis-associated virus named TT virus (TTV) has been isolated. However, its hepatotropism has not been proven. We have retrospectively analyzed the presence of TTV-DNA by polymerase chain reaction (PCR) and in situ hybridization in liver biopsies from 30 patients with liver disease (15 TTV-DNA-positive and 15 TTV-DNA-negative in serum), and prospectively in serum and liver from eight patients with normal liver histology. TTV-DNA was detected by PCR in the liver from the 15 patients with serum TTV-DNA and in serum and liver of two of the eight patients without liver disease. TTV-DNA titers in liver were 10 times higher than in serum, although no correlation between TTV-DNA titers in serum and liver were observed. In situ hybridization shows positive signals in the hepatocytes of the 17 patients infected by TTV but in none of the TTV-DNA-negative patients by PCR. No morphological changes were observed in the hepatocytes showing hybridization signals. The percentage of positive hepatocytes ranged from 2.1% to 30% and correlated with the TTV-DNA titers in liver (r = 0.54; P = 0.037). In conclusion, our results show that TTV is able to infect liver cells although they do not support a role for TTV in causing liver disease.

Figure 1.

Autoradiography of a 6% polyacrylamide gel showing the semiquantitation of the TTV-DNA titers in liver (L) and serum (S) of six patients. M, φX174 DNA digested with HinfI (Promega).

Figure 2.

Neighbor-joining tree constructed with the nucleotide sequences of the TTV isolates from serum and liver of seven patients with liver disease (patients 1–7), three patients with liver disease coinfected by HBV or HCV (patients 8–10), and two patients without liver disease (patients 11 and 12). The isolates are designated as the patient number plus S or B, indicating the origin from serum or liver, followed by the clone number. The GenBank accession number of the published TTV sequences included in the tree are: AB016942 and AB017767 (genotype 1a); AB017879 (genotype 1b); AB016952, AB017882, AB018904, AB017884, and AB060549 (genotype 2); AB017774 and AB018960 (genotype 3); AB017775 and AB017887 (genotype 4); AB017776 (genotype 5); AB017777 and AB017889 (genotype 6); AB017778 (genotype 7); AB017779 (genotype 8); AB017780 and AB017782 (genotype 9); AB017783 (genotype 10); AB017613 (genotype 11); AB021081 and AB021088 (genotype 12); AB021089 (genotype 13); AB021082 and AB021083 (genotype 14); AB021087 (genotype 15) and AB021084 (genotype 16). The bootstrap values are depicted in the nodes of the tree.

Figure 3.

Detection of TTV-DNA in liver biopsies. a: Hybridization of a liver section from a negative control (TTV-DNA-negative in serum). b: In situ detection of TTV-DNA in the liver biopsy from a patient with TTV-DNA in serum and liver by PCR. c, d: Hybridization in serial sections from a liver biopsy of a TTV-DNA-positive patient predigested (c) and nondigested (d) with DNase. The arrow shows an example of a hepatocyte in which DNA treatment precludes TTV-DNA detection. e–g: Intracellular distribution of TTV-DNA in the nucleus (e), perinuclear (f), and in the cytoplasm (g). Counterstained with safranine. Original magnifications: a–d, ×400; e–g, ×1000.

Figure 4.

In situ detection of TTV-DNA in the liver biopsy from a patient with hepatic steatosis showing that there is no topological relationship between the presence of TTV in the hepatocytes (solid arrows) and the lipid droplets (open arrows).