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From the Departments of Hepatology*
and
Pathology,
Fundación Jiménez
Díaz, and the Fundación Estudio Hepatitis
Virales,
Madrid, Spain
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Whether there is any association of the TTV infection and liver disease is a matter of debate.13,14 Consequently, to establish that TTV has a role in causing liver damage, it must be demonstrated that this is a hepatotropic virus. Okamoto et al9 have reported the presence of TTV-DNA by polymerase chain reaction (PCR) in the liver of TTV-infected patients with titers 10 to 100 times higher than those in serum. Furthermore, TTV secretion into bile has been demonstrated.15 All these findings could indicate that the virus may be hepatotropic. To confirm this finding, morphological evidence of the presence of TTV in the liver cells from infected patients should be proven. However, there is still no information available about the detection of TTV within individual liver cells by in situ hybridization or by immunohistochemistry.
In the present study, we have applied nonisotopic in situ hybridization for the detection of TTV genome in liver biopsies to identify the cell tropism, the intracellular location of the virus, and its relationship with the liver disease.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Fifteen of the patients had TTV-DNA in a serum sample collected when
the liver biopsy was obtained. Of them, seven patients had chronic
hepatitis C, one had chronic hepatitis B, and five had nonviral liver
diseases (three steatosis, one ductopenia, one siderosis), and two had
cryptogenic hepatitis. The remaining 15 patients were TTV-DNA-negative
in serum and were selected based on the criteria of having similar
clinical, histological, and epidemiological features as the
TTV-DNA-positive cases. Eight of them presented with chronic hepatitis
C, two with chronic hepatitis B, three with steatosis, and two with
cryptogenic hepatitis. Cryptogenic hepatitis was diagnosed on the basis
of its negativity to viral markers, autoimmunity, metabolic disease,
alcohol intake, and hepatotoxic drug exposure. No differences were
found with respect to age, gender, ALT, aspartate aminotransferase
(AST), and 
-glutamyltransferase levels or epidemiological
antecedents between TTV-positive and TTV-negative patients. The main
clinical characteristics of the patients are summarized in Table 1
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This study was approved by the ethics committee of our hospital and was conducted in accordance with the guidelines of "Good Clinical Practice," which underwrites the principles of the Declaration of Helsinki on human experimentation. Written informed consent according to local legal requirements was obtained from each patient participating in the study.
Detection of TTV-DNA in Serum and Liver by PCR
Total DNA from 200 µl of serum was isolated by proteinase K (1 mg/ml) and 1% sodium dodecyl sulfate digestion at 37°C for 2 hours. After phenol extraction and ethanol precipitation, the nucleic acid pellet was resuspended in 20 µl of distilled water.
For TTV detection in frozen liver samples, liver biopsies were homogenized in the presence of 1 x TSE buffer (10 mmol/L Tris-HCl, pH 7.5; 10 mmol/L NaCl; 2 mmol/L ethylenediaminetetraacetic acid) and digested with proteinase K and sodium dodecyl sulfate as described above. After phenol extraction and ethanol precipitation, the total DNA pellet was resuspended in 50 µl of distilled water. DNA concentration was estimated by measuring its absorbance at 260 nm. Ten µl of total DNA obtained from the 200 µl of sera or 1 µg of total DNA from liver biopsies were amplified by PCR using primers from ORF-1 from the untranslated region. PCR reactions were performed exactly as described by Okamoto et al9 (ORF-1 primers) and by Takahashi et al16 (untranslated region primers). In all cases, distilled-water serum samples previously known as negative for TTV-DNA and DNA extracted from the human hepatoma cell line HepG2 were used as negative controls for the DNA extraction and PCR. To confirm the specificity of the detection of TTV in liver, all PCR products were directly sequenced using the ALF Express DNA Sequencing System (Pharmacia Biotech AB, Uppsala, Sweden).
TTV-DNA Semiquantitation
TTV-DNA titers in serum and liver were semiquantitated by PCR
using the pCI-neo plasmid (Promega Co., Madison, WI) as external
standard. A 1:10 dilution of each positive serum sample and 0.1 µg of
total DNA from each positive liver sample were mixed with 1 pg of
pCI-neo. All of the mixtures were amplified by PCR with
-32P-dATP, using the primers TTSV (sense,
nucleotide position: 1021–1045) and SV2 (antisense, nucleotide
position: 1372–1395) deduced from the SV40 late polyA signal of
pCI-neo and the primers from TTV ORF-1 and untranslated region, as
described above. Primers for pCI-neo amplification were designed to
have a similar length and melting temperature as those used for TTV-DNA
amplification. The DNA fragments of 374 bp corresponding to pCI-neo and
271 or 199 bp corresponding to ORF-1 or untranslated region TTV-DNA
amplification, respectively, were resolved by 6% polyacrylamide gel
electrophoresis, followed by autoradiography of the dried gel.
Quantitation of the signals was performed by densitometric analysis (MD
ImageQuant Software version 3.3, Molecular Dynamics Ltd., Kemsing, UK).
The TTV-DNA titers were expressed as the intensity of the TTV-PCR
products, normalized with respect to the intensity of the pCI-neo-PCR
products.
Sequence Analysis
The PCR products from ORF-1 obtained from serum and liver samples of selected patients were cloned into the pCR-II-TOPO vector (TOPO TA Cloning kit, Invitrogene, San Diego, CA). Several clones from each sample were automatically sequenced in both directions using the ALF Express DNA Sequencing System (Pharmacia Biotech AB).
The TTV nucleotide sequences were aligned with the Clustal X program17 and edited with the GeneDoc program version 2.5.000 (Nicholas KB, Nicholas HB Jr, GeneDoc: a tool for editing and annotating multiple sequence alignments. 1997, distributed by the authors). The nucleotide sequences of the TTV-DNA amplified from our patients were compared with the TTV-DNA sequences retrieved from GenBank, corresponding to all of the published TTV genotypes. Phylogenetic analysis was performed using the DNADIST, NEIGHBOR, SEQBOOT, and CONSENSE programs from the Phylip package version 3.5c.18 The genetic distances were estimated by the Kimura two-parameters method and the unrooted phylogenetic tree was constructed by the neighbor-joining method. The final output of the tree was obtained using the Tree View program version 1.5.2.19 Bootstrap values were determined on 1000 resamplings of the data set. Values >70% were considered statistically significant for the observed grouping.
Nucleotide Sequence Accession Numbers
The GenBank accession numbers of the sequences reported in this article are from AF216391 through AF216432.
In Situ Hybridization
A PCR product of 271 bp in size corresponding to TTV ORF-1 was directly cloned into the pCR II-TOPO vector to obtain the pCTTV plasmid following the instructions supplied by the manufacturer. Verification of the insert orientation and nucleotide sequence was performed by automatic sequencing. The pCTTV plasmid was purified using the Wizard Plus SV Minipreps kit (Promega) and labeled with digoxigenin-11-dUTP (Boehringer Mannheim Biochemicals, Indianapolis, IN) by nick translation (Nick Translation System; GIBCO BRL, Grand Island, NY).
Paraffin-embedded liver sections (4 µm) were dewaxed in xylol and rehydrated through a series of ethanol dilutions. After digestion with proteinase K (1 µg/ml) at 37°C for 10 minutes, the sections were postfixed in a freshly-prepared solution of 4% paraformaldehyde in 0.1 mol/L phosphate-buffer at pH 7.0, dipped in distilled water, and acetylated in 0.5% (v/v) acetic anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 20 minutes at room temperature. Slides were rinsed in 2 x SSPE (20 x SSPE: 3 mol/L NaCl, 0.2 mol/L NaH2PO4, 16 mmol/L ethylenediaminetetraacetic acid, pH 7.4) and quickly dehydrated in ethanol. Hybridization was carried out in a solution consisting of 50% deionized formamide, 0.1 mol/L phosphate-buffer (pH 7.0), 4 x SSPE, yeast t-RNA (500 µg/µl), and 10% dextran sulfate containing 5 ng of heat-denatured labeled DNA probe per slide. After 16 hours at 50°C, slides were washed for 1 hour at 50°C with 1.5 x SSPE followed by an additional 1-hour wash at 50°C in 0.75 x SSPE. The digoxigenin-labeled hybrids were detected with a digoxigenin-antibody alkaline-phosphatase conjugate and an enzyme-substrate chromogen (nitroblue tetrazolium salt in 70% (v/v) dimethylformamide/5-bromo-4-chloro-3-indolyl-phosphate in dimethylformamide) according to the instructions supplied by the manufacturer (DIG Nucleic Acid Detection kit, Boehringer Mannheim Biochemicals). The slides were counterstained with 0.2% safranine in 5% ethanol.
The specificity of the signal was assessed by: 1) hybridization with the labeled plasmid without TTV-derived sequences; 2) digestion of the liver sections with RNase A (0.2 mg/ml; Sigma Chemical Co., St. Louis, MO) and DNase I (20 U/ml; Sigma) for 2 hours at 37°C, before the in situ hybridization; 3) competitive hybridization in the presence of an excess of unlabeled TTV probe; and 4) omission of the probe in the hybridization mixture.
Visualization of the in situ hybridization signals was performed using a NIKON Eclipse E400 light microscope (Nikon, Tokyo, Japan) using 20x, 40x, and 100x objectives. The images were captured using a high-resolution monochrome CCD camera (DIC-N; World Precision Instruments, Cambridge, UK).
The percentage of infected hepatocytes was determined by visual inspection, counting at least 2 x 103 cells per sample.
Histological Diagnosis
Slides from the liver biopsies analyzed were stained with hematoxylin and eosin for the histological diagnosis. The histological activity index was estimated according to Knodell et al20 and histological diagnosis was made according to Desmet et al.21
Statistical Analysis
Continuous variables were compared using the Student’s t-test. A difference with a P < 0.05 was considered significant. The Spearman correlation was used for correlations. The statistical analyses were performed using the SPSS package (SPSS for Windows release 6.0, SPSS Inc., Chicago, IL).
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). No statistical differences in TTV-DNA
titers in serum or in liver were found between patients with liver
disease coinfected with HBV or HCV, patients with liver disease
infected only by TTV and the two cases without evidence of liver
disease but with TTV-DNA in serum and liver (Table 2)
. Furthermore, no statistical
correlation between TTV-DNA titers in serum and liver was found
(r = 0.28; P = 0.535).
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although RNase pretreatment did not change the
hybridization pattern. Competitive hybridization with an excess of
unlabeled pCTTV plasmid diminished the signal intensity. The
specificity of the in situ hybridization technique was
further confirmed by the lack of positive signals when the slides were
hybridized with the plasmid without TTV-derived sequences and when the
probe was omitted in the hybridization mixture.
Regarding the liver cell types infected by TTV, in situ
hybridization revealed positive signals only in the hepatocytes of the
liver samples analyzed. The infected hepatocytes were randomly
distributed throughout the liver sections with no clustering of the
hybridization signals in any specific area of the liver biopsies. With
respect to the intracellular localization of the hybridization signals,
these were mainly detected in the cytoplasm and in the nucleus of the
hepatocytes although perinuclear staining was also observed in
scattered hepatocytes (Figure 3, e–g)
. The pattern of TTV distribution
and cellular localization patterns were similar in the 15 patients with
liver disease and in the two cases with normal liver histology. No
pathological features were observed in the hepatocytes showing
hybridization signals. Furthermore, no topographical correlation was
found between hepatocytes showing hybridization signals and the
hepatocyte steatosis, siderosis, or the inflammatory infiltrate in the
patients with cryptogenic hepatitis (Figure 4)
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The percentage of TTV-infected hepatocytes correlated significantly with the TTV-DNA titers in liver (r = 0.54; P = 0.037). A statistically significant correlation was observed between the percentage of TTV-infected hepatocytes in the 15 patients with liver disease infected by TTV and the ALT level (r = 0.73; P = 0.002). Interestingly, when the patients were divided into those having only TTV infection and TTV plus HCV or HBV infection, the statistically significant correlation between the percentage of infected hepatocytes and ALT levels was only maintained in the group of patients infected by TTV alone (r = 0.8; P = 0.031). Furthermore, in these patients intrahepatic TTV-DNA titers also correlated significantly with the ALT levels (r = 0.52; P = 0.04).
No correlation was observed between the percentage of TTV-infected hepatocytes and the histological activity index or its individual scores in TTV-infected patients coinfected with HCV or HBV.
Finally, no statistical differences were observed in the
histological activity index or in its scores between TTV-positive
patients coinfected with HCV or HBV and the patients infected with HCV
or HBV but without TTV (Table 3)
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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TTV-DNA was detected by PCR in the liver samples from the 15 patients with circulating TTV but not in the remaining 15 patients without the viral DNA in serum. Furthermore, TTV-DNA was detected in serum and liver from two of the eight cases without liver disease. The specificity of the detection was further confirmed by the data obtained from direct sequencing of the PCR products that demonstrated that the amplified DNA corresponded to the TTV genome. The semiquantitation of the PCR products shows, in agreement with previous reports, that the TTV-DNA titers were 10 times higher in liver (3.2 x 103 ± 1.9 x 103 density units) than in serum (3.2 x 102 ± 2.8 x 102 density units). However, no correlation was found between the viral DNA titers in liver and in serum. There are two possible explanations for this finding. First, that TTV may infect the liver cells but does not replicate in them, and second, that the main site of TTV replication may not be the liver. In this sense, TTV-DNA has been detected in peripheral blood mononuclear cells (PBMCs).23 The verifications of these hypotheses deserve future research.
When TTV detection was performed by in situ hybridization, positive signals were observed in all liver samples in which viral DNA was detected by PCR but not in the TTV-PCR-negative biopsies. The specificity of the in situ hybridization technique was further confirmed by the results obtained in the specificity test.
Regarding the cell types infected by TTV within the liver, hybridization signals were only observed in the hepatocytes, in both the patients with liver disease of viral or nonviral origin and the cases with normal liver histology. All considered, the data on PCR and in situ hybridization show that TTV can infect liver cells. However, hepatocytes with hybridization signals do not show any morphological changes, suggesting that TTV is not pathogenic for the infected cells.
The hybridization signals in the hepatocytes were detected mainly in the cytoplasm and in the nucleus. However, due to the method that was used for the visualization of the in situ hybridization signals (light microscopy) it was not possible to discriminate if the signals observed in the nucleus were inside the nucleus or in the thin cytoplasmic layer between the cell membrane and the nucleus.
The number of TTV-infected hepatocytes was similar in the patients with liver disease (from 2.1% to 30%) and in the two cases without liver diseases (8% and 10.3%) and these percentages correlate significantly with the viral titers in the tissue. On the other hand, no differences in the number of TTV-positive hepatocytes were found between those patients positive only for TTV and those also infected by HCV or HBV. This finding suggests that HCV or HBV infection did not inhibit TTV entry into the hepatocytes or TTV replication.
A statistically significant positive correlation between the ALT levels and the number of TTV-infected hepatocytes as well as with the intrahepatic TTV-DNA titers was found in the seven patients infected only by TTV. This finding suggests that TTV may have contributed to the liver disease in these patients. As the TTV was the only etiological agent of liver disease found in two of these patients, it could be assumed that TTV is responsible for some cases of cryptogenic hepatitis, as some other authors have suggested.9,13,23 The fact that TTV-infected cells did not show morphological changes argues against this notion. However, TTV may be pathogenic for the infected cells by mechanisms such as alteration of the cell membrane permeability that does not imply necessarily the alteration of the cell morphology. On the other hand, the remaining five patients had a well-known cause of liver disease (liver steatosis, ductopenia, and siderosis). In addition, no topological correlation between hepatocytes showing hybridization signals and the steatosis, siderosis, or the inflammatory infiltrate was observed. Furthermore, as mentioned above, TTV was also detected in the liver of two cases with normal liver histology without differences in the infection and distribution pattern with respect to the patients with liver disease. Considered as a whole, our results indicate that TTV may not be pathogenic for the liver in all cases.
It may be argued that the TTV strain or the complexity of the viral population are different in the patients with liver disease of nonviral origin and the cases with normal liver histology. However, the sequencing data obtained from the cloned PCR products do not support this hypothesis as the TTV subtype and the complexity of the viral populations infecting both groups were similar, at least in the ORF-1 of TTV-DNA. However, changes in other regions of the viral genome that may account for a different pathogenicity of distinct viral strains cannot be discarded.
Finally, no relation between the percentage of TTV-positive hepatocytes and ALT levels and the histological activity index was found in TTV-positive patients coinfected by HCV or HBV. Once again, this finding suggests that TTV has a minor role in causing liver disease.
In conclusion, using a specific in situ hybridization technique, in this study we have demonstrated that TTV can infect liver cells although our results do not support an important role for TTV in liver pathogenesis.
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Footnotes |
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Supported by a grant from the Fundación Hepatitis Virales (to E. R. I.). M.C. is a research fellow of the Fundación Conchita Rábago.
Accepted for publication January 6, 2000.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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X174 DNA digested with HinfI
(Promega).
