闽南地区TT病毒的变异及经输血传播的初步证据

TT virus(TTV)DNA was tested by nested-PCR from sera of hepatitis patients and volunteer blood donors in Minnan area. The amplified segment was a 189 base pair region in TTV ORF2. A total of six sequences were obtained from three non-A to G hepatits patients and two from volunteer blood donors. The sequences were found to be with 82.9% to 99.3% homology to TTV Japanese strain and Chinese strain. The divergence of sequence in these six segments varied from 0.7% to 17.1%, which indicated that the TTV had been existing for a long time in this area. In the serum of a non-A to G hepatitis patient who was negative for TTV DNA in the 14th day of disease course turned to be positive in the 30th day, two TTV sequences were obtained which showed 92.1% nucleotide homology. It indicated that different TTV strains can co exist in the same person. This patient's blood had been transfused ten times between the collection of his TTV negative sample and his positive serum sample. Seven of the blood donors were traced and sampled for sera, of which three were positive for TTV. For all 161 patients tested, the history of exposure to blood products was associated with an increased risk of TTV infection(relative risk, 3.0; 95% confidence intervals, 1.89～4.81).     

TT virus infection in nonhuman primates and characterization of the viral genome: identification of simian TT virus isolates

Newly discovered TT virus (TTV) is widely distributed in human populations. To understand more about the relationship between TTV and its hosts, we tested 400 sera from various nonhuman primates for the presence of TTV DNA by PCR assay. We collected serum samples from 24 different species of nonhuman primates. TTV DNA was determined by PCR with primers designed from the 5'-end region of the TTV genome. Nucleotide sequencing and phylogenetic analysis of viral genomes were also performed. TTV DNA was detected in 87 of 98 (89%) chimpanzees and 3 of 21 (14%) crab-eating macaques. Nucleotide sequences of the PCR products obtained from both animals were 80 to 100% identical between two species. In contrast, the sequences differed from TTV isolates in humans by 24 to 33% at the nucleotide level and 36 to 50% at the amino acid level. Phylogenetic analysis demonstrated that all TTV isolates obtained from simians were distinct from the human TTV isolates. Furthermore, TTV in simians, but not in humans, was classified into three different genotypes. Our results indicate that TTV in simians represents a group different from, but closely related to, TTV in humans. From these results, we tentatively named this TTV simian TTV (s-TTV). The existence of the s-TTV will be important in determining the origin, nature, and transmission of human TTV and may provide useful animal models for studies of the infection and pathogenesis of this new DNA virus.     

TT virus infection in children and adults who visited a general hospital in the south of Brazil for routine procedure

TT virus (TTV) is a newly described nonenveloped human virus, with a circular, negative-stranded DNA genome, that was first identified in the blood of a patient with posttransfusion hepatitis of unknown etiology. PCR primers and conditions used for TTV DNA amplification may greatly influence the level of TTV detection in serum. Three PCR assays, with different regions of the genome as targets, were used to test TTV DNA in 130 sera from children and adults visiting a hospital in the south of Brazil, most of them for routine procedure. Forty-four percent of adult sera and 73% of sera from children aged 0-10 years were TTV positive with at least one PCR assay. However, the three assays were able to detect only 33%, 35%, and 70% of the total positive samples. Our results showed a high prevalence of TTV infection in the south of Brazil, particularly among young children, and confirmed the necessity of performing several PCR assays to assess the true TTV prevalence in a determined population.     

Abnormal production of interleukin (IL)-11 and IL-8 in polycythaemia vera

We studied the production of interleukin (IL)-11 and IL-8, two cytokines known to affect erythropoiesis, in polycythemia vera (PV). In vivo, IL-11 was detected more frequently in serum and bone marrow (BM) plasma of PV patients than in controls (healthy donors and patients with idiopathic erythrocytosis (IE)). In addition, serum IL-11 levels of PV patients were higher than those of controls. IL-8 was elevated in serum of both PV and IE patients (respective median levels: 38.6 and 242pg/ml, vs 4.4pg/ml for healthy donors). BM plasma IL-8 levels of PV patients (508pg/ml) were significantly higher than those of IE patients (120pg/ml). In vitro, bone marrow (BM) stromal cells (BMSC) of PV patients produced significantly more IL-11 (x6.4) and IL-8 (x8.3) than BMSC of healthy donors or IE patients. In conclusion, both IL-11 and IL-8 are overproduced in PV, apparently by BMSC; IL-8 is also overproduced in IE, by cells other than BMSC.     

Circular double-stranded forms of TT virus DNA in the liver

TT virus (TTV) is an unenveloped, circular, and single-stranded DNA virus commonly infecting human beings worldwide. TTV DNAs in paired serum and liver tissues from three viremic individuals were separated by gel electrophoresis and characterized biophysically. TTV DNAs in sera migrated in sizes ranging from 2.0 to 2.5 kb. TTV DNAs in liver tissues, however, migrated at 2.0 to 2.5 kb as well as at 3.5 to 6.1 kb. Both faster- and slower-migrating forms of TTV DNAs in the liver were found to be circular and of the full genomic length of 3.8 kb. TTV DNAs migrating at 2.0 to 2.5 kb, from either serum or liver tissues, were sensitive to S1 nuclease but resistant to restriction endonucleases, and therefore, they were single-stranded. By contrast, TTV DNAs in liver tissues that migrated at 3.5 to 6.1 kb were resistant to S1 nuclease. They migrated at 3.7 to 4.0 kb after digestion with EcoRI, which suggests that they represent circular, double-stranded replicative intermediates of TTV. When TTV DNAs were subjected to strand-specific primer extension and then amplified by PCR with internal primers, those in serum were found to be minus-stranded DNAs while those in liver tissues were found to be a mixture of plus- and minus-stranded DNAs. These results suggest that TTV replicates in the liver via a circular double-stranded DNA.     

Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder. Several genetic/epigenetic abnormalities are deeply associated with the pathogenesis of MDS. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic abnormalities, BM aspiration is difficult to perform repeatedly to obtain serial samples because of pain and safety concerns. Here, we report that circulating cell-free DNAs from plasma and serum of patients with MDS can be used to detect genetic/epigenetic abnormalities. The plasma DNA concentration was found to be relatively high in patients with higher blast cell counts in BM, and accumulation of DNA fragments from mono-/di-nucleosomes was confirmed. Using serial peripheral blood (PB) samples from patients treated with hypomethylating agents, global methylation analysis using bisulfite pyrosequencing was performed at the specific CpG sites of the LINE-1 promoter. The results confirmed a decrease of the methylation percentage after treatment with azacitidine (days 3-9) using DNAs from plasma, serum, and PB mono-nuclear cells (PBMNC). Plasma DNA tends to show more rapid change at days 3 and 6 compared with serum DNA and PBMNC. Furthermore, the TET2 gene mutation in DNAs from plasma, serum, and BM cells was quantitated by pyrosequencing analysis. The existence ratio of mutated genes in plasma and serum DNA showed almost equivalent level with that in the CD34+/38- stem cell population in BM. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a safer and painless alternative to using BM cells.     

TT virus in Hungary: sequence heterogeneity and mixed infections

The majority of the viral hepatitis cases is caused by five hepatitis viruses (A,B,C,D,E). In 1997, TT virus was discovered. It was supposed that a number of the unknown hepatitis cases was caused by the TT virus. The aim of this study was to characterize TT viruses carried by healthy individuals and patients suffering from hepatitis of unknown origin in Hungary. TTV DNA was detected by seminested PCR with the commonly used N22 primers. Twenty of the 108 sera (18.5%) taken from healthy persons and 115 of the 228 sera (50.4%) of patients with hepatitis of unknown origin were found to be positive. The nucleotide sequences of 26 clones derived from 17 hepatitis patients and 15 clones from nine healthy persons were determined and a phylogenetic tree was constructed. Genotype 2 (group 1) was found to be the most frequent, but other group 1 genotypes (1, 6) and genotypes 8 and 17 of group 2 were also detected. Mixed TTV infections were found in eight cases (two healthy persons and six hepatitis patients). Variants belonging to the same group were carried in seven cases, and the presence of group 1 (genotype 2) and group 2 (genotype 8) TTV sequences were found in one single hepatitis patient.     

Sequencing a specific kinetoplast DNA fragment of Leishmania donovani for polymerase chain reaction amplification in diagnosis of leishmaniasis in bone marrow and blood samples

A set of oligonucleotide primers I and II was developed by analyzing the specificity of a cloned kinetoplast DNA (kDNA) fragment of Leishmania donovani and sequencing the fragment. Polymerase chain reaction (PCR) was conducted with the primers to amplify a minicircle kDNA fragment (297 bp) to detect L. donovani in the bone marrow (22 samples), whole blood (16 samples), and serum (17 samples) of 22 patients with visceral leishmaniasis. All of 22 patients were diagnosed by microscopic identification. Control samples of bone marrow, whole blood, and serum were obtained from patients with leukemia and from healthy volunteers. In addition, 12 dogs were infected with L. donovani promastigotes for the PCR test. The total number of patients positive by PCR testing was 95.5% (21/22), with 91.0% (20/22) from the bone marrow, 68.8% (11/16) from the blood, and 29.4% (5/17) from the sera. Similar results were obtained in infected dogs. No amplification products were seen in control samples from humans or dogs. Our results suggest that PCR may be useful in detecting kDNA in the bone marrow and blood of patients with visceral leishmaniasis.     

Visualization of TT virus particles recovered from the sera and feces of infected humans

TT virus (TTV) has not yet been cultured or visualized. We attempted to recover and visualize TTV-associated particles from the serum samples and feces of infected humans. Serum samples were obtained from 7 human immunodeficiency virus (HIV)-infected patients. Three patients had a high TTV DNA titer (10(8) copies/ml), three had a low TTV DNA titer (10(2) copies/ml), and one was negative for TTV DNA. Fecal supernatant was obtained from a different TTV-infected subject. The serum samples were fractionated by high-performance liquid chromatography, and TTV DNA-rich fractions were subjected to floatation ultracentrifugation in cesium chloride. Virus-like particles, 30-32 nm in diameter, were found in the 1.31-1.33 g/cm(3) fractions from each of the three serum samples with high TTV DNA titer, but not in any fraction from the four serum samples that either were negative for TTV DNA or had low TTV DNA titer. The TTV particles formed aggregates of various sizes, and immunogold electron microscopy showed that they were bound to human immunoglobulin G. Similar virus-like particles with a diameter of 30-32 nm banding at 1.34-1.35 g/cm(3) were visualized in fecal supernatant with TTV genotype 1a by immune electron microscopy using human plasma containing TTV genotype 1a-specific antibody.     

The prevalence of TT virus in cancer patients

Transfusion-transmitted virus (TTV) is a recently discovered transfusion-transmissible DNA virus. Its frequency and clinical impact has not been established in cancer patients in Turkey. In this study, we determined the prevalence of TTV DNA positivity, and its relationship with history of transfusion, amount of transfusion, age and sex in patients with hematological and solid malignancies. Sixty-one patients (35 male and 26 female) followed up for various malignancies and 45 healthy subjects were included in the study. ITV DNA was assayed by the polymerase chain reaction (PCR). TTV DNA was detected in 18 of 61 patients (29.5%) and in 5 of 45 control subjects (11.1%). In cancer patients, the prevalence of TTV DNA positivity was higher to comparison with control group. In addition, the prevalence of TTV DNA positivity was significantly higher in 22 patients who had a history of blood transfusion in the last 6 months than 39 patients who had no current or past history of transfusion (40.9% vs 23.0% respectively). These results suggest that the prevalence of TTV DNA is high and the parenteral route is an important mode of transmission for TTV in cancer patients. In addition, the high prevalence and persistence of TTV in cancer patients with parenteral risk exposure could be related to the immunodeficiency due to cancer and high viral loads by parenteral route.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

延边地区丙型肝炎患者合并TT病毒感染的检测

检测丙型肝炎患者血清标本中的TT病毒 (transfusiontransmittedvirus,TTV) ,了解延边地区丙型肝炎患者合并TTV感染状况。采用ELISA检测抗TTVIgG和巢式PCR检测丙型肝炎病毒 (HCV)感染患者血清中TTVDNA。采用全自动生化分析仪检测患者血清谷氨酸氨基转移酶 (ALT)和谷氨酸草酰乙酸氨基转移酶 (AST)。 4 5例丙型肝炎患者抗TTVIgG阳性率为 37.8% (17/45 ) ,巢式PCR阳性率为 4 2 .2 % (19/45 )。延边地区HCV感染患者重叠感染TTV较常见。     

Sequence heterogeneity of TT virus and closely related viruses

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a "swarm" of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.     

TT virus is distributed in various leukocyte subpopulations at distinct levels, with the highest viral load in granulocytes.

When TT virus (TTV) DNA was quantitated in whole blood and plasma aliquots from 27 viremic individuals by real-time detection PCR that can detect essentially all TTV genotypes, the TTV load was 6.9 +/- 3.5 (mean +/- standard deviation)-fold higher in the whole blood than in the plasma samples [P < 0.002 (paired t test)]. To clarify the reason for this difference, peripheral blood cells of various types including red blood cells, granulocytes (CD15+), B cells (CD19+), T cells (CD3+), monocytes (CD14+), and NK cells (CD3-/CD56+) were separated at a purity of 95.4-99.5% from each of three infected individuals with relatively high TTV viremia, and their TTV viral loads were determined. Red blood cells were uniformly negative, but the other cell types were positive for TTV DNA at various titers. In all three patients, the highest TTV load was found in granulocytes (4.2 x 10(4)-3.1 x 10(5) copies/10(6) cells), followed by monocytes (1.4-2.2 x 10(4) copies/10(6) cells) and NK cells (5.4-6.5 x 10(3) copies/10(6) cells); B and T cells were positive, with a low viral load (6.7 x 10(1)-2.7 x 10(3) copies/10(6) cells). These results indicate that TTV is distributed in various peripheral blood cell types at distinct levels, with the highest viral load in granulocytes, and that a significant proportion of the TTV DNA in peripheral blood is not identified by the standard plasma/serum DNA detection methods.     

Genomic characterization of a Brazilian TT virus isolate closely related to SEN virus-F

SEN virus (SENV) is a circular, single stranded DNA virus that has been first characterized in the serum of a human immunodeficiency virus type 1 (HIV-1)-infected patient. Eight genotypes of SENV (A-H) have been identified and further recognized as variants of TT virus (TTV) in the family Circoviridae. Here we describe the first genomic characterization of a SENV isolate (5-A) from South America. Using 'universal' primers, able to amplify most, if not all, TTV/SENV genotypes, a segment of > 3 kb was amplified by polymerase chain reaction from the serum of an HIV-1 infected patient. The amplicon was cloned and a 3087-nucleotide sequence was determined, that showed a high (85%) homology with the sequence of the Italian isolate SENV-F. Proteins encoded by open reading frames (ORFs) 1 to 4 consisted of 758, 129, 276, and 267 amino acids, respectively. By phylogenetic analysis, isolate 5-A was classified into TTV genotype 19 (phylogenetic group 3), together with SENV-F and TTV isolate SAa-38.     

Transmission of human TT virus of genotype 1a to chimpanzees with fecal supernatant or serum from patients with acute TTV infection

Fecal supernatant or serum containing TT virus (TTV) of genotype 1a (10(5) copies/ml) from patients with acute TTV infection was inoculated intravenously into two naive chimpanzees. Serum samples were obtained weekly and tested for TTV DNA by genotype 1-specific polymerase chain reaction. TTV DNA was detected in chimpanzee 228 at weeks 5-15 after inoculation with 0.5 ml of serum, and in chimpanzee 234 at weeks 7-19 after inoculation with 1 ml of fecal supernatant. The TTV DNA titer peaked at weeks 12 and 13 in chimpanzee 228 and at weeks 14-16 in chimpanzee 234. Mild biochemical and histological changes in biopsied liver samples were observed in both chimpanzees in association with the reduction in TTV titer. TTV DNA was transient in chimpanzee 228, but in chimpanzee 234 it reappeared at week 21 and persisted through week 30. These results indicate that TTV in feces is infectious and suggest that TTV has hepatitis-inducing capacity.     

Species-specific TT viruses and cross-species infection in nonhuman primates

Viruses resembling human TT virus (TTV) were searched for in sera from nonhuman primates by PCR with primers deduced from well-conserved areas in the untranslated region. TTV DNA was detected in 102 (98%) of 104 chimpanzees, 9 (90%) of 10 Japanese macaques, 4 (100%) of 4 red-bellied tamarins, 5 (83%) of 6 cotton-top tamarins, and 5 (100%) of 5 douroucoulis tested. Analysis of the amplification products of 90 to 106 nucleotides revealed TTV DNA sequences specific for each species, with a decreasing similarity to human TTV in the order of chimpanzee, Japanese macaque, and tamarin/douroucouli TTVs. Full-length viral sequences were amplified by PCR with inverted nested primers deduced from the untranslated region of TTV DNA from each species. All animal TTVs were found to be circular with a genomic length at 3.5 to 3.8 kb, which was comparable to or slightly shorter than human TTV. Sequences closely similar to human TTV were determined by PCR with primers deduced from a coding region (N22 region) and were detected in 49 (47%) of the 104 chimpanzees; they were not found in any animals of the other species. Sequence analysis of the N22 region (222 to 225 nucleotides) of chimpanzee TTV DNAs disclosed four genetic groups that differed by 36.1 to 50.2% from one another; they were 35.0 to 52.8% divergent from any of the 16 genotypes of human TTV. Of the 104 chimpanzees, only 1 was viremic with human TTV of genotype 1a. It was among the 53 chimpanzees which had been used in transmission experiments with human hepatitis viruses. Antibody to TTV of genotype 1a was detected significantly more frequently in the chimpanzees that had been used in transmission experiments than in those that had not (8 of 28 [29%] and 3 of 35 [9%], respectively; P = 0.038). These results indicate that species-specific TTVs are prevalent in nonhuman primates and that human TTV can cross-infect chimpanzees.