Infection with an unenveloped DNA Virus (TTV) associated with posttransfusion non-A to G hepatitis in hepatitis patients and healthy blood donors in Thailand

An unenveloped single-stranded DNA virus (TTV) has been reported in association with posttransfusion and acute and chronic hepatitis of unknown etiology. DNA of TTV was tested for by polymerase chain reaction with heminested primers in 127 patients with chronic liver disease and 105 healthy blood donors in Thailand. TTV DNA was detected in 23 (59%) of the 39 patients without hepatitis B surface antigen or RNA of hepatitis C virus, at a frequency significantly higher than the detection in 21 (36%) of the 59 patients with HBsAg (P < 0.05) or in 38 (36%) of the 105 blood donors (P < 0.05). Among patients with chronic liver disease, TTV DNA occurred in those with liver cirrhosis and hepatocellular carcinoma more frequently than in those with chronic hepatitis (35 of 65 or 54% vs. 20 of 62 or 32%, P < 0.05). There were no differences in age, sex, or markers of infection with hepatitis B, C and GBV-C/HGV viruses, indicating a mode of transmission of TTV different from those of the other hepatitis viruses. Phylogenetic analysis indicated three different genotypes of TTV with six distinct subtypes in Thailand. Based on these results, TTV would have a role in the development of chronic liver disease of unknown etiology in Thailand. J. Med. Virol. 56:234–238, 1998. © 1998 Wiley-Liss, Inc.     

地高辛标记探针原位杂交法检测肝病患者外周血单个核细胞中TTV-DNA

为检测肝病患者外周血单个核细胞(PBMC)中TTV-DNA(输血传播病毒脱氧核糖核酸), 以TTV ORF1为模板, 应用地高辛(Dig)作为标记物, 经聚合酶链反应(PCR)制备探针, 建立原位杂交方法进行检测.结果显示: 血清TTV-DNA阳性组, 双链探针检测PBMC中TTV-DNA, 阳性检出率为58.06%(18/31); 血清TTV-DNA阴性组, 双链探针检测PBMC中TTV-DNA, 阳性检出率为27.59%(8/29).双链探针阳性者, 再以负链探针检测其复制情况, 阳性率为22.2%(4/18). 结论: TTV可感染PBMC并在PBMC中复制.     

Genoprevalence in human tissues of TT-virus genotype 6

Summary. TT virus (TTV) is a newly discovered human virus of high genotypic diversity. TTV is widely distributed among humans, but the possible genotype-related differences in TTV biology are not well known. The prevalence and amount of TTV-DNA, especially of genotype 6, was determined by nested-PCR in various human tissues, and human parvovirus B19, another ssDNA virus, was used as a reference. TTV DNA was detected simultaneously in bile, peripheral blood mononuclear cells (PBMC) and plasma of 77% subjects, in 38% skin samples, in 38% synovial samples and in all (100%) adenoids, tonsils and liver samples. The relative concentrations of TTV-DNA did not vary significantly among the different samples. Genotype 6 TTV-DNA was detected in bile and plasma of one subject (3%), in skin and serum of one subject (8%) and in one liver (5%). The overall prevalence of TTV genotype 6 was 4% in subjects and 4% in sera. TTV genotype 6 was shown to occur in human tissues with no obvious tissue-type or symptom specificity. Parvovirus B19 DNA was detected overall in 38% subjects, and bile was the only sample type tested that did not persistently harbour B19 DNA.     

Transfusion-transmitted virus infection in HIV-1-seropositive patients

Objectives   To investigate the prevalence, persistence and genome heterogeneity of transfusion-transmitted (TTV) in HIV-1-infected patients, a group at high risk both of contracting blood-borne viruses and having viral persistence relating to immunodepression.
Methods   Plasma samples from 238 HIV-1 seropositive subjects and 226 healthy blood donors were examined for TTV-DNA both by polymerase chain reaction (PCR) using primers from the conserved regions in the N22 clone and PCR using primers deduced from the untranslated region (UTR). Direct DNA sequencing and phylogenetic analysis were used to characterize 27 TTV isolates from HIV-1 patients or healthy controls.
Results   Using PCR with the UTR primers, TTV DNA was detected in a very high percentage (> 80%) of samples both from HIV-1 seropositive subjects and from blood donors. Using PCR with N22 primers, shown to detect viral strains associated with hepatitis of unknown etiology, TTV DNA was found in 103 of 238 (43.3%) HIV-1-infected patients and in 22 of 226 (9.7%) blood donors. There was no difference in the prevalence of the TTV DNA in HIV seropositive subjects with regard to clinical features related to immunosuppression, markers of HCV infection or intravenous drug use; presence of TTV DNA was associated significantly only with male gender ( P = 0.003). Persistent or intermittent viremia was detected in plasma samples taken up over a period of 19 months in all (15 of 15) HIV-infected patients tested.
Conclusions   The persistence and high frequency of infection detected by PCR with N22 primers in HIV-1 seropositive patients suggest that further clinical investigation of immunocompromised hosts will provide information to clarify the pathogenic role of TTV.     

TT virus infection in healthy children,children after blood transfusion,and children with non-A to E hepatitis or other liver diseases in Taiwan

Serum samples from healthy and diseased children were studied for the presence of TTV DNA by nested PCR using primer sets generated from N-22 region and from the untranslated region (UTR) of the viral genome. N-22 positive TTV DNA was detectable in 33 (27%) of 122 healthy children, 47 (73.4%) of 64 polytransfused thalassemic children, 37 (46.3%) of 80 children who received transfusion during cardiac surgery, 8 (42.1%) of 19 non-A to E hepatitis, 10 (33.3%) of 30 HBV carrier children, and 5 (15.6%) of 32 infants with biliary atresia. A much higher prevalence of TTV DNA with rates varying from 78-100% in the above study groups was observed using the UTR primers. For children with N-22 positive TTV DNA, biochemical assessment of isolated TTV viremia in thalassemic children or children transfused during surgery showed no convincing association between raised ALT levels and TTV viremia. Coinfection with TTV in chronic HCV-infected or HBV-infected children did not result in higher peak ALT levels during follow-up, suggesting that TTV has no synergistic pathogenic effect. The phylogenetic analysis of the N-22 positive TTV DNA isolates revealed that most isolates from healthy children, children transfused during surgery, and non-A to E fulminant hepatitis children were type 1 TTV. These results indicate that TTV infection in children was significantly associated with transfusion. TTV infection is highly prevalent in early childhood in Taiwan but plays a minimal role in the induction of hepatitis in children.     

Detection of TT virus sequences in children with liver disease of unknown etiology

DNA sequences of TT virus (TTV) in 55 serum samples taken from 20 children with liver disease of unknown etiology (16 with acute liver disease, 2 with fulminant hepatitis, and 2 with chronic liver disease) and from 35 healthy children as controls were examined by using the hemi-nested polymerase chain reaction (PCR). The PCR was carried out using the established primers (NG059, NG061, NG063) to amplify TTV DNA sequences. The sequences were detected in 6 of the 20 patients (30.0%) with liver disease and in 5 of the 35 healthy children (14.2%) by direct gel analysis. There was no significant difference between the prevalence of liver disease patients and controls. However, both patients with fulminant hepatitis and both patients with chronic hepatitis had TTV DNA sequences. Four of the six TTV isolates from liver disease patients were genotype 1a, whereas only one of the five TTV isolates from controls was genotype 1a. Although the study population was small, it is possible that genotype 1a of TTV might be more pathogenic than other genotypes in children.     

Prevalence of TTV DNA among children with a history of transfusion or liver disease

The prevalence rates of serum TT virus (TTV) DNA among children with or without a history of transfusion or liver disease were studied by polymerase chain reaction (PCR) using either the Okamoto primer set or the Takahashi primer set developed more recently. Using Okamoto and Takahashi primer sets, the prevalence rates were 31.6% (12/38) and 78.9% (30/38), respectively, for children with a history of blood transfusion (including malignant and non-malignant groups) and 6.7% (2/30) and 60% (18/30), respectively, for children without a history of blood transfusion. Among pregnant women, these rates were 12.9% (4/31) and 61.3% (19/31), respectively. On the other hand, the prevalence rates were 0% (0/16) and 50% (8/16), respectively, in hepatitis B patients, 21.4% (3/14) and 71.4% (10/14), respectively, for hepatitis C patients, and 20.0% (9/45) and 57.8% (26/45), respectively, for non-A to C hepatitis patients (including 27 acute hepatitis patients, 5 fulminant patients and 13 chronic hepatitis patients). In this study, the prevalence rates determined by the Takahashi primer set tended to be 2-9 times higher than those determined using the Okamoto primer set. These results suggest that TTV infection is widespread among Japanese children. Furthermore, blood transfusion does not appear to be the major route of infection. The similar prevalence rates between control children and children with various types of hepatitis using the Takahashi primer system suggest that TTV infection does not play a direct causative role in the development of liver disease in children.     

Persisting TT virus (TTV) genogroup 1 variants in renal transplant recipients

Summary.  TT virus (TTV) genogroup 1 infection has an increased prevalence in solid organ transplant recipients. In this study, the presence of TTV in renal transplant recipients was examined by two PCR methods, one capable of detecting most TTV genotypes (UTR-PCR), the other specific to genogroup 1 (N22-PCR). The N22-PCR detected TTV in 57% (53/92) of the renal transplant patients and in 20% (13/66) of the healthy individuals, while the prevalence of TTV with the UTR-PCR was above 90% in both the control and the patient groups. The N22-PCR was used in longitudinal studies of 31 renal transplant recipients, these PCR products were sequenced and aligned. TTV status was not associated with the patients’ age at transplantation, male to female ratio and the time lag between kidney transplantation and the TTV test. During the follow-up consistent TTV status was found in 26 patients, while two initially TTV positive patients converted to negative and three initially negative patients converted to positive. The TTV variants varied among the tested patients, but were the same in the consecutive samples of each patient, indicating that TTV infection was persistent in renal transplant recipients and novel infection occured rarely in the post-transplant period. Received October 2, 2002; accepted December 27, 2002 Published online March 13, 2003     

Up-regulation of the monocyte chemotactic protein-3 in sera from bone marrow transplanted children with torquetenovirus infection

BackgroundTorquetenovirus (TTV) represents a commensal human virus producing life-long viremia in approximately 80% of healthy individuals of all ages. A potential pathogenic role for TTV has been suggested in immunocompromised patients with hepatitis of unknown etiology sustained by strong proinflammatory cytokines.ObjectivesThe aim of this study was to investigate the sera immunological profile linked to TTV infection in bone marrow transplant (BMT) children with liver injury.Study designTTV infection was assessed in sera from 27 BMT patients with altered hepatic parameters and histological features, by the use of quantitative real-time PCR, along with TTV genogroups and coinfection with HEV. The qualitative and quantitative nature of soluble inflammatory factors was evaluated studying a large set of cytokines using the Bioplex platform. As controls, sera from 22 healthy children negative for serological and molecular hepatitis markers including TTV and HEV, and for autoimmune diseases, were selected.Results and conclusionsTTV was detected in 81.4% of BMT patients with a viral load ranging from 10⁵ to 10⁹ copies/mL. All samples were HEV-RNA negative. A pattern of cytokines, IFN-γ, TNF-α, FGF-basic (p < 0.01) and MCP-3 (p < 0.001) was found significantly highly expressed in TTV-positive patients compared to TTV-negative and controls. Of note, MCP-3 chemokine showed the highest sera concentration independently of the amount of TTV load and the status of immune system deregulation (p < 0.001). In this pilot study for the first time, a positive association was found between TTV and increased level of MCP-3 suggesting a indirect role of TTV in liver injury.     

Mother-to-child transmission of TT virus: sequence analysis of non-coding region of TT virus in infected mother-infant pairs

Summary.  To investigate vertical transmission of TT virus, TTV-DNA was looked for in serum samples taken from 22 mothers and their 22 infants at birth and during nine months of follow-up. Sixteen mothers at delivery and six infants within nine months of age had TTV-DNA detected by the amplification of the non coding (NC) region. Two of these newborns had positive viremia at birth. Sequence analysis of the NC region of five mother-infant pairs revealed that the TTV strains detected at three and six months of age in two of the infants were closely related to that of their mothers, whereas two that became TTV-DNA positive at three moths had a different nucleotide sequence from that of their mothers. One of the two infants with detectable viremia at birth also had a different nucleotide sequence from her mother. These findings suggest that both in utero and perinatal transmission of TT virus may occur, and that the strain detected in the infants was not invariably dominant in the mothers at delivery. Received June 20, 2001 Accepted August 30, 2001     

Detection of dog filariasis in Marajo Island,Brazil by classical and molecular methods

Canine filariasis in domestic and wild dogs, foxes, and wolves is caused by several species of filarids. Although these filarial species inhabit different loci in the vertebrate definitive hosts, they generally release microfilariae into the bloodstream. Data about filarial infection in dogs in Brazil, especially on the Marajo Island, is scarce. For this reason, we conducted an analysis of 188 domestic dogs within two Marajo Island municipalities. The overall prevalence of microfilaremic was 32.45%; taken by blood smear and modified Knott’s method. No significant difference of positivity between male and female was observed (X ² Yates’s correction = 0.341; p = 0.559). Significant age–infection ratios were detected (X ² = 32.943; p < 0.0001). A high occult infection was detected (53.84%). PCR of rDNA and phylogenetic tree indicated that the microfilariae and adult worms found in domestic dogs from Marajo Island were Dirofilaria immitis.     

Detection of TT DNA virus (TTV) in blood donors and patients with hepatitis of unclear etiology]

A new human infective agent: TT virus (TTV) has been recently identified. The polymerase chain reaction detected TTV DNA in the sera of 5 (31.3%) out of 16 children with acute hepatitis, 5 (17.2%) out of 29 children and 3 (14.3%) out of 21 adults with liver diseases of unknown etiology, and 18 (13.2%) out of 136 free-of-charge blood donors. These results indicate a high prevalence of TTV infection in Russia and absence of an obvious correlation between this infection and nonA, nonB, nonC hepatitis in examined patients. Phylogenetic analysis of amplified fragments of viral DNA from 3 patients selected at random showed that the isolated strains belong to subtype 1a, most prevalent in the world.     

Immune Reconstitution Following Unmanipulated HLA-Mismatched/Haploidentical Transplantation Compared with HLA-Identical Sibling Transplantation

In this study, we prospectively investigated the immune reconstitution in patients with hematological malignancies after human leukocyte antigen (HLA)-mismatched/unmanipulated haploidentical transplantation (50 cases) and HLA-matched transplant (25 cases). Transplant-related mortality, relapse, leukemia-free survival, and overall survival were similar between the two transplant strategies, although the cumulative incidence of CMV antigenemia was significantly higher in haploidentical recipients than in HLA-matched recipients (49.9 ± 7.2% versus 13 ± 7%, P = 0.007). Compared with HLA-matched recipients, T-cell subset and dendritic cell subgroup cell counts in the first 90 days after grafting were lower in haploidentical recipients. The difference was most striking for CD4⁺ and CD4⁺ na?ve T cells. Reconstitution of B cells and monocytes was comparable between groups. T cells appeared equally functional in both groups among patients without graft-versus-host disease. Our results suggest that the clinical outcomes were not compromised by the early delayed immune reconstitution following haploidentical transplantation.     

Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus

Summary.  TT virus (TTV) is the only known human virus with single-stranded circular DNA, with a possible but yet unclear relationship to chicken anemia virus (CAV) of the family Circoviridae. Here we report a new human virus resembling TTV and CAV, designated TTV-like mini virus (TLMV). This non-enveloped virus was smaller (>30 nm) but had a similar density (1. 31–1.34 g/ml in CsCl) to TTV, when a TLMV/TTV-coinfected plasma was analyzed. Full-length sequencing revealed that the TLMV genome was a circular DNA comprising 2860 nt (isolate CBD231); significantly shorter than TTV (TA278, 3852 nt) but longer than CAV (CAECUX1, 2319 nt). A strand-specific hybridization assay using oligonucleotide-coated beads suggested TLMV was negative-stranded, like TTV and CAV. In genomic organization, TLMV was similar to both TTV and CAV. The untranslated region of TLMV resembled CAV in that both had direct repeats, whereas the sequence homology was more evident between TLMV and TTV. The predicted ORF1 protein of TLMV was rich in R/W/F residues at its amino terminus; the richness in W was shared by TTV, F by CAV, and R by both. ORF2 proteins of the three viruses had a common motif, WX₇HX₃CXCX₅H. Thus, TLMV is an intermediate between the remotely related TTV and CAV. Since CAV differs much from other circoviruses, it may better be classified together with TTV and TLMV under a new family: we would coin the Paracircoviridae. Accepted November 19, 1999/Received September 13, 1999     

Prevalence and Viral Persistence of TT Virus in Patients on Hemodialysis

 The aim of this study was toevaluate the prevalence of TT virus (TTV) in 107 hemodialysis patients and 100 healthy individuals. Sixteen percent of hemodialysis patients and 2% of the control population were TTV positive (P<0.001). Sex, time on dialysis, transfusions, and alanine aminotransferase levels were not related to the presence of TTV DNA. TTV was more frequent in patients 50 to 70 (12/51) years of age and persisted in the serum of six of nine patients 8 months later. TTV was found in peripheral blood mononuclear cells of all patients with chronic infection, and sequence variation was noted in the peripheral blood mononuclear cells of two patients. No relationship between TTV infection and hepatitis was observed. Viral persistence and nonparenteral transmission may be possible in patients on hemodialysis.     

Genotypes of TT virus (TTV) compared between liver disease patients and healthy individuals using a new PCR system capable of differentiating 1a and 1b types from others*

Summary.  TT virus (TTV) lacks obvious pathogenicity; almost all of the infected hosts are symptom-free. A possibility remains, however, that certain strains can cause liver disease while most others are non-pathogenic. Genotypes 1 a and 1 b have been proposed to contain such pathogenic strains. To test this possibility, we constructed a PCR system capable of detecting TTV of the 1 a and 1 b genotypes differentially from the other genotypes, and compared the frequencies of these genotypes between patients with liver disease of unknown etiology (n=42) and healthy individuals (n=50). The assay comprised 3 steps: i) PCR to amplify a 3.2-kb fragment using universal primers; ii) 2nd-round PCR, starting from the 3.2-kb amplicon, for a ∼280-nt fragment by use of genotype 1-specific primers; and iii) digestion of the ∼280-nt amplicon with MboI and BanI to discriminate between 1 a and 1 b. Eventually, 40 (95%) of the patients and 47 (94%) of the controls were positive for the 3.2-kb amplicon, and the 1 a, 1 b, 1 a+1 b, and non-1 genotypes of TTV were found in 2 (5%) vs 4 (9 %), 5 (13%) vs 4 (9%), 1 (3%) vs 1 (2%) and 32 (80%) vs 38 (81%) of the 40 patients and 47 controls, respectively: the distribution was almost identical between the two groups. The hypothesis that the genotype 1 of TTV may be more closely associated with liver disease than other genotypes was not supported by this study. Received June 11, 1999/Accepted July 25, 1999     

Anti-<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Antibodies are Frequent in Type 1 Diabetes

Anti-Saccharomyces cerevisiae antibodies (ASCA) have been described in many autoimmune diseases in which there is an increased intestinal permeability. Also in type 1 diabetes (T1D), there is an increased intestinal permeability. Since no data are available about ASCA in T1D, we evaluated, retrospectively, the frequency of ASCA in this disease. ASCA, IgG, and IgA, were determined by ELISA in sera of 224 T1D patients in which coeliac disease has been excluded and 157 healthy control group. The frequency of ASCA (IgG or IgA) was significantly higher in T1D patients than in the control group (24.5% vs. 2.5%, p < 10⁻⁷). The same observation was found in children and in adult patients when we compare them to healthy children and blood donors group respectively. Compared to children, adult patients with T1D showed significantly higher frequencies of ASCA of any isotype (38% vs. 13.7%, p < 10⁻⁴), both ASCA IgG and IgA (12% vs. 1.6%, p = 0.002), ASCA IgG (35% vs. 9.8%, p < 10⁻⁵) and ASCA IgA (15% vs. 5.6%, p = 0.001). The frequency of ASCA was statistically higher in females of all T1D than in males (30.8% vs.17.7%, p = 0.03), in girls than in boys (22% vs.6.2%, p = 0.017), and significantly higher in men than in boys (35.7% vs. 6.2%, p < 10⁻⁴). The frequency of ASCA IgG was significantly higher than that of ASCA IgA in all T1D patients (21% vs. 9.8%, p < 0.002), in all females (26.5% vs. 10.2%, p < 0.002), in women (37.9% vs. 12%, p < 0.001). The frequency of ASCA was significantly higher in all long-term T1D than in an inaugural T1D (29% vs. 14.5%, p = 0.019). The same observation was found in adults (45.8% vs. 17.8%, p = 0.01). In long-term T1D patients, ASCA were significantly more frequent in adults than children (45.8% vs. 14.5%, p < 10⁻⁴). The frequency of ASCA IgG was significantly higher in long-term T1D than in an inaugural T1D (25.2% vs. 11.6%, p = 0.03). Patients with T1D had a high frequency of ASCA.     

Prevalence, epidemiological aspects and clinical importance of TT virus infection in Slovakia

The aim of the study was to assess the prevalence, clinical impact and importance of different risk factors of transmission of TTV infection in Slovakia using two PCR methods. Sera of 426 adult persons were examined. TTV DNA was identified by PCR using primers from N22 and untranslated region (UTR) respectively. The established prevalence of TTV tested with N22 and UTR primers according to patients groups was: acute hepatitis of unknown etiology 4 resp. 28 of 37, acute hepatitis B 3 resp. 29 of 38, chronic hepatitis B 11 resp. 41 of 44, chronic hepatitis C 10 resp. 93 of 102, hemodialysis patients 13 resp. 72 of 72, health care workers 0 resp. 27 of 33, control group 8 resp. 83 of 100. Using N22 primers, TTV infection occurred more frequently in chronic hepatitis B group compared with health care workers, if UTR primers were used the group of hemodialysis patients differed significantly from both acute hepatitis groups, health care workers and controls (p < 0.05). From possible risk factors hemodialysis and transfusion count showed notable differences. Bilirubin and aminotransferase levels did not differ between TTV positive and negative groups. No pathogenetic role of TT virus in liver injury was confirmed.     

Fecal excretion of a nonenveloped DNA virus (TTV) associated with posttransfusion non-A-G hepatitis

Five patients with type B or C hepatocellular carcinoma were found to be infected with a nonenveloped DNA virus (TTV) associated with posttransfusion hepatitis of non-A–G etiology. Paired feces and serum samples from these patients were tested for TTV DNA by polymerase chain reaction with seminested primers and their sequences were compared. TTV DNA was detected in sera from all of the patients, while it was detected in feces from three patients, including two with high viral titers in serum. When feces and serum from one patient were subjected to floatation ultracentrifugation in CsCl, TTV in feces banded at a peak density of 1.35 g/cm³ and that in serum at 1.31–1.32 g/cm³. TTV isolates in three pairs of feces and serum had the identical sequence of 222 base pairs. The excretion of TTV into feces indicates that TTV would be transmitted not only parenterally but also nonparenterally by a fecal–oral route. J. Med. Virol. 56:128–132, 1998. © 1998 Wiley-Liss, Inc.     

Simultaneous detection of human bocavirus and adenovirus by multiplex real-time PCR in a Belgian paediatric population

Since the discovery of human bocavirus (hBoV), the virus has been detected worldwide in respiratory tract samples from young children by various polymerase chain reaction (PCR) assays and real-time PCRs (Q-PCR). Until now, no data have been reported on the presence of hBoV in Belgium and the detection of hBoV in a multiplex Q-PCR setting has not been described. The aim of this study was to develop a fast and reliable multiplex Q-PCR for the simultaneous detection of hBoV DNA and adenovirus (AdV) DNA. During the winter of 2004–2005, 445 nasopharyngeal aspirates (NPAs) were analysed from 404 Belgian children up to 5 years old with acute respiratory tract infections (ARTIs). (Co)infections with hBoV, AdV, respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and influenza A virus were investigated. A viral agent was detected in 61% (n = 272/445) of the NPAs. Multiplex Q-PCR found a prevalence of 11% (n = 51/445) hBoV and 13% (n = 58/445) AdV. Coinfections were more frequently found with AdV (62%; n = 36/58) than with hBoV (49%; n = 25/51). Follow-up samples were available from 22 patients with ARTIs. In three patients, hBoV DNA persisted for one month. Multiplex Q-PCR may help in closing the diagnostic gap by addressing a broader range of potential respiratory pathogens.