Distribution of TT virus (TTV), TTV-like minivirus,and related viruses in humans and nonhuman primates

TT virus (TTV) and TTV-like minivirus (TLMV) are small DNA viruses with single-stranded, closed circular, antisense genomes infecting man. Despite their extreme sequence heterogeneity (>50%), a highly conserved region in the untranslated region (UTR) allows both viruses to be amplified by polymerase chain reaction (PCR). TTV/TLMV infection was detected in 88 of 100 human plasma samples; amplified sequences were differentiated into TTV and TLMV by analysis of melting profiles, showing that both viruses were similarly prevalent. PCR with UTR primers also detected frequent infection with TTV/TLMV-related viruses in a wide range of apes (chimpanzees, gorillas, orangutans, gibbons) and African monkey species (mangabeys, drills, mandrills). These findings support the hypothesis for the co-evolution of TTV-like viruses with their hosts over the period of primate speciation, potentially analogous to the evolution of primate herpesviruses.     

Molecular epidemiology of TTV-like mini virus in Norway

Summary.  TT virus (TTV), the first human circovirus to be discovered, appears to be present in most people; less is known about the prevalence of the related TTV-like mini virus (TLMV). A sensitive nested PCR, specific for TLMV, detected the virus in 48% of 201 sera (Norwegian blood donors) previously found to have a 90% prevalence of TTV. More samples were either positive for both or negative for both viruses than what would have been expected from a random distribution (p = 0.08). Sequence analysis revealed considerable heterogeneity of Norwegian TLMV as compared to international sequences, suggesting that TLMV is efficiently dispersed in human populations. June 27, 2001 October 11, 2001     

Effect of immune modulation on TT virus (TTV) and TTV-like-mini-virus (TLMV) viremia

The present study was designed to investigate how two chronically replicating viruses, TT virus (TTV) and TTV-like mini virus (TLMV), interact with host defence systems. Successive serum samples from three groups of subjects, undergoing modifications of their antiviral defence, were tested by real-time PCR to measure changes in viral titers, and by sequence analyses to indicate whether increases in viremia could be attributed to infection with an unfamiliar strain: 1) in patients receiving immunosuppressants subsequent to kidney transplantation, viral titers tended to increase; 2) in soldiers undergoing extreme training known to cause immunosuppression, insignificant increases in titers were observed; and 3) interferon treatment of patients with hepatitis C virus caused a temporary decrease in TTV and TLMV titers. Increases in viremia were associated only occasionally with the appearance of novel strains. The above results add to knowledge on how these viruses are influenced by the host.     

High detection rates of TTV-like mini virus sequences in sera from Brazilian blood donors

TT virus (TTV) is an unenveloped virus with a single-stranded, circular DNA genome of 3,818-3,853 nucleotides (nt) that infects humans and non-human primates. Recently, the existence of a novel human virus, TTV-like mini virus (TLMV), that shows a genetic organization similar to that of TTV, but with smaller virion particle and genome, was proposed [Takahashi et al. (2000) Archives of Virology 145:979-993]. To date, no information is available with respect to the prevalence and pathogenicity of TLMV. A sensitive PCR assay was developed by using two oligonucleotide primers (LS2 and LA2) designed from the conserved non-coding region of the TLMV genome. One hundred thirty-seven sera from volunteer Brazilian blood donors were tested and 99 (72%) were TLMV DNA positive. No significant differences were observed between the groups of TLMV positive and negative subjects in relation to sex ratio, seroprevalence of TTV DNA, prevalence of anti-hepatitis A virus antibodies, area of residence, occurrence of daily contact with animals, family income, education level, and level of alanine aminotransferase. The specificity of the PCR assay was demonstrated after cloning of amplification products and determination of the nucleotide sequences (200-228 nt) of clones derived from 23 individuals. When DNAs extracted from TLMV/TTV-coinfected sera were submitted to PCR with LS2 and LA2 primers, the amplification products were derived exclusively from the TLMV genome. A markedly wide range of sequence divergence, even higher than that existent among TTV strains, was noted among TLMV isolates, with a maximum evolutionary distance of 0.80.     

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     

TLMV5‘非编码区基因的克隆与序列分析

目的 调查ＴＴＶ阴性的非甲￣庚型肝炎患者中ＴＴＶ－ｌｉｋｅ ｍｉｎｉ ｖｉｒｕｓ（ＴＬＭＶ）感染情况,对ＴＬＭＶ５’非编码区（５’ＮＣＲ）部分基因进行分子克隆与序列分析。方法 采用巢式ＰＣＲ技术检测５３例ＴＴＶ阴性的非甲－庚肝炎患者血清ＴＬＭＶ ＤＮＡ,对ＰＣＲ产物进行克隆、测序和分析。结果 ５３例病例中ＴＬＭＶ ＤＮＡ阳性３７例（６９．８％）,对其中８株ＴＬＭＶ基因克隆测序,并与Ｔａｋａｈａｓｈ     

TLMV5＇非编码区基因的克隆与序列分析

目的　调查TTV阴性的非甲～庚型肝炎患者中TTV～like mini virus(TLMV)感染情况,对TLMV5＇非编码区(5＇NCR)部分基因进行分子克隆与序列分析。方法　采用巢式PCR技术检测53例T T V阴性的非甲～庚肝炎患者血清TLMV DNA,对PCR产物进行克隆、测序和分析。结果　53例病例中TLMV DNA阳性37例(69.8％),对其中8株TLMV基因克隆测序,并与Takahashi报道的TLMV序列(GenBank Accession No ab 026930、ab026931)比较,其核苷酸序列同源性在64％～83％之间。结论　在T TV阴性的非甲～庚肝炎患者中存在TLMV感染。TLMV5＇NCR基因变异性较大。TLMV的致病性及其与非甲～庚肝炎的关系尚有待进一步研究。     

Progression towards AIDS leads to increased Torque teno virus and Torque teno minivirus titers in tissues of HIV infected individuals

Torque teno virus (TTV) and Torque teno minivirus (TTMV) are highly prevalent in the general population and although no disease has been associated with these viruses yet, co-infections with other pathological viruses are frequent. Both viruses are extremely heterogeneous, especially for DNA viruses, and the role of the immune system in controlling the infections has yet to be established. In this study the TTV/TTMV viral loads in HIV positive tissues have been investigated for the first time. The titers of both TTV and TTMV were compared in the bone marrow and spleen tissues from three groups: HIV negative individuals, HIV positive individuals and HIV positive individuals who had progressed to AIDS, leading to immunosuppression. Limiting dilution PCR using primers situated in the UTR region of the genome were used to semi-quantitate the virus, and TTV and TTMV were differentiated using melting curve analysis of the PCR product. The AIDS group had significantly higher titers compared with both the HIV positive and negative groups for both bone marrow (AIDS vs. HIV positive P = 0.006, AIDS vs. HIV negative P < 0.001) and spleen (AIDS vs. HIV positive P = 0.022, AIDS vs. HIV negative P < 0.001). Analysis of TTV/TTMV titer with CD4 T lymphocyte count showed a significant inverse correlation however neither HCV co-infection or type of Anellovirus infection (single TTV or TTMV, or mixed TTV/TTMV) showed any significant correlation with virus titer. The results show a link between deterioration of the immune system and increased the viral loads in studied tissues.     

Detection of porcine anelloviruses in pork meat and human faeces

Torque teno viruses (TTV) are icosahedral, single-stranded circular DNA viruses infecting several vertebrate species. Currently, these viruses are considered non-pathogenic although they are suggested to be co-factors in several diseases. Recently single-stranded circular DNA viruses have been found in human faeces. Considering the consumption of pork meat products and the ubiquitous nature of swine TTV (Torque tenosus virus, TTSuV), the human population is frequently exposed to these viruses. To determine if TTSuVs could be delivered through food, human faecal samples were analysed for their presence. Indeed, the results of this study show that up to 25% of faecal samples were positive for known TTSuVs by PCR and sequencing. Additionally, all commercially available pork products purchased in Spanish supermarkets contained DNA of TTSuV.     

Frequent detection of the replicative form of TT virus DNA in peripheral blood mononuclear cells and bone marrow cells in cancer patients.

The TT virus (TTV), a member of a family of human viruses related to the circoviridae viruses, was associated initially with acute and chronic liver diseases. TTV consists of a single-stranded, circular DNA genome of 3.8 kilobases (kb) and at least three open reading frames (ORFs). The objective of the present study was to determine whether or not TTV replicated in peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs). DNA was extracted from the PBMCs or BMCs of 153 cancer patients and from the PBMCs of 50 healthy blood donors (the controls). By using a single round of polymerase chain reaction (PCR), TTV was detected in 98.6% (141 of 143) of the PBMCs and in 90% (9 of 10) of the BMCs from cancer patients. TTV DNA was detected in significantly fewer control subjects at 86% (43 of 50; P < 0.05). Strand-specific PCR (SSPCR) targeting the ORF2 of the common genotypes of TTV was developed specifically to detect TTV positive or negative strand DNA and to examine TTV replication. TTV positive strand DNA, which may be an intermediate of viral replication, was detected in 55.3% (78 of 141) of the TTV-infected PBMCs of the cancer patients and in 7% (3 of 43) of the controls (P < 0.001). The replicative form of TTV was also detectable in 55.6% (5 of 9) of the TTV-infected BMCs. The existence of double-strand (positive and negative strands) TTV DNA in PBMCs and BMCs of the cancer patients was also supported by the finding that TTV DNA extracted from these cells was resistant to S1 nuclease. Using in situ hybridization, TTV DNA was also demonstrated to be present in the nucleus of PBMCs. It is concluded that replicative intermediate forms of TTV DNA are present in both PBMCs and BMCs, indicating that blood cells may be a site of TTV replication.     

Development of PCR assays with nested primers specific for differential detection of three human anelloviruses and early acquisition of dual or triple infection during infancy

We recently identified a novel human virus classifiable into a third group in the genus Anellovirus, tentatively designated torque teno midi virus (TTMDV), with a circular DNA genome of 3.2 kb and genomic organization resembling those of torque teno virus (TTV) (3.8 to 3.9 kb) and torque teno mini virus (TTMV) (2.8 to 2.9 kb). TTMDV was characterized by extreme genetic diversity similar to the TTV and TTMV genomes. Taking advantage of universal and virus species-specific primers derived from a highly conserved area located just downstream of the TATA box of the TTV, TTMDV, and TTMV genomes, a PCR method with simultaneous amplification of the genomic DNAs of these three anelloviruses in the first round and subsequent differential amplifications of these viruses in the second round was developed. High prevalence of TTMDV viremia was seen in adults (75/100 [75%]), comparable with the prevalences of TTV viremia (100%) and TTMV viremia (82%). Although none of 10 cord blood samples had detectable TTV, TTMDV, and TTMV DNAs, the prevalences of these three anelloviruses increased with the number of months after birth of the individual and reached 100% for individuals at one year of age. Dual or triple infection of TTV, TTMDV, and/or TTMV was seen in 10 (47.6%) of 21 infants 9 to 180 days of age and more frequently among infants 181 to 364 days of age (20/23 [86.9%]), comparable with the 93.1% (243/261) prevalence among subjects 1 to 81 years of age, indicating early acquisition of dual or triple anellovirus infection during infancy.     

GB virus C/hepatitis G virus and TT virus infections among high risk renal transplant recipients in India.

BACKGROUND: GB virus C/hepatitis G virus (GBV-C/HGV) and TT virus (TTV) have been widely reported in patients with high parenteral risk such as haemodialysis and renal transplant recipients. The occurrence of these agents in association with hepatitis B virus (HBV) and hepatitis C virus (HCV), in Indian renal transplant recipients, is yet unreported. STUDY DESIGN: Molecular and serological markers of GBV-C/HGV and TTV were examined in addition to those for HBV, HCV and hepatitis D virus (HDV) in a selected group of seventy renal transplant recipients. HGV RNA detection was achieved using primers specific for the 5'NCR and NS5a regions of the genome. Anti-GBV-C/HGV antibody was detected using the mu plate anti-HG env kit (Roche, Germany). TTV DNA PCR was performed using primers specific for the coding region (method A) of the genome. In 50% of patients, TTV DNA was also tested for using primers specific for the non-coding region (method B). Host related factors such as age, alanine aminotransferase (ALT) levels, number of transfusions, haemodialysis sessions, and months following transplantation were also studied. RESULTS: Exposure rates to GBV-C/HGV, TTV (method A), HBV, HCV and HDV were 58.6, 32.9, 52.9, 54.3 and 2.9%, respectively. 'Active' infection as measured by viraemia and/or virus-specific antigenaemia for GBV-C/HGV, TTV, HBV and HCV was 52.9, 32.9, 15.7 and 52.9%, respectively. The majority of GBV-C/HGV and TTV infections were seen as co-infections with other hepatitis viruses. Single infection with GBV-C/HGV and TTV was seen in ten (14.2%) and eight (11.4%) patients, and was not associated with ALT elevation when compared to uninfected blood donors. Using univariate analysis, GBV-C/HGV RNA was significantly associated with > or =20 haemodialysis sessions. TTV DNA occurrence was not associated with any risk factors. CONCLUSIONS: There is a high occurrence of GBV-C/HGV and TTV in this select group of renal transplant recipients in India. These viruses mostly occurred in the context of co-infections with other hepatitis viruses. Long term effects of multiple hepatotropic viral infections need to be carefully documented in such transplant populations.     

Molecular properties, biology, and clinical implications of TT virus, a recently identified widespread infectious agent of humans

TT virus (TTV) was first described in 1997 by representational difference analysis of sera from non-A to non-G posttransfusion hepatitis patients and hence intensively investigated as a possible addition to the list of hepatitis-inducing viruses. The TTV genome is a covalently closed single-stranded DNA of approximately 3.8 kb with a number of characteristics typical of animal circoviruses, especially the chicken anemia virus. TTV is genetically highly heterogeneous, which has led investigators to group isolates into numerous genotypes and subtypes and has limited the sensitivity of many PCR assays used for virus detection. The most remarkable feature of TTV is the extraordinarily high prevalence of chronic viremia in apparently healthy people, up to nearly 100% in some countries. The original hypothesis that it might be an important cause of cryptogenic hepatitis has not been borne out, although the possibility that it may produce liver damage under specific circumstances has not been excluded. The virus has not yet been etiologically linked to any other human disease. Thus, TTV should be considered an orphan virus.     

Mixed infections of adults and children with multiple TTV-like mini virus isolates

Testing of the DNA of TTV-like mini virus (TLMV) was done with serum samples obtained from 184 patients (children and adults) who visited different outpatient clinics at a university hospital in Florianopolis, south of Brazil. TLMV DNA was detected by PCR primers from the non-coding region of the genome. A global TLMV prevalence of 78% was found (94% among children below 11 years). PCR products from three serum samples (patients A-C) were cloned, and the sequences with a length of 201-227 nucleotides were determined for 16-19 clones derived from each of the sera. Among the 16 clones derived from patient C, 15 were identical, and the remaining one had a sequence homology of 99%. In contrast, eight different sequences were obtained among the 19 clones derived from patient A, and 10 distinct sequences were depicted among the 17 clones derived from the serum of patient B. Additionally, 13 clones derived from a saliva sample of patient B were sequenced, and seven different nucleotide sequences obtained. One particular sequence was predominant in both serum (8/17 clones) and saliva (7/13 clones) of patient B. On a phylogenetic tree, sequences derived from patient A (a 6-year-old boy), as well as those derived from patient B (a 24-year-old man), were located in five distinct evolutionary branches, taking a minimum divergence of 5% between branches. This suggested that adults and children are coinfected frequently with several TLMV isolates of different origins.     

Phylogenetically diverse TT virus viremia among pregnant women

Infections during pregnancy have been suggested to be involved in childhood leukemias. We used high-throughput sequencing to describe the viruses most readily detectable in serum samples of pregnant women. Serum DNA of 112 mothers to leukemic children was amplified using whole genome amplification. Sequencing identified one TT virus (TTV) isolate belonging to a known type and two putatively new TTVs. For 22 mothers, we also performed TTV amplification by general primer PCR before sequencing. This detected 39 TTVs, two of which were identical to the TTVs found after whole genome amplification.Altogether, we found 40 TTV isolates, 29 of which were putatively new types (similarities ranging from 89% to 69%). In conclusion, high throughput sequencing is useful to describe the known or unknown viruses that are present in serum samples of pregnant women.     

TT Virus as a Human Pathogen: Significance and Problems

In 1997 TTV was detected using representational difference analysis (RDA) in serum of a patient with posttransfusion hepatitis unrelated to known hepatitis viruses. The genome of TTV is a circular single-stranded DNA molecule of 3852 nt with negative polarity. TTV possibly can be grouped either into the existing family Circoviridae or into a recently established virus family Circinoviridae. Analysis of the complete DNA nucleotide sequence of TTV identified three partially overlapping open reading frames (ORFs). Neither DNA nucleotide nor corresponding amino acid sequences of TTV do show significant homologies to known sequences. TTV DNA nucleotide sequences amplified by PCR from sera of different patients show considerable sequence variations. Although the natural route of transmission of TTV is still unknown, there is clear evidence for a transmission of TTV through blood and blood products. TTV DNA can be detected in the feces of infected individuals suggesting that it may be possible to attract TTV infection from environmental sources. Since the discovery of TTV, numerous studies have investigated the prevalence of TTV infections in different human population groups all over the world. All these studies are based on PCR detection systems, but the technical aspects of the PCR systems vary significantly between the different investigators. The results of the epidemiological studies do not show a clear picture. The discovery of TTV as a viral agent and particularly the identification of a high percentage of infected carriers in the healthy human population raises the following questions: Firstly, what is the origin and molecular relatedness of TT virus. Secondly, what is the significance of TTV as a human pathogen. And thirdly, what are the exact molecular mechanisms of viral replication. To answer these questions it will be necessary to determine the primary structure and the coding capacity of several TTV patient isolates.     

High genetic diversity revealed by the study of TLMV infection in French hemodialysis patients

TT virus-like minivirus (TLMV) was recently discovered as a human circovirus. Little is known about its natural history and molecular epidemiology. A study of TLMV infection is described in a population of French hemodialysis patients. TLMV DNA was tested by seminested PCR system located in the noncoding region in 81 patients divided into seven groups according to the origin of their renal disease. Quantitation of TLMV DNA in serum was carried out. Sequences from 28 patients were compared with 40 sequences retrieved from databases and 53 TLMV sequences cloned from the serum of a single patient. The prevalence of TLMV DNA in hemodialysis patients was 95.1%. In this study, 24 samples (29.6%) presented viral loads of > 125 equivalents of plasmid (Ep)/ml, and only 6 (7.8%) had viral loads of > 125 x 10(2) Ep/ml. A significant correlation (P < 0.029) was found between viral loads of > 125 x 10(2) Ep/ml and the neoplastic origin of end-stage renal disease. Analysis of 53 sequences cloned from a single individual demonstrated high sequence variability, as shown by the genetic distance of 40.2%. This genetic distance is comparable to that between the most divergent sequences of TLMV reported to date (43.5%). These data suggest that TLMV viral load is possibly related to the level of immunocompetence of hemodialysis patients; the genetic diversity of TLMV is extremely high; and co-infection by different strains is possible.     

TT病毒在谷丙转氨酶升高的体检者和肝病患者中检 …

目的 通过研究ＴＴ病毒在谷丙转氨酶升高的体检人群和肝病患者中的意义。探讨其致病性。方法 收集１９例谷丙转氨酶升高体验者和４１例转氨酶正常的随机对照的血清,以及１８２例肝病患者的血清,采用ＰＣＲ方法检测ＴＴ病毒的ＤＮＡ。聚合酶链反应（ＰＣＲ）产物经限制性片段长度多态性（ＲＦＬＰ）分析验证。同时检测甲,乙,丙,戊,庚型肝炎病毒（ＨＡＶ,ＨＢＶ,ＨＣＶ和ＨＧＶ）感染标志。结果 １９例转氨酶升高体检者中,     

TT Virus Infection in French Hemodialysis Patients: Study of Prevalence and Risk Factors

The TT virus (TTV) is a recently discovered DNA virus which was first identified in patients with non-A to -G hepatitis following blood transfusion. In this study, we tested 150 attendees of two hemodialysis (HD) units of the public hospitals of Marseilles, France, for the presence of TTV genome by using a PCR-based methodology. The overall prevalence of TTV viremia was 28% (compared to 5.3% in blood donors from the same region). We demonstrated the existence of chronic infections and superinfections by strains belonging to different genotypes. The prevalence of infection was higher in patients originating from Africa, in patients with previous blood transfusion or organ transplantation, in patients with antibody to hepatitis B core antigen, and in those with diabetes mellitus. A high prevalence of TTV infection (50%) was also observed in a population of patients with diabetes mellitus but without renal disease. No significant relationship was found between TTV viremia and hepatitis C virus or GB virus C, transaminases, age, sex, and duration of HD treatment. The PCR amplification products (located in open reading frame 1 of the TTV genome) were sequenced. These genomic sequences were submitted to phylogenetic analysis by using the Jukes-Cantor algorithm for distance determination and the neighbor-joining method for tree building. In several instances, sequences from viruses isolated in a HD unit were grouped in the same phylogenetic cluster. These results together with the different distribution of cases in the two HD units suggest there is viral transmission within each.