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     

Real-time PCR methods for independent quantitation of TTV and TLMV

There is considerable interest in the possible clinical effects of the human circoviruses TT virus (TTV) and TTV-like mini virus (TLMV). Most people appear to have at least one of these viruses replicating actively in their bodies, thus mere correlation of the presence of virus and disease states are probably less informative than a quantitative analysis of viraemia. Real-time PCR based methods, with either SYBR Green or TaqMan probe, designed to quantitate selectively TTV and TLMV are described. The suggested TaqMan-based protocols were suitable for quantitation of viruses in the range of 10(2)-10(9) copies/ml of sample; and proved, by sequencing of PCR products, to be specific for each of the two viruses.     

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.     

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     

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.     

Detection and genotyping of TT virus in healthy and subjects with HBV or HCV in different populations in the United Arab Emirates

TT virus (TTV) and TTV-like viruses (TTVLs) have been reported to be associated with non-A-E hepatitis. To determine the rate of infection and genotypic characteristics of TTV in the United Arab Emirates (UAE), a total of 449 serum samples representing different populations in the UAE and comprising healthy as well as patients positive for HBsAg and HCV were screened. National subjects (n = 200) and non-nationals residing in the UAE (n = 249) were tested by PCR. The results obtained showed that the rate of TTV infection in healthy nationals, and those with HBsAg or antibody to HCV were 34.9, 97.9, and 95.7, respectively, compared to 89.1% (115/129), 89.2% (66/74), and 84.8% (39/46), respectively, in non-nationals. Sequence analysis of the untranslated region (UTR) using 71 clones generated from the PCR products of eight serum samples from healthy individuals (four nationals and four non-nationals) showed that 83.1% of the TTV clones were classified into groups 1-4, whereas 16.9% into possibly new genotype(s). The analysis also revealed that healthy national subjects carried multiple viruses. Phylogenetic analysis of representative sequences revealed clustering of clones into at least five major groups. Also, when compared to reference genotypes (from GenBank), two of our clones belonged to two previously identified genotypes. Non-significant gender differences were seen in all ethnic groups studied (P > 0.05). In conclusion, the rate of TTV infection in the UAE nationals is significantly lower (P < 0.05) than that of the non-nationals and several genotypes were isolated with common multi-infections.     

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。     

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.     

High prevalence of TT virus (TTV) and TTV-like minivirus in cervical swabs

Genomes of TT virus (TTV) and TTV-like minivirus DNA were detected in 80% and 61% of cervical swabs from healthy women, respectively, regardless of concurrent human papillomavirus infection. These results show that the potential exists for sexual transmission of TTV and related viruses.     

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.     

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.     

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,     

Full-length nucleotide sequence of a simian TT virus isolate obtained from a chimpanzee: evidence for a new TT virus-like species

Recently, we identified TT virus (TTV) isolates from nonhuman primates and named them simian TTV (s-TTV). To characterize the genomic structure of these isolates in more detail, the full-length nucleotide sequence of the s-TTV isolate (designated s-TTV CH65-1), recovered from a chimpanzee born in West Africa, was amplified by nested PCR with inverted primers deduced from the untranslated region of s-TTV DNA. CH65-1 was composed of 3899 nucleotides (nt) and had two open reading frames (ORF) spanning 2295 nt (ORF1) and 402 nt (ORF2). The sequence had only 52.3% similarity to the prototype TA278 human isolate. Phylogenetic analysis demonstrated that CH65-1 was distinct from the human TTV isolates. These results suggested that s-TTV may represent a new TTV-like viral species or genus.     

Related TT viruses in chimpanzees

A series of serum specimens obtained from two chimpanzees experimentally infected with hepatitis A virus (HAV), hepatitis C virus, and hepatitis G/GB-C virus were tested for TT virus (TTV) by polymerase chain reaction (PCR). All PCR fragments obtained from both animals were directly sequenced, and the nucleotide sequences were compared to each other and to all known TTV sequences. This comparison showed that both animals were infected simultaneously with four new TTV variants designated A, M1, M2, and M3. One chimpanzee was found to be infected with TTV only after HAV inoculation, whereas the other animal was infected with TTV before any experimental procedure was performed. A set of PCR primers specific for these four new TTV variants was used to amplify TTV-like sequences from nine naive chimpanzees. None of these animals was infected with the prototype TTV variant. Two of these animals, however, were infected with one of the new TTV variants, while one animal was infected with an additional new TTV variant designated T. Among 99 hepatitis patients, 29 were found to be infected with the prototype TTV variant. None of these human specimens was found to be positive by PCR specific for TTV variants A, M1, M2, and M3. Similarly, not a single specimen from a smaller subset of human serum samples was found to be positive for the TTV variant T. Phylogenetic analysis performed on all known TTV sequences demonstrated that TTV can be classified into 13 different, yet closely related TTV species, designated as TTV-I for the prototype variant through TTV-XIII. The new variants M1 and M2 were classified as two different genotypes of TTV-VI, variant M3 was classified as TTV-VII, variant A was classified as TTV-VIII, and variant T was classified as TTV-IX. Thus, the data obtained in this study suggest that TTV represents a large swarm of TTV-like species, some of which have not been detected in humans and circulate predominantly among chimpanzees.     

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.     

Distribution of TT virus genomic groups 1-5 in Brazilian blood donors, HBV carriers, and HIV-1-infected patients

Human isolates of the highly prevalent TT virus (TTV) have been classified into five major genomic groups (1-5). The geographical distribution of the groups throughout the world is not well known. Five different PCR assays were developed in an attempt to amplify specifically TTV DNAs of each genomic group. Serum samples collected from 72 Brazilian adults (24 voluntary blood donors, 24 hepatitis B virus (HBV) carriers, and 24 human immunodeficiency virus type 1 (HIV-1)-infected patients) were tested. TTV DNA from at least one genomic group was detected in 11 (46%) blood donors, 13 (54%) HBV carriers, and 24 (100%) HIV-1 patients. All five genomic groups were detected in the three populations, with the exception of group 2 in blood donors. Some samples, negative with all five specific assays, were positive with the commonly used untranslated region (UTR) PCR system. On the other hand, TTV DNA was detected in some samples by using specific assays but not with the UTR PCR. Mixed infections with 2-5 TTV isolates from different groups were detected in 21% blood donors, 29% HBV carriers, and 71% HIV-1 patients. Fifteen PCR products (three obtained with each assay) were sequenced. Most sequences showed high (>86%) homology with those of TTV isolates belonging to their presumed groups. However, three sequences had low homology with all TTV sequences available from the DNA databanks. In conclusion, TTV isolates belonging to all five known genomic groups circulate in Brazil, and the results suggest the existence of new and as yet uncharacterised major genomic groups.     

TTV, a new human virus with single stranded circular DNA genome

TT virus (TTV) was found in 1997 from a hepatitis patient without virus markers. However, the real impact of TTV on liver diseases remains uncertain to date. Due to the lack of suitable cell systems to support the growth of TTV, the biology of TTV is still obscure. This review tries to summarise the current status of TTV on aspects other than the taxonomic diversity of TTV. TTV was the first human virus with a single stranded circular DNA genome. TTV was considered to be a member of Circoviridae, but others suggested it conformed to a new family. TTV is distinct from ambisense viruses in the genus Circovirus, since the former genome is negative stranded. The genome structure of TTV is more related to chicken anaemia virus in the genus Gyrovirus, however, the sequence similarity is minimal except for a short stretch at 3816-3851 of TA278. Currently the working group is proposing the full name for TTV as TorqueTenoVirus and the TTV-like mini virus as TorqueTenoMiniVirus (TTMV) in a new genus Anellovirus (ring). TTVs are prevalent in non-human primates and human TTV can cross-infect chimpanzees. Furthermore, TTV sequences have been detected in chickens, pigs, cows and sheep. TTV can be transmitted by mother-to-child infection. However, within a year after birth, the prevalence reaches the same level for children born to both TTV-positive and TTV-negative mothers even without breast-feeding. The non-coding region surrounding a short 113 nt GC-rich stretch and occupying approximately one-third of the genome is considered to contain the putative replication origin. Three mRNAs are expressed by TTV, 3.0 and 1.2 and 1.0 kb species. A protein translated from the 3.0 kb mRNA is considered to be the major capsid protein as well as replicase. The nature of the proteins translated by the other two mRNAs are still putative.     

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.