Detecting recombination in TT virus: a phylogenetic approach

TT virus (TTV) has a remarkable genetic heterogeneity. To study TTV evolution, phylogenetic analyses were performed on 739 DNA sequences mapping in the N22 region of ORF1. Analysis of neighbor-joining consensus trees shows significant differences between DNA and protein phylogeny. Median joining networks phylogenetic clustering indicates that DNA sequence analysis is biased by homoplasy (i.e., genetic variability not originated by descent), indicative of either hypermutation or recombination. Statistical analysis shows that the significant excess of homoplasy is due to frequent recombination among closely related strains. Recombination events imply that the transmission of TTV is not clonal and provide the necessary basis to explain (i) the high degree of genetic divergence between TTV isolates, (ii) the lack of population structure on a world scale, and (iii) the number of highly divergent strains that seems typical of this virus. We show that recombination phenomena can be detected by phylogenetic analyses in very short sequences when a sufficiently large data set is available.     

PCR—微孔板杂交法检测不同人群TTV DNA

根据报道的TTV全序列设计引物和探针,建立PCR－微孔板杂交法,检测81例正常人群、92例职业献血员123例甲－庚型肝炎、32例非甲－庚型肝炎、48型发性肝癌患者的TTV DNA。结果表明TTV在以上五种人群中的阳性率分别为3．7％、4．3％、21．1％、28．1％、52．0％,前者与后三者比较有显著性差异（P＜0．05）,TTV合并HBV二重感染重叠感染的54．0％,这揭示不同人群均存在TTV感染,正常人群和职业献血员存在健康携带状态,甲－庚型肝炎和非甲－庚肝炎病人为高危人群,TTV可与各型肝炎存在重叠感染,TTV除经血传播外,存在其它传播途径,TTV感染与ALT及TBIL的升高密切相关。     

New DNA viruses identified in patients with acute viral infection syndrome

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.     

Prevalence of TT virus in patients with hepatitis B,C and hepatitis of unknown etiology

Discovery of TT virus in 1997 gave raise to intensive subsequent studies to learn about its structure, features and, what is the most important, about its role in pathogenesis of liver disease. The aim of the work was to analyze prevalence of TTV DNA in patients with diagnosed hepatitis B, C, that of unknown etiology and in healthy blood donors as well. Additionally the divergence of TTV sequence was estimated in selected cases. TTV DNA was detected by PCR technique using specific oligonucleotide primers for coding regions. TT virus has been detected in 25.6% (32/125) HBsAg positive patients and in 23.9% (51/213) HCV infected patients. In healthy blood donors the frequency of TTV was 24.3% (34/140) similarly to that found in HCV and HBV infected patients. The frequency of TTV DNA among patients with hepatitis of unknown etiology was 9.1%. This result was statistically significant lower than in the other groups. When detected sequences have been compared to these from NCBI base the homology result was 71% to 95%, and among different patients and groups of patients identity was 46% to 73%. On the basis of the obtained results it can be concluded that it is very unlikely that TTV coinfection plays any significant role in HCV or HBV infection. The hypothetical role of TTV infection in the etiopathogenesis of cryptogenic chronic hepatitis has not been confirmed. The results obtained in the small group of patients with hepatitis of unknown etiology are not conclusive and should be taken with some precaution. The final conclusion is the TTV coinfection does not contribute to the liver pathology. The divergence of TTV sequences may explain the various frequency of TTV viremia reported by other authors.     

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.     

Distinct Modes of Transmission of Tuberculosis in Aboriginal and Non-Aboriginal Populations in Taiwan

Tuberculosis incidence among aborigines is significantly higher than for Han Chinese in Taiwan, but the extent to which Mycobacterium tuberculosis (MTB) strain characteristics contribute to this difference is not well understood. MTB isolates from aborigines and Han Chinese living in eastern and southern Taiwan, the major regions of aborigines, were analyzed by spoligotyping and 24-loci MIRU-VNTR. In eastern Taiwan, 60% of aboriginal patients were ≤20 years old, significantly younger than the non-aboriginal patients there; aborigines were more likely to have clustered MTB isolates than Han Chinese (odds ratio (OR) = 5.98, p<0.0001). MTB lineages with high clustering were EAI (54.9%) among southern people, and Beijing (62.5%) and Haarlem (52.9%) among eastern aborigines. Resistance to first-line drugs and multidrug resistance (MDR) were significantly higher among eastern aborigines (≥15%) than in any other geographic and ethnic group (p<0.05); MDR was detected in 5 of 28 eastern aboriginal patients ≤20 years old. Among patients from the eastern region, clustered strains (p = 0.01) and aboriginal ethnicity (p = 0.04) were independent risk factors for MDR. The lifestyles of aborigines in eastern Taiwan may explain why the percentage of infected aborigines is much higher than for their Han Chinese counterparts. The significantly higher percentage of the MDR-MTB strains in the aboriginal population warrants close attention to control policy and vaccination strategy.     

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

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.     

Detection of TTV in peripheral blood cells from patients with altered ALT and AST levels

This work analyzes the prevalence of TTV DNA in peripheral blood cells from patients with hepatic alterations and healthy blood donors and measures levels of sodium, potassium, urea, creatinine, phosphatase alkaline, total and direct bilirubin, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in certain randomly selected patients. DNA samples from 111 individuals were evaluated. They were divided into two groups, "A" (study) and "B" (control), including 54 patients with liver enzyme alterations (ALT/AST) presenting non-B-non-C hepatitis and 57 blood donors, respectively. TTV DNA was determined by nested PCR. Certain products of the second-round PCR were sequenced. Serum biochemical assay was performed and disclosed TTV in 31.48% (17/54) of patients in group A and 5.26% (3/57) in the control group B. TTV prevalence was significantly higher in patients with liver disease than in healthy donors. In group A, sodium, potassium, urea, creatinine, phosphatase alkaline, total and direct bilirubin, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analyzed in certain randomly selected patients and no significant difference in biochemical levels (p>0.05) was found when TTV infected and noninfected individuals were compared. Knowledge related to TTV has rapidly increased, but many fundamental aspects remain unclear. This led us to question the role of TTV and doubt remains as to whether or not it is just a commensal virus. Further studies are necessary to confirm and extend these findings.     

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.     

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.     

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.     

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.     

Co-infections with hepatitis G and TT virus in patients with chronic hepatitis C in Hungary

The significance of co-infections with novel hepatitis viruses Hepatitis G (GBV-C, HGV) and TT virus (TTV) in chronic hepatitis C is not clear. We determined the prevalence of HGV RNA and TTV DNA in chronic hepatitis C patients and in asymptomatic hepatitis C virus (HCV) carriers, and assessed the influence of these agents on the course of HCV infection. Seventy-seven patients with chronic hepatitis C--50 of them treated with interferon (IFN)--and 33 HCV carriers with normal alanine aminotransferase have been investigated. Previous HBV infection was detected by testing serum HBsAg and aHBc. HGV RNA and TTV DNA were detected by PCR. In the healthy population, the prevalence of anti-HCV was 0.3%, HGV RNA 8.0% and TTV DNA 18.5%. In chronic hepatitis C HGV RNA occurred in 9.09% and TTV DNA in 40.25% of cases. In IFN-treated patients with sustained remission, the frequency of TTV was 20% vs. 45.7% found in non-responders. Among asymptomatic HCV-carriers, the prevalence of HGV RNA was 9.09% and TTV DNA 75.7%. Neither HGV RNA nor TTV DNA had apparent effect on the HCV infection. TTV was detected with the lowest frequency in persons with sustained remission due to IFN, suggesting antiviral effect of IFN on TTV.     

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.     

Detection and partial characterization of simian immunodeficiency virus SIVsm strains from bush meat samples from rural Sierra Leone

Human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that naturally infect sooty mangabeys (SMs; Cercocebus atys). In order to further investigate the relationship between HIV-2 and SIVsm, the SIV specific to the SM, we characterized seven new SIVsm strains from SMs sold in Sierra Leone markets as bush meat. The gag, pol, and env sequences showed that, while the viruses of all seven SMs belonged to the SIVsm-HIV-2 lineage, they were highly divergent viruses, in spite of the fact that most of the samples originated from the same geographical region. They clustered in three lineages, two of which have been previously reported. Two of the new SIVsm strains clustered differently in gag and env phylogenetic trees, suggesting SIVsm recombination that had occurred in the past. In spite of the fact that our study doubles the number of known SIVsm strains from wild SMs, none of the simian strains were close to the groups in which HIV-2 was epidemic (groups A and B).     

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).     

Multiple infection of TT virus (TTV) with different genotypes in Japanese hemophiliacs

To clarify the persistent TT virus (TTV) infection, we studied a possibility of multiple TTV infection by genotype analysis of isolated TTV obtained from seven Japanese hemophiliacs. The nucleotide sequences including 222 bp in the open reading frame 1 (ORF1) region of 10 TTV isolates from each patient were analyzed and classified into various TTV genotypes such as G1 to G6 by phylogenetic analysis using a N-J method. Multiple TTV genotypes were observed in all the hemophiliacs: three different TTV genotypes were found in three patients, whereas four different TTV genotypes were observed in the other three patients. The remaining patient was also infected with TTV of five different genotypes. Moreover, new TTV genotypes were found in these seven patients and tentatively designated as G7. The present findings indicate that multiple TTV infection with different TTV genotypes has occurred in Japanese hemophiliacs. They also provide valuable information to understand persistent TTV infection.     

Molecular epidemiology of 58 new African human T-cell leukemia virus type 1 (HTLV-1) strains: identification of a new and distinct HTLV-1 molecular subtype in Central Africa and in Pygmies.

To gain new insights on the origin, evolution, and modes of dissemination of human T-cell leukemia virus type I (HTLV-1), we performed a molecular analysis of 58 new African HTLV-1 strains (18 from West Africa, 36 from Central Africa, and 4 from South Africa) originating from 13 countries. Of particular interest were eight strains from Pygmies of remote areas of Cameroon and the Central African Republic (CAR), considered to be the oldest inhabitants of these regions. Eight long-term activated T-cell lines producing HTLV-1 gag and env antigens were established from peripheral blood mononuclear cell cultures of HTLV-1 seropositive individuals, including three from Pygmies. A fragment of the env gene encompassing most of the gp21 transmembrane region was sequenced for the 58 new strains, while the complete long terminal repeat (LTR) region was sequenced for 9 strains, including 4 from Pygmies. Comparative sequence analyses and phylogenetic studies performed on both the env and LTR regions by the neighbor-joining and DNA parsimony methods demonstrated that all 22 strains from West and South Africa belong to the widespread cosmopolitan subtype (also called HTLV-1 subtype A). Within or alongside the previously described Zairian cluster (HTLV-1 subtype B), we discovered a number of new HTLV-1 variants forming different subgroups corresponding mainly to the geographical origins of the infected persons, Cameroon, Gabon, and Zaire. Six of the eight Pygmy strains clustered together within this Central African subtype, suggesting a common origin. Furthermore, three new strains (two originating from Pygmies from Cameroon and the CAR, respectively, and one from a Gabonese individual) were particularly divergent and formed a distinct new phylogenetic cluster, characterized by specific mutations and occupying in most analyses a unique phylogenetic position between the large Central African genotype (HTLV-1 subtype B) and the Melanesian subtype (HTLV-1 subtype C). We have tentatively named this new HTLV-1 genotype HTLV-1 subtype D. While the HTLV-1 subtype D strains were not closely related to any known African strain of simian T-cell leukemia virus type 1 (STLV-1), other Pygmy strains and some of the new Cameroonian and Gabonese HTLV-1 strains were very similar (>98% nucleotide identity) to chimpanzee STLV-1 strains, reinforcing the hypothesis of interspecies transmission between humans and monkeys in Central Africa.