N22 Region Research Articles

Phylogenetic Analysis of TTV (TorqueTeno Virus) in Iraqi Woman Suffering of Failure Kidney Disease

There are still very few studies of the Torque Tenovirus infection rate between hemodialysis patients inIraq. Thus, the aim of this study was to assess the frequency of TTV viremia in hemodialysis Iraqi patients.This study has carried out in Baghdad city from October 2019 to February 2020 and included 150 patientswith kidney failure disease undergoing hemodialysis and 50 control subjects .Blood samples have beenobtained for serological TTV detection. The result shows PCR has been used to detect TTV, isolates havebeen identified through N22 region sequencing and phylogenetic analysis has also been performed. Of the150 patients, 2 (1 male and 1 female) were TTV positive.The results shows in ( isolate 14) substitutionfour Transversion A>C, C>G,A>T, and T>A three Transition T>C,A>G, and T>C of Specific region withinTTV(N-22) and showed 94% identified with a standard in Gene Bank while having 99% identified witha standard in Gene Bank with (isolate 17) substitution fiveTransversion A>T,A>C,T>A, A>Tand C>GthreeTransition A>G,A>G and T>C . The phylogenetic analysis showed that identical among themselvesand the world with 100% compatibility values.

High frequency of Torque Teno virus (TTV) among Egyptian hemodialysis patients

Very little reports are yet available for the infection rate of Torque Teno virus (TTV) among hemodialysis patients in Upper Egypt. Thus, the aim of this study was to assess the frequency and the possible genotypes of TTV in chronic renal failure patients undergoing dialysis. This cross-sectional study was carried out between August 2016 and February 2017 in three haemodialysis units in Minia, Egypt. Blood samples were collected for serological detection of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) and for detection of TTV. Nested polymerase chain reaction (PCR) was used to detect TTV. Isolates genotypes were identified by sequencing of the N22 region and phylogenetic analysis was also performed. Out of 100 dialysis patients, 76 were TTV positive (52 males and 24 females), with no significant association with gender. TTV was significantly more common among young adults than in older patients. Increased period of haemodialysis posed a high risk for acquiring TTV: Odd Ratio (OR) = 1 and 95% Confidence Interval (CI)= 1.01-1.03. No association was noted between TTV infection and either HCV or HBV infection. Genogroup 1, especially genotype 2 was the most frequently found type in hemodialysis patients. TTV is vastly predominant among Egyptian haemodialysis patients with no significant association with HCV or HBV. Further analyses are recommended to associate the renal failure outcome with the virus load. Key words: Torque Teno virus, dialysis, phylogenetic biogeography.

Prevalence and Genotyping of Torque Teno Virus in HBV/HIV and Chronic HBV Patients in Iran

Background:Torque teno virus (TTV) was the first human Anelloviridae detected in a Japanese patient with an unknown type of hepatitis in 1997. TTV is by far the first known single-stranded circular DNA virus infecting human. In spite of its widespread nature in human population, its pathogenesis is still unclear. In addition, information regarding TTV infection in Iranian population is limited. Therefore, we attempted to determine the prevalence and genotype ofTTV in three groups:HIV/HBV patients, chronic hepatitis B patients, and healthy individuals.Methods:The presence of TTV DNA in sera was investigated using PCR. The primer sets encompassing two 5’-UTR and N22 regions were used, and the positive products were collected for sequencing. Phylogenetic tree was generated based on N22 region and using the MEGA 7 software.Results:TTV DNA was detected in 452 patients with HIV/HBV and chronic hepatitis B, as well as in healthy control groups. The results from PCR indicated positive rates for these three groups 48%, 54%, and 49.3% using 5’-UTR primer and 15.1%, 12%, and 8% using N22 primer, respectively.Conclusion:Five genogroups were observed, which the second group was found to be the most frequent. The results of 5’-UTR primer showed more prevalence of TTV DNA comparing to N22 primer in patients and healthy control.

Development of an Immunoassay for Detection of Torque Teno Virus (TTV) Antibodies Using the N22 Expression Product from TTV Genotype 2

Aims: This study describes an immunoassay to detect anti-torque teno virus (TTV) antibodies using a peptide obtained from expression of the N22 region of TTV genotype 2. Methods: The N22 region (∼500 bp) of TTV genotype 2 was cloned in a pET-28a(+) vector and expressed in ZYM-5052 autoinduction medium. Following metal affinity chromatography, a purified polypeptide was used as an antigen for the development of an immunoassay to detect anti-TTV antibodies in human sera. Results: Recombinant protein (∼25-kDa) was obtained after 24 h of incubation at 25°C in ZYM-5052 autoinduction medium. A blot assay developed using this polypeptide as an antigen and TTV-positive sera as the primary antibody produced a distinct spot on the nitrocellulose membrane. Serum samples from 36 of 42 patients with renal disease and 29 of 48 patients with liver diseases produced a positive signal using this immunoassay. Simultaneously, 18 of 48 healthy controls were also detected to be positive for anti-TTV antibodies. These results were found to be comparable with TTV detection using PCR, and the assay showed a high sensitivity and specificity (i.e., 97.44 and 91.67%, respectively). Moreover, this assay could detect TTV infection irrespectively of the genotype, including cases of mixed infection. Conclusion: The present immunoassay using the N22 expression product may be used as an alternative to PCR to detect TTV infection in large populations.

THU-317 - N22 region based dot blot assay: an alternative to PCR for detection of Torque teno virus infection

THU-317 - N22 region based dot blot assay: an alternative to PCR for detection of Torque teno virus infection

New Phylogenetic Groups of Torque Teno Virus Identified in Eastern Taiwan Indigenes.

Torque teno virus (TTV) is a single-stranded DNA virus highly prevalent in the world. It has been detected in eastern Taiwan indigenes with a low prevalence of 11% by using N22 region of which known to underestimate TTV prevalence excessively. In order to clarify their realistic epidemiology, we re-analyzed TTV prevalence with UTR region. One hundred and forty serum samples from eastern Taiwanese indigenous population were collected and TTV DNA was detected in 133 (95%) samples. Direct sequencing revealed an extensive mix-infection of different TTV strains within the infected individual. Entire TTV open reading frame 1 was amplified and cloned from a TTV positive individual to distinguish mix-infected strains. Phylogenetic analysis showed eleven isolates were clustered into a monophyletic group that is distinct from all known groups. In addition, another our isolate was clustered with recently described Hebei-1 strain and formed an independent clade. Based on the distribution pattern of pairwise distances, both new clusters were placed at phylogenetic group level, designed as the 6th and 7th phylogenetic group. In present study, we showed a very high prevalence of TTV infection in eastern Taiwan indigenes and indentified new phylogenetic groups from the infected individual. Both intra- and inter-phylogenetic group mix-infections can be found from one healthy person. Our study has further broadened the field of human TTVs and proposed a robust criterion for classification of the major TTV phylogenetic groups.

Detection and Phylogenetic Analysis of Torque Teno Virus in Salivary and Tumor Biopsy Samples from Head and Neck Carcinoma Patients

Objectives: Because torque teno virus (TTV) has been implicated in tumorigenesis as a cocarcinogen, we studied TTV prevalence in saliva and biopsy samples from head and neck cancer (HNCC) patients, patients with premalignant lesions of oral cancer, and controls. We also wished to determine the TTV genotypes in HNCC patients. Methods: A seminested polymerase chain reaction (PCR) amplifying the N22 region of the TTV genome, as well as direct sequencing of PCR fragments, was used. Results: TTV prevalence was higher in HNCC patients (saliva: 27/71, 38%; tumor biopsy: 22/74, 30%) than in controls (saliva: 8/56, 14%; oral mucosa: 1/19, 5%). TTV prevalence was also high in patients with premalignant lesions of oral carcinoma (saliva: 9/18, 50%; biopsy: 5/21, 24%). By phylogenetic analysis, TTV belonging mostly to genotypes 1 and 2 was found in HNCC patients. In most of the cases, identical TTV strains were present in the biopsy and salivary sample of the same HNCC patient. In addition, the same TTV strain was detected in 2 laryngeal carcinoma biopsies obtained from 2 independent patients. Conclusions: Our data are compatible with the idea that TTV might act as a cocarcinogen in certain cases of HNCC. Alternatively, HNCC may facilitate either TTV replication or TTV entry into the saliva.

Prevalence of TT virus infection in Italian-dialysis patients

A novel DNA virus, transfusion-transmitted virus (TTV) is identified from the serum of a patient from acute hepatitis non-A-E. Few reports have been published on patients with renal disease. Patients on dialysis were at high risk of blood-borne viral infections and little is known about the prevalence of this virus in dialysis patients. In order to verify the prevalence of infection from TTV in these patients, we have examined the incidence of TTV in Italian patients with dialysis. Serum samples of 85 patients and 65 healthy individuals were examined. In order to evidence the presence of the TTV virus, a method of the seminested polymerase chain reaction (PCR) with TTV-specific primers derives from the N22 region deIl'ORF-1 of the virus has been used and products were analyzed by agarose-gel electrophoresis. All serum samples were also analyzed to markers HBV and HCV. The prevalence of TTV DNA in dialysis patients [35/85 (41.7%)] was significantly higher than in healthy population [7/65 (10.7%)]. Among TTV positive dialysis patients, HCV coinfection was present in six cases. The positivity rate for TTV-DNA tends to increase with age. Transfusion-transmitted virus had a high prevalence in Italian-dialysis patients. In our study the virus did not have an important clinical effect on patients; but remains the possibility that it may aggravate liver disease caused by HCV. However, the question of whether TTV infection might have a possible effect on dialysis patients requires further investigation in larger groups.

Torque teno virus: Its prevalence and isotypes in North India

To investigate the prevalence and genotype distribution of Torque teno virus (TTV) in patients with different liver diseases and chronic renal failure treated at a referral hospital in North India. Whereas prevalence of TTV was based on amplification of conserved region of ORF2 of TTV genome, the genotyping of TTV was carried out using restriction fragment length polymorphism (RFLP) procedure on the N22 region of ORF1. TTV-DNA was detected in 137 of 513 (26.7%) patients with liver diseases and 38 of 65 (58.5%) patients with chronic renal failure. TTV was also detected in 27% of healthy controls. The sequence analysis of the PCR product from 10 randomly selected cases failed to show a significant sequence divergence when compared with that of the TRM1 isolate of TTV genotype 1. The results of genotyping in 55 randomly selected patients showed the presence of genotype 1 (G1) in 53 (96.4%) and genotype 2 (G2) in 2 cases (3.6%), respectively. Other genotypes were not identified in this patient subgroup, suggesting that G1 is predominant in this area. The results of genotyping by RFLP were also supported by phylogenetic tree analysis, where G1 was found to be the major genotype. These results indicate that TTV is moderately present in Indian patients, with G1 to be the major genotype in North India. The pathogenicity and etiological role of TTV in different diseases is still a question mark and warrant further studies.

Distribution of TT Virus Genotypes and Genogroups in 69 Healthy and 59 Hepatitis B Virus Infected Korean Individuals

TT virus (TTV) infection is highly prevalent in general population and patients with hepatitis B virus (HBV) infection. The aim of the present study was to determine the distribution of the genotypes and genogroups of TTV in healthy and HBV-infected individuals in Korea. Distribution of TTV genotypes and genogroups was investigated in the serum samples of 69 healthy and 59 HBV-infected individuals. PCR products of N22 region were genotyped by sequence analysis. TTV genogroups were determined by 5 different genogroup-specific PCR assays. Among the 20 sequenced isolates, 9 (45%) were genotype 2, 8 (40%) were genotype 1, 2 (10%) were genotype 3, and 1 (5%) was genotype 4. TTV genogroup 4 was found most frequently (52/128), followed by genogroup 3 (42/128), genogroup 1 (35/128), genogroup 5 (32/128), and genogroup 2 (1/128). Mixed infections with different genogroups were frequent. TTV genotype 2 and 1 are predominant genotypes. TTV genotype 3 was detected for the first time in Korea. TTV genogroups 4 and 3 were predominant genogroups. No significant difference was observed in the distribution of TTV genogroups between healthy and HBV-infected individuals.

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.

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.

Phylogenetic classification of TT virus groups based on the N22 region is unreliable

Evolutionary studies on the recently discovered TT virus (TTV) are currently focused on approximately 220 nts of the N22 region, since this is the region for which most sequence information is available. Regarding the extensive sequence heterogeneity in this region, within group classification can be sufficiently reliable, however, between group classification becomes problematic. We observed high divergence at the nucleotide level between distant related strains (TTV groups) preventing unambiguous alignments, saturation in transitions within TTV groups and considerable phylogenetic noise due to conflicting signals within distinct genotypes. Consequently, analysing all TTV groups in one tree, using this 220 nt region provides unreliable results. Also within genotype analysis can produce conflicting results. On the other hand, this region is still suitable to some extent for within TTV group phylogenetic analysis. We suggest that care should be taken in future TTV phylogenetic analysis, in particular, larger and more conserved regions should be sequenced to allow between group comparisons.

TTV: prevalence and phylogenetic characterization of a novel blood-borne DNA virus in different groups from São Paulo, Brazil

SUMMARY OF THESIS* BASSIT, Leda Cattini – TTV: prevalencia e caracterizacao do novo virus de DNA transmissivel pelo sangue em diferentes populacoes de Sao Paulo.Sao Paulo, 2001. (Tese de Doutoramento – Faculdade de Ciencias Farmaceuticas da Universidade de Sao Paulo). TTV: PREVALENCE AND PHYLOGENETIC CHARACTERIZATION OF A NOVEL BLOOD-BORNE DNAVIRUS IN DIFFERENT GROUPS FROM SAO PAULO, BRAZIL Recently, an infection caused by a novel TT-virus (TTV), whichwas initially associated with post-transfusion non-A-G hepatitis, led usto investigate the TTV infection (clinic-epidemiological aspects,phylogenetic characterization and distribution of TTV genotypes) indifferent groups from Sao Paulo. The study included (i.) 138 childrenwithout history of blood transfusion or of liver disease, (ii.) 401 blooddonors and (iii.) 130 Patients with Bleeding Disorders (PBD). TTVinfection was investigated by PCR-based amplification of the untranslatedregion (UTR) and its prevalence was as high in low-risk groups (81-85%) as in PBD (98%). The distribution of this infection according tothe age groups only showed significant difference in the children group.Almost all the children aging 2 to 4 years (95%) were infected by TTV.However, this prevalence decreased continuously thereafter and reacheda lower prevalence of 47% in adolescents of male gender aging 15 to 17years. TTV DNA was also detected by a hemi-nested PCR amplificationdirected to the N22 region in blood donor and PBD samples; sequencingof these amplified products was also performed. Concerning PBD, ahigh frequency of N22 was found in 42% in comparison to 5.5% inblood donors. There was no association of N22 findings with ALT leveland epidemiological aspects in the prior group. Therefore, there was anassociation of N22 with patients co-infected with HCV, hemophilia Aand patients who had been treated with fresh blood products. Thedistribution of TTV genotypes was similar in both studied groups andgenotypes 1 and 2 of the Group I prevailed on the others. A less frequentGroup of TTV (Group II) was also observed in two patients. The TTVgenotype varied in the same patient when samples were collected in thecourse of time. The high prevalence of the UTR-specific isolates foundfor the low-risk groups indicates that the parenteral route of transmissiondoes not seem to play a major role for this infection. These findingswidely contributed for the epidemiological studies of TTV infection andfor its phylogenetic characterization in Brazil.

Changes of DNA titer and sequence variance of TT virus in hepatic disorders

TT virus (TTV) has been reported to occur in association with elevated alanine aminotransferase (ALT) levels in patients with posttransfusion hepatitis of unknown etiology. We examined whether the presence, change of DNA titer, or variation in sequence of this virus is associated with acute or chronic liver dysfunction in Japanese. We detected TTV by polymerase chain reaction (PCR) using primers generated from the conserved region of the TTV genome. Direct DNA sequencing of the original N22 region was used to characterize TTV isolates. We detected TTV DNA in 15 (25%) of 60 patients with liver dysfunction. Variants recovered from infected patients formed four genotypes/subtypes, corresponding to G1a, G1b, G2, and G4. Although TTV DNA titers in patients with G2 and G4 were lower than those with G1, TTV was consistently detected regardless of genotype/subtype. TTV infection continued for at least 1 year after normalization of ALT level in patients with acute liver dysfunction. Changes in DNA titer, substitutions of deduced amino acids, and variety of quasispecies of TTV were detected during the observation period, but no significant fluctuation in ALT level was found. We conclude that persistent infection, changes in DNA titer, and variation in sequence of this novel virus are not significantly related to hepatic disorders.

Molecular epidemiology of TT virus in Italy and phylogenesis of viral isolates from subjects at different risk for parenteral exposure

The prevalence and genotype distribution of human TT virus (TTV) in Italy were analysed in 593 subjects at different risk of parenteral infection who included blood donors, patients with chronic type C hepatitis (HCV), thalassemic patients, patients on haemodialysis, human immunodeficiency virus type 1 (HIV-1)-negative intravenous drug users (IVDUs), and HIV-1-infected subjects (IVDUs, heterosexual contacts and homosexual males). Plasma TTV-DNA was detected using nested PCR with primers deduced from the N22 region of the open reading frame 1 (ORF-1) and from the untranslated region (UTR) of the viral genome. Phylogenetic analysis of the sequences obtained from ORF-1 was also undertaken. A high prevalence of plasma TTV-DNA was observed using the UTR primers, with rates varying from 83–100% in the study groups. Using the N22 primers, HIV-1 positive IVDUs and homosexual males, haemodialysed patients and thalassemic patients had a significantly higher TTV prevalence (range: 23.0–86.1%) than blood donors, who displayed a high frequency of positivity (10.6%). Sequence analysis of 127 N22-positive isolates revealed that 42.5% were of type 1, 53.5% of type 2, 2.4% of type 3, and that two isolates (1.6%) were closely related to genotypes 1-2 but distinct from the other major genotypes. TTV-2 was significantly more prevalent in patients at high risk for parenteral infection and in HIV-1 positive homosexuals. In sequential samples from 15 TTV-infected subjects, N22 sequences were detectable persistently in 12 (80.0%) and UTR sequences persisted in all 15 patients over a mean period of 29.6 months. This data indicates that TTV is widespread in Italy in parenterally exposed subjects, and that the infection frequently persists. J. Med. Virol. 63:76–84, 2001. © 2001 Wiley-Liss, Inc.

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

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

Detection of TT virus DNA in patients with liver disease and recipients of liver transplant.

TT virus (TTV) is transfusion-transmissible but its involvement in post-transfusion hepatitis is uncertain. To investigate the potential association of TTV with liver diseases, the prevalence of TTV DNA was tested by semi-nested PCR in 113 carriers of hepatitis C virus (HCV), 10 patients with acute liver failure, 11 patients with cryptogenic cirrhosis and 200 control blood donors. Thirty-seven of these patients underwent liver transplantation and were tested pre- and post-transplantation. TTV DNA was semi-quantified in serial samples from seven patients with unexplained post-transplant hepatitis. TTV genotyping was performed on samples from 28 patients by sequence analysis. The prevalence of TTV DNA in blood donors was 1.5% and 17% in HCV infected haemophiliacs. In patients with acute or chronic liver disease or hepatitis, 6 to 27% prevalence was observed. After liver transplantation, the prevalence of TTV DNA increased from 16 to 46% (P < 0.01). In patients who developed unexplained hepatitis post-transplantation, TTV viraemia did not parallel ALT levels. TTV DNA either increased in titre or became detectable shortly after transplantation, suggesting that either TTV was transfusion-transmitted, or, more likely, that immunosuppression caused a recurrence of low level or undetectable TTV viraemia. TTV had considerable genomic diversity in the N22 region, corresponding to at least 4 genotypes. Genotype 2 was found in 14/28 patients.

Molecular epidemiology of TT virus in Italy and phylogenesis of viral isolates from subjects at different risk for parenteral exposure

The prevalence and genotype distribution of human TT virus (TTV) in Italy were analysed in 593 subjects at different risk of parenteral infection who included blood donors, patients with chronic type C hepatitis (HCV), thalassemic patients, patients on haemodialysis, human immunodeficiency virus type 1 (HIV-1)-negative intravenous drug users (IVDUs), and HIV-1-infected subjects (IVDUs, heterosexual contacts and homosexual males). Plasma TTV-DNA was detected using nested PCR with primers deduced from the N22 region of the open reading frame 1 (ORF-1) and from the untranslated region (UTR) of the viral genome. Phylogenetic analysis of the sequences obtained from ORF-1 was also undertaken. A high prevalence of plasma TTV-DNA was observed using the UTR primers, with rates varying from 83-100% in the study groups. Using the N22 primers, HIV-1 positive IVDUs and homosexual males, haemodialysed patients and thalassemic patients had a significantly higher TTV prevalence (range: 23.0-86.1%) than blood donors, who displayed a high frequency of positivity (10.6%). Sequence analysis of 127 N22-positive isolates revealed that 42.5% were of type 1, 53.5% of type 2, 2.4% of type 3, and that two isolates (1.6%) were closely related to genotypes 1-2 but distinct from the other major genotypes. TTV-2 was significantly more prevalent in patients at high risk for parenteral infection and in HIV-1 positive homosexuals. In sequential samples from 15 TTV-infected subjects, N22 sequences were detectable persistently in 12 (80.0%) and UTR sequences persisted in all 15 patients over a mean period of 29.6 months. This data indicates that TTV is widespread in Italy in parenterally exposed subjects, and that the infection frequently persists.

Marked Genomic Heterogeneity and Frequent Mixed Infection of TT Virus Demonstrated by PCR with Primers from Coding and Noncoding Regions

A nonenveloped, single-stranded, and circular DNA virus designated TT virus (TTV) has been reported in association with hepatitis of unknown etiology. TTV has a wide sequence divergence (∼52%), by which it is classified into at least 16 genotypes separated by an evolutionary distance of >0.30. Therefore, the detection of TTV DNA by polymerase chain reaction would be influenced by primers deduced from conserved or divergent regions of the genome. Of the 30 sera from healthy individuals, up to 17% tested positive with primers deduced from coding region, much less frequently than up to 93% testing positive with primers from noncoding region. These differences were not attributable to the sensitivity of detection, because a cloned TTV DNA of genotype 1a was detected sensitively (up to 1 copy per test) with primers deduced from either the coding or the noncoding region of the same genotype. Sera testing positive only with noncoding region primers, or those showing higher titers with noncoding than coding region primers, contained TTV DNA strains with sequence divergence of 47–53% from the TA278 isolate of genotype 1a within the N22 region spanning 222–231 nucleotides. Some of the sera contained two or three TTV DNA strains of distinct genotypes. These results indicate TTV strains with extremely high sequence divergence prevailing in healthy individuals and frequent mixed infection with TTV strains of distinct genotypes.