Torque teno virus:Its prevalence and isotypes in North India

AIM： 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. METHODS： Whereas prevalence of TFV was based on amplification of conserved region of ORF2 of TTV genome, the genotyping of TFV was carried out using restriction fragment length polymorphism （RFLP） procedure on the N22 region of ORFI. RESULTS： 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. Trv was also detected in 2/7% 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. CONCLUSION： 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.

High-throughput sequencing exclusively identified a novel Torque teno virus genotype in serum of a patient with fatal fever

Torque teno virus(TTV) has been found to be prevalent world-wide in healthy populations and in patients with various diseases, but its etiological role has not yet been determined. Using high-throughput unbiased sequencing to screen for viruses in the serum of a patient with persistent high fever who died of suspected viral infection and prolonged weakness, we identified the complete genome sequence of a TTV(isolate Hebei-1). The genome of TTV-Hebei-1 is 3649 bp in length, encoding four putative open reading frames, and it has a G+C content of 49%. Genomic comparison and a BLASTN search revealed that the assembled genome of TTV-Hebei-1 represented a novel isolate, with a genome sequence that was highly heterologous to the sequences of other reported TTV strains. A phylogenetic tree constructed using the complete genome sequence showed that TTV-Hebei-1 and an uncharacterized Taiwan Residents strain, TW53A37, constitute a new TTV genotype. The patient was strongly suspected of carrying a viral infection and died eventually without any other possible causes being apparent. No virus other than the novel TTV was identified in his serum sample. Although a direct causal link between the novel TTV genotype infection and the patient's disease could not be confirmed, the findings suggest that surveillance of this novel TTV genotype is necessary and that its role in disease deserves to be explored.

Total Chemical Synthesis, Assembly of Human Torque Teno Virus Genome

Torque teno virus(TTV) is a nonenveloped virus containing a single-stranded,circular DNA genome of approximately 3.8kb.We completely synthesized the 3 808 nucleotides of the TTV(SANBAN isolate) genome,which contains a hairpin structure and a GC-rich region.More than 100 overlapping oligonucleotides were chemically synthesized and assembled by polymerase chain assembly reaction(PCA),and the synthesis was completed with splicing by overlap extension(SOEing).This study establishes the methodological basis of the chemical synthesis of a viral genome for use as a live attenuated vaccine or gene therapy vector.

Torque teno virus in liver diseases and after liver transplantation

Torque teno virus(TTV)has been proposed as a surrogate biomarker for immune monitoring in different patient cohorts.Historically,TTV has been associated with different liver diseases such as post-transfusion hepatitis,hepatitis B,and hepatitis C,but the virus's pathogenicity is controversial.TTV is a ubiquitous DNA virus,highly prevalent and mostly indolent in the general population.Thus,TTV viral load is more relevant than prevalence to understand TTV infection.In the context of liver transplantation,TTV viral load is modulated by the immune,viral,and inflammatory status.After liver transplantation,the TTV viral load positively correlates with the intensity of immunosuppression(IS),and low TTV viral burden is a predictor of acute rejection episodes,making it an attractive marker for the efficacy of IS.However,the TTV role as a single or a panel biomarker needs to be evaluated in further independent prospective trails.

PCR Detection and Sequence Analysis of Torque Teno Sus Virus Strains from Guangdong Province

[Objective] Torque teno virus （TIT） is a novel virus with negative single-strand DNA discovered in recent years, which is ubiquitous and nonpatho- genie. Torque teno sus virus （TrsuV） is widely prevalent in swine populations, which is considered to be associated with some diseases such as post-weaning multi- systemic wasting syndrome （PMWS）. This study aimed to provided data for epidemiology of Tl＇suV in Guangdong Province. [Method] PCR primers were synthe- sized based on untranslated region （UTR） segment of TYsuV genome, to conduct PCR detection of 14 swine serum samples from two swine farms in Guangdong Province. A total of four PCR products of TrsuV1 and TrsuV2 from two swine farms were selected for cloning, sequencing and analysis. [ Result] Ten TrsuVl pos- itive samples （71% ） and eight TrsuV2 positive samples （57%） were obtained by PCR, including five double-positive samples （36%）. Sequence analysis of PCR products and reference strains showed that the UTR segments of samples GDTI-1 and GDT1-2 were both 305 bp, sharing 90.2% -95.1% similarity with Tl＇suVl reference strain, and the UTR segments of samples GDT2-1 and GDT2-2 were respectively 259 bp and 248 bp, sharing 67.3% - 100% similarity with TrsuV2 refer- ence strain. [ Conclusion] These results conformed that there are at least two types of Tl＇suV in Guangdong Province, with relatively high detection rates in some swine farms. Despite the putative harmlessness of Tl＇suV, the public health significance of TTsuV is noticeable due to its potential pathogenicity.

一种基于real-time PCR技术的TTV检测方法的建立及应用

目的:本研究旨在开发一种具有更高灵敏度和特异性的TTV检测技术,为揭示TTV在多种疾病过程中的作用提供重要的技术支持。方法:为了更精确、灵敏的检测TTV,本研究分析了目前公布的所有亚型的TTV基因序列,在此基础上建立了一种基于UTR区域的real-time PCR检测方法,并与文献报道应用较为广泛的PCR检测方法进行了对比。结果:本研究建立的方法在1×10^(7)~1×10^(1) copies/μL标准品浓度范围内具有良好的线性关系,相关系数为1.000,斜率为-3.446,检测下限为1×10^(1) copies/μL。重复性试验结果显示,组内变异系数为7.22%,表明本方法重复性、稳定性较强。针对30份临床样本,使用本研究建立的real-time PCR检测方法及目前被多个研究所使用的4套引物进行对比。结果表明,本研究所建立的方法灵敏度显著高于文献中报道的4种方法(P<0.01);Sanger测序结果表明,本方法检测出的30份阳性样本均为TTV,检测特异性为100%。结论:本研究采用基于TaqMan探针的real-time PCR检测方法,检测灵敏性高、覆盖基因型范围广,尤其对于TTV病毒载量较低的情况下能够进行定量检测,对于TTV病毒的致病性及作为免疫标志物的应用提供重要的技术支持。

TT virus infection in patients with chronic hepatitis B and response of TTV to lamivudine

AIM: To investigate the responses of TT virus (TTV) and hepatitis B virus (HBV) to a long-term lamivudine therapy.METHODS: Sixteen patients infected with both TTV and HBV were treated with lamivudine 100 mg daily for 30 months. Blood samples were drawn at the beginning of the therapy and subsequently at month 3, 6, 9, 12 and 30.Serum TTV was quantified by real time PCR and serum HBV was detected by hybridization assay and nested polymerase chain reaction.RESULTS: TTV infection was detected in 100 % of HBV-infected patients. Loss of serum TTV DNA after one year of treatment occurred in 1/16 (6 %) patients. At the end of therapy, TTV DNA was positive in 94 % of them. The decline of HBV viremia was evident at 3 months after therapy and the response rate was 31%, 44 %, 63 %, 50 % and 50 %at month 3, 6, 9, 12 and 30, respectively.CONCLUSION: TTV replication is not sensitive to lamivudine and is highly prevalent in HBV-infected patients.

TT virus and hepatitis G virus infections in Korean blood donors and patients with chronic liver disease

AIM:To determine the prevalences of TTV and HGV infections among blood donors and patients with chronic liver disease in Korea,to investigate the association of TTV and HGV infections with blood transfusion,and to assess the correlation between TTV and HGV viremia and hepatic damage. METHODS:A total of 391 serum samples were examined in this study.Samples were obtained from healthy blood donors(n=110),hepatitis B surface antigen(HBsAg)-positive donors(n=112),anti-hepatitis C virus(anti-HCV)-positive donors(n=69),patients with type B chronic liver disease (n=81),and patients with type C chronic liver disease(n=19). Trv DNA was detected using the hemi-nested PCR.HGV RNA was tested using RT-PCR.A history of blood transfusion and serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were also determined. RESULTS:TTV DNA was detected in 8.2%of healthy blood donors,16.1%of HBsAg-positive donors,20.3%of anti- HCV-positive donors,21.0%of patients with type B chronic liver disease,and 21.1%of patients with type C chronic liver disease.HGV RNA was detected in 1.8%of healthy blood donors,1.8%of HBsAg-positive donors,17.4%of anti-HCV-positive donors,13.6%of patients with type B chronic liver disease,and 10.5%of patients with type C chronic liver disease.The prevalence of TTV and HGV infections in HBV- or HCV-positive donors and patients was significantly higher than in healthy blood donors(P<0.05), except for the detection rate of HGV in HBsAg-positive donors which was the same as for healthy donors.There was a history of transfusion in 66.7%of TTV DNA-positive patients and 76.9%of HGV RNA-positive patients(P<0.05).No significant increase in serum ALT and AST was detected in the TTV or HGV-positive donors and patients. CONCLUSION:TTV and HGV infections are more frequently found in donors and patients infected with HBV or HCV than in healthy blood donors.However,there is no significant association between TTV or HGV infections and liver injury.

Coinfection of TT virus and response to interferon therapy in patients with chronic hepatitis B or C

AIM: To investigate the serum positive percentage of TT virus (TTV) in patients with chronic hepatitis B or C and the response of the coinfected TTV to interferon (IFN) during IFN therapy for chronic hepatitis B and C. METHODS: We retrospectively studied the serum samples of 70 patients with chronic hepatitis who had received IFN-alpha therapy from January 1997 to June 2000, which included 40 cases of hepatitis B and 30 hepatitis C. All the patients had been followed up for at least 6 months after the end of IFN therapy. The serum TTV DNA was detected using the polymerase chain reaction (PCR) before and every month during the course of IFN treatment. RESULTS: TTV infection was detected in 15% (6/40) of the chronic hepatitis B group and 30% (9/30) of the chronic hepatitis C group. Loss of serum TTV DNA during IFN therapy occurred in 3 of 6 patients (50%) and 6 of 9 (67%) of hepatitis B and C groups, respectively. Seronegativity of TTV was found all during the first month of IFN therapy in the 9 patients. There was no correlation between the seroconversion of TTV and the biochemical changes of the patients. CONCLUSION: TTV is not infrequently coinfected in patients with chronic hepatitis B and C in Taiwan, and more than half of the TTV infections are IFN-sensitive. However, the loss of serum TTV DNA does not affect the clinical course of the patients with chronic hepatitis B or C.

Response of TT virus to IFN plus ribavirin treatment in patients with chronic hepatitis C

AIM:TT virus (TTV) is a newly described DNA virus related to postransfusion hepatitis that produces persistent viremia in the absence of clinical manifestations.PEG-IFN plus ribavirin have been useful in the treatment of chronic hepatitis C infection.This study investigated the responses of TT virus (TTV) and hepatitis C virus (HCV) to PEG-IFN plus ribavirin therapy. METHODS:Fifteen patients infected with HCV were treated with PEG-IFN(0.5 μg/body weight/week) and ribavirin (1000 mg-1 200 mg/daily) for 48 weeks,Blood samples were drawn at the beginning and the end of the therapy.Serum TTV DNA and HCV RNA were quantified by real time PCR. RESULTS:At the beginning of treatment,TTV infection was detected in 10/15 (66.6%) of HCV-infected patients.Loss of serum TTV DNA at the end of therapy occurred in 6/10 (60%) patients.Out of these 6 patients,4 (67%) became positive for TTV DNA after 6 months of therapy.Regarding HCV viremia,11/15 (73%) patients were negative for serum HCV RNA after 48 weeks of therapy,7/11 (64%) of these cases also became negative for TTV DNA following the combined treatment.In the 3/4 (75%) patients who were positive for HCV RNA at the end of therapy,TTV DNA was detected as well.Sustained HCV response at 6 months after treatment was 53% (8/15). CONCLUSION:No TTV sustained response can be achieved in any patient after PEG-IFN plus ribavirin administration.

Identification of a novel torque teno mini virus in cerebrospinal fluid from a child with encephalitis

Dear Editor, Anelloviruses, which are all single-strand circular DNA viruses, have extremely heterogeneous genetic features. To date, the family Anelloviridae is divided into 1 l genus, and the most dominated species include torque teno virus （TTV）, torque teno mini virus （TTMV） and torque teno midi virus （TTMDV）, belonging to the genus Alphatorquevirus, Betatorquevirus and Gammatorquevirus, respectively. Most of these viruses are detected in the body fluid from the majority of human populations, although their disease associations are still elusive （de Oliveira, 2015; Focosi et al., 2016）.

High frequencies of HGV and TTV infections in blood donors in Hangzhou

AIM: To determine the frequencies of HGV and TTV infections in blood donors in Hangzhou. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed. RESULTS: Thirty-two (15.8%) and 30 (14.8%) of the 203 serum samples were positive for HGV RNA and TTV DNA, respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA. Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3 serum samples compared with the reported HGV and TTV sequences was 89.36%, 87.94%, 88.65% and 63.51%, 65.77% and 67.12%, respectively. CONCLUSION: The infection rates of HGV and/or TTV in blood donors are relatively high, and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.

Clinicopathological study on TTV infection in hepatitis of unknown etiology

AIM: To investigate the state of infection, replication site, pathogenicity and clinical significance of transfusion transmitted virus (TTV) in patients with hepatitis, especially in patients of unknown etiology. METHODS: Liver tissues taken from 136 cases of non-A non-G hepatitis were tested for TT virus antigen and nucleic acid by in situ hybridization (ISH) and nested-polymerase chain reaction (PCR). Among them, TT virus genome and its complemental strand were also detected in 24 cases of autopsy liver and extrahepatic tissues with ISH. Meanwhile, TTV DNA was detected in the sera of 187 hepatitis patients by nested-PCR. The pathological and clinical data of the cases infected with TTV only were analyzed. RESULTS: In liver, the total positive rate of TTV DNA was 32.4% and the positive signals were located in the nuclei of hepatocytes. In serus, TTV DNA was detected in 21.4% cases of hepatitis A-G, 34.4% of non-A non-G hepatitis and 15% of healthy donors. The correspondence rate of TTV DNA detection between liver tissue with ISH and sera with PCR was 63.2% and 89.3% in the same liver tissues by ISH and by PCR, respectively.Using double-strand probes and single-strand probes designed to detect TTV genome, the correspondence rate of TTV DNA detected in liver and extrahepatic tissues was 85.7%. Using single-strand probes, TTV genome could be detected in liver and extrahepatic tissues by PCR, but its complemental strands (replication strands) could be observed only in livers. The liver function of most cases infected with TTV alone was abnormal and the liver tissues had different pathological damage such as ballooning, acidophilia degeneration, formation of apoptosis bodies and focus of necrosis, but the inflammation in the lobule and portal area was mild. CONCLUSION: The positive rate of TTV DNA among cases of hepatitis was higher than that of donors, especially in patients with non-A non-G hepatitis, but most of them were coinfected with other hepatitis viruses. TTV can infect not only hepatocytes, but also extrahepatic tissues. However, the chief replication place may be liver. The infection of TTV may have some pathogenicity. Although the pathogenicity is comparatively weak, it can still damage the liver tissues. The lesions in acute hepatitis (AH) and chronic hepatitis (CH) are mild, but in severe hepatitis (SH), it can be very serious and cause liver function failure, therefore, we should pay more attention to TTV when studying the possible pathogens of so-called &quot;liver hepatitis of unknown etiology&quot;.

Tissue tropism of the TTV in experimentally infected rhesus monkeys

OBJECTIVE: To determine whether the transfusion-transmitted virus (TTV) is hepatotropic. METHODS: Total DNA was extracted from various tissues of 5 experimentally infected Rhesus monkeys during the viremic period. A dot hybridization was done with viral double stranded DNA probes or single antisense probes. RESULTS: The double-stranded probe was hybridized with DNA from the liver, bone marrow, spleen,stomach, small intestine and colon. The single-stranded antisense probe was hybridized with DNA from the liver, small intestine and bone marrow of all 5 monkeys, but not with that from other tissues. CONCLUSIONS: As the viral genome was of negative polarity, the plus-stranded fragment identified in our study might be a replicative intermediate, and was only demonstrated in the liver, small intestine, and bone marrow by dot blot hybridization with single-stranded antisense probes. It is suggested that the TTV replicates in the liver, bone marrow and small intestine, and TTV might be hepatotropic.