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 of TTV 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.


INTRODUCTION
uman torque teno virus (TTV) is a recent identified infectious agent that has been assigned to the Anelloviridae family [1] . The first TTV was isolated from the serum of a patient with post-transfusion hepatitis of unknown etiology in 1997 [2] . It has a negative-sense, single-stranded circular DNA genome of approximately 3.8 kb and an unenveloped, small, spherical particle with a diameter of 50 ± 30 nm [2] . Sequence diversity of TTV is extremely wide, which is classified into at least 23 genotypes with sequence divergence of more than 30% for into five phylogenetic groups [2] . These phylogenic groups have a very high prevalence in the general population [1] . TTV can be transmitted through blood transfusions, intravenous drugs, and fecal oral routes. TTVs co-infections with HIV or HBV are common since they share similar transmission routes as HIV and HBV [3] . Since the discovery of human Anelloviruses, TTV has been suggested to be associated with various pathological conditions, such as hepatitis, cancer, respiratory diseases, as well as hematological and autoimmune disorders, with few arguments for their direct involvement [1] . Three strains of TTVs recently shown to more infect humans [4] are reclassified in H Anellovirus as well [5,6] . Detection of TTV is mainly performed by PCR. The PCR targeting the N22 region within the largest open reading frame 1 (ORF1) can mainly detect 1 to 5 TTVs in 10%-30% of the population, while the PCR directed for the UTR region can find most known TTVs in over 90% of the population [6,7] .
TTV has been reported to be associated with hepatitis of unknown etiology [6] . Nucleotide sequence variations between TTV genotypes can be more than 50%, which leads to 39 genotypes [5] . Before the discovery of TTV, nohuman virus with such a small genome accompanied with a high mutation rate has been reported. TTV genome variation is higher at ORF levels than in the UTRs regions [5] . In a study, Takayama et al. [8] analyzed 50 sera samples from hemophilic patients for TTV DNA with 44.4% detection rate. The N22 gene of the virus was present in 1.9% to 42.4% of blood donors in different countries, and for UTR gene, the rates were 69% to 94% [9][10][11][12][13][14] . The goal of this study was to determine the prevalence and genotyping of TTV in Iran among three groups: HIV/HBV patients, chronic hepatitis B patients, and healthy individuals.

Patients
Subjects for this study consisted of 152 HIV/HBV co-infected patients, 150 chronic HBV patients, as well as 150 uninfected individuals who were collected from Iran between April 2012 and February 2014. Samples related to HIV/HBV co-infected patients were gathered by collaboration with Lorestan University of Medical Sciences (Lorestan, Iran). However, samples regarding chronic HBV patients were obtained from patients referred by a specialist in HBV infection diagnosis to Virology Diagnostic Laboratory of Golestan University of Medical Sciences (Golestan, Iran). All patients were tested for serological marker of HBV (HBsAg) using a commercial ELISA kit from DIALAB (Austria), and Western blotting was employed as a serological test for HIV detection. Sera were separated from a total of 452 blood samples and kept in -70 °C for further analysis. All the samples were tested by PCR on the virus N22 and 5΄-UTR regions. The study was approved by the Ethics Committee of Golestan University of Medical Sciences under ethic no. Ir. goums.REC.1394.102.

Detection of TTV with N22 region primers
The TTV PCR reaction for N22 region contained 100 ng of template DNA, 1 μL (15 pM stock) of each amplification primer, 1 μL of dNTP (10 mM stock), 0.5 μL of Taq DNA polymerase (GeNet Bio, Korea), 2 mM of MgCl 2 , and 2.5 μL of 10× PCR buffer. The following thermal cycles were set for genome amplification: initial denaturation at 94 °C for 7 min, followed by 35 cycles at 94 °C for 30 s, 60 °C for 40 s, and 72°C for 40 s, with a final elongation at 72 °C for 7 min. The PCR products were separated on 1.5% agarose gel containing safe stain, Eva green (Cinaclone, Iran; Fig .1).

TTV detection based on UTR region amplification
The PCR reaction for 5΄-UTR region included 100 ng of template DNA, 10 pmol of each forward and reverse primer, 0.6 mM of dNTPs, 1.25 U of Taq DNA polymerase (GeNet Bio), 2 mM of MgCl 2 , and 1 PCR buffer. Thermal amplifications were as follows: initial denaturation at 94 °C for 5 min and 40 cycles at 94 °C for 20 s, 57 °C for 25 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min (Fig. 2).

Sequence data mining
In total, 44 DNA sequences of TTV isolates were obtained from the GenBank database and used as a reference sequences for further analysis.

Phylogenetic analysis
Phylogenetic tree was constructed based on TTV N22 amplicon sequenced data and compared to the TTV reference sequence with accession number AB008394. Before comparison, the sequenced data were trimmed and aligned by BioEdit (5.0.9) and ClustalW (1.81) software, respectively. Bootstrap resampling was carried out on 1000 replicates to ensure the reliability of the tree. Based on phylogenetic analysis, 5 genogroups (1, 2, 3, 4, and 5) were identified. The analysis of 15 N22 sequenced isolates demonstrated that genogroup 2 was significantly more prevalent among all the patients (Fig. 3). Also, the five genotypes were found among HIV/HBV, chronic HBV patients, and uninfected individuals in Iran. Demographic and paraclinical data are shown in Tables 1 and 2. Fig. 3. Phylogenetic tree of human Anelloviruses. The tree was built using the MEGA 7 software with the neighbor-joining method [16] of the nucleotide sequence of ORF1 in 15 TTV isolates. The phylogenic tree includes 140 isolates obtained from the International Committee on Taxonomy of Viruses (ICTV). Available complete ORF1 sequences of TTV were downloaded from the GenBank with accession number AB008394. The isolated viruses in the present study are highlighted in yellow.

DISCUSSION
TTV was first reported in Japan in 1997 by Nishizawa in patients with fulminant hepatitis and chronic liver disease of unknown etiology. This virus was later identified in a large number of patients, who mostly suffered from acute and chronic hepatitis, in several countries [17,18] . The association between TTV infection and hepatitis is argumentative as there is no evidence of causative effect of TTV to date [19][20][21] .
Concomitant infections with TTV and HBV are common; nonetheless, the effect of TTV infection on patients with chronic HBV infection is unknown [22] . TTV has been found to be highly common in men worldwide [23] . Currently, only PCR assay is available for TTV virus [24] , but the accurate detection of all TTV genotypes depends on the selection of the amplification region. Current primers are designed for either N22 or 5΄-UTR regions. N22 primers only recognize and amplifiy some of the genotypes, but 5΄-UTR primers are able to identify and amplify all types of virus genotype [25] . Here, we showed that TTV infection cannot be frequently detected by 5΄-UTR primers in chronic HBV patients. Based on our results, it can be concluded that both N22 and 5΄-UTR primers can detect the TTV infection; however, the prevalence may be in a wide range of 5 to 92% due to the sensitivity of the primers. Several studies support this wide range of the reported prevalence of TTV in a single population based on the applied primers.
Conservation of 5΄-UTR locus within different genotypes may explain the high incidence of TTV detection based on 5΄-UTR primers. It was generally observed that the prevalence of TTV infection based on N22 region amplification was below 15% in healthy subjects . The N22-based PCR method generally had a lower detection rate than UTR PCR, due to its limited genotype specificity. This finding has also previously been reported by Ergünay group [26] . TTV prevalence among Brazilian blood donors, by using different primers, was reported to be 11.9% to 50.5%. Accordingly, TTV prevalence in Saudi Arabia has been reported to be up to 100%, when 5΄-UTR primers were used [27] . Because of a very high genetic variety, several studies have been conducted to investigate the prevalence of TTV. These studies are affected by the viral, amplified region and sensitivity of its primers [28] . Phylogenetic analysis of TTV was firstly performed on N22 region, as it was suggested that this region would exploit enough diversity for phylogenetic analysis. However, TTV genome amplification has revealed higher genetic variety than other DNA viruses.   [29] semi-nested PCR 26.9% (liver tissue); 23.5% (plasma samples of transplanted patients with cryptogenic cirrhosis); 25.9% (liver tissue), 11.1% (plasma samples of patients with determined cirrhosis)
The results of the present study in comparison with other reports demonstrate that different primers chosen in studies could affect the findings. To date, there is little or no data covering the incidence and genotype distribution of human TTV in Iranian population afflicted by HIV/HBV co-infections, CHBV infection, and or healthy individuals. To achieve this goal, further investigations with larger population samples are needed. Accordingly, the patients have to be selected from different parts of the country.
Likewise the previous studies, the present work shows that the 5΄-UTR primer-based PCR may report more prevalence of TTV DNA comparing to N22 primers either in patients or healthy control (Tables 3  and 4).
Although our results suggested that TTV incidence was higher among HIV/HBV and chronic HBV patients comparing healthy population, TTV frequency was not significantly different between HIV/HBV coinfection and chronic HBV cases. Further investigations are needed to evaluate the risk of TTV infection in those patients.  [30] PCR 64% (HIV); 34% (healthy control) 2014