Al-Qahtani et al. Virology Journal (2016) 13:208 DOI 10.1186/s12985-016-0664-6 RESEARCH Open Access Prevalence of anelloviruses (TTV, TTMDV, and TTMV) in healthy blood donors and in patients infected with HBV or HCV in Qatar Ahmed A. Al-Qahtani1,2,3, Enas S. Alabsi4,5, Raed AbuOdeh6, Lukman Thalib4 and Gheyath K. Nasrallah4,5* Abstract Background: Anelloviruses (TTV, TTMV, and TTMDV) have been associated with non A-G hepatitis. The goal of the current study was to estimate the prevalence of these anelloviruses in Qatar. Methods: A total of 607 blood samples (500 healthy donors, and 53 HBV-and 54 HCV-positive patients) representing different nationalities were tested for the presence of TTV, TTMV, and TTMDV DNA by nested PCR. Results: Prevalence rates for the three viruses were high in all studied groups, and exceeding 95% in the HBV group (for TTV and TTMDV). Infection with more than one type of viruses was common and significant in most of the positive patients (p < 0.05) and ranging from 55.4% for TTV/TTMV and TTMV/TTMDV co-infections in the healthy group, to 96.3% for TTV/TTMV co-infections in the HBV group. Further, and as with most previous studies, no significant association was found between anelloviruses infections and age, nationality, or gender (p > 0.05) albeit the detection of higher infection rates among females and Qatari subjects. Conclusion: This was the first published study to look at prevalence of Anellowviruses in the Middle East. High prevalence rates of the three viruses in all studied groups was noted. Further studies are needed to explore and compare the different genotypes of these viruses in the region. Keywords: Anelloviruses, HBV, HCV, PCR Background to Alphatorquevirus, Betatorquevirus,andGammator- Human infections with anelloviruses (small ssDNA vi- quevirus, genera respectively [6]. ruses) are widely spread [1, 2]. A prominent member of The Alphatorquevirus genera (TTV) alone has 29 spe- the Anelloviridae family is the Torque teno virus (TTV), cies. Much less is known about phylogenetic groups for reported for the first time in 1997 in a Japanese patient TTMV and TTMDV [7]; TTMV ORF1 sequence com- with post-transfusion hepatitis of unknown etiology [3]. parison showed significant divergence, and complete Subsequently (in 2000), another small DNA virus, desig- genome analysis of TTMDV also shows sequences clus- nated Torque teno mini virus (TTMV), remotely similar tering [7]. to TTV was discovered [4]. In 2007 a third addition to The three viruses share a similar genome organization the anellovirus genus was reported; the new virus had a (UTR region, open reading frame region followed by genome of 3.2 kb (TTV and TTMV were around 3.8 short G/C rich region). Further, the three viruses are ex- and 2.8 kb, respectively), hence the designation Torque tremely divergent in the ORF regions both at the nucleo- teno midi virus (TTMDV) [5]. The Anelloviridae family tide and amino acid levels [6]. contains 12 genera; TTV, TTMV, and TTMDV belong Furthermore, infection with anelloviruses can be highly common reaching a 90% prevalence rate [8]. Re- ports on the prevalence of TTMV in humans indicated * Correspondence: [email protected] high rates of infection in French [9], Brazilian [10, 11] 4Department Health Sciences, College of Arts and Sciences, Qatar University, PO Box 2713, Doha, Qatar and in Norwegian [12] blood donors and also in French 5Biomedical Research Center, Qatar University, Doha, Qatar hemodialysis patients (95%) [13]. Further, TTMV DNA Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Al-Qahtani et al. Virology Journal (2016) 13:208 Page 2 of 6 has been detected in amniotic fluid, cord blood and A total of 500 samples from healthy blood donors breast milk [10, 14]. TTMV DNA has also been detected were used in the present study. These samples were col- in cervical swabs of healthy women [15], women diag- lected from the blood donation unit during the period nosed with cervical cancers, peripheral blood mono- from June to August 2013. In addition, blood samples nuclear cells, feces and saliva [16]. from 54 infected HBV to 53 HCV patients were also ob- TTMDV DNA was detected in plasma and saliva sam- tained from the diagnostic virology laboratory at the ples of French blood donors [17], in healthy and hepa- HMC. titis C Italian serum samples [18], in healthy blood donors, hepatitis C patients and HIV patients in Iran [19], and in Korean chronic hepatitis patients and blood Plasma DNA extraction μ products [20]. TTMDV was also reported in Hungarian DNA was extracted from 200 l aliquots of plasma using children with acute respiratory disease [21]. a standard commercial kit for viral DNA extraction from Since TTV was first detected in blood samples it was body fluids (PureLink® Viral RNA/DNA Kits - Life Tech- referred to as transfusion-transmitted (TT) virus [3]. nologies; Grand Island, New York, USA). Subsequent studies suggested the existence of other ways of TTV transmission including parenteral, sexual, Whole genome amplification of DNA vertical and others [22]. Due to small sample volume, extracted DNA was ampli- TTV has been detected in almost every human tissue fied using REPLI-g Mini Kit (Catalog # 150025, Qiagen, type or body fluid, the newly discovered TTMV can be USA) following manufacturer’s protocol. Amplified DNA transmitted from mother to baby; similar virus se- was stored at −20 °C until use. quences in mother and baby were detected in umbilical cord blood, breast milk, amniotic fluid, and sera of new- borns [7]. Detection of TTMDV and TTMV Finally, viremic infections with multiple genotypes of Both viruses were detected using nested PCR. For the any of the three viruses at any one time is not uncom- first round PCR setup, the following components were mon [12, 17, 23]. used: one μl of whole genome amplified DNA, 10 pmol The aim of the current study was to investigate the each of forward and reverse primers (universal primers) rates of infection of TTV, TTMV, and TTMDV in Qatar, as described previously [24] and GoTaq Green Master and to examine the association of these virus infections, Mix (Promega, Madison, WI, USA) in a reaction volume if any, with HBV or HCV. of 50 μl(μl). The mixture was preheated at 95°C for 5 min followed by 35 cycles with parameters as follows: Methods 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final ex- Ethical approval tension of 72 °C for 5 min. For the second round, two Prior to samples collection from patients and blood donors, PCR reactions to detect TTMDV and TTMV were sep- Institutional Review Board of Hamad Medical Corporation arately prepared. For the detection of TTMDV, the reac- μ (HMC) (Protocol No.13422/13) and Qatar University (IRB tion mixture consisted of 5 l of the amplification No. QU-IRB289-A/14) approvals were obtained. Further- product of the first round, 15 pmol of NG795/NG796 more, because this research involves no risk to the partici- primers [24] and GoTaq Green Master Mix with a total μ pants, a waiver of all the consent requirements was volume of 40 l. The mixture was preheated at 95°C for obtained. The participants’ rights and welfare were not 5 min followed by 30 cycles of 95°C for 20 s, 62°C for harmfully affected, because personal information related to 20 s, 72°C for 20 s, and a final extension of 72°C for participants was not claimed. Blood samples obtained from 5 min. The same conditions were used to detect TTMV the blood bank and the hospital were anonymous. The only except that the primers NG792/NG793/NG794 as sense information collected related to the participants were na- primers and NG791 as antisense primer were used [24]. μ tionality, sex, and age. Information concerning collected Fifteen l of the amplified products were electropho- data was kept confidential. resed in 2.5% agarose gel, stained with GreenView Plus (GeneCopoeia, USA), and photographed under UV light. Blood samples collection The samples that were used in this project to detect Statistical analysis TTMDV and TTMV DNA were exactly the same ones Data were analyzed using SPSS software. The chi-square we used in our previous TTV published project [8], and test was used to compare proportions between the the data from it were used again in this study for com- groups. Differences were considered to be statistically parison purposes between the three anelloviruses. significant at was p < 0.05. Al-Qahtani et al. Virology Journal (2016) 13:208 Page 3 of 6 Results Study population analysis In the present study, a total of 607 plasma samples were screened for the presence of anellovirsuses (TTV, TTMDV, TTMV) DNA using nested PCR. The majority of samples were from healthy blood donors and non- Qatari male nationals. Age, gender and nationality distri- butions were not significantly different in healthy blood donors and HBV- or HCV-positive patients. Blood do- nors in Qatar were predominantly males, and so did those tested positive for HBV and HCV.