Background: Torque teno virus is a small chronically persisting circular negative ssDNA virus reaching near 100% prevalence. It is reported to be a marker for immune function in immunocompromised patients. The possibility of vertical maternal-fetal transmission remains controversial but incidence rate of TTV DNA in children increased with age. TTV dynamics well studied for allogeneic hematopoietic stem cell transplantation as a predictor of post- transplant complications but there is no viral proliferation kinetics data for other patient groups or healthy individuals. The aim of this study was to determine TTV dynamics during the first year of life of healthy infants. Methods: Ninety eight clinically healthy breastfeeding infants (1–12 months of age) were analyzed by quantitative PCR for the whole blood TTV load with the test sensitivity of about 1000 viral copies per milliliter of blood (total number of samples including repeatedly tested infants was 109). Results: 67% of all analyzed samples were TTV-positive demonstrating significant positive correlation between age and TTV load (r = 0.81, p < 0.01). Conclusions: This is the first study to suggest that viral load increases during the first year of life reaching a plateau after 6 months with strong proliferation for the first 60 days. Our data well correlates with TTV dynamics in patients following allogeneic hematopoietic stem cell transplantation. Keywords: Torque teno virus, Transfusion-transmitted virus, TTV, Viral load dynamics, Neonatal period, Infants, TORCH infections Background with age [13, 15, 19], but there is no information about Torque teno virus (TTV) is a small chronically persisting the viral load during the first months of life. circular negative ssDNA virus reaching near 100% preva- TTV dynamics well studied for allogeneic hematopoietic lence [1, 2]. TTV is transmitted in all ways including con- stem cell transplantation as a predictor of post-transplant tact and respiratory . It was suggested that presence of complications [21, 22]. But there is no viral proliferation TTV can cause several diseases such as acute respiratory kinetics data for other patient groups or healthy diseases , liver diseases [5, 6] and cancer , but this individuals. data did not have any convincing support. It is reported to The aim of this study was to determine TTV dynamics be a marker for immune function in immunocomprom- during the first year of life of healthy breastfeeding ised patients . infants. The routes of mother-to-child transmission of TTV have not been fully elucidated and the possibility of ver- Methods tical maternal-fetal transplacental transmission remains Patients and blood samples collection controversial [9–20]. Also, several authors demonstrated This prospective single-center study included 98 clinic- that incidence rate of TTV DNA in children increased ally healthy breastfeeding infants (1–12 months of age, number per month as 9; 6; 13; 8; 11; 14; 6; 9; 10; 6; 4; 2 * Correspondence: firstname.lastname@example.org Kulakov National Medical Research Center for Obstetrics, Gynecology and accordingly). 10 infants were tested repeatedly (2 or 3 Perinatology, Oparina 4, Moscow 117513, Russia times), so the total number of samples was 109. The ex- Pirogov Russian National Research Medical University, Ostrovityanova 1, clusion criteria were as follows: any infectious or genetic Moscow 117997, Russia © The Author(s). 2018 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. Tyschik et al. Virology Journal (2018) 15:96 Page 2 of 4 Fig. 1 Torque teno virus dynamics during the first year of life. Data for 0 months from . Numbers correspond to sample size disease, any immunological deviations, voluntary refusal DNA extraction room (Zone 1). To prevent cross-contam- of research. Two separate aliquots of each capillary blood ination of the samples, all procedures were carried out in sample were collected into Microvette 200 K3EDTA the UV-equipped PCR-box using sterile disposable tubes (Sarstedt, Germany) between June 2017 and January and aerosol-resistant tips. 2018 at the Kulakov National Medical Research Center for Obstetrics, Gynecology, and Perinatology (Moscow, TTV quantification Russia). Samples were stored at − 20 °C for 1–7 days qPCR was performed using the DTprime Real-Time until DNA extraction. PCR Cycler (DNA-Technology, Russia) as described in , with the test sensitivity of about 1000 viral copies DNA extraction per milliliter of blood. qPCR of the unique human genome DNA was extracted from 50 μl aliquots of thawed whole fragment (in a separate PCR tube) was used as DNA ex- blood using a standard commercial silica-sorbent kit for traction control. To prevent PCR contamination by previ- DNA extraction from body fluids (Probe-GS DNA Extrac- ous reactions or biological samples, the reactions were tion Kit, DNA-Technology, Russia). To prevent exogenous combined using aerosol-resistant tips in UV-equipped contamination, DNA isolation was performed in a separate PCR-box in a separate PCR-preparation room (Zone 2). Fig. 2 Percent of TTV DNA positive infants during the first months of life. Data from [15, 19] based on serum analysis Tyschik et al. Virology Journal (2018) 15:96 Page 3 of 4 Also, no electrophoresis of TTV PCR products or other the first months of life by serum analysis [15, 19]. Our procedures that would require PCR-tube opening were whole blood results correlates with previous serum data, performed in the building. All the negative controls and expectantly showing a greater percentage of positive surface washings were negative. samples (see Fig. 2). Breast milk is often positive for TTV (23.3–67.3%) [13, Data analysis 14, 26] and it has been suggested to be one of the major qPCR data were analyzed using the DTprime Real-Time routes of Torque teno virus transmission for babies. So, PCR Cycler Software v.7.7 (DNA-Technology, Russia). the newborn TTV progression may be a consequence of Microsoft Office Excel 2016 (Microsoft Corporation, the mother’s breast milk TTV or immune system changes USA) and GraphPad Prism 6 (GraphPad Software, USA) during the neonatal period. were used for statistical analysis. Conclusions Results This is the first study to suggest that TTV viral load in- TTV whole blood viral load was quantified for 98 infants creases during the first months of healthy infants develop- at 1–12 months of age (see. Fig. 1). Because of logistic ment reaching a plateau after 3–6months with strong difficulties only 10 infants were tested repeatedly (2 or 3 proliferation for the first 60 days. Fast viral load increasing times) (blue lines at Fig. 1) and only several mother-child correlates with previous data on TTV DNA prevalence. pairs were examined for TTV load (data not shown). No Also, neonatal TTV dynamics is similar to TTV prolifera- breast milk samples were tested. tion in patients following allogeneic hematopoietic stem 67% of all analyzed samples were TTV-positive (higher cell transplantation, demonstrating the possible similarity than 10 copies per 1 mL of whole blood as a test sensitiv- of intracellular mechanisms of viral progression. ity) with median 5 × 10 viral genomes per 1 mL (range of Abbreviations median values: 0–1,6 × 10 ) demonstrating significant PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction; positive correlation between age and TTV load (r =0.81, ssDNA: Single stranded deoxyribonucleic acid; TTV: Torque teno virus; p <0.01). UV: Ultraviolet For 10 repeatedly tested infants 3 did not show TTV Authors’ contributions for both tests, 1 was unchanged and other 6 show more EAT and ASR conducted molecular studies and drafted the manuscript, AVD TTV in a second analysis (see Fig. 1). performed the sampling, DVR and GTS designed the study and drafted the manuscript. All authors read and approved the final version of the manuscript. Discussions Ethics approval and consent to participate Despite more than twenty years of TTV research, the The study protocol was reviewed and approved by the Ethics Committee of routes of mother-to-child transmission have not been the Pirogov Russian National Research Medical University (Protocol No.2017/23); the study was conducted in accordance with the Declaration of Helsinki. fully elucidated and the possibility of transplacental TTV All participants (children’s parents) provided written informed consent. transmission remains controversial [9–20]. Some authors demonstrate the absence of TTV in cord blood or Competing interests baby blood after delivery [10, 13, 20], but other show The authors declare that they have no competing interests. 13.8–48.1% of TTV-DNA positive cord blood samples [9, 11, 14, 18]. Such differences can be explained by Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in the low sensitivity of PCR (for studies where the virus published maps and institutional affiliations. did not detected) or by PCR-product contamination (since the cord blood TTV was usually detected by Received: 16 March 2018 Accepted: 21 May 2018 contamination-friendly Nested-PCR technique). In any case, it can be argued that even if the virus passes the References transplacental barrier, the cord blood viral concentra- 1. Vasilyev EV, Trofimov DY, Tonevitsky AG, Ilinsky VV, Korostin DO, Rebrikov tion is very low and does not depend on the mothers DV. 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Published: May 30, 2018