Nucleic Acid-Based Screening of Maternal Serum to Detect Viruses in Women with Labor or PROM

  • Ankit A. Shah
  • David Wang
  • Emmet HirschEmail author
Original Article


The purpose of this study was to determine whether timing of the initiating event of spontaneous labor (either uterine contractions with intact fetal membranes or rupture of membranes prior to labor (PROM)) is associated with maternal viral infection. It was a prospective case control study of women with either spontaneous labor or PROM occurring < 37 weeks’ gestation (“cases”) or at term (“controls”). An initial unbiased screen for viruses was performed with next-generation sequencing (NGS) in serum pooled from eight cases delivered by C/S and represents a range of gestational ages, membrane rupture status, and presence or absence of chorioamnionitis. Custom PCR was used to query individual patient samples from the original cohort. The NGS screen generated 15 million reads. Seven unique viral sequences were detected in two cases, all identified as torque teno virus (TTV), an ubiquitous DNA anellovirus of no known pathogenicity. Using nested and semi-nested PCR, sera from 72 patients (47 cases and 25 matched controls, stratified by ROM status) were screened for the 3 subtypes of anelloviruses (TTV, TTMDV, or TTMV). These were found in 43/47 cases (91%) and 16/25 controls (64%) (p = 0.012, OR = 5.9 (95% CI = 1.4–29.9)). In logistic regression, pregnant women with at least one type of anellovirus were more likely to experience preterm labor than those with no anellovirus (p = 0.03, aOR = 4.6, CI = 1.2–18.7). Among women experiencing a spontaneous initiating event of labor, TTV virus was more likely to be present in the serum of preterm than term patients. TTV may have a role in determining the timing of parturition.


High-throughput sequencing Preterm labor Parturition Viral infection 



We thank Justin Barr, Integrated DNA Technologies, Coralville, Iowa, for the assistance of designing the “G-Block” template containing anellovirus DNA sequences used to test the degenerate primer sets. We thank Sabrina Gaiazov, MPH, Epidemiologist at INOVA Translational Medicine Institute, for assisting with statistical analysis.

Funding Information

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that there is no conflict of interest.


  1. 1.
    2017 Premature birth report card. White Plains, NY: March of Dimes Foundation; 2017.Google Scholar
  2. 2.
    Klein LL, Gibbs RS. Use of microbial cultures and antibiotics in the prevention of infection-associated preterm birth. Am J Obstet Gynecol. Jun 2004;190(6):1493–502.CrossRefGoogle Scholar
  3. 3.
    Offenbacher S, Katz V, Fertik G, et al. Periodontal infection as a possible risk factor for preterm low birth weight. J Periodontol Oct. 1996;67(10 Suppl):1103–13.CrossRefGoogle Scholar
  4. 4.
    Romero R, Miranda J, Chaemsaithong P, et al. Sterile and microbial-associated intra-amniotic inflammation in preterm prelabor rupture of membranes. The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. Aug 2015;28(12):1394–409.CrossRefGoogle Scholar
  5. 5.
    Shim SS, Romero R, Hong JS, Park CW, Jun JK, Kim BI, et al. Clinical significance of intra-amniotic inflammation in patients with preterm premature rupture of membranes. Am J Obstet Gynecol. Oct 2004;191(4):1339–45.CrossRefGoogle Scholar
  6. 6.
    Yoon BH, Oh SY, Romero R, Shim SS, Han SY, Park JS, et al. An elevated amniotic fluid matrix metalloproteinase-8 level at the time of mid-trimester genetic amniocentesis is a risk factor for spontaneous preterm delivery. Am J Obstet Gynecol. Nov 2001;185(5):1162–7.CrossRefGoogle Scholar
  7. 7.
    Mosby LG, Rasmussen SA, Jamieson DJ. 2009 pandemic influenza a (H1N1) in pregnancy: a systematic review of the literature. Am J Obstet Gynecol. Jul 2011;205(1):10–8.CrossRefGoogle Scholar
  8. 8.
    Raj RS, Bonney EA, Phillippe M. Influenza, immune system, and pregnancy. Reprod Sci. Dec 2014;21(12):1434–51.CrossRefGoogle Scholar
  9. 9.
    Short CE, Douglas M, Smith JH, Taylor GP. Preterm delivery risk in women initiating antiretroviral therapy to prevent HIV mother-to-child transmission. HIV medicine. Apr 2014;15(4):233–8.CrossRefGoogle Scholar
  10. 10.
    Liu J, Zhang S, Liu M, Wang Q, Shen H, Zhang Y. Maternal pre-pregnancy infection with hepatitis B virus and the risk of preterm birth: a population-based cohort study. The Lancet Global health Jun. 2017;5(6):e624–32.CrossRefGoogle Scholar
  11. 11.
    Huang QT, Huang Q, Zhong M, Wei SS, Luo W, Li F, et al. Chronic hepatitis C virus infection is associated with increased risk of preterm birth: a meta-analysis of observational studies. J Viral Hepat. Dec 2015;22(12):1033–42.CrossRefGoogle Scholar
  12. 12.
    Cardenas I, Means RE, Aldo P, et al. Viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor. J Immunol. Jul 15 2010;185(2):1248–57.CrossRefGoogle Scholar
  13. 13.
    Cardenas I, Mor G, Aldo P, Lang SM, Stabach P, Sharp A, et al. Placental viral infection sensitizes to endotoxin-induced pre-term labor: a double hit hypothesis. Am J Reprod Immunol. Feb 2011;65(2):110–7.CrossRefGoogle Scholar
  14. 14.
    Ilievski V, Hirsch E. Synergy between viral and bacterial toll-like receptors leads to amplification of inflammatory responses and preterm labor in the mouse. Biol Reprod. Nov 2010;83(5):767–73.CrossRefGoogle Scholar
  15. 15.
    De Vlaminck I, Khush KK, Strehl C, et al. Temporal response of the human virome to immunosuppression and antiviral therapy. Cell. Nov 21 2013;155(5):1178–87.CrossRefGoogle Scholar
  16. 16.
    Gaynor AM, Nissen MD, Whiley DM, et al. Identification of a novel polyomavirus from patients with acute respiratory tract infections. PLoS pathogens. May 4 2007;3(5):e64.CrossRefGoogle Scholar
  17. 17.
    Lim ES, Cao S, Holtz LR, Antonio M, Stine OC, Wang D. Discovery of rosavirus 2, a novel variant of a rodent-associated picornavirus, in children from The Gambia. Virology. Apr 2014:454, 25–5, 33.CrossRefGoogle Scholar
  18. 18.
    Wylie KM, Mihindukulasuriya KA, Sodergren E, Weinstock GM, Storch GA. Sequence analysis of the human virome in febrile and afebrile children. PLoS One. 2012;7(6):e27735.CrossRefGoogle Scholar
  19. 19.
    Hirsch E, Goldstein M, Filipovich Y, Wang H. Placental expression of enzymes regulating prostaglandin synthesis and degradation. Am J Obstet Gynecol. Jun 2005;192(6):1836–42 discussion 1842-1833.CrossRefGoogle Scholar
  20. 20.
    Finkbeiner SR, Kirkwood CD, Wang D. Complete genome sequence of a highly divergent astrovirus isolated from a child with acute diarrhea. Virol J. 2008;5:117.CrossRefGoogle Scholar
  21. 21.
    Zhao G, Wu G, Lim ES, Droit L, Krishnamurthy S, Barouch DH, et al. VirusSeeker, a computational pipeline for virus discovery and virome composition analysis. Virology. Mar 2017;503:21–30.CrossRefGoogle Scholar
  22. 22.
    Handley SA, Thackray LB, Zhao G, et al. Pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome. Cell. Oct 12 2012;151(2):253–66.CrossRefGoogle Scholar
  23. 23.
    Li R, Yu C, Li Y, et al. SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. Aug 1 2009;25(15):1966–7.CrossRefGoogle Scholar
  24. 24.
    Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. May 2008;18(5):821–9.CrossRefGoogle Scholar
  25. 25.
    Fassler J, Cooper P. BLAST Glossary. 2011 Jul 14. In: BLAST® Help [Internet]. Bethesda: National Center for Biotechnology Information (US); 2008.Google Scholar
  26. 26.
    Ninomiya M, Takahashi M, Nishizawa T, Shimosegawa T, Okamoto H. Development of PCR assays with nested primers specific for differential detection of three human anelloviruses and early acquisition of dual or triple infection during infancy. J Clin Microbiol. Feb 2008;46(2):507–14.CrossRefGoogle Scholar
  27. 27.
    Biagini P. Classification of TTV and related viruses (anelloviruses). Curr Top Microbiol Immunol. 2009;331:21–33.PubMedGoogle Scholar
  28. 28.
    Focosi D, Antonelli G, Pistello M, Maggi F. Torquetenovirus: the human virome from bench to bedside. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases. Jul 2016;22(7):589–93.CrossRefGoogle Scholar
  29. 29.
    Constantin CM, Masopust D, Gourley T, et al. Normal establishment of virus-specific memory CD8 T cell pool following primary infection during pregnancy. J Immunol. Oct 1 2007;179(7):4383–9.CrossRefGoogle Scholar
  30. 30.
    Lissauer D, Choudhary M, Pachnio A, Goodyear O, Moss PA, Kilby MD. Cytomegalovirus sero positivity dramatically alters the maternal CD8+ T cell repertoire and leads to the accumulation of highly differentiated memory cells during human pregnancy. Hum Reprod. Dec 2011;26(12):3355–65.CrossRefGoogle Scholar
  31. 31.
    Arenas-Hernandez M, Romero R, Xu Y, et al. Effector and activated T cells induce preterm labor and birth that is prevented by treatment with progesterone. J Immunol. May 1 2019;202(9):2585–608.CrossRefGoogle Scholar
  32. 32.
    Bonney EA, Johnson MR. The role of maternal T cell and macrophage activation in preterm birth: cause or consequence? Placenta. Mar 9 2019.Google Scholar
  33. 33.
    Biagini P, Gallian P, Cantaloube JF, Attoui H, de Micco P, de Lamballerie X. Distribution and genetic analysis of TTV and TTMV major phylogenetic groups in French blood donors. J Med Virol. Feb 2006;78(2):298–304.CrossRefGoogle Scholar
  34. 34.
    Bzhalava D, Ekstrom J, Lysholm F, et al. Phylogenetically diverse TT virus viremia among pregnant women. Virology. Oct 25 2012;432(2):427–34.CrossRefGoogle Scholar
  35. 35.
    Hino S, Miyata H. Torque teno virus (TTV): current status. Rev Med Virol. Jan-Feb 2007;17(1):45–57.CrossRefGoogle Scholar
  36. 36.
    Okamoto H. History of discoveries and pathogenicity of TT viruses. Curr Top Microbiol Immunol. 2009;331:1–20.PubMedGoogle Scholar
  37. 37.
    McElvania TeKippe E, Wylie KM, Deych E, Sodergren E, Weinstock G, Storch GA. Increased prevalence of anellovirus in pediatric patients with fever. PLoS One. 2012;7(11):e50937.CrossRefGoogle Scholar
  38. 38.
    Tshering C, Takagi M, Deguchi E. Detection of torque teno sus virus types 1 and 2 by nested polymerase chain reaction in sera of sows at parturition and of their newborn piglets immediately after birth without suckling colostrum and at 24 hr after suckling colostrum. J Vet Med Sci. Mar 2012;74(3):315–9.CrossRefGoogle Scholar
  39. 39.
    Bagaglio S, Sitia G, Prati D, Cella D, Hasson H, Novati R, et al. Mother-to-child transmission of TT virus: sequence analysis of non-coding region of TT virus in infected mother-infant pairs. Arch Virol. Apr 2002;147(4):803–12.CrossRefGoogle Scholar
  40. 40.
    Tyschik EA, Shcherbakova SM, Ibragimov RR, Rebrikov DV. Transplacental transmission of torque teno virus. Virology journal. May 8 2017;14(1):92.CrossRefGoogle Scholar

Copyright information

© Society for Reproductive Investigation 2020

Authors and Affiliations

  1. 1.Department of Obstetrics and GynecologyNorthShore University Health SystemEvanstonUSA
  2. 2.Department of Obstetrics and Gynecology, Pritzker School of MedicineUniversity of ChicagoChicagoUSA
  3. 3.Departments of Molecular Microbiology and Pathology & ImmunologyWashington University School of MedicineSt. LouisUSA

Personalised recommendations