Low rate shedding of HSV-1 DNA, but not of infectious virus from human donor corneae into culture media

J Med Virol. 1997 Jul;52(3):320-5. doi: 10.1002/(sici)1096-9071(199707)52:3<320::aid-jmv14>3.0.co;2-m.


Fluid samples derived from 451 organ cultured corneae were tested for the presence of HSV-1 DNA after electroseparation and amplification for fragments of the glycoprotein D- and thymidine kinase-encoding genes. Of the culture media, 134 were processed immediately after withdrawal (Group 1); 100 were stored at ambient temperature for 6 to 60 weeks (Group 2); 90 were stored at -8 degrees C for 4 to 9 weeks (Group 3); and 127 were stored at -20 degrees C for 2 to 30 weeks (Group 4). The degradation of human DNA (marker gene, betaglobin) under these different storage conditions and of human and HSV-1 DNA as a sequential function of time at ambient temperature was gauged by the loss of a detectable signal for the respective component. Endothelial cell density within each of the corneal discs was determined before and after organ culture. In 7/451 culture fluid samples, HSV-1 DNA corresponding to either the glycoprotein D- or thymidine kinase-encoding genes was detected. In culture fluid samples derived from Group 2 at ambient temperature, for 6 to 60 weeks) and 3 (at -8 degrees C, for 4 to 9 weeks), complete degradation precluded the detection of human DNA, and hence probably also of HSV-1 DNA; only at -20 degrees C did DNA remain stable for protracted periods of time. Even so, HSV-1 DNA was detected in only 2% of those media in which no degradation was to be expected; additionally, there existed no correlation between its presence in culture fluid samples and the loss of endothelial cells or cytopathic changes. DNA can be extracted successfully and concentrated twenty-fold from high-volume samples by electroseparation. When shed into culture fluid, it is remarkably prone to a time and temperature dependent degradation, which may lead to false negative results. It is concluded that there is no infectious virus to be expected in the specimens; the occurrence of HSV-1 DNA in donor corneae would not appear to be an important factor influencing their biological quality during the period of organ culture.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Viral / genetics
  • Cell Count
  • Cornea / virology*
  • Corneal Transplantation
  • Culture Media
  • DNA, Viral / analysis*
  • Epithelial Cells
  • Herpesvirus 1, Human / genetics*
  • Humans
  • Organ Culture Techniques
  • Thymidine Kinase / genetics
  • Tissue Donors
  • Viral Envelope Proteins / genetics
  • Virus Shedding*


  • Antigens, Viral
  • Culture Media
  • DNA, Viral
  • Viral Envelope Proteins
  • glycoprotein D, Human herpesvirus 1
  • Thymidine Kinase