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

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°C for 4 to 9 weeks (Group 3); and 127 were stored at −20°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 Groups 2 at ambient temperature, for 6 to 60 weeks) and 3 (at −8°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°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. J. Med. Virol. 52:320–325, 1997. © 1997 Wiley‐Liss, Inc.