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 fo r fragments of the glycoprotein D- and thymidine kinase-encoding genes . Of the culture media, 134 were processed immediately after withdrawa l (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 differe nt storage conditions and of human and HSV-1 DNA as a sequential funct ion of time at ambient temperature was gauged by the loss of a detecta ble signal for the respective component. Endothelial cell density with in each of the corneal discs was determined before and after organ cul ture. In 7/451 culture fluid samples, HSV-1 DNA corresponding to eithe r the glycoprotein D- or thymidine kinase-encoding genes was detected. In culture fluid samples derived from Groups 2 (at ambient temperatur e, for 6 to 60 weeks) and 3 (at -8 degrees C, for 4 to 9 weeks), compl ete degradation precluded the detection of human DNA, and hence probab ly 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; additional ly, 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-v olume samples by electroseparation. When shed into culture fluid, it i s remarkably prone to a time and temperature dependent degradation, wh ich 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 cultu re. (C) 1997 Wiley-Liss, Inc.