title: Low rate shedding of HSV-1 DNA, but not of infectious virus from human donor corneae into culture media.
abstract: 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.
authors.affiliation: Department of Ophthalmology, University of Bern, Switzerland.
authors.forename: J G
authors.initials: JG
authors.lastname: Garweg
authors.forename: M
authors.initials: M
authors.lastname: Boehnke
mesh_terms: D000956:Antigens, Viral / Q000235:genetics; D002452:Cell Count; D003315:Cornea / Q000821:virology*; D016039:Corneal Transplantation; D003470:Culture Media; D004279:DNA, Viral / Q000032:analysis*; D004847:Epithelial Cells; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D009924:Organ Culture Techniques; D013937:Thymidine Kinase / Q000235:genetics; D014019:Tissue Donors; D014759:Viral Envelope Proteins / Q000235:genetics; D017201:Virus Shedding
chemical_list: D000956:Antigens, Viral; D003470:Culture Media; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; D013937:Thymidine Kinase