M.J. Welsh and A.E. Smith, RAC Application 
Retroviral Particles - The presence of retroviral particles will be assessed by electron 
microscopic analysis of fixed, embedded, and sectioned 293 cell pellets. 
In Vitro Viral Test - The presence of viral contamination will be evaluated by cultivation of 
293 cell lysates on MRC-5 (ATCC CCL171) and VERO (ATCC CCL81) cells. The 
presence of viral contaminants will be assessed by screening for cytopathic effects (CPE) 
and hemagglutination/hemadsorption after 14 days of culture. To enhance sensitivity of 
this assay, blind passage of inoculated cell cultures will also be performed. 
In Vivo Viral Test - The presence of inapparent viral contamination will be evaluated using 
a number of in vivo indicator systems, including adult mice, suckling mice, guinea pig, and 
embryonated hen eggs (yolk sac and allantoic routes of administration). These tests will be 
made by direct inoculation for all test systems, as well as blind passage into additional 
suckling mice and embryonated eggs. All animals and eggs will be monitored for 
morbidity and mortality. 
Bovine Viruses - The presence of bovine viral contamination will be evaluated by 
cultivation of 293 cell lysate on BT cells (bovine turbinate, ATCC CLR 1390). This test 
will detect the presence of bovine viral diarrhea virus, infectious bovine rhinotracheitis, 
bovine adenovirus, bovine parvovirus, and parainfluenza 3. After 14 days, the cultures will 
be examined for CPE and screened for the presence of viral antigens with 
immunofluorescence localization techniques. 
Porcine Parvovirus - The presence of porcine parvovirus originating from trypsin used to 
passage cells will be evaluated by the cultivation of 293 cell lysate on PT-1 (porcine 
testicular) cells. After 14 days, the cultures will be examined for CPE and screened for the 
presence of viral antigen with immunofluorescence localization techniques. 
Human Epstein - Barr Virus - The presence of EBV contamination will be evaluated by 
Southern blot analysis. 
Human cytomegalovirus - The presence of CMV contamination will be evaluated by the 
cultivation of 293 cell lysate on MRC-5 cells. Cultures will be examined for CPE and 
screened for the presence of viral antigen with immunofluorescence localization 
techniques. Blind passage of inoculated cultures will be performed at 21-28 days. 
Human Hepatitis B - The presence of Hepatitis B contamination will be determined by 
testing of 293 cell culture supernatants for Hepatitis B surface antigen with a monoclonal 
ELISA technique. 
Human Immunodeficiency Virus - The presence of HIV will be evaluated by the 
inoculation of 293 cell lysates onto phytohemagglutinin stimulated human peripheral blood 
lymphocytes. The cultures will be monitored for CPE and syncytium formation. Culture 
supernatants will be evaluated by ELISA for the presence of HIV-1 p24 antigen. 
Human parvoviruses - The presence of adeno-associated virus and B19 contamination will 
be evaluated by Southern Blot analysis. 
4. Tests for the 293 Working Cell Bank 
The WCB will be tested for sterility and mycoplasma. 
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Recombinant DNA Research, Volume 16 
