100 Applied Biosafety, 12(2) pp. 100-108 ABSA 2007 Abstract When the first wave of the SARS epidemic seemed to have reached its conclusion, it was completely unclear how the spread of the virus would evolve. Taking into account all the uncertainties and anticipating the worst-case scenario, many laboratories and vaccine manufacturers started working on a vaccine approach against SARS infection. The results presented here describe the evaluation of decontamination practices performed within the framework of a SARS- Coronavirus (SARS-CoV) vaccine development project. We show that it takes 45 days at room temperature to fully inactivate the human SARS-CoV, whereas an enveloped virus such as the rabies virus, when treated in a similar way, is totally inactivated in three days. Moreover, the SARS-CoV is very resistant to alkaline treatment and, even more surpris- ingly, formaldehyde fumigation is not efficacious on the dried virus (under the conditions tested). Only heat (autoclave) and hypochlorite chloride treatments are efficacious treatments for the decontamination of SARS-CoV. Introduction In the 1990s, vaccine manufacturers faced a huge increase in the need and demand for vaccines. Existing production processes needed to be optimized to meet the international demands. Amongst the different solutions, the establishment of sequential vaccine production cam- paigns was an attractive strategy, requiring no additional investment in terms of facilities. However, the issue of cross-contamination between two different viruses han- dled successively in the same building needed careful at- tention. The validation of the decontamination of infec- tious agents became a prerequisite. One of the first steps was to determine the time nec- essary for a virus to become inactivated at room tempera- ture. As expected, there are viruses that are relatively resis- tant, e.g., polio virus (Milstien et al., 1997) and viruses that are very fragile, e.g., rabies virus (Saluzzo Klamm et al., 1991; Sorodoc et al., 1979), but very resistant when the stabilizer (solution of sugar and amino acids), used in the final vaccine formulation, is added (Melnick, 1996). A similar observation was made for influenza virus in cell culture medium solution, which was very sensitive to dry- ing, but more resistant when recovered from allantoic fluid of an embryonated egg (Schaffer et al., 1976). More- over, these studies show the relative resistance of certain viruses (polio virus, hepatitis A virus) for alkaline treat- ment (NaOH) (Salo Rutala et al., 2000). The principle lesson learned from these experiments is the extreme diversity of viral characteristics to different decontaminating conditions. Consequently, it is neces- sary to validate the decontamination conditions for each new virus. In this context, and in reference to the previ- ous published work on viral decontamination practices (Sofer et al., 2003), we present here an evaluation of de- contamination practices for the human SARS-CoV. Materials and Methods Virus and Cells The UTAH SARS-Coronavirus was kindly provided by Pat Campbell, from the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, U.S.A. At the CDC, the virus was cloned and passed twice on Vero cells. Vero cells were grown in flasks to which the virus was added three days later. The harvest was performed three to four days later after infection followed by a 0.8-0.2 ?m filtration. The decontamination studies presented here were performed either with crude SARS-CoV infected Vero cell harvest (in culture medium) or on tenfold con- centrated harvest diafiltered in PBS buffer. The different studies were performed at room temperature (22C +: 3C) with a relative humidity RH of 10-25%. Article Evaluation of SARS-Coronavirus Decontamination Procedures Anne-Marie Pagat, Raphaelle Seux-Goepfert, Charles Lutsch, Valrie Lecouturier, Jean-Franois Saluzzo, and Inca C. Kusters Sanofi Pasteur, Marcy LEtoile, France 101 Determination of Virus Titer The viral quantification was performed in 96 well plates seeded with Vero cells at a density of 8000 cells per well. Tenfold dilutions of the viral suspension to be ti- trated were serially performed in Iscoves 4% FCS (Foetal Calf Serum) medium and 200 ?l of each dilution was added to the Vero cell monolayers (100 ?l per well), with a repetition of six wells per dilution. After three days in- cubation at 37C, with 5% CO2, the cells were examined for cytopathic effects (CPE). The infectious titer was cal- culated using an in-house method (adapted from Spear- man and Krber) and expressed in TCID50 (50% tissue culture infectious dose) units. Virus Amplification Technique To ensure the absence of any residual infectious par- ticle after the decontamination treatment, viral amplifica- tion testing was performed: 6.25 ml of viral suspension to be tested was placed in a 75 cm flask seeded with 2x106 Vero cells the day before. The flask was incubated for 7 days a