Evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products

Abstract

Background: Severe acute respiratory syndrome coronavirus (SARS-CoV) has been detected in the blood of infected individuals, which may have the potential to contaminate donated blood and plasma-derived products in the event of a future outbreak. Effective methods for inactivating the SARS-CoV in protein solutions are described in this report.

Study design and methods: Heat, ultraviolet (UV) irradiation, octanoic acid, and solvent/detergent (S/D) methods were tested individually for their ability to inactivate SARS-CoV in protein solutions appropriately mimicking blood-derived products. Treated samples were tested for inactivation in a tissue culture growth assay.

Results: Viral inactivation by heat treatment at 60 degrees C required 15 to 30 minutes to inactivate the SARS-CoV. UVC efficiently inactivated SARS-CoV in 40 minutes, whereas UVA required the addition of psoralen to enhance inactivation of the virus. The presence of bovine serum albumin limited the ability of UVC and UVA to inactivate SARS-CoV and octanoic acid treatment does not reduce the infectivity of SARS-CoV-spiked protein solutions. S/D treatment required 2, 4, and up to 24 hours for Triton X-100, Tween 80, and sodium cholate inactivation, respectively.

Conclusion: Heat, UVC irradiation, and S/D treatments effectively inactivate SARS-CoV, whereas octanoic acid treatment is insufficient for inactivation of the virus.

Figure 1

Inactivation of SARS‐CoV by heat treatment of (A ) human serum and (B ) BSA solutions. SARS‐CoV was diluted 1:10 in human serum, PBS, or BSA solutions. The virus/diluent mixtures were incubated in a water bath at (A) 56°C (○) and 65°C (▪) and (B) 60°C, and samples were removed at designated time points. Samples were frozen and analyzed simultaneously by TCID₅₀ assay. The broken line shows the limit of detection for the assay. (□) PBS; (▪) 10 percent BSA; ( ) 16 percent BSA; ( ) 25 percent BSA.

Figure 2

Inactivation of SARS‐CoV by ultraviolet light. (A ) Virus was incubated in PBS, with and without BSA, and exposed to UVC light for the indicated times. (□) PBS; (▪) 10 percent BSA; ( ) 16 percent BSA; ( ) 25 percent BSA. (B ) Virus was mixed into DMEM, with and without 10 percent BSA. (□) DMEM, psoralen; (▪) 10 percent BSA, psoralen; ( ) DMEM, no psoralen; ( ) 10 BSA, no psoralen. Psoralen was added to half of the viral samples. All viral samples were analyzed for TCID₅₀ after exposure to UVA. The broken line shows the limit of detection for the assay.

Figure 3

Octanoic acid treatment of virus. (A ) SARS‐CoV was diluted into 10 percent (□), 16 percent ( ), and 25 percent (▪) BSA solutions with octanoic acid (20 mmol/L) or PBS. (B ) Octanoic acid (40 mmol/L) was added to virus stocks (RSV or HSV‐1) in DMEM and then analyzed for infectivity by TCID₅₀ assay. The broken line shows the limit of detection for the assay. (□) Time 0; ( ) 1 hour; (▪) 24 hours.

Figure 4

S/D treatment of SARS‐CoV. Virus was diluted in PBS or BSA solutions. The S/D concentrate (20×) was added and samples were removed at designated time points and frozen immediately. The detergents used were (A ) Triton X‐100, (B ) Tween 80, and (C ) sodium cholate. The broken line shows the limit of detection for the TCID₅₀ assay. (□) PBS; (▪) 10 percent BSA; ( ) 16 percent BSA; ( ) 25 percent BSA.