Inactivation of VACV and MPXV
For inactivation experiments, published protocols for virus inactivation are used as a starting point [22, 23]. Here, detergents were added to blood products to inactivate enveloped viruses by removal of the viral membrane in combination with heat inactivation. To this aim, VACV VR-1536 (ATCC #VR-1536) or MPXV clade IIb (in-house isolate) were diluted 1:4 in 20 % human serum albumin (HSA, PAN Biotech) to simulate a viraemic serum sample. Inactivation reagent was prepared as a stock solution in PBS and diluted 1:3 in simulated viraemic serum to a final concentration of 0.3 % tri-n-butyl-phosphate (TnBP, Merck)/0.3 % polysorbate 80 (Tween-80, Sigma-Aldrich)/1 % octoxynol-9 (Triton X-100, Sigma-Aldrich) or 1.5 % Tween-20/1.5 % Triton X-100 and incubated either at room temperature, 30 min at 56 °C or 1 h at 60 °C. As a control an equal amount of buffer (PBS) was added to the simulated serum sample. Efficacy of the inactivation treatment was tested by the reduction of infectious doses in cell culture. To do this, the treated samples were filtered through DetergentOUT GBS10-5000 spin columns (G-Biosciences) according to the manufacturers’ recommendations to remove interfering detergents. Subsequently, the flow-through was diluted in cell culture medium (DMEM, with 10 % FCS), and 100 µL/well of dilutions ranging from 100 to 10-7 were added to 96-well plates pre-seeded with Vero E6 cells. After 7 days of incubation at 37 °C, 5 % CO2, the cells were analysed for CPE by light microscopy, and the wells with CPE were counted to calculate the TCID50/mL. Additionally, for verification of inactivation, three replicates of simulated viraemic VACV serum treated with 1.5 % Tween-20/1.5 % Triton X-100 were then heated for 30 min at 56 °C and were passaged three times in cell culture on Vero E6 cells. For this purpose, the supernatants of the dilutions 100 to 10-5 of the first titration step were pooled and the corresponding cells trypsinised and added to the supernatant. 1 mL of supernatant/cell suspension was added to prepared 6-well plates with non-confluent Vero E6 cells and incubated for seven days. After incubation, the cells were trypsinised and pooled with the corresponding supernatants and 0.5 mL were added to fresh cells in a 6-well plate. This procedure was repeated once again for a total of three rounds of passaging. Finally, the cells were lysed by three freeze/thaw cycles and the DNA was extracted with the Qiagen DNA Blood kit according to the manufacturers’ recommendations. The amount of poxvirus DNA was determined using a pan-OPXV real-time-PCR targeting the rpo gene as described elsewhere [24].