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].