Neutralization test
Micro-neutralization tests (NT) were set up with three different OPXV strains: MPXV clade IIb (in-house isolate), VACV Lister-Elstree Bavarian Nordic (LELS-2003-007, Bavarian Nordic GmbH) and CPXV strain HumGri07/1 (in-house isolate) [16]. For this purpose, Vero E6 cells (#85020206, European Collection of Authenticated Cell Cultures (ECACC)) in DMEM (Gibco) with 10 % FCS (Sigma) were infected at MOI ⁓ 0.1. After four to seven days — depending on the morphological cell status — the infected cells were lysed by three freeze-thaw cycles and the virus suspension was vortexed for 10 sec. Cell debris was pelleted for 10 min at 1300×g, and from the supernatant a stock virus solution with a target titre of 1000 TCID50/mL was prepared in cell culture medium and stored in aliquots at -80 °C. The stock titre was confirmed by triplicate titration. For detection of neutralising antibodies, the patient sera were diluted in medium (DMEM) to six two-fold dilution steps, resulting in dilutions of 1:10 to 1:320. 500 µL of virus stock were added to 500 µL of each respective serum dilution and mixed by pipetting up and down. Following incubation at RT for one hour, 100 µL of each virus/serum dilution were added per well in eight replicates to 96-well plates containing Vero E6 cells seeded the previous day (1.5×104 cells/well in 100 µL of medium) and incubated at 37 °C, 5 % CO2, for 7 days. In each experiment, the virus stock used was titrated as a control. For this purpose, 1 mL of the virus dilution was mixed with 1 mL of medium and incubated at RT for 1 h. Decimal dilutions were prepared in medium, resulting in dilutions of 1:101 to 1:109, then 100 µL of each virus dilution were added per well in eight replicates to a 96-well plate pre-seeded with Vero E6 cells (1.5×104cells/well in 100 µL of medium). As a negative control, 100 µL of medium/well was added to eight wells. After 7 days the cells were inspected by microscopy for CPE. The titre of the virus stock was calculated according to the following formula: TCID50/mL = 10^((n/8+0.5))/0.5 where n = number of wells with CPE. The titre of the patient sera was calculated according to the following formula: Titre (1:x) =10×2^((n/8+0.5)) where n = number of wells without CPE. In case that not all wells in the last dilution step showed a CPE, the titre was indicated as “≥”.