Effect of nonhuman primate sera on MV neutralization of freshly prepared and freeze/thawed preparations of monkeypox virus. (A) A general outline of the experimental design. (B) Fresh preparations of monkeypox virus were split and incubated with either cynomolgus (Cyno) serum or HI serum and anti-L1 ab (7D11) or PBS (n = 3). (C) This was repeated with AGM serum (n = 2). A portion of these samples were freeze/thaw/sonicated before the addition of serum, antibody, or PBS control (B,C). (D,E) Our animal virus stock (crude stock) that has undergone multiple freeze/thaw and sonication cycles was treated similarly, n = 3. Statistical differences, using a two-way ANOVA and Sidak’s multiple comparisons (Graphpad Prism), were noted (**** p < 0.0001, *** p < 0.001 and ** p < 0.01). Consolidated table of neutralization values are given for cynomolgus macaque sera (F) and AGM sera (G). There was no statistical difference between serum, crude, and any of the freeze thaw groups (p > 0.05). There was also no statistical difference between analogous cynomolgus macaque and AGM treatment groups (p > 0.05).