Evaluation of Virulence in Cynomolgus Macaques Using a Virus Preparation Enriched for the Extracellular Form of Monkeypox Virus

Abstract

The 2022 global human monkeypox outbreak emphasizes the importance of maintaining poxvirus research, including enriching a basic understanding of animal models for developing and advancing therapeutics and vaccines. Intravenous administration of monkeypox virus in macaques is arguably one of the best animal models for evaluating the efficacy of medical countermeasures. Here we addressed one criticism of the model, a requirement for a high-titer administration of virus, as well as improving our understanding of monkeypox virus pathogenesis. To do so, we infected macaques with a challenge dose containing a characterized inoculum enriched for the extracellular form of monkeypox virus. Although there were some differences between diseases caused by the enriched preparation compared with a relatively similar unpurified preparation, we were unable to reduce the viral input with the enriched preparation and maintain severe disease. We found that inherent factors contained within the serum of nonhuman primate blood affect the stability of the monkeypox extracellular virions. As a first step to study a role of the extracellular form in transmission, we also showed the presence of this form in the oropharyngeal swabs from nonhuman primates exposed to monkeypox virus.

Keywords: countermeasures; dissemination; extracellular virion; model; monkeypox; morphogenesis; nonhuman primates; orthopoxvirus; smallpox; transmission; viremia.

Figure 1

Evaluation of fresh preparations of monkeypox virus from supernatants using different multiplicities of infections and harvest times. High-multiplicity of infections (3 and 30 pfu/cell) were utilized for synchronization of viral infection on confluent Vero E6 cells plated in T175 flasks. Three experiments were conducted for each MOI (A) and time point (B). Titers are given (PFU/mL) as well as variability, as determined by % coefficient of variation (CV), and are shown in text boxes on the graph.

Figure 2

Immunoelectron microscopy of a fresh preparation of monkeypox virus using either an anti-EV antibody (10F10) or anti-MV antibody (5D8). To confirm the relative absence or presence of EV in our crude and/or fresh preparations of monkeypox virus, immunoelectron microscopy was utilized. (A) Crude preparation with anti-MV antibody; (B) fresh preparation with anti-EV antibody. Scale bars are 100 nM.

Figure 3

Titration of inoculum in the presence or absence of anti-MV antibody. (A) Back titration of the inoculum was performed by either pipetting or expunging the inoculum through a syringe subsequent to plaque assay. Antibody known to neutralize the MV form of monkeypox virus (7D11) was used to evaluate the morphogenic content (EV and MV). (B) The inoculums subsequently underwent four sequences of a freeze/thaw and three sonication cycles, after which a neutralization assay was performed.

Figure 4

Assessment of lesion burden and QPCR. Lesions (A), viremia (QPCR) (B) over time are shown. Mean with standard error of the mean is shown for lesion counts (A) and median and error are shown for QPCR data (B). Animal #3 (EV) was shown to have extravasation of the challenge inoculum and is shown in black (square symbol). Green line, lower limit of detection. Horizontal dotted line, lower limit of quantitation. Blood obtained previous to exposure is shown on the X-axis as “Pre-exposure”. Blood obtained within 2 min of intravenous exposure was on Day 0 and is shown on the X-axis as “Exposure”.

Figure 5

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).

Figure 6

Effect of serum on monkeypox virus comet formation. (A,B) Two concentrations of monkeypox virus were adsorbed on Vero E6 cells for 2 h and unattached virus was subsequently removed by washing. A final concentration of 10% cynomolgus serum or heat-inactivated serum in the presence or absence of anti-L1 antibody (1:500) was used to overlay the cells and a 1.5% methylcellulose overlay with and without anti-L1 antibody was utilized as a no-comet control and to determine the effect the antibody on the primary plaques. (C) Percent primary plaque reduction in the presence of anti-L1 antibody within each matrix listed on the X axis. (D) Percent primary plaque reduction of serum using either the heat-inactivated serum or media to normalize. High virus concentration was a target of 100 pfu/well, whereas intermediate virus concentration was a target of 50 pfu/well.

Figure 7

IEM of EV detected in oropharyngeal swabs of intravenously infected macaques. Oral swabs were collected three days post-exposure (A–C,G) or cell culture (D–F) with monkeypox virus prepared and stained with antibody against EV (A–E,G) or MV (F) for IEM. Cell cultures of monkeypox virus were either prepared with (D,E) or without (F) purification. Measurements of individual virions are given for (B). For (A–D,G), scale bars are 100 nM; 500 nM for (E,F).