Experimental infection of cynomolgus macaques (Macaca fascicularis) with aerosolized monkeypox virus

Abstract

Monkeypox virus (MPXV) infection in humans results in clinical symptoms very similar to ordinary smallpox. Aerosol is a route of secondary transmission for monkeypox, and a primary route of smallpox transmission in humans. Therefore, an animal model for aerosol exposure to MPXV is needed to test medical countermeasures. To characterize the pathogenesis in cynomolgus macaques (Macaca fascicularis), groups of macaques were exposed to four different doses of aerosolized MPXV. Blood was collected the day before, and every other day after exposure and assessed for complete blood count (CBC), clinical chemistry analysis, and quantitative PCR. Macaques showed mild anorexia, depression, and fever on day 6 post-exposure. Lymphadenopathy, which differentiates monkeypox from smallpox, was observed in exposed macaques around day 6 post-exposure. CBC and clinical chemistries showed abnormalities similar to human monkeypox cases. Whole blood and throat swab viral loads peaked around day 10, and in survivors, gradually decreased until day 28 post-exposure. Survival was not dose dependent. As such, doses of 4 × 10(4) PFU, 1 × 10(5) PFU, or 1 × 10(6) PFU resulted in lethality for 70% of the animals, whereas a dose of 4 × 10(5) PFU resulted in 85% lethality. Overall, cynomolgus macaques exposed to aerosolized MPXV develop a clinical disease that resembles that of human monkeypox. These findings provide a strong foundation for the use of aerosolized MPXV exposure of cynomolgus macaques as an animal model to test medical countermeasures against orthopoxviruses.

Figure 1. Clinical disease scores, survival, temperature and weight.

A) Changes in average clinical scores and B) percent survival of macaques exposed to different doses of aerosolized MPXV. C) Average body temperature of macaques exposed to different doses of aerosolized MPXV. The temperature data was collected every 15 min from implanted telemetry devices. The dashed line indicates the average baseline body temperature; n: number of animals. D) Changes in average body weight of survivors and non-survivors exposed to aerosolized MPXV. n: number of animals.

Figure 2. Average number of leukocytes and platelets in macaques after exposed to aerosolized MPXV.

The dotted lines indicate the normal reference range; n: number of animals. Graphs are shown for A) total white blood cells (WBC), B) percentage of granulocytes (GR), C) percentage of lymphocytes (LY), D) platelets (PLT) for all MPXV dosage groups, and survivors versus non-survivors (right).

Figure 3. Serum chemistries in macaques exposed to aerosolized MPXV.

The dotted lines indicate the normal reference range; n: number of animals. Graphs show average A) total protein, B) albumin, C) lactate dehydrogenase (LDH), D) C-reactive protein, E) aspartate transaminase (AST), F) and alanine transaminase (ALT), G) urea nitrogen.

Figure 4. Average number of MPXV viral genomes in whole blood and throat swabs.

The average number of MPXV viral genomes in A) whole blood, and B) throat swabs from different MPXV dosage groups, and survivors versus non-survivors (right). The X-axis is at the limit of detection  = 5000 genomes/ml; n: number of animals.

Figure 5. Viral load in tissues.

A) Tissue viral load of macaques exposed to different doses of aerosolized MXPV and euthanized during the acute phase of disease (survivors are not included). B) Tissue viral load in survivors and non-survivors. The X-axis is at the limit of detection = 5000 genomes/g; n: number of animals.

Figure 6. Pathology and presence of MPXV antigen in lung tissue.

Figures A–D are histological sections of lung tissues from cynomolgus macaques infected via aerosolized MPXV. Positive immunoreactivity for orthopoxvirus antigen, shown as brown staining, is associated with necrotizing lesions primarily concentrated around bronchi and bronchioles. [Immunoperoxidase method using rabbit polyclonal antibody to vaccinia virus; original magnification ×40 (Figure 6A) or ×20 (Figure 6 B, C, D)]. A) 4×10⁴ PFU (day 10 post-exposure). B) 1×10⁵ PFU (day 8 post-exposure). C) 4×10⁵ PFU (day 11 post-exposure). D) 1×10⁶ PFU (day 9 post-exposure). E) Percent immunoreactivity in the lungs of non-survivors by dosage group, measured by digital microscopy image analysis.