Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Aerosolized Monkeypox Virus

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 4×104 PFU, 1×105 PFU, or 1×106 PFU resulted in lethality for 70% of the animals, whereas a dose of 4×105 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

Pathogenesis of aerosolized Eastern Equine Encephalitis virus infection in guinea pigs

Mice and guinea pigs were experimentally exposed to aerosols containing regionally-distinct strains (NJ1959 or ArgM) of eastern equine encephalitis virus (EEEV) at two exclusive particle size distributions. Mice were more susceptible to either strain of aerosolized EEEV than were guinea pigs; however, clinical signs indicating encephalitis were more readily observed in the guinea pigs. Lower lethality was observed in both species when EEEV was presented at the larger aerosol distribution (> 6 μm), although the differences in the median lethal dose (LD50) were not significant. Virus isolation and immunohistochemistry indicated that virus invaded the brains of guinea pigs within one day postexposure, regardless of viral strain or particle size distribution. Immunohistochemistry further demonstrated that neuroinvasion occurred through the olfactory system, followed by transneuronal spread to all regions of the brain. Olfactory bipolar neurons and neurons throughout the brain were the key viral targets. The main microscopic lesions in infected guinea pigs were neuronal necrosis, inflammation of the meninges and neuropil of the brain, and vasculitis in the brain. These results indicate that guinea pigs experimentally infected by aerosolized EEEV recapitulate several key features of fatal human infection and thus should serve as a suitable animal model for aerosol exposure to EEEV

Determination of Antibiotic Efficacy against Bacillus anthracis in a Mouse Aerosol Challenge Model

An anthrax spore aerosol infection mouse model was developed as a first test of in vivo efficacy of antibiotics identified as active against Bacillus anthracis. Whole-body, 50% lethal dose (LD(50)) aerosol challenge doses in a range of 1.9 × 10(3) to 3.4 × 10(4) CFU with spores of the fully virulent Ames strain were established for three inbred and one outbred mouse strain (A/J, BALB/c, C57BL, and Swiss Webster). The BALB/c strain was further developed as a model for antibiotic efficacy. Time course microbiological examinations of tissue burdens in mice after challenge showed that spores could remain dormant in the lungs while vegetative cells disseminated to the mediastinal lymph nodes and then to the spleen, accompanied by bacteremia. For antibiotic efficacy studies, BALB/c mice were challenged with 50 to 100 LD(50) of spores followed by intraperitoneal injection of either ciprofloxacin at 30 mg/kg of body weight (every 12 h [q12h]) or doxycycline at 40 mg/kg (q6h). A control group was treated with phosphate-buffered saline (PBS) q6h. Treatment was begun 24 h after challenge with groups of 10 mice for 14 or 21 days. The PBS-treated control mice all succumbed (10/10) to inhalation anthrax infection within 72 h. Sixty-day survival rates for ciprofloxacin and doxycycline-treated groups were 8/10 and 9/10, respectively, for 14-day treatment and 10/10 and 7/10 for 21-day treatment. Delayed treatment with ciprofloxacin initiated 36 and 48 h postexposure resulted in 80% survival and was statistically no different than early (24 h) postexposure treatment. Results using this mouse model correlate closely with clinical observations of inhalational anthrax in humans and with earlier antibiotic studies in the nonhuman primate inhalational anthrax model

Pathology and presence of MPXV antigen in lung tissue.

<p>Figures <b>A–D</b> 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012880#pone-0012880-g006" target="_blank">Figure 6A</a>) or ×20 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012880#pone-0012880-g006" target="_blank">Figure 6</a> B, C, D)]. <b>A</b>) 4×10⁴ PFU (day 10 post-exposure). <b>B</b>) 1×10⁵ PFU (day 8 post-exposure). <b>C</b>) 4×10⁵ PFU (day 11 post-exposure). <b>D</b>) 1×10⁶ PFU (day 9 post-exposure). <b>E</b>) Percent immunoreactivity in the lungs of non-survivors by dosage group, measured by digital microscopy image analysis.</p

Key histopathologic lesions in cynomolgus macaques exposed to aerosolized MPXV.

<p>* = lesion seen in survivors<b>.</b></p

Serum chemistries in macaques exposed to aerosolized MPXV.

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

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

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

Summary of inhaled doses, fever, and disease outcome in cynomolgus macaques exposed to aerosolized MPXV.

a<p>Defined as the first day with >8 h of significant temperature elevation (as determined by ARIMA modeling).</p>b<p>Calculated as the number of days (converted to hours) with 12 or more h of significant temperature elevation.</p>c<p>Calculated as the sum of the significant temperature elevations.</p>d<p>The maximum change in temperature.</p>e<p>Calculated by dividing fever hours by fever duration in hours.</p>f<p>Mean time-to-death.</p