The cytokines used in this experiment 
- Interferon (IFN)-γ
- IL-1β
- IL-2
- IL-4
- IL-6
- IL-8
- IL-10
- IL-12p70
- IL-13
- tumor necrosis factor-α

Tulane University

- Virus
- Animals and Procedure
- Histopathologic Scoring
- Immunohistochemistry
- Cytokine Production in Plasma
- Detection of Binding IgG Antibodies in Plasma
- Statistical Analysis

Materials and Methods

Blair, R. V., Vaccari, M., Doyle-Meyers, L. A., Roy, C. J., Russell-Lodrigue, K., Fahlberg, M., . . . Rappaport, J. (2021). Acute respiratory distress in aged, sars-cov-2–infected african green monkeys but not rhesus macaques. The American Journal of Pathology, 191(2), 274-282. doi:10.1016/j.ajpath.2020.10.016

Acute Respiratory Distress in Aged, SARS-CoV-2–Infected African Green Monkeys but Not Rhesus Macaques

Animals were observed twice-daily for 28 days. Samples include blood, cerebrospinal fluid, feces, urine, bronchoalveolar lavage and mucosal swabs. These samples were collected at pre-exposure and post-exposure days and necropsy. Physical examination and imaging (radiography, PET, computed tomography) occurred 7 days prior to exposure and a week after exposure. Following the euthanization of the animals, samples from the left cranial and caudal lung lobes were processed in the media and analyzed. Right lung lobes contained in a fixative to preserve the samples. 

Animals and Procedure

Pulmonary pathology, a measurement of the severity of ARDs, was scored using random forest tissue segmentation algorithms. These algorithms recognized fibrin, edema and cellular inflammation using HALO software. Tissues samples from each of the right lung lobes were analyzed to determine the percentage of inflamed tissue. The percentage of inflammation was converted to a pathology score based on the following scoring system: fibrin and edema score of 0, 0% to 2%; 1, 2% to 5%; 2, 5% to 15%; 3, 15% to 30%; and 4, >30%; and cellular inflammation score of 0, 0% to 0.5%; 1, 0.5% to 3%; 2, 3% to 6%; 3, 6% to 12%; and 4, >12%.

Histopathologic Scoring

The lung samples, specifically 5 micrometer sections, were incubated for 1 hour with antibodies. These include
- SARS-CoV-2 nucleoprotein, mouse IgG1
- ACE2, rabbit polyclonal
- Iba-1, rabbit polyclonal
- Pancytokeratin, rabbit polyclonal

Secondary antibodies dyed with Alexa Fluor fluorochromes and diluted in NGS. 


Immunohistochemistry

Cytokine Production in Plasma

Analysis of serum samples included testing for IgG antibodies against SARS-CoV-2 S1/S2 using an enzyme-linked immunosorbent assay kit. Positive samples were indicated as a difference of ≥0.300 between absorbance values of the positive viral antigen and those of the negative control antigen (positive samples had an MFIA score of ≥3.0). 

 Detection of Binding IgG Antibody in Plasma

Statistical softwares such as GraphPad Prism was used to analyze the data. In order to compare viral loads between RMs and AGMs, the Mann-Whitney U-Test was performed. The Pearson correlation test determined a correlational relationship between cytokines and viral loads. 

Statistical Analysis

In this experimental procedure, the virus used was SARS-Cov-2. Viral stock was prepared in Vero E6 cells and analyzed using deep sequencing.

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