key: cord-0919394-pj1pg3px
authors: Mulka, Kathleen R.; Beck, Sarah E.; Solis, Clarisse V.; Johanson, Andrew L.; Queen, Suzanne E.; McCarron, Megan E.; Richardson, Morgan R.; Zhou, Ruifeng; Marinho, Paula; Jedlicka, Anne; Guerrero-Martin, Selena; Shirk, Erin N.; Braxton, Alicia; Brockhurst, Jacqueline; Creisher, Patrick S.; Dhakal, Santosh; Brayton, Cory F.; Veenhuis, Rebecca T.; Metcalf Pate, Kelly A.; Karakousis, Petros C.; Klein, Sabra L.; Jain, Sanjay K.; Tarwater, Patrick M.; Pekosz, Andrew S.; Villano, Jason S.; Mankowski, Joseph L.
title: Progression and Resolution of SARS-CoV-2 Infection in Golden Syrian Hamsters
date: 2021-06-28
journal: bioRxiv
DOI: 10.1101/2021.06.25.449918
sha: 011d21943c4db0e78761669b7055fee4006d8c3a
doc_id: 919394
cord_uid: pj1pg3px

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

In December 2019, a novel beta coronavirus was isolated from patients that presented trachea, lung (left lobe), esophagus, stomach, small intestine, cecum, large intestine, 174 brain, heart, kidney, liver, gallbladder, spleen, adrenal gland, reproductive organs, 175 urinary bladder, lymph nodes, salivary glands, bone, haired skin, skeletal muscle, bone 176 marrow, and decalcified cross sections of the head.

In situ hybridization and immunohistochemistry 179 In situ hybridization to detect SARS-CoV-2 RNA 180 In situ hybridization (ISH) was performed on 5µm-thick sections of formalin-fixed lung 181 mounted on charged glass slides using the Leica Bond RX immunostaining, the create thresholder function was applied to detect levels of DAB 215 above a threshold that was designated as positive within a given annotated area, or 216 region of interest (ROI). The percent positive ROI was calculated using positive area 217 quantitated by the thresholder divided by total area of the ROI. For SARS-CoV-2 ISH 218 quantitation, the train pixel classifier tool was used. Within an ROI, annotations were 219 created and designated as either positive or ignore, which allowed QuPath to correctly 220 identify areas of positive staining. Percent positive ROI was calculated using positive 221 area detected by the classifier divided by total area of the ROI.

To quantitate consolidation of 7 DPI lungs, the wand tool was used to outline each 224 scanned section of lung, creating an annotation. Superpixels were generated using the 225 DoG superpixel segmentation function in QuPath. These detections were then selected, 226 and intensity features were added. Next, areas within the ROI were annotated using 227 multiple slides of infected and control animals that were designated as "Consolidation", 228 "Non-consolidated", "Atelectasis", or "Ignore". This allowed the classifier to successfully 229 detect areas of affected tissue, while ignoring areas that were unaffected, densely 230 stained due to normal tissue architecture, or collapsed due to variable formalin infusion 231 of the lungs. Percent consolidation was calculated using the number of superpixels 232 identified as consolidated divided by the total number of superpixels detected for a 233 given slide. test was used to evaluate differences between two groups. Infectious virus titers and 266 viral RNA copies were log transformed and compared using two-way ANOVA with 267 mixed-effects analysis followed by Bonferroni's multiple comparison test.

The most robust finding following SARS-CoV-2 inoculation was decreased body weight 272 (Fig 1A) . Animals inoculated with SARS-CoV-2 progressively lost body weight in the 273 first week of infection until the 6 DPI nadir (-13.8% decline in group mean body weight 274 from baseline; 19.3 % lower than control animal group mean at 6 DPI, P < 0.001) before 275 gradually rebounding over the course of the following 3 weeks. At day 28 DPI, control 276 and infected group body weights were not significantly different (P=0.095). Sex CoV-2, and those differences were explored previously 28 .

SARS-CoV-2-inoculated hamsters did not consistently develop clinical signs of 280 respiratory disease; only mild nasal discharge or slight increased respiratory effort was 281 observed intermittently in very few animals.

Peak infectious viral titers in the nasal turbinates, trachea, and lungs were present at 2 284 DPI, then decreased at 4 DPI (Fig. 2) . The highest levels of infectious SARS-CoV-2 285 were found in nasal turbinates at 2 DPI (5.0 x 10 7 TCID50/mL); lung levels were highest at 286 2 DPI (7.33 x 10 6 TCID50). While infectious virus was cleared from the respiratory tract The nasal cavity still contained eosinophilic proteinaceous exudate mixed with abundant 323 degenerate neutrophils in all infected animals and none of the uninfected animals.

Olfactory epithelium was multifocally eroded with degenerate and necrotic epithelium; 325 inflammatory cells consisted of predominantly neutrophils infiltrating into the mucosa.

The tracheal submucosa only contained mild scattered infiltrates of mononuclear cells in 327 one infected animal. 

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