key: cord-0983085-h3f9dvv1
authors: dos Santos Pereira Andrade, Ana Cláudia; Campolina-Silva, Gabriel Henrique; Queiroz-Junior, Celso Martins; de Oliveira, Leonardo Camilo; de Souza Barbosa Lacerda, Larisse; Pimenta, Jordane Clarisse; de Souza, Filipe Resende Oliveira; de Meira Chaves, Ian; Passos, Ingredy Beatriz; Teixeira, Danielle Cunha; Bittencourt-Silva, Paloma Graziele; Costa Valadão, Priscila Aparecida; Rossi-Oliveira, Leonardo; Antunes, Maisa Mota; Almeida Figueiredo, André Felipe; Wnuk, Natália Teixeira; Temerozo, Jairo R.; Ferreira, André Costa; Cramer, Allysson; Oliveira, Cleida Aparecida; Durães-Carvalho, Ricardo; Arns, Clarice Weis; Guimarães, Pedro Pires Goulart; Costa, Guilherme Mattos Jardim; de Menezes, Gustavo Batista; Guatimosim, Cristina; da Silva, Glauber Santos Ferreira; Souza, Thiago Moreno L.; Barrioni, Breno Rocha; de Magalhães Pereira, Marivalda; de Sousa, Lirlândia Pires; Teixeira, Mauro Martins; Costa, Vivian Vasconcelos
title: A suitable murine model for studying respiratory coronavirus infection and therapeutic countermeasures in BSL-2 laboratories
date: 2021-05-29
journal: bioRxiv
DOI: 10.1101/2021.05.28.446200
sha: 3f935bd03f0b9c4a43e672f9c88c201f170741d3
doc_id: 983085
cord_uid: h3f9dvv1

Several animal models are being used to explore important features of COVID-19, nevertheless none of them recapitulates all aspects of the disease in humans. The continuous refinement and development of other options of in vivo models are opportune, especially ones that are carried out at BSL-2 (Biosafety Level 2) laboratories. In this study, we investigated the suitability of the intranasal infection with the murine betacoronavirus MHV-3 to recapitulate multiple aspects of the pathogenesis of COVID-19 in C57BL/6J mice. We demonstrate that MHV-3 replicated in lungs 1 day after inoculation and triggered respiratory inflammation and dysfunction. This MHV-model of infection was further applied to highlight the critical role of TNF in cytokine-mediated coronavirus pathogenesis. Blocking TNF signaling by pharmacological and genetic strategies greatly increased the survival time and reduces lung injury of MHV-3-infected mice. In vitro studies showed that TNF blockage decreased SARS-CoV-2 replication in human epithelial lung cells and resulted in the lower release of IL-6 and IL-8 cytokines beyond TNF itself. Taken together, our results demonstrate that this model of MHV infection in mice is a useful BSL-2 screening platform for evaluating pathogenesis for human coronaviruses infections, such as COVID-19.

morphology and trigger inflammation-associated tissue damage (Fig. 2a) . 179

Compared to mock controls, a higher number of cells stained for the pan-180 leukocyte marker CD45 was found in the lung sections of infected mice after 1 181 day and especially 3 days after inoculation (Fig. 2b) injury. As such, approximately 50% of mice had discrete inflammatory cell 189 infiltration in close association with areas of alveolar edema and hyperemic 190 vessels at 1 day after MHV-3 infection (Fig. 2 d-f ). At 3 dpi, the inflammatory 191 infiltrate became robust and widespread throughout the lung tissue, leading to 192 greater histopathological changes. Inflammation foci were frequently seen in the 193 vicinity of bronchioles, in perivascular areas, and in the lumen of hyperemic 194 vessels ( Fig. 2 b , d-f). Some blood vessels presented eosinophilic fibrillar material 195 adherent to the vascular wall, which is characteristic of fibrin thrombi. In some 196 samples, areas of necrosis and hemorrhage were also observed ( Fig. 2 d) . 197

Conversely, the inflammation-associated lung damage in the mouse lung at 5 dpi 198 was milder and encompassed the changes herein portrayed for day 1 after 199 infection ( Fig. 2 d-f ), suggesting that inflammation was resolving. 200

The intrapulmonary concentration of major chemokines (CCL2, CCL3, 201 CCL4, CCL5) and cytokines (TNF, IL-6, IL-1β, IL-12, IFNγ) was markedly 202 increased at 1 dpi and/or 3 dpi and lowered thereafter (Fig. 2 g) . In contrast, high 203 levels of the inflammatory mediators (IL-6, IL-10, TNF, IL-1β and IFNγ) were only 204 detected in the blood at 5 dpi (Fig. 2 h) . These data suggest that the current 205 infection model exhibits transient pulmonary pneumonia followed by systemic 206

Next, we asked whether these histopathological changes could impact on 208 lung function. By using the whole-body plethysmography method, two control pre-209 whether MHV-3 could trigger testicular damage. Interestingly, the percentage of 245 altered seminiferous tubules raised significantly after inoculation. The altered 246 sites displayed epithelium sloughing, elevated germ cell apoptosis, and retention Increasing concentrations of TNF have previously been associated with 255 tissue damages triggered by MuCov and human coronaviruses and are currently 256 thought to aggravate COVID-19 severity (6,27,28). Once this cytokine was 257 presently found along with other pro-inflammatory mediators at higher levels 258 within the lung of infected mice (Fig. 2g) , we next interrogated the contribution of 259 TNF signaling for the pathogenesis of MHV-3 infection. To this end, we infected 260 mice genetically deficient for TNF receptor type 1 (TNFR1, also known as p55) 261 with 10³ PFU of MHV-3 intranasally and compared with wild type (WT) mice. 262

Strikingly, TNFR1 knockout mice (TNFR1 KO) were protected from abrupt weight 263 loss and lethality, with 100% of mice surviving by the end of 14-days follow-up 264 period (Fig. 5 a, b) . The progressive leukopenia and thrombocytopenia 265 phenotype observed in WT infected mice were not found in TNFR1 KO (Fig. 5c) . 266

Moreover, compared to WT, the TNFR1 KO group had significantly lower viral 267 loads (Fig. 5d ), inflammation and injury in their lungs (Fig. 5 e-g) . 268

To verify whether such a protective profile against MHV-3 could also be 269 achieved using pharmacological approaches, WT mice were first infected with 270 10³ PFU intranasally and then treated with etanercept, a selective TNF inhibitor. 271

The Etanercept treatment started after 24 hours of infection and was given via 272 two routes: (i) intranasal; or (ii) intraperitoneal (Fig 6 a) . Regardless the treatment Similar findings were seen when survival rate was compared among groups, with 275 etanercept-treated mice living on average by 3 days longer than the untreated 276 ones (vehicle group) (Fig. 6 c) . It is worth noting that both the local and systemic 277 treatment with etanercept were capable of inhibiting MHV-3 replication in the 278 lungs, with infectious viruses no longer detected in the majority of animals ( Fig. 6 results showed that etanercept treatment reduced the SARS-Cov-2-mediated 293 cellular damage in a dose-dependent manner (Fig. 7 b) . Etanercept at 5 and 10 294 ng/mL had also a slight but statistically inhibitory effect on SARS-CoV-2 (Fig. 7c) . 295

Moreover, the overproduction of pro-inflammatory cytokines triggered by SARS-296

CoV-2 infection was significantly reduced in Calu-3 cells treated with etanercept 297 (Fig. 7d) . These results suggest that TNF blocking by pharmacologic approaches 298 might also be beneficial against SARS-CoV-2 infection in human lung epithelial 299 cells. 

Mice were divided into two groups (mock n=9 and infected n=11) and three 566 days post infection animals were deeply anesthetized until respiratory arrest. 567

Mice were tracheostomized and a polyethylene tube (P50) was inserted into the 568 trachea. The pressure-volume curve was made by injecting air volume in a step-569 wise manner (using the 3 mL glass syringe), with 0.1 mL increments until 570 intratracheal pressure peaked at approximately 35 cmH2O. In the deflation limb, 

Graph Pad Prism 8.0 software was used for statistical analysis. First, data 611 distribution was assessed by the Shapiro-Wilk test and Q-Q plots. Parametric 612 comparisons between two or more groups were done using Student-t test or one-613 way ANOVA, respectively, or by using Mann-Whitney or Kruskal-Wallis test to 614 assess differences between two or more non-parametric datasets. Survival rates 615 10, TNF, IL-1b and IFNg in the plasma of infected mice at 5dpi (n=5). ns= not 868 significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. 869 One-way repeated measures ANOVA plus Tukey's post hoc test were applied to 874 assess differences between pre-infection and post-infection periods (n=8). c 875

Analysis of pulmonary compliance showing reduced compliance and vital 876 capacity in infected mice compared to mock (unpaired t test, n=9-11). Static 877 compliance was calculated from the steepest point of the deflation part of the were normalized to mock group and presented as mean + S.E.M percent of mock 932 controls. Assessed by Mann-Whitney test. n=7-8. ns= not significant (P > 0.05). 933 *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. 934 

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