Rafaela das Dores Pereira

The COVID-19 pandemic caused by SARS-CoV-2 has increased the attention to viral diseases due to the global rapid dissemination, high transmissibility, and potential lethality. In these situations, the development of pre-clinical models is crucial to better understand the molecular aspects of the pathogenesis of such condition. Thus, animal models of Betacoronavirus infection that mimic disease features observed during COVID-19 in humans are fundamental tools to uncover disease mechanisms. In this chapter, we describe in detail the animal models and the pathogenesis of the disease caused by different Betacoronaviruses in rodents. The main differences between the murine models used for coronavirus studies are discussed, such as the mouse model for mouse hepatitis virus (MHV) infection as well as humanized model (K18-hACE2), as well as hamster models for infection with SARS-CoV-2. Methods concerning virus manipulation and animal infection are also described in detail, along with important notes on different techniques to assess clinical outcomes and protocols of sample collection for further biochemical, immune, microbiological and histopathological examination.

The E3 ubiquitin ligase Smurf1 catalyzes the ubiquitination and proteasomal degradation of several protein substrates related to inflammatory responses and antiviral signaling. This study investigated the role of Smurf1 in modulating inflammation induced by Betacoronavirus infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 (wild-type) or Smurf1-deficient (Smurf1^−/−) mice were infected with MHV-A59 to evaluate the inflammatory response in vitro. Smurf1 was found to be required to downregulate the macrophage production of pro-inflammatory mediators, including TNF, and CXCL1; to control viral release from infected cells; and to increase cell viability. To assess the impact of Smurf 1 in vivo, we evaluated the infection of mice with MHV-A59 through the intranasal route. Smurf1^−/− mice infected with a lethal inoculum of MHV-A59 succumbed earlier to infection. Intranasal inoculation with a 10-fold lower dose of MHV-A59 resulted in hematological parameter alterations in Smurf1^−/− mice suggestive of exacerbated systemic inflammation. In the lung parenchyma, Smurf1 expression was essential to promote viral clearance, downregulating IFN-β mRNA and controlling the inflammatory profile of macrophages and neutrophils. Conversely, Smurf1 did not affect IFN-β mRNA regulation in the liver, but it was required to increase TNF and iNOS expression in neutrophils and decrease TNF expression in macrophages. In addition, Smurf1^−/− mice exhibited augmented liver injuries, accompanied by high serum levels of alanine aminotransferase (ALT). These findings suggest that Smurf1 plays a critical role in regulating the inflammatory response in macrophages and attenuating systemic inflammation during Betacoronavirus infection.

Plasmodium berghei ANKA (PbA) infection in mice resembles several aspects of severe malaria in humans, such as cerebral malaria and acute respiratory distress syndrome. Herein, the effects of N-(coumarin-3-yl)cinnamamide (M220) against severe experimental malaria have been investigated. Treatment with M220 proved to protect cognitive abilities and lung function in PbA-infected mice, observed by an object recognition test and spirometry, respectively. In addition, treated mice demonstrated decreased levels of brain and lung inflammation. The production and accumulation of microglia, and immune cells that produce the inflammatory cytokines TNF and IFN-γ, decreased, while the production of the anti-inflammatory cytokine IL-10 by innate and adaptive immune cells was enhanced. Treatment with M220 promotes immunomodulatory, neuroprotective, and lung function-preserving effects during experimental severe malaria. Therefore, it may be an interesting therapeutic candidate to treat severe malaria effects.

Exacerbated inflammatory responses are a hallmark of severe coronavirus disease 2019 (COVID-19). Zileuton (Zi) is a selective inhibitor of 5-lipoxygenase, an enzyme involved in the production of several inflammatory/pro-resolving lipid mediators. Herein, we investigated the effect of Zi treatment in a severe acute respiratory syndrome (SARS) model. Mouse hepatitis virus (MHV)3-infected mice treated with Zi significantly improved the clinical score, weight loss, cardiopulmonary function, and survival rates compared with infected untreated animals. The protection observed in Zi-treated mice was associated with a lower inflammatory score, reduced dendritic cell-producing tumor necrosis factor (TNF), and increased neutrophil-producing interleukin (IL)-10 in the lungs three days after infection (dpi). At 5 dpi, the lungs of treated mice showed an increase in Th2-, Treg CD4⁺-, and Treg CD8⁺-producing IL-10 and reduced Th1 infiltrating cells. Furthermore, similar results were found upon Zi treatment after SARS-CoV-2 infection in transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2), significantly improving the clinical score, weight loss, and lung inflammatory score compared with untreated animals. Our data suggest that Zi protects against developing severe lung disease during SARS induced by betacoronavirus without affecting the host’s capacity to deal with infection.

Exacerbated inflammatory responses are a hallmark of severe coronavirus disease 2019 (COVID-19). Zileuton (Zi) is a selective inhibitor of 5-lipoxygenase, an enzyme involved in the production of several inflammatory/pro-resolving lipid mediators. Herein, we investigated the effect of Zi treatment in a severe acute respiratory syndrome (SARS) model. Mouse hepatitis virus (MHV)3-infected mice treated with Zi significantly improved the clinical score, weight loss, cardiopulmonary function, and survival rates compared with infected untreated animals. The protection observed in Zi-treated mice was associated with a lower inflammatory score, reduced dendritic cell-producing tumor necrosis factor (TNF), and increased neutrophil-producing interleukin (IL)-10 in the lungs 3 days after infection (dpi). At 5 dpi, the lungs of treated mice showed an increase in Th2-, Treg CD4+-, and Treg CD8+-producing IL-10 and reduced Th1 infiltrating cells. Furthermore, similar results were found upon Zi treatment after SARS-CoV2 infection in transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2), significantly improving the clinical score, weight loss, and lung inflammatory score compared with untreated animals. Our data suggest that Zi protects against developing severe lung disease during SARS induced by betacoronavirus without affecting the host’s capacity to deal with infection.

O objetivo deste estudo foi desenvolver farinhas produzidas com coprodutos de frutas e a aplicação das mesmas em cookies. Foram desenvolvidas seis farinhas: casca de abacaxi, casca de manga, casca de banana, casca de laranja, entrecasca de melancia e amêndoa de manga. Foi estudada a formulação padrão do cookie e as demais substituindo 15% da farinha de trigo por cada farinha produzida. As farinhas foram caracterizadas pelos parâmetros de rendimento, pH e cor, enquanto que os cookies por volume específico, cor, textura e aceitação sensorial, sendo analisados pela correlação de Pearson. As farinhas apresentaram pH ácido e diferentes cores, sendo a de casca de banana a mais escura e a de amêndoa da manga a mais clara. O rendimento das farinhas variou entre 9,8% (casca de banana) e 52,2% (amêndoa da manga). Os volumes específicos das formulações não apresentaram diferença, sendo o valor de 1,69±0,03cm3.g-1 para a amostra padrão. A dureza variou entre 26,22±3,24N (casca de laranja) e 52,37±4,55N (entrecasca de melancia). Os parâmetros de cor dos cookies sofreram influência das farinhas adicionadas, sendo o Delta E maior naquele produzido com farinha de casca de banana. Os cookies apresentaram boas características sensoriais nos atributos aparência e sabor. Na correlação de Pearson foi possível observar que o volume específico interfere na percepção de sabor e que os mesmos esperam produto com aparência semelhante ao padrão. Neste contexto, existe a possibilidade de elaborar cookies nutritivos e com boa aceitação a partir da utilização de partes usualmente descartadas de frutas.