RESUMO
Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Peixe-Zebra , Macrófagos , PeptídeosRESUMO
This study evaluates a possible correlation between multidrug-resistant Klebsiella pneumoniae strains and virulence markers in a Danio rerio (zebrafish) model. Whole-genome sequencing (WGS) was performed on 46 strains from three Brazilian hospitals. All of the isolates were colistin-resistant and harbored blaKPC-2. Ten different sequence types (STs) were found; 63% belonged to CC258, 22% to ST340, and 11% to ST16. The virulence factors most frequently found were type 3 fimbriae, siderophores, capsule regulators, and RND efflux-pumps. Six strains were selected for a time-kill experiment in zebrafish embryos: infection by ST16 was associated with a significantly higher mortality rate when compared to non-ST16 strains (52% vs. 29%, p = 0.002). Among the STs, the distribution of virulence factors did not differ significantly except for ST23, which harbored a greater variety of factors than other STs but was not related to a higher mortality rate in zebrafish. Although several virulence factors are described in K. pneumoniae, our study found ST16 to be the only significant predictor of a virulent phenotype in an animal model. Further research is needed to fully understand the correlation between virulence and sequence types.
RESUMO
Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.
