RESUMEN
This study compares the effects of virus-cell interactions among SARS-CoV-2 variants of concern (VOCs) isolated in Brazil in 2021, hypothesizing a correlation between cellular alterations and mortality and between viral load and transmissibility. For this purpose, reference isolates of Alpha, Gamma, Zeta, and Delta variants were inoculated into monolayers of Vero-E6 cells. Viral RNA was quantified in cell supernatants by RTâPCR, and infected cells were analyzed by Transmission Electron Microscopy (TEM) for qualitative and quantitative evaluation of cellular changes 24, 48, and 72 hours postinfection (hpi). Ultrastructural analyses showed that all variants of SARS-CoV-2 altered the structure and function of mitochondria, nucleus, and rough endoplasmic reticulum of cells. Monolayers infected with the Delta variant showed the highest number of modified cells and the greatest statistically significant differences compared to those of other variants. Viral particles were observed in the cytosol and the cell membrane in 100 % of the cells at 48 hpi. Alpha showed the highest mean particle diameter (79 nm), and Gamma and Delta were the smallest (75 nm). Alpha and Gamma had the highest particle frequency per field at 48 hpi, while the same was observed for Zeta and Delta at 72 hpi and 24 hpi, respectively. The cycle threshold of viral RNA varied among the target protein, VOC, and time of infection. The findings presented here demonstrate that all four VOCs evaluated caused ultrastructural changes in Vero-E6 cells, which were more prominent when infection occured with the Delta variant.
Asunto(s)
COVID-19 , Citología , Humanos , SARS-CoV-2 , ARN Viral/genéticaRESUMEN
Abstract This study compares the effects of virus-cell interactions among SARS-CoV-2 variants of concern (VOCs) isolated in Brazil in 2021, hypothesizing a correlation between cellular alterations and mortality and between viral load and transmissibility. For this purpose, reference isolates of Alpha, Gamma, Zeta, and Delta variants were inoculated into monolayers of Vero-E6 cells. Viral RNA was quantified in cell supernatants by RT‒PCR, and infected cells were analyzed by Transmission Electron Microscopy (TEM) for qualitative and quantitative evaluation of cellular changes 24, 48, and 72 hours postinfection (hpi). Ultrastructural analyses showed that all variants of SARS-CoV-2 altered the structure and function of mitochondria, nucleus, and rough endoplasmic reticulum of cells. Monolayers infected with the Delta variant showed the highest number of modified cells and the greatest statistically significant differences compared to those of other variants. Viral particles were observed in the cytosol and the cell membrane in 100 % of the cells at 48 hpi. Alpha showed the highest mean particle diameter (79 nm), and Gamma and Delta were the smallest (75 nm). Alpha and Gamma had the highest particle frequency per field at 48 hpi, while the same was observed for Zeta and Delta at 72 hpi and 24 hpi, respectively. The cycle threshold of viral RNA varied among the target protein, VOC, and time of infection. The findings presented here demonstrate that all four VOCs evaluated caused ultrastructural changes in Vero-E6 cells, which were more prominent when infection occured with the Delta variant.
RESUMEN
Dengue virus (DENV) infection by one of the four serotypes (DENV-1 to 4) may result in a wide spectrum of clinical manifestations, with unpredictable evolution and organ involvement. Due to its association with severe epidemics and clinical manifestations, DENV-2 has been substantially investigated. In fact, the first emergence of a new lineage of the DENV-2 Asian/American genotype in Brazil (Lineage II) in 2008 was associated with severe cases and increased mortality related to organ involvement. A major challenge for dengue pathogenesis studies has been a suitable animal model, but the use of immune-competent mice, although sometimes controversial, has proven to be useful, as histological observations in infected animals reveal tissue alterations consistent to those observed in dengue human cases. Here, we aimed to investigate the outcomes caused by two distinct lineages of the DENV-2 Asian/American genotype in the lung, heart and skeletal muscle tissues of infected BALB/c mice. Tissues were submitted to histopathology, immunohistochemistry, histomorphometry and transmission electron microscopy (TEM) analysis. The viral genome was detected in heart and skeletal muscle samples. The viral antigen was detected in cardiomyocytes and endothelial cells of heart tissue. Heart and lung tissue samples presented morphological alterations comparable to those seen in dengue human cases. Creatine kinase serum levels were higher in mice infected with both lineages of DENV-2. Additionally, statistically significant differences, concerning alveolar septa thickening and heart weight, were observed between BALB/c mice infected with both DENV-2 lineages, which was demonstrated to be an appropriate experimental model for dengue pathogenesis studies on lung, heart and skeletal muscle tissues.
RESUMEN
Este trabalho é uma revisão bibliográfica sobre as interferências dos tubos de coleta com gel separador em análises bioquímicas, comparando estes resultados com os obtidos em tubos com separador mecânico (Barricor). Para tanto, buscamos artigos nas bases de dados PubMed, Google acadêmico, Scielo e LILACS. Após a aplicação dos critérios de inclusão e exclusão, foram selecionados 21 artigos para a escrita da revisão. Estes trabalhos mostraram que o gel pode causar instabilidade nas dosagens de íons e de diversas substâncias orgânicas. Além disso, em alguns casos a análise da amostra fica impossibilitada devido ao fenômeno de formação do gel flutuante. No entanto, através do uso do tubo com separador mecânico foi possível minimizar essas interferências e instabilidades. Outra vantagem está relacionada ao tempo reduzido de centrifugação da amostra com o tubo de separador mecânico (3 min. vs. 10 min.). Este tipo de separação também permite uma centrifugação mais eficaz da amostra, com menor contaminação do plasma com células e plaquetas, quando comparada à centrifugação do soro obtido nos tubos tradicionais com gel separador. Por estas razões, fica evidente que as amostras de plasma obtidas em tubos de separação mecânica demonstram ser de melhor qualidade para as análises bioquímicas.
This work is a bibliographic review about the interferences of the collection tubes with separating gel in biochemical analyzes, comparing these results with those obtained with a mechanical separator (Barricor). For that, we searched for articles in the databases Pubmed, Google scholar, Scielo, and LILACS. After applying the inclusion and exclusion criteria, we selected 21 papers to write the review. These studies showed that the gel might cause instability in the dosage of ions and various organic substances. Besides, in some cases, the analysis of the sample is impossible due to the phenomenon of floating gel formation. However, through the use of the tube with a mechanical separator, it was possible to minimize these interferences and instabilities. Another advantage is related to the reduced time of centrifugation of the sample with the mechanical separator tube (3 min vs. 10 min). This type of separation also allows more efficient centrifugation of the specimen, with less contamination of the plasma with cells and platelets when compared to the serum obtained in traditional tubes with separator gel. For these reasons, the plasma samples obtained in mechanical separation tubes prove to be of better quality for biochemical analyzes.
