First detection of dengue virus in the saliva of immunocompetent murine model.

The lack of an experimental animal model for the study of dengue pathogenesis is a limiting factor for the development of vaccines and drugs. In previous studies, our group demonstrated the susceptibility of BALB/c mice to infection by dengue virus (DENV) 1 and 2, and the virus was successfully isolated in several organs. In this study, BALB/c mice were experimentally infected intravenously with DENV-4, and samples of their saliva were collected. Viral RNA extracted from the saliva samples was subjected to qRT-PCR, with a detection limit of 0.002 PFU/mL. The presence of DENV-4 viral RNA was detected in the saliva of two mice, presenting viral titers of 109 RNA/mL. The detection of DENV RNA via saliva sampling is not a common practice in dengue diagnosis, due to the lower detection rates in human patients. However, the results observed in this study seem to indicate that, as in humans, detection rates of DENV RNA in mouse saliva are also low, correlating the infection in both cases. This study reports the first DENV detection in the saliva of BALB/c immunocompetent mice experimentally infected with non-neuroadapted DENV-4.

Low impact to fixed cell processing aiming transmission electron microscopy.

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.

Ultrastructure of Zika virus particles in cell cultures.

Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient's blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.

Comparative analysis of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Zeta (P.2) and Delta (B.1.617.2) in Vero-E6 cells: ultrastructural characterization of cytopathology and replication kinetics

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.

Caracterização físico-química de três xenoenxertos utilizados na implantodontia / Physicochemical characterization of three xenografts used in implant dentistry

Os biomateriais de origem xenógena, utilizados como substitutos ósseos, têm sido alvo de grande interesse clínico, devido às suas propriedades de biocompatibilidade e osteocondução. As propriedades físico-químicas dos biomateriais exercem uma grande influência sobre sua interação com as células e subsequentemente desempenham um papel importante na resposta biológica in vivo dos materiais. As características físicas envolvem características microestruturais de forma e tamanho das partículas, porosidade, densidade e área de superfície. A caracterização em termos da química envolve a determinação da composição química e a distribuição dos elementos no biomaterial. Sendo assim, o objetivo deste trabalho foi avaliar comparativamente as propriedades físico-químicas de três xenoenxertos de origem bovina comercialmente disponíveis no mercado nacional para tratamento de perdas ósseas na Odontologia. A Microscopia Eletrônica de Varredura (MEV) evidenciou discrepâncias no tamanho das partículas dos biomateriais estudados quando relacionados com os dados fornecidos pelo fabricante. Os difratogramas obtidos pela Difração de Raios X (DRX), apontaram dois biomateriais mais amorfos, Bio-Oss® e Lumina-Bone Porous®, e um mais cristalino Extra Graft XG13®, corroborando com a análise Termogravimétrica que evidenciou significativa perda de massa nos dois biomateriais amorfos. O biomaterial Extra Graft XG13® não apresentou porosidade detectável pelo método de análise Área superficial específica e porosidade (Brunauer, Emmett and Teller BET). Os materiais Bio-Oss® e Lumina Bone®, mostraram presença significativa do grupo hidroxila na análise do infravermelho, confirmando a presença de água no biomaterial. Os biomateriais estudados apresentaram grandes discrepâncias quanto a cristalinidade, porosidade e área superficial entre si, essas características impactam na resposta biológica após a implantação e, por isso, devem ser avaliadas de acordo com a indicação clínica.

Estudos ultraestruturais de células Vero-E6 infectadas com SARS-CoV-2 e tratadas com moléculas de ação antiviral

Em dezembro de 2019, um novo coronavírus (SARS-CoV-2) foi identificado em Wuhan, China, e associado a uma infecção respiratória grave, hoje conhecida como COVID-19. Apesar do impacto na saúde pública mundial e de diferentes esforços, até o presente momento não há droga antiviral específica a ser utilizada. Nesse contexto, um sistema in vitro padronizado e bem caracterizado para produção de massa viral, para testes de candidatos à fármacos e vacinas contra o novo coronavírus é de extrema importância. Assim, este estudo objetivou: I) avaliar a susceptibilidade e permissividade das células de linhagem de rim de macaco verde africano (Vero-E6) para o SARS-CoV-2 e caracterizar o seu ciclo replicativo por microscopia eletrônica de transmissão (MET) e de varredura de alta resolução (MEV); e II) verificar o impacto na biossíntese do vírus em células Vero-E6 mediante o tratamento com antivirais. Para isso, monocamadas de células Vero-E6 foram inoculadas com MOI (multiplicidade de infecção) 0,01 de SARS-CoV-2 e analisadas por MET e MEV para os estudos de susceptibilidade, permissividade e morfogênese. Para os estudos de inibição da biossíntese viral, as monocamadas infectadas foram tratadas com mefloquina e ao daclatasvir a 10 µM e, posteriormente, processadas por técnicas padrão para análises por MET. A partir do sobrenadante, o RNA viral foi quantificado por RT-qPCR e o título viral determinado por contagem de placas unidades formadoras de placa (PFU/mL). Diversas alterações ultraestruturais foram observadas nas células infectadas, incluindo intensa ativação celular evidenciada por filopódios, formação de sincícios, acúmulo de corpúsculos lipídicos, proliferação de vesículas de dupla membrana (VDM), degeneração de mitocôndrias, aumento no número de ribossomos elétron-densos e espessamento do retículo endoplasmático rugoso (RER). Com relação à biossíntese viral, observou-se internalização das partículas do SARS-CoV-2 majoritariamente por endocitose, parte da síntese viral ocorrendo no lúmen das VDMs e liberação da progênie viral por exocitose. O ensaio de qRT-PCR demonstrou aumento da progênie viral em log2 em 72 horas pós-infecção. Os estudos referentes ao objetivo II revelaram que a mefloquina a 10 µM promoveu inibição da replicação viral em 100%, enquanto o tratamento com daclatasvir inibiu em 80%, porém com este tratamento as células permanecem com viabilidade acima de 90%, enquanto com mefloquina esse número reduz para 9%. Os dados apresentados comprovam em nível ultraestrutural a susceptibilidade e a permissividade das células de linhagem Vero-E6 para o SARS-CoV-2 reforçando a utilização desta linhagem como um sistema in vitro para a produção de massa viral e testes pré-clínicos de drogas

Low impact to fixed cell processing aiming transmission electron microscopy

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.

Ultrastructure of Zika virus particles in cell cultures

Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible.