Cytotoxicity and antiviral activity evaluation of Cymbopogon spp hydroethanolic extracts

Cymbopogon citratus and C. nardus are noteworthy among the several existing plant species displaying medicinal properties, due to the potential pharmacological activity of these species, including antiviral, antibacterial, antifungal and anti-trypanosomal activities. The objective of this study was to carry out in vitro toxicity tests of plant extracts from both species and analyze potential antiviral activity against Human mastadenovirus serotype 5 (HAdV-5). Two cell lines (A549 and VERO) were used and mitochondrial and lysosomal viability were determined by the MTT and neutral red assay, respectively, after two exposure times (24 hours and six days). The aim of these assays was to counteract the behavior of the extracts against the different cell lines and determine their non-toxic concentration range, in order to evaluate possible antiviral activity against HAdV-5. Plaque reduction and inhibition index of viral titer assays were performed using the maximum non-cytotoxic concentrations (MNCC) of each extract. The results indicate MNCC at 625 μg/mL for all extracts, except for Cymbopogon nardus obtained with 80% ethanol (CN80), which showed toxicity at concentrations higher than 312.5 μg/mL. CN80 was the only extract that displayed potential activity against HAdV-5, at a concentration of 75 μg/mL, becoming a candidate for extract fraction purification and/or the isolation of substances related to the observed antiviral activity

Alternative inactivated poliovirus vaccines adjuvanted with quillaja brasiliensis or quil-a saponins are equally effective in inducing specific immune responses

Inactivated polio vaccines (IPV) have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV). An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE) and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control), Quil-A (50 mg), AE (400 mg) or QB-90 (50 mg). Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-c and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A

Presença de partículas virais infecciosas em amostras de água de diferentes tipos e localidades do Rio Grande do Sul, Brasil

The parameters for drinking water currently are based only on the analysis of bacteria; nevertheless the absence of coliforms does not exclude viral contamination. Enteric viruses may resist conventional water and sewage treatment methods, due to its physical and chemical stability. Although laborious and time consuming, analysis by viral isolation in cell culture allows to evaluate the viability of viral particles, advantageous feature when compared to molecular biology techniques. Sixty-four water samples from surface and tap water from various Rio Grande do Sul’s locations were collected and inoculated into 24-well microplates containing CER cell line cells. The microplates were stored in 37°C oven, 5% CO2 for seven days and observed daily for possible detection of cytopathic effect (CPE) in inverted microscope. CPE consistent with viral presence was observed in 12 (21.8%) of 55 samples, with higher prevalence in tap water, indicating the need to revise our current legislation on drinking water.Os parâmetros de potabilidade da água atualmente baseiam-se apenas na análise de bactérias; porém a ausência de coliformes não exclui a contaminação viral. Vírus entéricos podem resistir aos métodos convencionais de tratamento de água e esgoto, devido à suas características de estabilidade físico-química. Apesar de laboriosa e demorada, a análise virológica por isolamento em cultivo celular permite avaliar a viabilidade das partículas virais presentes, aspecto vantajoso quando comparado a técnicas de biologia molecular. Sessenta e quatro amostras de água superficial e de torneira de diversas localidades do Rio Grande do Sul foram coletadas e inoculadas em microplacas de 24 poços, contendo células da linhagem CER. As microplacas foram armazenadas em estufa 37ºC, 5% de CO2 por sete dias, sendo observadas diariamente para possível detecção de efeito citopático (ECP) em microscópio invertido. Foi observado ECP condizente com presença viral em 12 (21,8%) das 55 amostras analisadas, com maior prevalência em águas de torneira, indicando a necessidade de revisar a legislação vigente sobre potabilidade da água

Early detection of SARS-CoV-2 P.1 variant in Southern Brazil and reinfection of the same patient by P.2

Multiple variants of the Severe Acute Respiratory Syndrome coronavirus 2 virus (SARS-CoV-2) have been constantly reported across the world. The B.1.1.28 lineage has been evolving in Brazil since February 2020 and originated the P.1 variant of concern (VOC), recently named as the Gamma variant by the newly WHO nomenclature proposal, and P.2 as a variant of interest (VOI). Here we describe an early case of P.1 primary infection in Southern Brazil in late November 2020, soon after the emergence of the variant in Manaus, Northern Brazil. The same male patient was reinfected by another B.1.1.28 variant, namely P.2, in March, 2021. The genomic analysis confirmed genetically significant differences between the two viruses recovered in both infections, the P.1 lineage in the first episode and P.2 in the reinfection. Due the very early detection of P.1, we have also investigated the circulation of P.1 in the same region by differential RT-qPCR, showing that this was an isolated case of P.1 at the time of detection, and this variant has disseminated and became prominent from late January to the end of March, 2021. SARS-CoV-2 recent reports of reinfection have raised critical questions on whether and how well a first infection protects against reinfection

Genomic epidemiology of SARS-CoV-2 in Esteio, Rio Grande do Sul, Brazil

Background: Brazil is the third country most affected by Coronavirus disease-2019 (COVID-19), but viral evolution in municipality resolution is still poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We aimed to track molecular evolution and spread of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Esteio (Southern Brazil) using phylogenetics and phylodynamics inferences from 21 new genomes in global and regional context. Importantly, the case fatality rate (CFR) in Esteio (3.26%) is slightly higher compared to the Rio Grande do Sul (RS) state (2.56%) and the entire Brazil (2.74%). Results: We provided a comprehensive view of mutations from a representative sampling from May to October 2020, highlighting two frequent mutations in spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in spike Receptor Binding Domain (RBD) characteristic of the B.1.351 and P.1 lineages, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). E484K was found in two genomes from mid-October, which is the earliest description of this mutation in Southern Brazil. Lineages containing this substitution must be subject of intense surveillance due to its association with immune evasion. We also found two epidemiologicallyrelated clusters, including one from patients of the same neighborhood. Phylogenetics and phylodynamics analysis demonstrates multiple introductions of the Brazilian most prevalent lineages (B.1.1.33 and B.1.1.248) and the establishment of Brazilian lineages ignited from the Southeast to other Brazilian regions. Conclusions: Our data show the value of correlating clinical, epidemiological and genomic information for the understanding of viral evolution and its spatial distribution over time. This is of paramount importance to better inform policy making strategies to fight COVID-19

Chemical composition, adjuvant activity evaluation and propagation studies of quillaja brasiliensis

Quillaja brasiliensis (A. St.-Hil. &Tul.) Mart. é uma espécie nativa do Rio Grande do Sul, conhecida popularmente como pau-sabão, devido à capacidade de suas folhas e cascas formarem abundante espuma em água. A espécie congênere chilena, Q. saponaria, é uma das principais fontes industriais de saponinas, as quais são utilizadas, entre outros, como adjuvantes em vacinas. Tendo em vista a presença de saponinas em Q. brasiliensis, métodos por cromatografia em camada delgada (CCD) e cromatografia líquida de alta eficiência (CLAE) foram desenvolvidos para a caracterização e o doseamento de fração purificada de saponinas, a partir do extrato aquoso de folhas, denominada QB-90. Ensaios para verificar a toxicidade subcutânea e o perfil dose-dependente para atividade adjuvante também foram realizados com esta fração. Para o doseamento de QB-90 no extrato aquoso foi desenvolvido e validado um método por CLAE empregando coluna de fase reversa C8, sistema isocrático acetonitrila:água, fluxo de 0,8 ml/min e detecção a 214 nm. Na validação do método, foram avaliados os parâmetros de linearidade e intervalo de variação, precisão, exatidão, limite de detecção, limite de quantificação e robustez. Em relação à toxicidade subcutânea de QB-90 em camundongos, não foram observados efeitos sistêmicos no intervalo de doses de 50 a 400 μg. Vacinas experimentais preparadas com herpesvírus bovino tipo 1 (BoHV-1) como antígeno e QB-90 (50-200 μg) foram capazes de aumentar a resposta imunológica em camundongos de modo comparável às saponinas de Q. saponaria (QUIL-A®, 100 μg). Com vistas à potencial utilização sustentável da espécie brasileira na obtenção de saponinas de interesse industrial, protocolos básicos de obtenção de plantas de Q. brasiliensis por micropropagação e por germinação de sementes foram desenvolvidos. Para melhor compreender o perfil de acúmulo de QB-90, seu conteúdo foi investigado em diferentes órgãos vegetais, em diferentes estações do ano e durante o desenvolvimento de plântulas, assim como em resposta a fatores de estresses bióticos e abióticos. O conteúdo de QB-90 não foi afetado pela aplicação de ácido salicílico exógeno (5 mM). No entanto, foi aumentado pela aplicação exógena de ácido jasmônico (40 μM e 400 μM), bem como pela exposição à radiação UVC. Tendência de aumento no teor de QB-90 foi observada com a aplicação de dano mecânico controlado e com a exposição à radiação UVB. A distribuição órgão-específica de QB-90 foi avaliada, detectando-se maiorconcentração nas folhas do que em raízes e caules de plantas propagadas em laboratório. O teor de QB-90 também foi analisado nas diferentes estações climáticas ao longo de dois anos, indicando que a redução da insolação, geralmente associada a períodos de baixa pluviosidade, está associada à maior produção de QB-90.Quillaja brasiliensis (A. St.-Hil. & Tul.) Mart. is a native tree of Rio Grande do Sul, the Southern state of Brazil, commonly known as soap tree due to the capacity of their leaves and barks to produce abundant foam in water. The related Chilean species, Q. saponaria, is one of the main sources of industrial saponins which are used as adjuvant formulation for vaccines. Considering the presence of saponins in Q. brasiliensis, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods were developed to characterize and quantify the purified saponin fraction named QB-90, obtained from the aqueous extract of leaves. Additionally, the subcutaneous toxicity and dose-response profile to adjuvant activity of QB-90 were evaluated in mice. An HPLC method was developed and validated to quantify QB-90 content in aqueous extract, employing RP-8 column, mobile phase acetonitrile:water, flow rate of 0.8 ml/min and detection at 214 nm. The validation parameters evaluated were linearity and range, precision, accuracy, detection limit, quantitation limit and robustness. In relation to QB-90 subcutaneous toxicity in mice, systemic effects were not observed in doses ranging from 50-400 μg. Experimental vaccines prepared with bovine herpesvirus type I (BoHV-1) antigen and QB-90 (50- 200 μg) were able to enhance the immune responses of mice in a comparable manner to saponins from Q. saponaria (QuilA, 100 μg). Considering the potential sustainable utilization of the Brazilian species as a source of saponins of industrial use, basic protocols for obtaining Q. brasiliensis plants by micropropagation and seed germination were developed. To better understand the accumulation patterns of QB-90, we investigated its content in different plant organs; throughout the seasons and during seedling development, as well as in response to potential biotic and abiotic stress factors. Content of QB-90 wasn’t affected by exogenous application of salicylic acid (5mM). However, it was increased by exogenous application of jasmonic acid (40 μM and 400 μM), as well as by exposure to UVC. Trends toward increase in QB-90 content were observed with exposure to UVB and wounding. The organ-specific QB-90 distribution was evaluated and higher amounts were observed in leaves than roots and stems of plants propagated in the laboratory. Variations inQB-90 content in different seasons for two years showed that lower insolation, generally combined with low precipitation periods, were associated with higher QB- 90 content

Análise da produção de saponinas de Quillaja brasiliensis através de CLAE

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Análise da produção de saponinas de Quillaja brasiliensis através de CLAE

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Análise química de saponinas de Quillaja brasiliensis

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