Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

<p>Abstract</p> <p>Background</p> <p>The yellow fever virus, a member of the genus <it>Flavivirus</it>, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.</p> <p>Results</p> <p>YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.</p> <p>Conclusion</p> <p>This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow <it>in vivo </it>studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.</p

Upstream and downstream process development of a Vero cell-based yellow fever vaccine

Yellow fever (YF) is a lethal viral disease that is endemic in some tropical regions of South America, Central America and Africa. An attenuated vaccine produced in embryonated eggs is available since the 1930’s and is known to be highly effective and safe. However, after large vaccination campaigns in the 2000’s, reports of rare, but serious adverse events have stimulated Biomanguinhos/FIOCRUZ, who produces the current attenuated 17DD vaccine, to develop a new, inactivated vaccine. Over the last years, through a partnership of the Federal University of Rio de Janeiro and Biomanguinhos/FIOCRUZ, both upstream and downstream processes were developed. The upstream process was established based on Vero cell cultivation on microcarriers in serum-free medium, using stirred-tank bioreactors. The first studies were carried out in spinner flasks to select the microcarrier type and the serum-free medium. Also, statistical DOE tools were used to study the infection step, varying the moiety of infection and the time of infection. This process was then scale-up to stirred-tank bioreactors and further optimized regarding microcarrier concentration, stepwise medium addition, dissolved oxygen level/sparging intensity, impeller configuration and time of harvest. The final upstream process that was established results in virus titers of 10^8 pfu/mL within a time frame 144h post inoculation of the cells in the bioreactor. The downstream process was designed priorizing chromatographic techniques, aiming at achieving high purity levels and extensive removal of process-related critical contaminants, such as DNA and host-cell proteins (HCP), as preconized by the regulatory authorities. For the capture step, both cation- and anion-exchange chromatographies were evaluated. A Q membrane adsorber process was selected and the best operational conditions in terms of pH, temperature, buffers and washing strategies were determined. For the second purification step, three techniques were evaluated: multimodal chromatography, ultrafiltration/diafiltration, and hydrophobic interaction chromatography using a HIC membrane adsorber. The multimodal resin showed the best results, and operational conditions of this step were further optimized. The final 2-step yellow-fever virus purification process resulted in an overall yield of 52% and residual HCP of 350 ppm (0.05%). Residual DNA was 1.2 ng per dose, considering the dose established based on animal studies, and is in agreement with the limit recommended by the World Health Organization (\u3c10 ng/dose). Electrophoretic analysis (SDS-PAGE) of the purified samples showed a band corresponding to 96% of identified proteins with molecular mass of 56 kDa, which is the expected mass for the virus envelope protein (E). Anti-E Western blot (WB) showed a single band, confirming the identity of the samples. No band was revealed in the anti-HCP blot, confirming the low HCP levels quantified. The developed process allows the production of a new, high-purity yellow-fever vaccine through a scalable technology, which is better suited than egg-based technology to meet emergency demands in case of epidemics and is useful in the current scenario of increasing worldwide demand YF vaccine

Thermal, mechanical and chemical analysis of poly(vinyl alcohol) multifilament and braided yarns

Poly(vinyl alcohol) (PVA) in multifilament and braided yarns (BY) forms presents great potential for the design of numerous applications. However, such solutions fail to accomplish their requirements if the chemical and thermomechanical behaviour is not sufficiently known. Hence, a comprehensive characterisation of PVA multifilament and three BY architectures (6, 8, and 10 yarns) was performed involving the application of several techniques to evaluate the morphological, chem- ical, thermal, and mechanical features of those structures. Scanning electron microscopy (SEM) was used to reveal structural and morphological information. Differential thermal analysis (DTA) pointed out the glass transition temperature of PVA at 76 °C and the corresponding crystalline melt- ing point at 210 °C. PVA BY exhibited higher tensile strength under monotonic quasi-static loading in comparison to their multifilament forms. Creep tests demonstrated that 6BY structures present the most deformable behaviour, while 8BY structures are the least deformable. Relaxation tests showed that 8BY architecture presents a more expressive variation of tensile stress, while 10BY of- fered the least. Dynamic mechanical analysis (DMA) revealed storage and loss moduli curves with similar transition peaks for the tested structures, except for the 10BY. Storage modulus is always four to six times higher than the loss modulus.This work was funded by European Regional Development funds (FEDER) through the Competitiveness and Internationalization Operational Program (POCI)—COMPETE andby Na-tional Funds through Portuguese Fundação para a Ciência e Tecnologia (FCT) under the project UID/EMS/50022/2020, UID/EEA/04436/2019 andUID/ CTM/00264/2019. Andrea Zille acknowledges financial support of the FCT through the project PTDC/CTM-TEX/28295/2017,and Nuno Dourado acknowledges financial support of the FCT through the project PTDC/EME-SIS/28225/2017. M.F.S.M. de Moura acknowledges the ‘Laboratório Associado de Energia, Transportes e Aeronáutica’ (LAETA) for the financial support

Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection

Simultaneous Separation of Antioxidants and Carbohydrates From Food Wastes Using Aqueous Biphasic Systems Formed by Cholinium-Derived Ionic Liquids

project CICECO-Aveiro Institute of Materials, UID/CTM/50011/2019. Associate Laboratory for Green Chemistry-LAQV, FCT Ref. UID/QUI/50006/2019. POCI-01-0145-FEDER-016403. Investigator FCT project IF/00621/2015. Programa Mais Centro under project CENTRO-07-ST24-FEDER-002008. COMPETE: PEst-C/SAU/UI0709/2011.The food industry produces significant amounts of waste, many of them rich in valuable compounds that could be recovered and reused in the framework of circular economy. The development of sustainable and cost-effective technologies to recover these value added compounds will contribute to a significant decrease of the environmental footprint and economic burden of this industry sector. Accordingly, in this work, aqueous biphasic systems (ABS) composed of cholinium-derived bistriflimide ionic liquids (ILs) and carbohydrates were investigated as an alternative process to simultaneously separate and recover antioxidants and carbohydrates from food waste. Aiming at improving the biocompatible character of the studied ILs and proposed process, cholinium-derived bistriflimide ILs were chosen, which were properly designed by playing with the cation alkyl side chain and the number of functional groups attached to the cation to be able to create ABS with carbohydrates. These ILs were characterized by cytotoxicity assays toward human intestinal epithelial cells (Caco-2 cell line), demonstrating to have a significantly lower toxicity than other well-known and commonly used fluorinated ILs. The capability of these ILs to form ABS with a series of carbohydrates, namely monosaccharides, disaccharides and polyols, was then appraised by the determination of the respective ternary liquid-liquid phase diagrams at 25 degrees C. The studied ABS were finally used to separate carbohydrates and antioxidants from real food waste samples, using an expired vanilla pudding as an example. With the studied systems, the separation of the two products occurs in one-step, where carbohydrates are enriched in the carbohydrate-rich phase and antioxidants are mainly present in the IL-rich phase. Extraction efficiencies of carbohydrates ranging between 89 and 92% to the carbohydrate-rich phase, and antioxidant relative activities ranging between 65 and 75% in the IL-rich phase were obtained. Furthermore, antioxidants from the IL-rich phase were recovered by solid-phase extraction, and the IL was recycled for two more times with no losses on the ABS separation performance. Overall, the obtained results show that the investigated ABS are promising platforms to simultaneously separate carbohydrates and antioxidants from real food waste samples, and could be used in further related applications foreseeing industrial food waste valorization.publishersversionpublishe

USE OF COCONUT BAGASSE AS ALTERNATIVE ADSORBENT FOR SEPARATION OF COPPER (II) IONS FROM AQUEOUS SOLUTIONS: ISOTHERMS, KINETICS, AND THERMODYNAMIC STUDIES

The use of coconut bagasse as an adsorbent for the treatment of Cu(II) ions from aqueous solutions has been investigated. The adsorbent was characterized by infrared spectroscopy, including zeta potential and pH effects. To assess the possibility of removing Cu(II) from aqueous solutions by this sorbent the effects of contact time, initial metal ion concentration, and temperature were studied at pH 5.5. Kinetic studies showed that the amount adsorbed increased with initial Cu(II) concentration, and the equilibrium was established in 120 min. The kinetic data were analyzed using a pseudo second-order equation. Adsorption equilibrium data were investigated using the Langmiur, Freundlich, D–R, Temkin and Halsey isotherm models. The adsorption of Cu(II) on the coconut bagasse was endothermic (H◦ 86.2 kJ/mol), resulting in an increase in entropy (S◦ 339 J/mol/K) and a decrease in Gibbs free energy (G -16.34 to -22.44 kJ/mol) in the temperature range of 301-313 K. A reduction in adsorption capacity with an increase in heat of adsorption revealed an ion exchange mechanism for Cu(II) adsorption

The Brazilian Developments on the Regional Atmospheric Modeling System (BRAMS 5.2): An Integrated Environmental Model Tuned for Tropical Areas

We present a new version of the Brazilian developments on the Regional Atmospheric Modeling System where different previous versions for weather, chemistry and carbon cycle were unified in a single integrated software system. The new version also has a new set of state-of-the-art physical parameterizations and greater computational parallel and memory usage efficiency. Together with the description of the main features are examples of the quality of the transport scheme for scalars, radiative fluxes on surface and model simulation of rainfall systems over South America in different spatial resolutions using a scale-aware convective parameterization. Besides, the simulation of the diurnal cycle of the convection and carbon dioxide concentration over the Amazon Basin, as well as carbon dioxide fluxes from biogenic processes over a large portion of South America are shown. Atmospheric chemistry examples present model performance in simulating near-surface carbon monoxide and ozone in Amazon Basin and Rio de Janeiro megacity. For tracer transport and dispersion, it is demonstrated the model capabilities to simulate the volcanic ash 3-d redistribution associated with the eruption of a Chilean volcano. Then, the gain of computational efficiency is described with some details. BRAMS has been applied for research and operational forecasting mainly in South America. Model results from the operational weather forecast of BRAMS on 5 km grid spacing in the Center for Weather Forecasting and Climate Studies, INPE/Brazil, since 2013 are used to quantify the model skill of near surface variables and rainfall. The scores show the reliability of BRAMS for the tropical and subtropical areas of South America. Requirements for keeping this modeling system competitive regarding on its functionalities and skills are discussed. At last, we highlight the relevant contribution of this work on the building up of a South American community of model developers

The Brazilian developments on the Regional Atmospheric Modeling System (BRAMS 5.2): an integrated environmental model tuned for tropical areas

We present a new version of the Brazilian developments on the Regional Atmospheric Modeling System (BRAMS), in which different previous versions for weather, chemistry, and carbon cycle were unified in a single integrated modeling system software. This new version also has a new set of state-of-the-art physical parameterizations and greater computational parallel and memory usage efficiency. The description of the main model features includes several examples illustrating the quality of the transport scheme for scalars, radiative fluxes on surface, and model simulation of rainfall systems over South America at different spatial resolutions using a scale aware convective parameterization. Additionally, the simulation of the diurnal cycle of the convection and carbon dioxide concentration over the Amazon Basin, as well as carbon dioxide fluxes from biogenic processes over a large portion of South America, are shown. Atmospheric chemistry examples show the model performance in simulating near-surface carbon monoxide and ozone in the Amazon Basin and the megacity of Rio de Janeiro. For tracer transport and dispersion, the model capabilities to simulate the volcanic ash 3-D redistribution associated with the eruption of a Chilean volcano are demonstrated. The gain of computational efficiency is described in some detail. BRAMS has been applied for research and operational forecasting mainly in South America. Model results from the operational weather forecast of BRAMS on 5 km grid spacing in the Center for Weather Forecasting and Climate Studies, INPE/Brazil, since 2013 are used to quantify the model skill of near-surface variables and rainfall. The scores show the reliability of BRAMS for the tropical and subtropical areas of South America. Requirements for keeping this modeling system competitive regarding both its functionalities and skills are discussed. Finally, we highlight the relevant contribution of this work to building a South American community of model developers.CNPqFAPESPEarth System Research Laboratory at the National Oceanic and Atmospheric Administration (ESRL/NOAA), Boulder, USAInst Nacl Pesquisas Espaciais, Ctr Previsao Tempo & Estudos Climat, Cachoeira Paulista, SP, BrazilDiv Ciência da Computação, Instituto Tecnológico de Aeronáutica, São José dos Campos, SP, BrazilUniv Estadual Paulista Unesp, Fac Ciencias, Bauru, SP, BrazilCtr Meteorol Bauru IPMet, Bauru, SP, BrazilUniv Fed Sao Paulo, Dept Ciencias Ambientais, Diadema, SP, BrazilUniv Sao Paulo, Inst Astron Geofis & Ciencias Atmosfer, Sao Paulo, SP, BrazilUniv Fed Campina Grande, Dept Ciencias Atmosfer, Campina Grande, PB, BrazilEmbrapa Informat Agr, Campinas, SP, BrazilUniv Fed Sao Paulo, Inst Ciencia & Tecnol, Sao Jose Dos Campos, SP, BrazilUniv Fed Rio Grande do Norte, Dept Ciencias Atmosfer & Climat, Programa Pos Grad Ciencias Climat, Natal, RN, BrazilInst Nacl Pesquisas Espaciais, Ctr Ciencias Sistema, Sao Jose Dos Campos, SP, BrazilUniv Fed Sao Joao Del Rei, Dept Geociencias, Sao Joao Del Rei, MG, BrazilInst Nacl Pesquisas Espaciais, Lab Associado Computacao & Matemat Aplica, Sao Jose Dos Campos, BrazilUniv Evora, Inst Ciencias Agr & Ambientais Mediterr, Evora, PortugalUniv Lusofona Humanidades & Tecnol, Ctr Interdisciplinar Desenvolvimento Ambient Gest, Lisbon, PortugalUniv Fed Pelotas, Fac Meteorol, Pelotas, RS, BrazilUnive Tecnol Fed Parana, Londrina, PR, BrazilNASA, Goddard Space Flight Ctr, Univ Space Res Assoc, Goddard Earth Sci Technol & Res Global Modeling &, Greenbelt, MD USAUniv Fed Sao Paulo, Inst Ciencia & Tecnol, Sao Jose Dos Campos, SP, BrazilUniv Fed Sao Paulo, Inst Ciencia & Tecnol, Sao Jose Dos Campos, SP, BrazilCNPq: 306340/2011-9FAPESP: 2014/01563-1FAPESP: 2015/10206-0FAPESP: 2014/01564-8Web of Scienc

IL-17 Produced during Trypanosoma cruzi Infection Plays a Central Role in Regulating Parasite-Induced Myocarditis

Chagas disease is caused by the intracellular parasite Trypanosoma cruzi. This infection has been considered one of the most neglected diseases and affects several million people in the Central and South America. Around 30% of the infected patients develop digestive and cardiac forms of the disease. Most patients are diagnosed during the chronic phase, when the treatment is not effective. Here, we showed by the first time that IL-17 is produced during experimental T. cruzi infection and that it plays a significant role in host defense, modulating parasite-induced myocarditis. Applying this analysis to humans could be of great value in unraveling the elements involved in the pathogenesis of chagasic cardiopathy and could be used in the development of alternative therapies to reduce morbidity during the chronic phase of the disease, as well as clinical markers of disease progression. The understanding of these aspects of disease may be helpful in reducing the disability-adjusted life years (DALYs) and costs to the public health service in developing countries