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      Inhibitory effect of microalgae and cyanobacteria extracts on influenza virus replication and neuraminidase activity

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          Abstract

          Background

          The influenza virus can cause seasonal infections with mild to severe symptoms, circulating worldwide, and it can affect people in any age group. Therefore, this infection is a serious public health problem that causes severe illness and death in high-risk populations. Every year, 0.5% of the world’s population is infected by this pathogen. This percentage can increase up to ten times during pandemics. Influenza vaccination is the most effective way to prevent disease. In addition, anti-influenza drugs are essential for prophylactic and therapeutic interventions. The oseltamivir (OST, a neuraminidase inhibitor) is the primary antiviral used in clinics during outbreaks. However, OST resistant viruses may emerge naturally or due to antiviral pressure, with a prevalence of 1–2% worldwide. Thus, the search for new anti-influenza drugs is extremely important. Currently, several groups have been developing studies describing the biotechnological potential of microalgae and cyanobacteria, including antiviral activity of their extracts. In Brazil, this potential is poorly known and explored.

          Methods

          With the aim of increasing the knowledge on this topic, 38 extracts from microalgae and cyanobacteria isolated from marine and freshwater biomes in Brazil were tested against: cellular toxicity; OST-sensitive and resistant influenza replications; and neuraminidase activity.

          Results

          For this purpose, Madin-Darby Canine Kidney (MDCK)-infected cells were treated with 200 μg/mL of each extract. A total of 17 extracts (45%) inhibited influenza A replication, with seven of them resulting in more than 80% inhibition. Moreover, functional assays performed with viral neuraminidase revealed two extracts (from Leptolyngbya sp. and Chlorellaceae) with IC 50 mean < 210 μg/mL for influenza A and B, and also OST-sensitive and resistant strains. Furthermore, MDCK cells exposed to 1 mg/mL of all the extracts showed viability higher than 80%.

          Discussion

          Our results suggest that extracts of microalgae and cyanobacteria have promising anti-influenza properties. Further chemical investigation should be conducted to isolate the active compounds for the development of new anti-influenza drugs. The data generated contribute to the knowledge of the biotechnological potential of Brazilian biomes that are still little explored for this purpose.

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          Most cited references51

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          Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology.

          Land-adapted plants appeared between about 480 and 360 million years ago in the mid-Palaeozoic era, originating from charophycean green algae. The successful adaptation to land of these prototypes of amphibious plants - when they emerged from an aquatic environment onto the land - was achieved largely by massive formation of "phenolic UV light screens". In the course of evolution, plants have developed the ability to produce an enormous number of phenolic secondary metabolites, which are not required in the primary processes of growth and development but are of vital importance for their interaction with the environment, for their reproductive strategy and for their defense mechanisms. From a biosynthetic point of view, beside methylation catalyzed by O-methyltransferases, acylation and glycosylation of secondary metabolites, including phenylpropanoids and various derived phenolic compounds, are fundamental chemical modifications. Such modified metabolites have altered polarity, volatility, chemical stability in cells but also in solution, ability for interaction with other compounds (co-pigmentation) and biological activity. The control of the production of plant phenolics involves a matrix of potentially overlapping regulatory signals. These include developmental signals, such as during lignification of new growth or the production of anthocyanins during fruit and flower development, and environmental signals for protection against abiotic and biotic stresses. For some of the key compounds, such as the flavonoids, there is now an excellent understanding of the nature of those signals and how the signal transduction pathway connects through to the activation of the phenolic biosynthetic genes. Within the plant environment, different microorganisms can coexist that can establish various interactions with the host plant and that are often the basis for the synthesis of specific phenolic metabolites in response to these interactions. In the rhizosphere, increasing evidence suggests that root specific chemicals (exudates) might initiate and manipulate biological and physical interactions between roots and soil organisms. These interactions include signal traffic between roots of competing plants, roots and soil microbes, and one-way signals that relate the nature of chemical and physical soil properties to the roots. Plant phenolics can also modulate essential physiological processes such as transcriptional regulation and signal transduction. Some interesting effects of plant phenolics are also the ones associated with the growth hormone auxin. An additional role for flavonoids in functional pollen development has been observed. Finally, anthocyanins represent a class of flavonoids that provide the orange, red and blue/purple colors to many plant tissues. According to the coevolution theory, red is a signal of the status of the tree to insects that migrate to (or move among) the trees in autumn. Copyright © 2013 Elsevier Masson SAS. All rights reserved.
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            IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion

            Introduction The recently identified interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses [1]–[3]. Ectopic expression of IFITM1, -2 and -3 restricts a growing number of unrelated viruses, including IAV [1], [2], [4]–[7]. IFITM3 has been shown to potently restrict infection by IAV and the Respiratory Syncytial Virus in vivo [8]–[10]. In contrast, arenaviruses and some retroviruses, such as murine leukemia virus (MLV), are resistant to IFITM restriction [2], [6]. The IFITMs have been reported to inhibit HIV-1 entry, albeit less potently than IAV and apparently in a cell type-dependent manner [11]–[13]. The mechanism by which IFITMs inhibit infection of diverse viruses is not fully understood. IFITM2 and -3 are predominantly found in late endosomes (LE) and lysosomes [13], [14], whereas IFITM1 is also found at the cell periphery [4], [15]. Different membrane topologies of IFITMs have been proposed [16], but recent data suggests that IFITM3 is a type II transmembrane protein [17]. Accumulating evidence implies that IFITMs may interfere with virus-endosome fusion [1], [2], [5], [13], [14]. The fact that IFITMs seem to expand acidic intracellular compartments [13] indicates that the fusion block is downstream of the low pH trigger. Effective restriction of viruses that enter from the LE, such as IAV, Ebola virus (EBOV) and SARS coronavirus seems consistent with the cellular localization of IFITM2 and -3 proteins. However, these proteins also restrict Vesicular Stomatitis Virus (VSV) that appears to fuse with early endosomes [18]. IFITMs have been reported to curtail viral infection by modifying properties of cellular membranes, such as fluidity and spontaneous curvature [3], [5], [14]. These effects could be related, in part, to the accumulation of cholesterol in LE as a result of IFITM-mediated disruption of the interaction between the vesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) [14]. Since lipids play an important role in membrane fusion, these findings offer an attractive paradigm for a broad antiviral defense mechanism that involves altering the lipid composition of cellular membranes. The recent finding that amphotericin B, which forms complexes with sterols [19], rescues IAV infection in IFITM2- and IFITM3-expressing cells [20] is in line with the notion that cholesterol may be directly or indirectly involved in IAV restriction. However, lipid composition-based models do not readily explain the lack of restriction of amphotropic MLV and arenaviruses, which enter cells via distinct endocytic routes [21], [22]. These findings indicate that IFITMs may restrict virus entry from a subset of intracellular compartments. In order to define the mechanism of IFITM restriction, it is important to identify the viral entry step(s) targeted by these proteins, define compartments in which restriction occurs, and elucidate potential changes in intracellular membranes that may be responsible for this phenotype. Here, we examined the mechanism of IFITM3 restriction of IAV using single particle imaging and a direct virus-cell fusion assay. Our results show that IFITM3 does not inhibit the lipid mixing stage of IAV fusion but blocks the release of viral contents into the cytosol, and that this phenotype does not correlate with cholesterol accumulation in intracellular compartments. Specifically, IFITM3 inhibits the conversion of hemifusion to fusion through a mechanism that does not rely on cholesterol accumulation. Together these findings reveal a previously unappreciated view of IFITM-mediated restriction and suggest new avenues of investigation to delineate the mechanism by which these proteins block infection. Results Virus- and cell type-dependent restriction of viral fusion by IFITM3 We chose to focus on IFITM3 to study the mechanism of IAV restriction because this protein potently inhibits infection in vitro and in vivo [8]–[10]. Since published data suggest that IFITM3 likely inhibits the viral fusion step, a direct virus-cell fusion assay was employed to evaluate the extent of restriction in different cell lines [23]. HIV-1 particles carrying the β-lactamase-Vpr (BlaM-Vpr) chimera and pseudotyped with the influenza HA and NA proteins from the H1N1 A/WSN/33 strain (referred to as IAVpp) were allowed to fuse with cells transduced with an empty vector or with an IFITM3-expressing vector. The resulting cytosolic BlaM activity was measured as previously described [24]. Out of several cell lines tested, A549 and MDCK cells over-expressing IFITM3 were least permissive to IAVpp fusion (Fig. 1A). In agreement with the previous reports [2], [13], we found that IFITM3 over-expression partially inhibited VSV G glycoprotein-mediated fusion of pseudoviruses (VSVpp) carrying the BlaM-Vpr chimera (Fig. 1A). Similar to inhibition of IAVpp fusion, the IFITM3-mediated restriction of VSVpp was most potent in A549 and MDCK cells. As expected, fusion of particles pseudotyped with the Lassa fever virus glycoprotein (LASVpp), which directs virus entry through an IFITM3-resistant pathway [2], [6], was not considerably affected by IFITM3 over-expression. 10.1371/journal.ppat.1004048.g001 Figure 1 IFITM-mediated restriction of virus-endosome fusion in different cell types. (A) IFITM3-mediated inhibition of viral fusion with different cell types. BlaM-Vpr carrying pseudoviruses (IAVpp, VSVpp and LASVpp, MOI = 1) were bound to IFITM3- or vector-transduced A549, MDCK, CV1, HeLaH1 or CHO cells in the cold. Fusion was allowed to proceed for 90 min at 37°C and was measured by the BlaM assay, as described in Materials and Methods. ND, not determined. Data are means and SEM from 2 independent triplicate experiments. (B) IFITM3 expression patterns in A549, MDCK and CHO cells transduced with an empty vector (left) or IFITM3 (right). Cells were fixed, permeabilized and immunostained for IFITM3 (red), as described in Materials and Methods. The nuclear stain, Hoechst-3342, is shown in blue. (C) IFITM3 restricts fusion of influenza virus-like particles containing β-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1). Experiments were carried out as described above. Data are means and SEM from 2 independent triplicate experiments. (D) Exposure to low pH overcomes the IFITM3-mediated block of IAVpp fusion. To force pseudovirus fusion at the plasma membrane, A549 cells transduced with IFITM1, IFITM3 or an empty vector were pretreated with 50 nM BafA1 for 30 min at 37°C or left untreated. IAVpp/BlaM-Vpr pseudoviruses (MOI = 1) were bound to cells of in the cold and exposed to either a pre-warmed pH 5.0 MES-citrate buffer or neutral buffer for 10 min at 37°C and further incubated in growth medium (with or without BafA1) for 90 min at 37°C. Data are means and SEM from 2 independent triplicate experiments. ***, P 0.2. (C) Ensemble averages of initial vDiD dequenching profiles. The dequenching traces were aligned at the onset of hemifusion and averaged for each time point. Error bars are SEM. (D) The extent of vDiD dequenching was calculated based on I2/I1 ratio, as illustrated in Fig. 2B. Importantly, IAV lipid mixing was readily detected in IFITM3+ A549 and MDCK cells (Figs. 2D–G and 3A). Not only was lipid mixing not inhibited in A549-IFITM3 cells, but a >3-fold greater fraction of particles released vDiD in these cells compared to control cells (Fig. 3A, P 0.5). These results indicate that IFITM3 over-expression does not affect the properties of fusion intermediates responsible for vDiD redistribution, such as the size and/or architecture of a hemifusion site (e.g., [34], [35]). We then asked whether the rate of vDiD dequenching varied depending on the cell type. The average rate of vDiD fluorescence increase in MDCK cells was ∼2-fold greater than in A549 cells (Figs. 3C and S4B, P 690 particles) for each cell line. Error bars are standard deviations. (G) Dose-dependence of U18666A effect on viral fusion. A549 cells were pre-incubated for 18 h with indicated concentrations of U18666A or DMSO (control). BlaM-Vpr-carrying pseudoviruses (MOI = 1) were allowed to fuse with cells for 90 min at 37°C in the presence of U18666A or DMSO. Data are means and SEM from 2 triplicate experiments. **, P = 0.005. By comparison, pretreatment of A549-Vector cells with U18666A, which inhibits transport of LDL-derived cholesterol from LE/lysosomes (reviewed in [38]), resulted in a dramatic shift in the filipin staining pattern from the plasma membrane to endosomes (Fig. 5B). Aberrant accumulation of cholesterol in LE is also known to occur in cells lacking the functional NPC1 cholesterol transporter [39]. We therefore knocked down NPC1 expression in A549 cells using shRNA (shNPC1, Fig. 5D) and examined the resulting cholesterol distribution (Fig. 5B). Reduced NPC1 expression correlated with excess cholesterol in intracellular compartments, which was also much more pronounced than endosomal filipin staining in A549-IFITM3 cells. We next asked whether the cholesterol accumulation induced by U18666A pretreatment or by down regulation of NPC1 can phenocopy the IFITM3-mediated restriction of viral fusion. Neither IAV lipid mixing (vDiD dequenching) nor fusion (BlaM signal) was inhibited by silencing NPC1 in A549 cells (Fig. 5E, F). VSVpp also fused with shNPC1-transduced cells as efficiently as with control cells (Fig. 5E). These results show that excess cholesterol does not inhibit viral fusion or hemifusion. In control experiments, silencing the NPC1 expression potently suppressed fusion of Ebola GP-pseudotyped particles (EBOVpp, Fig. 5E), which use NPC1 as a receptor [40], [41]. Similar to the NPC1 knockdown phenotype, pretreatment of A549 cells with 10 µM U18666A, which caused cholesterol buildup in endosomes (Fig. 5B), did not inhibit fusion of IAVpp or VSVpp (Fig. 5G). As will be shown below for MDCK cells, higher doses of U18666A can inhibit viral fusion (Fig. 5G), but this effect is due to elevation of endosomal pH as opposed to cholesterol accumulation in endosomes. To generalize the effects of excess cholesterol in A549 cells, we tested whether endosomal cholesterol can inhibit viral fusion in MDCK cells. As in A549 cells, IFITM3 over-expression in MDCK cells caused moderate accumulation of cholesterol in endosomes (Fig. 6A), while pre-treatment with U18666A caused a much more dramatic buildup of intracellular cholesterol (Fig. 6B). However, unlike A549 cells, IAVpp and VSVpp fusion was significantly inhibited in U18666A-treated MDCK cells (Fig. 6C). Since prolonged exposure to U18666A has been reported to raise endosomal pH [42], we sought to determine if insufficiently acidic pH could prevent IAV hemifusion/fusion with pretreated MDCK cells. 10.1371/journal.ppat.1004048.g006 Figure 6 IFITM3-mediated restriction of IAV fusion is not related to cholesterol accumulation in endosomes of MDCK cells. (A) Sub-cellular distributions of cholesterol (filipin staining) and IFITM3 (antibody staining) in MDCK-Vector and MDCK-IFITM3 cells. Images show confocal sections through the middle of cells. (B) Filipin staining of MDCK cells pretreated with 20 µM U18666A for 18 h or mock-treated cells. (C) Dose-dependence of U18666A effect on viral fusion. MDCK-Vector cells were pretreated for 18 h with indicated concentrations of U18666A or DMSO (control). BlaM-Vpr-carrying pseudoviruses (MOI = 1) were allowed to fuse with cells for 90 min at 37°C in the presence of U18666A or DMSO. Data are means and SEM from 2 triplicate experiments. (D–F) pH distributions in IAV-carrying endosomes of MDCK cells measured using AF488- and CypHer5E-labeled viruses. Viruses were pre-bound to cells in the cold and incubated at 37°C for 45 min before acquiring images. Calculated pH values are shown for MDCK cells without (D) and with pretreatment with 20 µM U18666A for 18 h (E), as well as for MDCK-IFITM3 cells (F). Data are from 10 image fields each. The pH in IAV-carrying endosomes was measured using virions co-labeled with the pH-insensitive AF488 (green) and CypHer5E (red), which fluoresces brighter at acidic pH [28] (Fig. S7A). Cells were incubated with viruses for 45 min, and the red/green signal ratio from individual particles was measured (Fig. S7B). The average pH in virus-containing endosomes of MDCK-IFITM3 cells was slightly less acidic than in control cells: 5.38±0.03 (n = 498) vs. 4.98±0.04 (n = 242), respectively (Fig. 6D and F, P 20 tracks. *, P 30 cells per field. Particle-to infectivity ratio was calculated from the fraction of infected cells and the average number of virions bound to cells. Live cell imaging experiments (n = 10 for A549 and n = 6 for MDCK cells) yielded the number of cells receiving at least hemifusion event. (PDF) Click here for additional data file. Figure S6 Subcellular distribution of cholesterol and levels of total and free cellular cholesterol. (A) Total cellular filipin was estimated by calculating the filipin fluorescence intensity over the entire image field (after subtracting the background signal) and normalizing by the number of cells per field. Data are means and standard deviations for 4 and 6 fields for A549 and A549-IFITM3 cells (131 and 184 cells), respectively. (B, C) Total and free cellular cholesterol (in µg/106 cells) were measured by a fluorimetric enzymatic assay using the Cholesterol Kit from Sigma-Aldrich. Data are means and standard deviations from 2 measurements performed with duplicate samples. ***, P<0.001; *, P<0.03. (PDF) Click here for additional data file. Figure S7 Calibration of labeled IAV as a pH-sensor. AF488- and CypHer5E- labeled IAV particles were attached to poly-L-lysine coated coverslips, and the ratio of two fluorescence signals was measured in citrate-phosphate buffers of different acidity. (A) Top and bottom panels are images of labeled IAV at neutral pH and low pH, respectively. (B) The total signal for each dye was determined after thresholding and the CypHer5E/AF488 ratio at different pH are plotted. Error bars are standard deviations for 3 different imaged fields for each pH value. The line indicates a first order polynomial fit to the data, which served as a pH calibration curve. (PDF) Click here for additional data file. Figure S8 An example of single IAV lipid mixing event in CHO cells. (A) Image panels show entry of an AF488 (green) and vDiD (red) labeled virus into a CHO cell that culminates in vDiD dequenching (arrow). (B) Fluorescence intensity profiles of AF488 and vDiD obtained by tracking the virion shown in panel A. (PDF) Click here for additional data file. Figure S9 pH distribution in IAV carrying endosomes of CHO cells. Shown are the distributions of endosomal pH in CHO cells pretreated with 40 µM of U18666A for 12 h or left untreated. Cells were incubated with AF488/Cypher5E-labeled IAV, and endosomal pH was measured as described in Materials and Methods. U18666A increased endosomal acidity (P<0.001). (PDF) Click here for additional data file. Figure S10 Incoming IAV tends to colocalize with IFITM3-positive endosomes. A549-IFITM3 cells were allowed to internalize IAV for 90 min at 37°C and immunostained for the IAV-NP using mouse antibody (Millipore, Billerica, MA) and for IFITM3. The enlarged boxed area is shown on the right. IAV and IFITM3 puncta were identified by thresholding and object identification. The extent of colocalization was estimated by counting IAV puncta, which exhibited a volumetric overlap of at least 50% with IFITM3 puncta, and normalizing over all IAV puncta. The number in the right corner is the mean % colocalization and standard deviation for 7 image fields. (PDF) Click here for additional data file. Figure S11 A line-fitting approach to determining the onset and the initial rate of vDiD dequenching in single IAV fusion experiments. Fitting the vDiD dequenching traces with two straight lines yields the time of hemifusion (TH) and the initial slope of dequenching. (PDF) Click here for additional data file. Methods S1 Description of additional methods employed in this study. (DOCX) Click here for additional data file. Movie S1 Lipid mixing between single vDiD-labeled IAV and an endosome in A549 cells. IAV co-labeled with AF488 (green) and vDiD (red) was incubated with A549 cells at 37°C. The lipid mixing event (hemifusion) is manifested in marked increase of vDiD fluorescence. The numbers in the upper right corner show time after raising the temperature (min∶sec∶msec). Scale bar is 10 µm. For details, see Fig. 2A, B. (AVI) Click here for additional data file. Movie S2 Lipid mixing between single vDiD-labeled IAV and an endosome in A549-IFITM3 cell. IAV co-labeled with AF488 (green) and vDiD (red) was incubated with cells at 37°C. The lipid mixing event (hemifusion) is manifested in marked increase of vDiD fluorescence. The numbers in the upper right corner show time after raising the temperature (min∶sec∶msec). For details, see Fig. 2D, E. (AVI) Click here for additional data file. Movie S3 Lipid mixing upon entry of single vDiD-labeled IAV into an MDCK-IFITM3 cell. IAV co-labeled with AF488 (green) and vDiD (red) was incubated with cells at 37°C. Lipid mixing (hemifusion) is seen as marked increase in the vDiD signal. The numbers in the upper right corner show time after raising the temperature (min∶sec∶msec). For details, see Fig. 2F, G. (AVI) Click here for additional data file. Movie S4 IAVpp fusion with an endosome in A549 cell. A single IAV pseudovirus co-labeled with YFP-Vpr (green) and Gag-iCherry (red) releases its content marker (iCherry) after entering the cell. The numbers in the upper right corner show time after raising the temperature (min∶sec∶msec). For details, see Fig. 4A, B. (AVI) Click here for additional data file. Movie S5 IAVpp fusion with an MDCK cell. A single IAV pseudovirus co-labeled with YFP-Vpr (green) and Gag-iCherry (red) releases its content marker (iCherry) after entering the cell. The numbers in the upper right corner show time after raising the temperature (min∶sec∶msec). For details, see Fig. 4C, D. (AVI) Click here for additional data file.
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              An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.

              A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.
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                Author and article information

                Contributors
                Journal
                PeerJ
                PeerJ
                peerj
                peerj
                PeerJ
                PeerJ Inc. (San Francisco, USA )
                2167-8359
                26 October 2018
                2018
                : 6
                : e5716
                Affiliations
                [1 ]Laboratório de Vírus Respiratórios e do Sarampo, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz , Rio de Janeiro, Brazil
                [2 ]Laboratório de Fitoplâncton Marinho, Instituto de Biologia, Universidade Federal do Rio de Janeiro , Rio de Janeiro, Brazil
                [3 ]Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro , Rio de Janeiro, Brazil
                [4 ]Laboratório de Microbiologia Marinha, Instituto de Biologia, Universidade Federal do Rio de Janeiro , Rio de Janeiro, Brazil
                [5 ]Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz , Rio de Janeiro, Brazil
                [6 ]Centro de Desenvolvimento Tecnológico em Saúde, Fundação Oswaldo Cruz , Rio de Janeiro, Brazil
                Article
                5716
                10.7717/peerj.5716
                6204821
                30386690
                ac37963c-d224-468d-9640-b330232cace3
                ©2018 Silva et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

                History
                : 23 April 2018
                : 10 September 2018
                Funding
                Funded by: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
                Funded by: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)
                Funded by: Instituto Oswaldo Cruz - Fiocruz
                This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Oswaldo Cruz - Fiocruz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Marine Biology
                Virology
                Freshwater Biology

                cyanobacteria,microalgae,neuraminidase inhibition,anti-influenza extracts,ost-sensitive and resistant influenza viruses

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