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Biological and Biochemical Analysis of Bacteria and Viruses
Published in Christopher S. Cox, Christopher M. Wathes, Bioaerosols Handbook, 2020
Andreas Hensel, Klaus Petzoldt
Conditions and containers for growth and maintenance of cell cultures may vary and depend mainly on the diagnostic needs of laboratories. Multiplication of a virus in a sensitive cell line can be evaluated by visible reactions, but it may be necessary to subculture the infected cells before a viral effect can be found. The following morphologic changes related to virus infection may occur: a cytopathic effect in the cell culture that may have a typical appearance (e.g., Poliovirus, Foot-and-mouth-disease virus, Herpes simplex virus), an inhibition of metabolism in the cell (e.g., Enterovirus), an ability to bind erythrocytes due to virus-specific hemagglutinin in the cell membrane (e.g., Influenza virus, Parainfluenza virus), an appearance of complement-fixing antigen (Adenovirus, Pseudorabies virus), and a transformation or interference with a non-pathogenic virus (e.g., BVD virus) or an oncogenic virus (e.g., SV 40, Rous sarcoma virus). When such a cell culture is examined, the viral effects may be known, but the name of the virus is not. Hence, virus identification based on infectivity assays needs confirming by a second test system, e.g., immunofluorescence, enzyme immunoassay, or neutralization test.76,78,79
Evidence for a Role of Infections in the Activation of Autoreactive T Cells and the Pathogenesis of Autoimmunity
Published in Richard K. Burt, Alberto M. Marmont, Stem Cell Therapy for Autoimmune Disease, 2019
J. Ludovic Croxford, Stephen D. Miller
The mechanisms by which a virus infection can induce demyelination have recently been reviewed9,10 and include: a direct cytopathic effect of the virus on infected cells such as oligodendrocytes, lysis of infected cells by antibody-mediated mechanisms and/or cytotoxic T cells, or bystander demyelination due to toxic levels of proinflammatory mediators such as TNF-a or reactive oxygen species. It is possible that the induction of MS may involve a nonspecific infection of the CNS and, therefore, the genetic susceptibility of each individual to a variety of infectious pathogens holds the key as to whether the infectious agent is capable of inducing autoimmune disease. Furthermore, studies need to address whether the presence of certain infectious agents correlate to the different temporal characteristics associated with relapsing-remitting or primary-progressive MS. Most of the evidence supporting viral infection in the pathogenesis of MS has been elucidated from animal models and will be discussed later.
Vectored vaccines
Published in Amine Kamen, Laura Cervera, Bioprocessing of Viral Vaccines, 2023
Zeyu Yang, Kumar Subramaniam, Amine Kamen
The primary characterization of AdV are viral particle units (VPs) and infectious viral particle units (IVPs). The most common method to physically determine the total viral particles relies on the absorbance reading at 260 nm. A more precise method to quantify the AdV is the anion-exchange–high-performance liquid chromatography (AE–HPLC), in which the VPs were detected by photodiode array detector at 260 nm [53]. For example, the AdV serotype 5 particles can be measured by AE-HPLC method using a UNO Q column. The virus peak was eluted at 450 mM NaCl in about 8 min. Another quantification method for detecting the VPs uses quantitative polymerase chain reaction (qPCR), which serves as a gold standard method. Compared with qPCR, the digital-droplet PCR (ddPCR) is more robust and has higher throughput since it provides absolute quantification with higher sensitivity, omits the use of a standard, and requires less sample volume [54]. For the quantification of IVP, the titers were usually determined by fifty-percent tissue culture infective dose (TCID50) assay. The TCID50 quantifies the amount of virus which can kill 50% of host cells or produce a cytopathic effect in 50% of cultured cells [36,55]. For replication-competent AdV (RCA), determinations are generally performed on pre-clinical and clinical studies. Results can be observed by cytopathic effect on permissive cells such as A-549 and Hela cells after multiple dilutions. In addition, using PCR can enhance the sensitivity of the method. To standardize the results of different quantitation methods and facilitate transfer of pre-clinical and clinical data, a reference material for AdV vector has been generated [56]. For more details, refer to chapter 9.
Intraoperative storage of saphenous vein grafts in coronary artery bypass grafting
Published in Expert Review of Medical Devices, 2019
Catherine J. Pachuk, Sophie K. Rushton-Smith, Maximilian Y. Emmert
There was no microscopic evidence of cytotoxicity after 30 minutes of exposure to DuraGraft or saline (Table 2). By 60 minutes of exposure, 30% of the saline-exposed cells in all three replicate samples had lysed (Grade 2) and failed to recover after the 24-hour recovery period (Table 3). By comparison, no cytopathic effect was observed in DuraGraft-exposed cells at 60 minutes of exposure, and there was no lysis or evidence of reactivity in any of the replicate samples (Table 2). After 5 hours of exposure, 90% of saline-exposed cells had lysed (Grade 4) with nearly complete or complete destruction of cell monolayers in all three replicate samples (Table 2), with no recovery observed after 24 hours (Table 3). In contrast, there was no lysis or evidence of reactivity in DuraGraft-exposed cells at 5 hours. After 24 hours of exposure, saline-exposed cells were 100% lysed in all three replicate samples (Grade 4), while DuraGraft-exposed cells in all three replicates exhibited only sight reactivity (Grade 1), with 10% lysis Table 3.
Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus
Published in Preparative Biochemistry and Biotechnology, 2019
Jun Zhao, Hai-Yang Yu, Yu Zhao, Feng-Hua Li, Wei Zhou, Bin-Bin Xia, Zhi-Yuan He, Jason Chen, Guo-Tuo Jiang, Ming-Li Wang
The protective effect of the recombinant protein on CEF cells against VSV infection was evaluated by the cytopathic effect inhibition based IFN-α bioassay.[24] Briefly, CEF cells were seeded in a 96-well cell culture plate containing 100 μl of DMEM medium at the concentration of 1.0 × 105 cells/ml. After incubation at 37 °C overnight, the medium was discarded and 100 μl of a serially two-fold diluted solution of the rChIFN-α fusion protein or recombinant human interferon-α (rHuIFN-α) standard (served as a positive control) were added to each well. Following another 18 h of incubation, each well was added with 50 μl VSV at m.o.i = 0.1 or DMEM medium. 24 h later, the cytopathic effect (CPE) degree of CEF cells in each well was checked separately using an optical microscope. Then, the cells were fixed in a solution containing 4% PFA (pH 7.4) and stained with crystal violet. After rinsing and drying, 100 μl pure methanol was added to each well. The plate was gently shaken, and the optical density (OD) value of each solution at 570 nm was read using a Microplate Reader (Bio-Tek Model EL311SX). All samples were tested independently three times. The unit of data was set to U/ml, of which one unit of IFN biological activity is defined as the reciprocal of the dilution that inhibited 50% CPE. Finally, the titer of rChIFN-α was calculated by the Reed–Muench method as previously described.[25,26] The Reed–Muench method is a simple method for determining 50% endpoints in experimental biology.[26] That is, the concentration of a test substance that produces an effect of interest in half of the test units. Examples include LD50 (the median lethal dose of a toxin or pathogen), EC50 and IC50 (half maximal effective or inhibitory concentration, respectively, of a drug), and TCID50 (50% tissue culture infectious dose of a virus). The reason for using 50% endpoints is that many dose-response relationships in biology follow a logistic function that flattens out as it approaches the minimal and maximal responses, so it is easier to measure the concentration of the test substance that produces a 50% response.