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The Viruses
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
Viral infection is initiated following virus adsorption, penetration, and uncoating (Figure 16.2). The nucleic acids of DNA viruses, except those of the poxviruses are reproduced in the cell nucleus. Poxviruses are reproduced in the cytoplasm. The nucleic acids of RNA viruses, except for retroviruses and certain orthomyxoviruses such as influenza A virus are reproduced in the cytoplasm. The viral genomic element formed early during viral nucleic acid synthesis acts as a template for synthesis of new viral genomes and messenger RNA transcription and is called the replicative form (RF). The RF gives rise to progeny viral nucleic acids, detected as replicative intermediates in addition to viral mRNA. Translation of viral mRNA gives rise to the viral proteins. The processes of assembly, maturation, and release of progeny virions from cells are generally associated with cytopathic effects such as the formation of inclusion bodies, cytolysis, and cell fusion.
Anti-HSV and Cytotoxicity Properties of Three Different Nanoparticles Derived from Indian Medicinal Plants
Published in P. Mereena Luke, K. R. Dhanya, Didier Rouxel, Nandakumar Kalarikkal, Sabu Thomas, Advanced Studies in Experimental and Clinical Medicine, 2021
K. Vasanthi, G. Reena, G. Sathyanarayanan, Elanchezhiyan Manickan
Vero cells were plated on 96-well plate, at a density of 10,000 cells/well for 70–80% confluency. Cells was infected with HSV-1 and HSV-2 and incubated for 90 min at 37°C. Varying concentrations of the different nanoparticles were added to the virus infected 96 well plates and incubate for 7 days at 37°C in 5% CO2 environment. Cytopathic effects (CPE) and their reduction were observed for 7 days (Figure 13.3). Cell control and Virus control, Drug control were put along with the assay.
Determination of Antiviral Activity
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
The initial or primary screening tool for antiviral studies is the cell culture test. Such tests utilize living cells susceptible to the targeted virus. The cells are cultivated in a defined synthetic medium, usually supplemented by some type of serum (such as fetal bovine, calf, or horse), a buffer, and sometimes bacterial- and fun gal-inhibiting antibiotics. In most antiviral tests, the cells are allowed to divide to become a thinly confluent monolayer, at which time the medium is changed and new medium containing a pretitered concentration of virus is added to the cells, usually shortly following, at the same time as, or shortly before addition of a test substance to the cells. The virus infection will usually subsequently alter the cell, causing a visibly discernible cytopathic effect. Such effects can be manifested as alteration in cell shape, size, in the actual destruction of the cell, or in the cell detaching from the monolayer. Most of these effects are readily seen microscopically; major cell destruction can be seen as plaques visible to the naked eye, particularly if an agar overlay is added to the cells to isolate the infection. pH alteration usually occurs in such virally infected cells, resulting in a color change in the medium due to an indicator dye incorporated into the medium. More subtle viral infections can be demonstrated by specific immunofluorescence procedures [25]. We have used this latter technique successfully for antiviral studies with rotavirus [26,27].
Pulmonary delivery of favipiravir inhalation solution for COVID-19 treatment: in vitro characterization, stability, in vitro cytotoxicity, and antiviral activity using real time cell analysis
Published in Drug Delivery, 2022
Ayca Yildiz Pekoz, Ozlem Akbal Dagistan, Hanan Fael, Meltem Culha, Aybige Erturk, Nur Sena Basarir, Gokben Sahin, Muge Serhatli, Gamze Cakirca, Saban Tekin, Leyla Semiha Sen, Mustafa Sevim, Lutfiye Mulazimoglu Durmusoglu, Berrak C. Yegen
For this study, a high-throughput, quantitative Real Time Cell Analysis (RTCA) assay was performed using xCELLigence RTCA MP (Agilent Technologies, CA, USA) to determine favipiravir’s effects on the virus by quantifying the viral cytopathic effect and examining progression in infected cells. The xCELLigence RTCA MP system evaluates the effects of viruses on cells by measuring the changes in cell numbers through cell index (CI) and electronic impedance (Teng et al., 2013; Fang et al., 2011). Furthermore, the RTCA system allows simple detection and observation of cellular death due to both the viruses or the antiviral impact of medications (Teng et al., 2013; Fang et al., 2011). The suppression of SARS-CoV-2-induced cytopathic effect was categorized as either ‘completely’ or ‘partially’ antiviral. The test sample was considered ‘completely’ antiviral, if the SARS-CoV-2-induced cytopathic effect was inhibited at the highest concentration tested. On the other hand, it was considered ‘partially’ antiviral, if the effect was delayed and did not entirely inhibit the cytopathic effect of the highest concentration (Zost et al., 2020).
Quantification methods for viruses and virus-like particles applied in biopharmaceutical production processes
Published in Expert Review of Vaccines, 2022
Keven Lothert, Friederike Eilts, Michael W. Wolff
A classical approach for the determination of the infective virus titer is the TCID50 assay. Consecutive dilutions of the sample material are used to infect suitable host cells, usually in a 96-well plate format. The concentration at which fifty percent of the samples are infected is the measure for statistically calculating virus titers [6]. For the read-out, cytopathic effects are evaluated. Additionally, the virus of interest could directly express a fluorophore, such as the green fluorescent protein (GFP), in infected cells. This enables the analysis by fluorescence microscopy or plate readers. For products that cannot express fluorophores on their own, an additional staining procedure is possible, for example, using the alamar blue dye, which stains cells showing cytopathic effects, e.g. due to viral infection [7,8]. Staining can also be performed by using a fluorescently labeled antibody [9].
Potent neutralization of SARS-CoV-2 by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold
Published in mAbs, 2020
Zehua Sun, Chuan Chen, Wei Li, David R. Martinez, Aleksandra Drelich, Du-San Baek, Xianglei Liu, John W. Mellors, Chien-Te Tseng, Ralph S. Baric, Dimiter S. Dimitrov
The standard live virus-based microneutralization assay was used for measurement.22 Serially diluted test antibodies were prepared in 96-well microtiter plates with a final volume of 60 μl per well before adding 120 infectious units of SARS-CoV or SARS-CoV-2 in 60 μl to individual wells. After 2 h culturing at room temperature, 100 μl of the antibody-virus mixtures were transferred into designated wells of confluent Vero E6 cells grown in 96-well microtiter plates. Vero E6 cells with or without virus infection served as positive and negative controls, and Vero E6 cells treated with the MERS-CoV RBD-specific neutralizing m336 mAb were included as an additional control. Virus-induced formation of cytopathic effect was detected by microscopy after four days of incubation at 37°C. The work was performed in the Department of Microbiology & Immunology, Centers for Biodefense and Emerging Diseases, Galveston National Laboratory, 301 University Blvd, Galveston, Texas 77550, USA.