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Introduction to Cells, DNA, and Viruses
Published in Patricia G. Melloy, Viruses and Society, 2023
Virologists are scientists who study viruses. They typically conduct their work in laboratory settings, although they occasionally conduct their research outside the laboratory and engage in more field work. A virologist might conduct experiments using live animal models (in vivo research) or work with mammalian or other animal cells grown in culture in the laboratory (in vitro research). One commonly used technique to study viral growth in the lab is known as a plaque assay. In this technique, host cells are added to media in a petri dish, and then the cells are infected with virus for several days. Scientists then monitor the clearings in the cell layer created by the virus killing cells in that local area. The clearing is known as a plaque. The number of plaques can be counted to quantify the amount of virus present (Lostroh 2019). The virologist uses all the tools that a typical cell and molecular biologist might use to analyze genes and gene expression into a protein. DNA sequencing is used to compare one virus to another. A powerful technique used to amplify DNA sequences is known as polymerase chain reaction (PCR), and this tool can be used for medical diagnostics to look for viral nucleic acid as well (Nobel Media AB 1993). Techniques such as northern blot and western blot are used to study RNA and protein expression respectively. Scientists may also use online databases or tools to study viral DNA sequences or protein structure.
Non-Photocatalytic and Photocatalytic Inactivation of Viruses Using Antiviral Assays and Antiviral Nanomaterials
Published in Devarajan Thangadurai, Saher Islam, Charles Oluwaseun Adetunji, Viral and Antiviral Nanomaterials, 2022
Suman Tahir, Noor Tahir, Tajamal Hussain, Zubera Naseem, Muhammad Zahid, Ghulam Mustafa
Plaque assay, the process typically utilised for virion absorption examination, can identify the influence of NPs on virion (Rigotto et al. 2011). Using plaque assay, virus infectivity can be measured and the antiviral capability of functional NPs can be assessed. In the usual assay, the stock of virus is diluted 10 times, and 0.1 mL of aliquot dilution is inoculated on monolayers of vulnerable cells. After the incubation phase, a virus is enabled to bind to the cells, and nutrient medium agar is coated on the monolayers. After a longer incubation phase, the original affected cells discharge viral progeny, and the existence of the gel limits their spreading to adjacent cells; this results in the development of the zone of affected cells termed as plaque, which become huge, sufficient and noticeable to bare eye in room temperature circumstances. Titer of stock virus can be estimated in PFU (plaque forming units), which is accurate value of virus contagion. Virucidal capability of NPs can precisely be established through analysing PFU value of viral previously and afterward attaching with antiviral NPs.
Immunomodulation in Gene Therapeutics
Published in Thomas F. Kresina, Immune Modulating Agents, 2020
Andreas Block, Susan S. Rich, Shu-Hsia Chen, Savio L. C. Woo
The recombinant adenovirus expressing murine interleukin 2 was synthesized by calcium coprecipitation of the plasmids pJM17 and pAd.RSV-mIL-2 in 293 transformed human kidney cells. pJM17 is a plasmid containing the complete human pathogen Ad5 adenovirus genome, and pAd.RSV-mIL-2 was generated by insertion of the rous sarcoma virus long-terminal repeat promotor and the gene for murine interleukin 2 into pXCJL.l, kindly provided by Frank Graham, McMaster University. Coprecipitation results in an Ad5 adenovirus with deletion of the early gene region 1 (El) and replacement by the interleukin 2 gene. Thus these recombinant adenoviruses can only replicate in transformed 293 cells providing the El gene and are replication-deficient in normal and tumor tissue. To reduce the risk of recombination with wild-type adenovirus, resulting in replication-competent recombinant adenovirus, the E3 region was point mutated in first-generation and deleted in second-generation recombinant adenoviruses. Ad.RSV-IL-2 was amplified from a single plaque and purified by using discontinuous cesium gradient ultracentrifugation. The titer of infectious particles was determined as plaque forming units (p.f.u.) utilizing a plaque assay in 293 cells. The synthesis of the suicide gene adenovirus Ad.RSV-TK was similar to synthesis of the cytokine expressing adenovirus and has been reported [96,97].
Multivalent IgM scaffold enhances the therapeutic potential of variant-agnostic ACE2 decoys against SARS-CoV-2
Published in mAbs, 2023
Meghan M. Verstraete, Florian Heinkel, Janessa Li, Siran Cao, Anh Tran, Elizabeth C. Halverson, Robert Gene, Elizabeth Stangle, Begonia Silva-Moreno, Sifa Arrafi, Jegarubee Bavananthasivam, Madeline Fung, Mariam Eji-Lasisi, Stephanie Masterman, Steve Xanthoudakis, Surjit Dixit, John Babcook, Brandon Clavette, Mark Fogg, Eric Escobar-Cabrera
Viral genomic RNA was quantified by real time-PCR to the target viral envelope gene as previously described.77 Five days after infection, bronchoalveolar lavage (BAL) and lung tissue necropsy were collected for measurement of infectious viral particles by plaque assay. qRT-PCR was also performed to measure viral RNA in the samples using the method described.77 Plaque assay was carried out as previously described.78 In brief, centrifuge clarified supernatant from homogenized lung tissues were diluted in a 1 in 10 serial dilution in infection media. Virus was adsorbed on Vero cells for 1 h at 37°C before inoculum was removed and overlaid with infection media containing 0.6% ultrapure, low-melting point agarose. Infected cells were incubated at 37°C/5% CO2 for 72 h. After incubation, cells were fixed with 10% formaldehyde and stained with crystal violet. Plaques were enumerated and PFU was determined per gram of lung tissue or per mL of BAL.
Antiviral and antibacterial potential of electrosprayed PVA/PLGA nanoparticles loaded with chlorogenic acid for the management of coronavirus and Pseudomonas aeruginosa lung infection
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2023
Asmaa Saleh, Dalia H. Abdelkader, Thanaa A. El-Masry, Duaa Eliwa, Badriyah Alotaibi, Walaa A. Negm, Engy Elekhnawy
The in vitro antiviral activity of the free CGA, PVA/PLGA NPs loaded with CGA, and blank NPs was investigated against the low pathogenic human coronavirus (HCoV-229E) and MERS-CoV (NRCEHKU270) using plaque assay. Plaque assay is a commonly used quantitative method for determining the number of infectious viruses [35] by determination of the number of the formed plaques in the cell culture following infection with the studied virus [36]. In the current study, PVA/PLGA NPs loaded with CGA had significantly lower (p < .05) values of the half maximal inhibitory concentration (IC50) against the low pathogenic human coronavirus (HCoV-229E) and MERS-CoV (NRCEHKU270). The value of IC50 represents the drug concentration needed to achieve an in vitro 50% inhibition. Therefore, lower values of IC50 indicate more potent antiviral activity [36]. A future study is needed to investigate the effectiveness of the formula against the tested viruses in vivo.
Identification and validation of Sertoli cell homing peptides as molecular steering for testis targeted drug delivery
Published in Journal of Drug Targeting, 2023
Yugandhara Jirwankar, Vikas Dighe
Plaque assay was performed according to the New England Biolab’s instruction manual. Blue-colored plaques were picked and amplified in E. coli ER2738 host cells. Amplified phage clones were isolated with PEG/NaCl precipitation, and ssDNA was isolated according to the protocol mentioned by Green et al. [34]. PCR was performed to amplify the product using forward primer: 5′-TGG TTG TTG TCA TTG TCG GC-3′ and reverse primer: 5′-GCA AGC CCA ATA GGA ACC CA-3′. PCR reaction mixture of 50 µL consisted of 25 µL 2x DreamTaq Green PCR Master Mix, 2.5 µL each of forward and reverse primer (10 µM stock), 15 µL nuclease-free water, and 5 µL template. PCR conditions were initial denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and Final extension at 72 °C for 5 min. The reaction was performed on Agilent’s Sure Cycler 8800. The PCR product was run on 1.8% agarose gel, extracted using PureLink Quick gel extraction kit; Sanger sequencing was performed with -96gIII reverse sequencing primer provided in the PhD-12 phage library kit.