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Detection Assays and Techniques Against COVID-19
Published in Hanadi Talal Ahmedah, Muhammad Riaz, Sagheer Ahmed, Marius Alexandru Moga, The Covid-19 Pandemic, 2023
Shahzad Sharif, Maham Saeed, Javed Hussain Shah, Sajjad Hussain, Ahmad Adnan, Hanadi Talal Ahmedah, Muhammad Riaz
Further analyzing strategies are elaborated as the techniques involved in characterization or structure elucidation like EM, XRD, and AFM that exhibit a full range of techniques that can be strongly employed to detect drug discovery, analysis, and ultimate results for ensuring public health and welfare. The use of advanced technology for scientific development, for example, the improvement of Corona detection bio-sensors, there should be a strong contribution in shaping a new era utilizing the best technical tools for future utilizations. Hence, differences must be highlighted with similarities among the different viral RNA infections and the COVID-19 disease. This will serve the understanding between the present technology and that which will sensitize and characterize the SARS-CoV2 virus [14].
The Challenge of Parasite Control
Published in Eric S. Loker, Bruce V. Hofkin, Parasitology, 2023
Eric S. Loker, Bruce V. Hofkin
Several newer vaccine types have been developed, some of which are acellular vaccines in that they do not include whole organisms. An example is the subunit vaccine, in which the vaccine consists of particular immunostimulatory antigens only. The hepatitis B vaccine, for instance, is composed only of viral surface proteins. Our understanding of the stimulatory role of T cells in a humoral response has resulted in the development of conjugate vaccines (Figure 9.28). These vaccines rely on a combination of antigens that stimulate both B and T cells. The Haemophilus influenzae vaccine, for instance, combines polysaccharides found in the bacterial capsule with peptides recognized by antigen-specific T cells. The result is a much stronger antibody response then could be elicited with the polysaccharides alone. And most recently, used clinically only since late 2020, are the mRNA vaccines used against the SARS-CoV-2 virus that causes Covid-19. These vaccines consist of viral mRNA that encodes an antigenic viral peptide. The RNA is surrounded by a lipid-based vesicle, which fuses with host cells, allowing the RNA to enter these cells. The viral mRNA is subsequently translated by host translation machinery and the resulting viral peptide is released from the cell where it stimulates an immune response. See the web callout associated with this section to learn about other vaccine types, including those based on nucleic acid.
The Viruses
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
The neuraminidase enables influenza virus to penetrate mucous secretions by virtue of its enzymatic activity. Neuraminidase also promotes the release of the virions as they bud from the cell surface. The envelope hemagglutinin serves to attach the virus to cells by binding to cell receptors. The virus then enters the cell in an endosomal vesicle. As the pH of the vesicle becomes acidic, the hemagglutinin changes conformation and allows fusion of the viral envelope with the endosomal membrane, resulting in uncoating and release of the viral nucleocapsid into the cell cytoplasm. Influenza viruses, unlike most RNA viruses, replicate in the cell nucleus rather than in the cytoplasm. The influenza virus has a negative stranded RNA, which is not translated directly by the host cell. Initiation of replication is possible because the virus encodes and packages its own RNA-dependent RNA polymerase. The viral RNA consists of eight different single-stranded segments, each coding for at least one of the major viral proteins. If two strains of influenza A virus infect the same cell, an interchange of entire genomic segments can occur (reassortment). Unlike classical genetic recombination, splicing and rejoining of the nucleic acid is not required in this process. Related influenza A viruses also infect animals of a variety of species, including pigs and many types of birds. These viral strains represent potential pools of genetic material for pathogenic human influenza strains by reassortment of genomic segments between animal and human influenza strains that infect a common host.
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.
Advances in RT-LAMP for COVID-19 testing and diagnosis
Published in Expert Review of Molecular Diagnostics, 2023
Gihoon Choi, Taylor J. Moehling, Robert J. Meagher
The most straightforward RT-LAMP assay format is to use compatible additives or none at all. Raw sample specimens are added directly to the fully formulated master mix (including enzymes), and the reaction is heated to initiate RT-LAMP. This single-step, ‘one-pot’ technique releases viral genomes into the LAMP master mix via thermal lysis at the reaction temperature. The exact temperature at which RNA begin to release from the particle is not well characterized. Meanwhile, RNases act to degrade viral RNA as soon as it is released from the viral particle. We characterize this process as a race between RNase and RT (Figure 3a): as the reaction heats up, viral particles release RNA into the solution, exposing them to RNase. The BIP primer must bind to the viral RNA and the RT enzyme must synthesize the first strand of cDNA before RNase degrades the viral RNA, and before the RT enzyme loses its activity. Once the first strand of cDNA is synthesized, the task of the RT enzyme is complete. Any residual RNase activity is irrelevant because the remainder of the LAMP process proceeds purely with DNA. We are not aware of much literature detailing this kinetic race, except that if the starting viral load is high enough, the RT outcompetes the RNase, leading to successful amplification. Work by Sun et al. with digital RT-LAMP suggests that the efficiency of the RT step is significantly below 100%, so more than one copy of RNA must survive to trigger exponential amplification and a positive result [28].
Modern vaccine strategies for emerging zoonotic viruses
Published in Expert Review of Vaccines, 2022
Atif Ahmed, Muhammad Safdar, Samran Sardar, Sahar Yousaf, Fiza Farooq, Ali Raza, Muhammad Shahid, Kausar Malik, Samia Afzal
The structure of self-amplifying (saRNA) closely resembles that of naRNA. Unlike naRNA vaccines, which contain only genes for antigen expression, saRNA-based vaccines contain viral RNA replication genes along with the genes for antigen expression to stay for a prolonged duration and enhanced antigen production even with a negligible dose of RNA [48]. When saRNA is delivered into the cytosol, it initially expresses the non-structural genes to assemble the replication complex quickly. The newly assembled replication machinery then transcribes the full-length negative-sense RNA into full-length genomic RNA and shorter sub-genomic RNAs. The entire stretch of genomic RNA mediates the translation of more replicates for auto-replication, while the expression of sub-genomic RNAs occurs at tremendously high levels for antigen protein production to stimulate vigorous immune responses in the host [10]. Apart from immense expression, the size of the transcript and the high degree of secondary structure limits the elevated production. The trans-amplifying method based on the bipartite RNA vector system was utilized to prepare the small-length RNAs and enhance stability [49,50]. In this technique, a replicase-encoding gene is present in one vector cassette, while the second molecule contains a gene to encode antigenic peptides. As a result, nano-gram doses of mRNA are sufficient to elicit a robust and protective immune response in vaccinated individuals [49].