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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].
Production of Clinical Lots of Gene Therapy Vectors Using Good Manufacturing Practice: Experience in a University Setting
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Viral vectors must be tested for replication competent viruses. For instance, adenovirus vectors need to be free of replication competent adenovirus (RCA) at a level of one plaque forming unit (PFU) of RCA per patient dose. This testing is done by passage of the recombinant adenovirus vector on a cell line that does not support the replication of vector virus such that only RCA can replicate. In the case of recombinant adenovirus vectors deleted in region El, since such vectors can grow only in the 293 packaging cell line that supplies El function, the vector is passaged on a cell line such as A549 (human lung carcinoma) which does not supply El function, to test for RCA.
The discovery of novel antivirals for the treatment of mpox: is drug repurposing the answer?
Published in Expert Opinion on Drug Discovery, 2023
Ahmed A. Ezat, Jameel M. Abduljalil, Ahmed M. Elghareib, Ahmed Samir, Abdo A. Elfiky
The use of nanoparticles and nanotechnology in the medical field has grown intensively in the last few decades due to their unique properties compared to their bulk materials. One of those promising nanoparticles is silver-included nanoparticles that have shown effective antimicrobial and antiviral properties against many organisms [79,80]. Rogers et al. have utilized silver nanoparticles (polysaccharide-coated and non-coated with different diameters) and silver nitrate (with different concentrations) as inhibitors for MPXV infectivity. Using a plaque reduction assay, they evaluated the antiviral activity of Ag-NPs and AgNO3. They found out that Ag-NPs (10 nm) coated with polysaccharide (PS) and all concentrations of AgNO3 (except for 100 µg/mL) exhibited a reduced number of plaque-forming units (PFU) compared to the controls. For Ag-NP (80 nm), Ag-NP (55 nm), and Ag-PS-NPs (25 nm), there was a dose-dependent decrease in plaque formation with the lowest results at 12.5 µg/mL. No cellular cytotoxicity was observed upon using silver nanoparticles or AgNO3, except at an AgNO3 concentration of 100 µg/mL. They also observed an exciting increase in the mean number of PFU compared to control at 50 and 100 µg/mL concentrations. The mechanisms that lead to viral inhibition by silver nanoparticles could not be fully understood. The viral inhibition could be attributed to the obstruction of nanoparticles to the viral host binding or disruption of intracellular pathways that may lead to attenuation in viral replication [75].
Oncolytic adenovirus promotes vascular normalization and nonclassical tertiary lymphoid structure formation through STING-mediated DC activation
Published in OncoImmunology, 2022
Teng He, Zhixing Hao, Mingjie Lin, Zhongwei Xin, Yongyuan Chen, Wei Ouyang, Qi Yang, Xiaoke Chen, Hui Zhou, Wanying Zhang, Pin Wu, Feng Xu
To evaluate the effects of Ad and Ad-IL15, unilateral B16-F10 and MC-38 tumor models were established in immunocompetent mice. In these models, 2*105 B16-F10 cells or 1*106 MC-38 cells were subcutaneously injected into the right flanks of the mice. Unless otherwise indicated, the mice were randomly divided into a treatment group and a control group when the tumors reached approximately 50 mm3. Approximately 5 × 108 plaque-forming units (PFU) of virus suspension or sterile phosphate-buffered saline (PBS) as a control (40 µL/injection) was injected into each tumor every other day, for a total of three doses. Eleven days after the last treatment, the mice were sacrificed, and tumor tissues were collected for follow-up experiments. To evaluate the effect of Ad and Ad-IL15 on tumor VN and TLS formation, the treatment regimen was performed as described above, and tumor tissues, blood and spleens were collected on the 7th day after the last treatment. To study of functional tumor vessels, 200 µL of 1 mg/mL Alexa Fluor 488-conjugated Lycopersicon esculentum (a.k.a. lectin) (Vector Laboratories) was injected into the caudal vein before the mice were euthanized.
Bacteriophage therapy cures a recurrent Enterococcus faecalis infected total hip arthroplasty? A case report
Published in Acta Orthopaedica, 2021
Ann-Sophie Neuts, Hanneke J Berkhout, Anita Hartog, Jon H M Goosen
Our patient travelled to Georgia to retrieve his ampoules of Pyophages and IntestiPhages. They were delivered in 10-mL vials as an oral suspension. He started using them on June 13, 2018, and continued for 19 days. The Pyophages were taken in the morning and Intestiphages in the evening. After a 2-week pause, the bacteriophage therapy was restarted for another 19 days. Unfortunately, the exact composition of these phages and their concentration, expressed as plaque-forming units (PFUs), remain unknown to us and cannot be deduced from the patient leaflet. As recommended in the available literature, our patient received daily antibiotics during the 2 short periods of phage therapy. During the first period of phage therapy, he used oral amoxicillin 1,000 mg 4 times a day. During the second period, he used oral doxycycline 200 mg once a day. In December 2019, all antibiotics were stopped. He had no hip complaints when we saw him in our outpatient clinic in February 2021. No new cultures have been obtained up to the time of writing (July 2021). A time schedule of the treatment is presented in Figure 2.