Nanomedicine "New Food for an Old Mouth": Novel Approaches for the Treatment of COVID-19

Coronavirus disease (COVID-19) is an infectious disease caused by coronavirus. Devel-oping specific drugs for inhibiting replication and viral entry is crucial. Several clinical trial studies are underway to evaluate the efficacy of anti-viral drugs for COVID-19 patients. Nanomedicine formulations can present a novel strategy for targeting the virus life cycle. Nano-drug delivery systems can modify the pharmacodynamics and pharmacokinetics properties of anti-viral drugs and reduce their adverse effects. Moreover, nanocarriers can directly exhibit anti-viral effects. A number of nanocarriers have been studied for this purpose, including liposomes, dendrimers, exosomes and decoy nanoparticles (NPs). Among them, decoy NPs have been considered more as nanodecoys can efficiently protect host cells from the infection of SARS-CoV-2. The aim of this review article is to highlight the probable nanomedicine therapeutic strategies to develop anti-viral drug delivery systems for the treatment of COVID-19.Copyright © 2023 Bentham Science Publishers.

Trace Elements Zinc and Selenium: Their Significance in the Conditions of the Covid-19 Pandemic

Within the conditions of the ongoing COVID-19 pandemic, when many questions regarding prevention and treatment strategies remain unsolved and the search for the best antiviral agents is underway, attention should be paid to the role of trace elements zinc and selenium in increasing the body's resistance to viral infections and their direct antiviral activity against SARS-CoV-2. Experimental data show that trace elements zinc and selenium not only actthrough regulating the immune response at all levels of humoral and cellular immunity, but also can play a significant role in adjuvant therapy for viral diseases. This is especially relevant in the case of COVID-19. Studies of the direct antiviral effect of these micro-elements testify to its 3 main ways to SARS-Cov-2: I - counteraction to virus replication and its transcription through: (i) their covalent binding to the SH-group of the cysteine of the main protease M(Pro) of the virus;(ii) inhibition of its RNA polymerase activity by zinc;II - preventing the penetration of the virus into cells due to blocking SH-groups of protein disulfide isomerase (RDI) of the protein of its spikes (peplomers);III - decreasing the adsorption capacity of the virus due to the blocking of the electrostatic interaction of SARS-CoV-2 peplomers and angiotensin-converting enzyme (ACE-2) in ultra-low, uncharacteristic oxidation states (Zn+1and Se-2). The intensity of the antiviral action of these trace elements may depend on their chemical form. It was found that zinc citrate (a five-membered complex of zinc with citric acid) and monoselenium citric acid obtained with the help of nanotechnology have a greater intensity of action and higher chemical purity. Taking into account the immunostimulating and direct antiviral effect of zinc and selenium, their use in the form of pharmaceuticals and dietary supplements should be considered as adjunctive therapy for SARS-CoV-2 in patients, or as a preventive strategy for uninfected people from risk groups during the spread of COVID-19.Copyright © Publisher PH <<Akademperiodyka>> of the NAS of Ukraine, 2023.

Withania somnifera phytochemicals possess SARS-CoV-2 RdRp and human TMPRSS2 protein binding potential.

Abstract: Coronavirus disease-19 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has infected approximately 26 million people and caused more than 6 million deaths globally. Spike (S)-protein on the outer surface of the virus uses human trans-membrane serine protease-2 (TMPRSS2) to gain entry into the cell. Recent reports indicate that human dipeptidyl peptidase-4 inhibitors (DPP4 or CD26) could also be utilized to check the S-protein mediated viral entry into COVID-19 patients. RNA dependent RNA polymerase (RdRp) is another key virulence protein of SARS-CoV-2 life cycle. The study aimed to identify the potential anti-SARS-CoV-2 inhibitors present in Withania somnifera (Solanaceae) using computer aided drug discovery approach. Molecular docking results showed that flavone glycoside, sugar alcohol, and flavonoid present in W. somnifera showed - 11.69, - 11.61, - 10.1, - 7.71 kcal/mole binding potential against S-protein, CD26, RdRp, and TMPRSS2 proteins. The major standard inhibitors of the targeted proteins (Sitagliptin, VE607, Camostat mesylate, and Remdesivir) showed the - 7.181, - 6.6, - 5.146, and - 7.56 kcal/mole binding potential. Furthermore, the lead phytochemicals and standard inhibitors bound and non-bound RdRp and TMPRSS2 proteins were subjected to molecular dynamics (MD) simulation to study the complex stability and change in protein conformation. The result showed energetically favorable and stable complex formation in terms of RMSD, RMSF, SASA, Rg, and hydrogen bond formation. Drug likeness and physiochemical properties of the test compounds exhibited satisfactory results. Taken together, the present study suggests the presence of potential anti-SARS-CoV-2 phytochemicals in W. somnifera that requires further validation in in vitro and in vivo studies. Supplementary information: The online version contains supplementary material available at 10.1007/s42535-022-00404-4.

Molecular Epidemiology of SARS-CoV-2 Omicron Sub-Lineages Isolated from Turkish Patients Infected with COVID-19.

Early detection and characterization of new variants and their impacts enable improved genomic surveillance. This study aims to evaluate the subvariant distribution of Omicron strains isolated from Turkish cases to determine the rate of antiviral resistance of RdRp and 3CLpro inhibitors. The Stanford University Coronavirus Antiviral & Resistance Database online tool was used for variant analyses of the strains uploaded to GISAID as Omicron (n = 20.959) between January 2021 and February,2023. Out of 288 different Omicron subvariants, B.1, BA.1, BA.2, BA.4, BE.1, BF.1, BM.1, BN.1, BQ.1, CK.1, CL.1, and XBB.1 were the main determined subvariants, and BA.1 (34.7%), BA.2 (30.8%), and BA.5 (23.6%) were reported most frequently. RdRp and 3CLPro-related resistance mutations were determined in n = 150, 0.72% sequences, while the rates of resistance against RdRp and 3CLpro inhibitors were reported at 0.1% and 0.6%, respectively. Mutations that were previously associated with a reduced susceptibility to remdesivir, nirmatrelvir/r, and ensitrelvir were most frequently detected in BA.2 (51.3%). The mutations detected at the highest rate were A449A/D/G/V (10.5%), T21I (10%), and L50L/F/I/V (6%). Our findings suggest that continuous monitoring of variants, due to the diversity of Omicron lineages, is necessary for global risk assessment. Although drug-resistant mutations do not pose a threat, the tracking of drug mutations will be necessary due to variant heterogenicity.

Is Remdesivir-Induced Symptomatic Bradycardia Persistent? A Two Case Report of COVID-19 Infection

As of May 2022, a total of over 528 million cases of coronavirus 19 disease (COVID-19) worldwide with over 6 million deaths. Remdesivir is a broad-spectrum antiviral medication approved worldwide;it acts by inhibiting the RNA-dependent RNA polymerase, used for moderate-to-severe COVID-19 which requires supplemental oxygen but not intubation. Not shown to improve mortality but shorten the recovery time, especially if given within the first 10 days of symptom initiation. Despite its worldwide use, its cardiovascular safety profile has not been determined as yet. Herein, we report two cases of COVID-19 infection who develop symptomatic bradycardia on a 5-day course of remdesivir.Copyright © 2022 Annals of Clinical Cardiology Published by Wolters Kluwer - Medknow.

Favipiravir pharmacokinetics in immunocompromised infants and children with chronic RNA viral infections

IntroductionFavipiravir selectively inhibits RNA polymerase responsible for single-stranded viral replication. It is licensed for treating influenza and repurposed to treat other diseases such as Ebola and COVID-19. It is metabolised by hepatic aldehyde oxidase (AO) and is an AO inhibitor with complex pharmacokinetics. We have used favipiravir, in combination with other antivirals, in severely immunocompromised children with life-threatening RNA virus infections. As an unlicensed indication, favipiravir pharmacokinetics were routinely monitored at our institution. Population pharmacokinetic model is used to describe the favipiravir pharmacokinetic properties, drug exposure and sources of variability in these children.MethodsRoutine favipiravir plasma levels of 9 patients (0.8–11yrs, mean age=5.3yrs;median weight=15kg) were analysed retrospectively (62 samples). All patients received favipiravir 200mg or 400mg tds and had at least one plasma level 45min (peak), 3h and 8h (trough) post-dose. Parameter estimation and model simulation properties (visual predictive check) were assessed using R language (v 4.1.2) and RStudio (2022.02.0+443).ResultsA one-compartment model with weight as covariate best describes the data, with (1) elimination clearance=1L/h and volume of distribution=7.54L, both allometric scaled centring at median weight, and (2) estimated t1/2=5.17h with Cmax = 24µg/mL at 200mg and 41µg/mL at 400mg.ConclusionsTo our knowledge this is the first report of favipiravir pharmacokinetic parameters in infants and young children. Weight significantly improves the model fit as a covariate. Reported EC50 for norovirus in vitro was 19–39µg/mL and enterovirus 71 was 23µg/mL, indicating higher favipiravir doses or combination with other antivirals are required.

Clinical Effectiveness and Adverse Events of Adjunctive Remdesivir Administration Vs Standard of Care in Adults with Severe and Critical Covid-19

Intro: The unavailability of specific treatment for COVID-19 prompted the empirical use of remdesivir, a viral RNA polymerase inhibitor. Since evidences present conflicting results, this study aims to determine the clinical effectiveness and adverse events of adjunctive remdesivir administration vs standard of care (non-remdesivir) in COVID-19 adult patients in a tertiary hospital in Baguio City, Philippines. Method(s): We performed a single-center, retrospective study of severe to critical COVID-19 patients admitted from September 2020 to September 2021. Stratified random sampling was employed and data collection was through chart review. Analysis was done with R Statistical Software version 4, utilizing paired T-test and McNemar test, with p-value of <0.05 considered as significant. Finding(s): A total of 318 patients were reviewed and classified into the remdesivir (n=159) and standard of care (non-remdesivir) (n=159) groups. Baseline characteristics were comparable except for co-morbidities (p<0.05). There were no noted significant differences between both groups in terms of morality (p=0.885) and reduction in chest radiograph infiltrates (p=0.182). However, the average number of days to clinical improvement (7 days vs 12 days) and recovery (16 days vs 21 days) were statistically lesser in the remdesivir group (p=0.00). Also, those who experienced diarrhea (p=0.33) and transaminitis (p=0.003) were significantly higher in those given remdesivir. Conclusion(s): There was no significant difference in terms of mortality in those given remdesivir vs standard of care alone. Nevertheless, remdesivir administration is associated with significantly faster time to clinical improvement and recovery. The drug is thought to facilitate faster lung viral load clearance and improved pulmonary function through inhibition of RNA polymerase. Though not potentially life-threatening, the drug may cause diarrhea and elevation in transaminases.Copyright © 2023

Molecular Detection and Genetic Characterization of Bovine Kobuvirus (BKV) in Diarrhoeic Calves in a Central Italy Herd

Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.

Attacking the SARS-CoV-2 Replication Machinery with the Pathogen Box's Molecules

Introduction: The world is currently facing a pandemic initiated by the new coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus. Viral transcription and replication are the fundamental processes of any virus. They allow the synthesis of genetic material and the consequent multiplication of the virus to infect other cells or organisms.Methods: The most important protein in SARS-CoV-2 is the RNA polymerase (RdRp or nsp12), responsible for both processes. The structure of this protein (PDB ID: 6M71) was used as a target in the application of computational strategies for a drug search, like virtual screening and molecular docking. Here, the Pathogen Box database of chemical compounds was used together with Remdesivir, Beclabuvir, and Sofosbuvir drugs as potential inhibitors of nsp12.Results: The results showed Top10 potential target inhibitors with binding energy (ΔG) higher than those of the positive controls, of which TCMDC-134153 and TCMDC-135052, both with ΔG = −7.53 kcal/mol, present interactions with three important residues of the nsp12 catalytic site.Conclusion: These proposed ligands would be used for subsequent validation by molecular dynamics, where they can be considered as drugs for the development of effective treatments against this new pandemic.

Safety Profile of Molnupiravir with Significant Effect on COVID-19: A Review

Background: As the COVID era unfolds, researchers reveal that rapid changes in viral genetic material allow viruses to circumvent challenges triggered by the host immune system and resist anti-viral drugs, potentially leading to persistent viral manifestations in host cells. Molnupiravir (RNA-dependent RNA polymerase inhibitor) is a novel anti-viral medicine promising a vital role in coming setbacks.Objectives: This review aims to clarify the safety and efficacy of the molnupiravir molecule in light of existing case studies. As a result, it is intended to explore and discuss the molecular structure, mechanism of action, discovery and development process, preclinical research, clinical investigations, and other subtopics.Methods: A total of 75 publications were searched using multiple engines, such as Google Scholar, PubMed, Web of Science, Embase, Cochrane Library, ClinicalTrials.gov, and others, with a constraint applied to exclude publications published over 11 years ago. Molnupiravir, safety, efficacy, COVID- 19, RdRp, PK-PD, and clinical study were utilized as keywords.Results: Clinical results on molnupiravir are supported by investigations that were recently disclosed in a study on both sex volunteers (male and female) with an age restriction of 19 to 60 years, followed by a Phase-3 Clinical Trial (NCT04575584) with 775 randomly assigned participants and no fatalities reported due to treatment.Conclusion: Molnupiravir proved a high level of safety, allowing it to be tested further. This review supports the safety and efficacy of this molecule based on the established evidence, which claims the most anticipated employment of molnupiravir in COVID protocol.

Identification of FDA-approved drugs against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) through computational virtual screening.

The SARS-CoV-2 coronavirus is responsible for the COVID-19 outbreak, which overwhelmed millions of people worldwide; hence, there is an urgency to identify appropriate antiviral drugs. This study focuses on screening compounds that inhibit RNA-dependent RNA-polymerase (RdRp) essential for RNA synthesis required for replication of positive-strand RNA viruses. Computational screening against RdRp using Food and Drug Administration (FDA)-approved drugs identified ten prominent compounds with binding energies of more than - 10.00 kcal/mol, each a potential inhibitor of RdRp. These compounds' binding energy is comparable to known RdRp inhibitors remdesivir (IC50 = 10.09 µM, SI = 4.96) and molnupiravir (EC50 = 0.67 - 2.66 µM) and 0.32-2.03 µM). Remdesivir and molnupiravir have been tested in clinical trial and remain authorized for emergency use in the treatment of COVID-19. In docking simulations, selected compounds are bound to the substrate-binding pocket of RdRp and showed hydrophobic and hydrogen bond interaction. For molecular dynamics simulation, capmatinib, pralsetinib, ponatinib, and tedizolid phosphate were selected from the initial ten candidate compounds. MD simulation indicated that these compounds are stable at 50-ns MD simulation when bound to RdRp protein. The screen hit compounds, remdesivir, molnupiravir, and GS-441524, are bound in the substrate binding pocket with good binding-free energy. As a consequence, capmatinib, pralsetinib, ponatinib, and tedizolid phosphate are potential new inhibitors of RdRp protein with potential of limiting COVID-19 infection by blocking RNA synthesis. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02072-1.

Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses.

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC). Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs. Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection. Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

In-silico studies of active phytochemicals from siddha medicinal herbs of karisalai chooranam against SARS-CoV-2 main protease (3 CLpro), RNA dependent RNA polymerase and angiotensin-converting enzyme II receptor

Aim: The contagious disease COVID 19 is a recently out-broken pandemic situation which threatens humankind all over the world. Siddha system of medicine is one of the traditional medical systems of India, which has provided a novel remedy for many epidemics like Dengue, Chicken guinea earlier. On evaluating the literature evidence and considering the mortality and severity of the disease, we have attempted to identify the possible inhibition of viral replication by "Karisalai Chooranam" - a polyherbal Siddha formulation which contains herbs like Karisalai (Wedelia chinensis), Thoodhuvelai (Solanum trilobatum), Musumusukai (Melothria maderaspatana) and Seeragam (Cuminum cyminum). The aim of this study was to identify the bioactive components present in Karisalai chooranam and pin down the components that inhibit COVID 19 protease by In Silico molecular docking analysis. Material and methods: The study was performed for the active compounds present in the herbs (Wedelia chinensis - Benzoic acid, Solanum trilobatum- Disogenin, Melothria maderaspatana- beta-sitosterol, Cuminum cyminum L- Coumaric acid and Limonene) with three potential targets, PDB id: 6LU7 3-chymotrypsin-like protease (3CLpro), PDB id: 6-NUR RNA dependent RNA polymerase and PDB id: 2AJF Angiotensin-converting enzyme II (ACE2) receptor using Autodock Vina. Key findings: The active phytocomponents present in "Karisalai chooranam" was found to inhibit the target 3CL proenzyme and hereby halt the formation of 16 non-structural proteins (nsp1-nsp16) that are highly essential for viral replication and there by prevents viral survival in the host environment. The phytocomponents also inhibited the target RNA dependent RNA polymerase (PDB)-6NUR RdRp which possess versatile action in mediating nonstructural protein (nsp 12) essential for viral replication. A significant binding against the target Angiotensin-converting enzyme II (ACE2) receptors - PDB- 2AJF was found which was recognized as a binding site for novel coronavirus to cause its pathogenesis. Among the five active components present in the herb, the binding ability of Disogenin and beta-sitosterol with COVID19 protease suggests a possible mechanism of protease inhibition and thus preventing viral replication. Significance: The results strongly suggest that phytocomponents of "Karisalai chooranam" may act as a potential therapeutic agent for the management of COVID-19 and related symptoms. Further, the efficacy of the active compounds should be tested in vitro before being recommended as a drug.

The SARS-CoV-2 Nsp13 Helicase Required for Coronavirus Replication Interacts with Structured Nucleic Acids in a Strand-Specific Manner and Catalyzes a Novel Protein-RNA Remodeling Activity Implicated in the Proofreading Mechanism of the Replication-Transc

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded, positive-sense RNA virus responsible for COVID-19, requires a set of virally encoded nonstructural proteins that compose a replication-transcription complex (RTC) to replicate its 30 kilobase genome. One such nonstructural protein within the RTC is Nsp13, a highly conserved molecular motor ATPase/helicase. Upon purification of the recombinant SARS-CoV-2 Nsp13 protein expressed using a eukaryotic cell-based system, we biochemically characterized the enzyme by examining its catalytic functions, nucleic acid substrate specificity, and putative protein-nucleic acid remodeling activity. We determined that Nsp13 preferentially interacts with single-stranded (ss) DNA compared to ssRNA during loading to unwind with greater efficiency a partial duplex helicase substrate. The binding affinity of Nsp13 to nucleic acid was confirmed through electrophoretic mobility shift assays (EMSA) by determining that Nsp13 binds to DNA substrates with significantly greater efficiency than RNA. These results demonstrate strand-specific interactions of SARS-CoV-2 Nsp13 that dictate its ability to load and unwind structured nucleic acid substrates. We next determined that Nsp13 catalyzed unwinding of double-stranded (ds) RNA forked duplexes on substrates containing a backbone disruption (neutrally charged polyglycol linker (PGL)) was strongly inhibited when the PGL was positioned in the 5' ssRNA overhang, suggesting an unwinding mechanism in which Nsp13 is strictly sensitive to perturbation of the translocating strand sugar-phosphate backbone integrity. Furthermore, we demonstrated for the first time the ability of the coronavirus Nsp13 helicase to disrupt a high-affinity nucleic acid-protein interaction, i.e., a streptavidin tetramer bound to biotinylated RNA or DNA substrate, in a uni-directional manner and with a preferential displacement of the streptavidin complex from biotinylated ssDNA versus ssRNA. In contrast to the poorly hydrolysable ATP-gamma-S or non-hydrolysable AMP-PNP, ATP supports Nsp13-catalyzed disruption of the nucleic acidprotein complex, suggesting that nucleotide binding by Nsp13 is not sufficient for protein-RNA disruption and the chemical energy of nucleoside triphosphate hydrolysis is required to fuel remodeling of protein bound to RNA or DNA. Our results build upon structural studies of the SARS-CoV-2 RTC in which it was suggested that Nsp13 pushes the RNA polymerase (Nsp12) backward on the template RNA strand. Experimental evidence from our studies demonstrate that Nsp13 helicase efficiently remodels a large high affinity protein-RNA complex in a manner dependent on its intrinsic ATP hydrolysis function. We proposed that this novel biochemical activity of Nsp13 is relevant to its role in SARS-CoV-2 RNA processing functions and replication. It was proposed that Nsp13 facilitates proofreading during coronavirus replication when a mismatched base is inadvertently incorporated into the SARS-CoV-2 genome during replication to reposition the RTC so that the proofreading nuclease complex (Nsp14-Nsp10) can gain access and remove the nascently synthesized nucleotide to ensure polymerase fidelity. Our findings implicate a direct catalytic role of Nsp13 in protein-RNA remodeling during coronavirus genome replication beyond its duplex strand separation or structural stabilization of the RTC, yielding new insight into the proofreading mechanism. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Sirna-A Potential Therapy for Covid-19 Infection

Background: The deadly virus COVID-19 has affected more than 1 crore people and claimed more than 5 lakh lives worldwide according to the World health organization. Though there are numerous treatment modalities available including anti-bacterials, antivirals, vaccines etc., none of them can be considered as effective cure for SARS CoV-2 virus as they are mostly non-specific in action. Aim(s): siRNA therapy can be considered as a significant treatment modality due to its specificity in action. The aim of this review is to explore siRNA as a potential treatment strategy for the treatment of COVID-19. Material(s) and Method(s): In this review we shall explore the targets of siRNA therapy which includes viral RNA-dependent RNA polymerase, helicase, protease and nucleoprotein N. siRNA related patents provide solutions for novel RNAi techniques, high expense of chemically synthetic siRNA, techniques for restraining SARS-CoV by disturbing RNA etc., siRNA-based drug delivery systems and limitations of nanocarrier delivery system were reviewed. siRNA is a gene silencer that targets highly conserved sequences which codes for protease 3CL (nsp5) and viral helicase (from 16-18 kbp). Conclusion(s): Thus, siRNA-based therapy is considered highly efficacious as it can hit the highly conserved regions of SARS-CoV-2 RNA.Copyright © 2023, Advanced Scientific Research. All rights reserved.

Reverse Transcription Recombinase Polymerase Amplification integrated with CRISPR Technology and LFA for point-of-care detection of SARS-CoV-2 virus

The field-deployable point-of-care diagnostic test for rapid detection of SARS-COV-2 is needed for implementation of the control measures. In this direction, recently developed CRISPR technology combined with isothermal recombinase polymerase amplification assay is a versatile highly sensitive detection platform for rapid diagnosis of infectious diseases. Here we report the development of RT-RPA-CRISPR based LFA assay for detection of SARS-CoV-2 targeting conserved RdRp and E genes. Various sets of primers and gRNAs were designed targeting conserved regions of the RdRp and E genes of different lineages of SARS-CoV-2 viruses. The isothermal RT-RPA based amplification reactions were standardized using invitro transcribed RNAs of the target regions. The optimum amplification was observed at 42degreeC for 30 min as confirmed by visualization of the amplicons in agarose gel. Subsequently, CRISPRCAS12 reaction was implemented for specific detection of amplicons. Different sets of gRNAs targeting RdRp and E genes were designed and synthesized by in-vitro transcription. The CRISP/CAS12-gRNA complex and single stranded fluorescence probe were added to the RT-RPA amplicons for cleavage of fluorescence probe in positive reaction. Subsequently, the cleaved probes were detected in precoated LFA strips. Upon probe cleavage reaction, the product was mixed with buffer and loaded into LFA strips. In positive reaction, test line showed strong band in test line and light band in control line. The standardized RT-RPA-CRISPR-LFA assay was tested for detection of SARS-CoV-2 using previously isolated RNAs from clinical cases of human SARS-CoV-2 infections. The developed assay successfully detected the positive cases. In conclusion, the developed assay could serve as versatile POC platform for rapid detection of SARS-CoV-2 nucleic acids in human as well as animals.

Abstracts of the papers presented in the International Conference of Indian Virological Society, VIROCON 2022 on "Emerging and Re-emerging Viral Infections Impacting Humans, Animals, Plants, Fish and Environment"

The proceedings contain 206 papers. The topics discussed include: influenza: experiences from Kashmir;outbreaks of different viral etiologies amidst COVID-19 pandemic;development of a colorimetric isothermal (LAMP) assay for rapid detection of monkeypox virus;circulation of genetically diverse non-polio enteroviruses in respiratory samples during COVID-19 pandemic period (2021-22);evolutionary analysis of all eleven genes of species C rotaviruses circulating in humans and domestic animals;molecular characterization of dengue viruses circulating in Pune district, Maharashtra from 2009-2022;isolation and genomic characterization of cell fusing agent virus from aedes aegypti mosquitoes from Assam, India;structure-based identification and evaluation of antiviral activity of potent small molecule inhibitors targeting alphavirus RNA-dependent RNA polymerase;integration of HBV receptor NTCP into hepatoma cell using grnome editing;and hepatitis B virus genome targeting using CRISPR/Cas9based gene editing tool.

In Vitro and In Vivo Therapeutic Potential of 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea on Cells and K18-hACE2 Mice Infected with SARS-CoV-2.

We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6'-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-κB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and/or its molecular derivatives against this and other related viruses.

Current trends in RNA virus detection through metatranscriptome sequencing data.

With advances in sequencing technology, metatranscriptome sequencing from a variety of environmental and biological sources has revealed the existence of various previously unknown RNA viruses. This review presents recent major RNA virome studies sampled from invertebrate and vertebrate species as well as aquatic environments. In particular, we focus on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and related RNA virus identification through metatranscriptome sequencing analyses. Recently developed bioinformatics software and databases for RNA virus identification are introduced. A relationship between newly identified RNA viruses and endogenous viral elements in host genomes is also discussed.