ABSTRACT
Over the last few decades, the emergence and re-emergence of various pathogenic viruses have significantly impacted human health. The continuous rise in cases with increasing mortality rates has driven the chase for effective treatment options and early diagnosis to combat this global health issue. Currently, used laboratory techniques for virus detection require complex equipment, trained personnel, and, most importantly, are time-consuming. In times of outbreaks and epidemics like COVID-19 and Ebola, easy-to-use and point-of-care tests, especially for developing and underdeveloped countries, are indispensable. This chapter explicitly discusses the availability of the detection methods for various human viral pathogens with their shortcomings and recent advancements in biosensors. With the ongoing improvement in biosensors, these hold important avenues for rapid, sensitive, and scalable devices for viral diagnostic purposes. The effectiveness of previously known and current approaches/devices/methods that utilize different principles for detection has also been reviewed here, with the listing of all the available tests for various human pathogens. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023.
ABSTRACT
Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.
ABSTRACT
SARS-CoV-2 is a positive-sense RNA virus that contains open reading frame 1ab (ORF1ab) to produce 16 nonstructural proteins (nsps). Five stem-loops (SL) are found in the 5' UTR of the RNA that are involved in myriad viral functions and are labeled SL1 through SL5. SL1 is crucial to viral replication. Upon viral infection, nsp1 binds the ribosomal 40S subunit to inhibit all host mRNA translation. Upon SL1 binding to nsp1, viral mRNA can be processed by the ribosome, allowing viral proteins to be produced. In this study, we are examining small DNA oligonucleotides that bind to SL1-mimetic DNA in order to block SL1-nsp1 interactions. We designed a DNA analog of the SL1 hairpin and two small DNA oligonucleotides that are complementary to either the helical stem or the loop region of SL1. The binding of these oligonucleotides to the SL1 hairpin should allow the formation of either an alternate duplex or a triplex structure. Isothermal titration calorimetry (ITC) and circular dichroism (CD) techniques were performed in 1 MKCl and 10 mM MgCl2 at two different pH (5.5 and 7.0) to examine structural and thermodynamics of binding. ITC of the two oligonucleotides showed modest binding. Results from DNA binding experiments, thermal denaturation, and CD show the hairpin structure is thermodynamically more favored and mostly remains intact under the conditions examined.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Introduction In this study, we aimed to monitor anti-spike and anti-nucleocapsid antibodies positivity in healthcare workers (HCWs) vaccinated with two doses of inactivated CoronaVac (Sinovac, China) vaccine. Methods Overall, 242 volunteer HCWs were included. Of the participants, 193 were HCWs without history of prior documented COVID-19 (Group 1), while 49 had history of prior documented COVID-19 before vaccination (Group 2). The participants were followed up for SARS-CoV-2 antibodies positivity at four different blood sampling time points (immediately before the second vaccine dose and at the 1st, 3rd months and 141-150 days after the second dose). We investigated the serum IgG class antibodies against SARS-CoV-2 RBD region and IgG class antibodies against SARS-CoV-2 nucleocapsid antigen by chemiluminescent microparticle immunoassay (CMIA) method using commercial kits. Results We found positive serum anti-RBD IgG antibody in 76.4% of the participants (71% in Group 1;98% in Group 2) 28 days after the first dose. When the antibody levels of the groups were compared at the four blood sampling time points, Group 2 anti-RBD IgG levels were found to be significantly higher than those in Group 1 at all follow-up time points. Although anti-RBD IgG positivity persisted in 95.6% of all participants in the last blood sampling time point, a significant decrease was observed in antibody levels compared to the previous blood sampling time point. Anti-nucleocapsid IgG antibody was positive in 12 (6.2%) of participants in Group 1 and 32 (65.3%) in Group 2 at day 28 after the first dose. At the fourth blood sampling time point, anti-nucleocapsid antibodies were found to be positive in a total of 20 (9.7%) subjects, 10 (6.1%) in Group 1 and 10 (23.8%) in Group 2. Conclusions In this study, it was determined that serum antibody levels decreased in both groups after the third month after the second dose in HCWs vaccinated with CoronaVac vaccine.Copyright © GERMS 2022.
ABSTRACT
Of various SARS-CoV-2 variants, some have drawn special concern or interest because of their heightened disease threat. The mutability of individual SARS-CoV-2 genes/proteins presumably varies. The present study quantified gene/protein mutations in 13 major SARS-CoV-2 variants of concern/interest, and analyzed viral protein antigenicity using bioinformatics. The results from 187 carefully perused genome clones showed significantly higher mean percent mutations in the spike, ORF8, nucleocapsid, and NSP6 than in other viral proteins. The ORF8 and spike proteins also tolerated higher maximal percent mutations. The omicron variant presented more percent mutations in the NSP6 and structural proteins, whereas the delta featured more in the ORF7a. Omicron subvariant BA.2 exhibited more mutations in ORF6, and omicron BA.4 had more in NSP1, ORF6, and ORF7b, relative to omicron BA.1. Delta subvariants AY.4 and AY.5 bore more mutations in ORF7b and ORF8 than delta B.1.617.2. Predicted antigen ratios of SARS-CoV-2 proteins significantly vary (range: 38-88%). To overcome SARS-CoV-2 immune evasion, the relatively conserved, potentially immunogenic NSP4, NSP13, NSP14, membrane, and ORF3a viral proteins may serve as more suitable targets for molecular vaccines or therapeutics than the mutation-prone NSP6, spike, ORF8, or nucleocapsid protein. Further investigation into distinct mutations of the variants/subvariants may help understand SARS-CoV-2 pathogenesis.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Antigens, Viral , Mutation , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
The rapid rate of mutation of the RNA genome of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the emergence of viral variants, leading to the enhanced survivability of the virus. Hence, searching for new drugs that can restrict new viral infections by interacting with wild-type and mutated viral proteins is important. However, new drug development's economic and time-constraining nature makes drug repurposing a more viable solution to address the problem. In this work, we conducted a computational study to screen 23 Non-Steroidal Anti-Inflammatory Drugs (NSAID) interactions with 5 major viral proteins of SARS-CoV-2 that are mainly involved in host infection. Our in-silico results establish a database that shows that different NSAID ligands interact with the different viral proteins with good binding affinities. Stabilizing point mutations were introduced within the conserved amino acids involved in ligand-protein interactions. Redocking the NSAID ligands with these mutated viral proteins showed that the NSAID ligands could bind with the mutated and wild-type viral proteins with comparable binding affinities. We conclude that the NSAID ligands could be repurposed as therapeutic drugs against the SARS-CoV-2 virus. Additionally, our work generated a repository that includes binding affinities, possible modes of interaction, and specific interacting residues of the protein (wild-type and mutated) ligand complexes that could be used for future validation studies. Further, our results point to the potential of these drugs to treat other viral infections with similar disease etiology.Copyright All © 2023 are reserved by International Journal of Pharmaceutical Sciences and Research.
ABSTRACT
Context: The coronavirus disease 2019 (COVID-19) pandemic is still a cause of worldwide health concern. Diabetes and its associated comorbidities are risk factors for mortality and morbidity in COVID-19. Selecting the right antidiabetic drug to achieve optimal glycemic control might mitigate some of the negative impacts of diabetes. Metformin continues to be the most widely administered antidiabetic agent. There is evidence of its beneficial outcome in COVID-19 independent of its glucose-lowering effect. Evidence Acquisition: A thorough literature search was conducted in PubMed, Google Scholar, Scopus, and Web of Science to identify studies investigating metformin in COVID-19. Result(s): Several overlapping mechanisms have been proposed to explain its antiviral properties. It could bring about conformational changes in the angiotensin-converting enzyme-2 receptor and decrease viral entry. The effects on the mammalian target of the rapamycin pathway and cellular pH have been proposed to reduce viral protein synthesis and replication. The immunomodulatory effects of metformin might counter the detrimental effects of hyperinflammation associated with COVID-19. Conclusion(s): These findings call for broader metformin usage to manage hyperglycemia in COVID-19.Copyright © 2023, International Journal of Endocrinology and Metabolism.
ABSTRACT
Background: BST2/Tetherin is an interferon-stimulated gene with antiviral activity against enveloped viruses. Particularly, BST2 tethers virions at their site of assembly, preventing their release and spread. In addition to this primary role, BST2 is as an important bridge between the innate and adaptive immune system, since (i) BST2 routes tethered particles to lysosomes, which generates viral breakdown products that engage pattern recognition receptors;and (ii) trapped virions facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). In turn, viruses have evolved mechanisms to bypass BST2, either by targeting BST2 for proteasomal/lysosomal degradation or by removing BST2 from sites of virion assembly. However, the role of BST2 in SARS-CoV-2 replication, spread, evolution, and pathogenesis remains largely unknown. Method(s): The antiviral potential of BST2 against SARS-CoV-2 was investigated by infecting different SARS-CoV-2 isolates (Hong Kong, Alpha, Beta, Delta, and Omicron) in BST2+ and BST2- cells. Culture supernatants were collected to assess virion production by ELISA and infectivity by TCID50. Infected cells were analyzed by western blot and flow cytometry to examine viral and cellular protein levels, including BST2. Transfection of individual SARS-CoV-2 ORFs and mutagenesis studies allowed us to identify the genes that the virus uses to downregulate BST2. Immunoprecipitation assays revealed protein-protein interactions and changes in ubiquitination patterns. Experiments with proteasomal and lysosomal inhibitors furthered our mechanistic understanding of how SARS-CoV-2 counteracts BST2. Finally, fluorescence microscopy studies uncovered changes in the subcellular distribution of BST2 in SARS-CoV-2 infected cells. Result(s): While BST2 reduces virion release, SARS-CoV-2 has evolved to counteract this effect. Specifically, SARS-CoV-2 uses the Spike to interact with BST2, sequester the protein at perinuclear locations, and ultimately route it for lysosomal degradation. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that each variant is more efficient than the previously circulating strain at downregulating BST2 and facilitating virion production, and that mutations in the Spike account for their enhanced BST2 antagonism. Conclusion(s): As part of its adaptation to humans, SARS-CoV-2 is improving its capacity to counteract BST2, highlighting that BST2 antagonism is important for SARS-CoV-2 infectivity and transmission.
ABSTRACT
The SARS-CoV-2 replication and transcription complex (RTC) is made up of nine distinct non-structural viral proteins encoded by the ORF1ab gene. These proteins house seven enzymatic sites that synthesize new viral genomic and subgenomic RNA, proofread and correct errors in the synthesis, add a 5'-cap to the nascent RNA, and truncate the intermediate negative sense 5'-poly-U tail. While x-ray crystallography and cryo-EM have provided high resolution structures of each of the individual proteins of the RTC and have shed light on how subsets of the proteins associate, a full picture of the RTC has remained elusive. Using molecular modeling tools, including protein-protein docking, we have generated a model of the RTC centered around hexameric nsp15, which is capped on two faces by trimers of nsp14/nsp16/(nsp10)2. A conformational change of nsp14, necessary to facilitate binding to nsp15, then recruits six nsp12/nsp7/(nsp8)2 polymerase subunits. To this, six nsp13 subunits are distributed around the complex. The resulting superstructure is composed of 60 subunits total and positions the nsp14 exonuclease and nsp15 endonuclease sites in line with the dsRNA exiting the nsp12 polymerase site. Nsp10 acts to separate the RNA strands, directing the nascent strand to the nsp12 NiRAN site, where a transiently associated nsp9 facilitates the first step in mRNA capping. The RNA is then directed to the nsp14 N7-methyltransferase site and the nsp16 2'O-methyltransferase site to complete the capping. Additionally, template switching during transcription is proposed to be facilitated by positioning of the TRS-L RNA-bound N-protein above the polymerase active site, between two subunits of nsp13. The model, while constructed based on structural considerations, offers a unifying set of hypotheses to explain the diverse set of processes involved in coronavirus genome replication and transcription. All work presented was funded by Gilead Sciences.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Background: A major consequence of COVID-19 is long-term metabolic complications (metabolic PASC or Long COVID) following acute disease resolution leading to hyperglycemia, increased risk of diabetes or defects in glucose metabolism. However, the mechanisms underlying the links between COVID-19 and glycometabolic disruptions remain unclear. Method(s): 15 African green monkeys (AGM;Chlorocebus aethiops) were infected with SARS-CoV-2 (Wuhan stain) and divided into two groups: unvaccinated (n=10) and vaccinated (BNT162b2 (Pfizer) 4-days post infection;n=5). Subgenomic SARS-CoV-2 mRNA (sgRNA) reflecting active replication was quantified in nasal and pharyngeal swabs, and blood chemistry analysis was performed longitudinally up to 18 weeks post-infection. We quantified liver glycogen at necropsy using Periodic acid-Schiff staining. Finally, we longitudinally analyzed 96 plasma proteins using a proximity extension assay (Olink). STRING was used to identify enriched protein networks. Comparisons between the two groups over time were performed using PERMANOVA. Result(s): All animals had detectable sgRNA ( >3.64x106) at day 3, and only two were undetectable at week 5. Post-infection BNT162b2 vaccination partially inhibited the SARS-CoV-2 mediated disruption of glucose levels (P=0.001, Fig. 1A). Liver glycogen levels following necropsy correlated positively with blood glucose levels at week 12 (r=0.74, P =0.003). Histopathological analysis revealed no marked evidence of long-term inflammation or fibrosis of pancreatic islets. Using the plasma proteomic data, we identified a signature of 15 SARS-CoV-2-modulated plasma proteins coinciding with early onset hyperglycemia during acute infection (P=0.001, Fig. 1B). These proteins are enriched for biological processes linked to chemotaxis (FDR=1.38E-06), and viral protein interaction with cytokines (FDR=1.01E-12) (Fig. 1C). Of these, CCL25 and glial cell derived neurotrophic factor (GDNF) remained persistently elevated post-acute infection and correlated with blood glucose levels (r=0.57, P=0.0003;and r=0.64, P<0.0001, respectively, Fig. 1D). Conclusion(s): Our AGM model validates phenotypes of metabolic PASC and offers an opportunity to mechanistically study the manifestations of PASC. Our preliminary data suggest that vaccine-preventable early insults by metabolicregulating immune factors may contribute to long-term dysregulated liver and systemic glucose homeostasis during PASC. These immune factors warrant further investigation for their mechanistic links to PASC. (Figure Presented).
ABSTRACT
RNA viruses are the major class of human pathogens responsible for many global health crises, including the COVID-19 pandemic. However, the current repertoire of U.S. Food and Drug Administration (FDA)-approved antivirals is limited to only nine out of the known 214 human-infecting RNAviruses, and almost all these antivirals target viral proteins. Traditional antiviral development generally proceeds in a virus-centric fashion, and successful therapies tend to be only marginally effective as monotherapies, due to dose-limiting toxicity and the rapid emergence of drug resistance. Host-based antivirals have potential to alleviate these shortcomings, but do not typically discriminate between infected and uninfected cells, thus eliciting unintended effects. In infected cells where host proteins are repurposed by a virus, normal host protein functions are compromised;a situation analogous to a loss-of-function mutation, and cells harboring the hypomorph have unique vulnerabilities. As well-established in model systems and in cancer therapeutics, these uniquely vulnerable cells can be selectively killed by a drug that inhibits a functionally redundant protein. This is the foundation of synthetic lethality (SL). To test if viral induced vulnerabilities can be exploited for viral therapeutics, we selectively targeted synthetic lethal partners of GBF1, a Golgi membrane protein and a critical host factor for many RNA viruses including poliovirus, Coxsackievirus, Dengue, Hepatitis C and E virus, and Ebola virus. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. A genome-wide chemogenomic CRISPR screen identified synthetic lethal partners of GBF1 and revealed ARF1 as the top hit. Disruption of ARF1, selectively killed cells that synthesize poliovirus 3A alone or in the context of a poliovirus replicon. Combining 3A expression with sub-lethal amounts of GCA - a specific inhibitor of GBF1 further exacerbated the GBF1-ARF1 SL effect. Together our data demonstrate proof of concept for host-based SL targeting of viral infection. We are currently testing all druggable synthetic lethal partners of GBF1 from our chemogenomic CRISPR-screen, in the context of dengue virus infection for their abilities to selectively kill infected cells and inhibit viral replication and infection. Importantly, these SL gene partners of viral-induced hypomorphs only become essential in infected cells and in principle, targeting them will have minimal effects on uninfected cells. Our strategy to target SL interactions of the viral-induced hypomorph has the potential to change the current paradigm for host-based therapeutics that can lead to broad-spectrum antivirals and can be applied to other intracellular pathogens. This work is supported by National Institutes of Health grants R01 GM112108 and P41 GM109824, R21 AI151344 and foundation grant FDN-167277 from the Canadian Institutes of Health Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
The transmembrane domains of viral proteins are highly conserved and crucial to normal viral function. Oligomeric transmembrane domains present novel opportunities for drug development, as their disruption can prevent the assembly of the virus. The Reichart lab is particularly interested in developing retro-inverso peptide inhibitors. Retro-inverso peptides are peptides using D-amino acids mirroring a region of target protein, which allows the peptide to inhibit viral assembly, but they are also significantly less likely to be catabolized by natural metabolic or immunologic processes. The efficacy of these inhibitors is governed largely by the extent to which they mirror the target protein, making highly conserved regions, such as transmembrane domains, ideal target regions for these inhibitors. The primary technique in the literature for the investigation of oligomerization states uses fluorescence spectroscopy. We are now working on developing a novel alternative system to evaluate protein oligomerization using spin-labeled peptides that are directly incorporated into the peptide sequence. Direct incorporation of the spin-label into the peptide sequence is a more powerful technique than the standard procedures used in the literature. In particular, the ability to incorporate spin labels in various positions within the protein can give novel insights into the relative depth of the protein within a membrane, which is very difficult to study using other techniques and not possible using the fluorescence technique. The transmembrane domains of proteins with known and well-characterized monomer and trimer standard oligomerization states were synthesized using an Fmoc Solid- Phase Peptide Synthesis (SPPS) procedure incorporating an Fmoc-2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, (Fmoc-TOAC) instead of an alanine. Direct incorporation of stable N-oxide spin labels, which can be contrasted to labeling cysteine residues after the protein synthesis, has been used for the investigation of the secondary structure of proteins for decades, but the application of this spin labeling technique to study the oligomerization states of transmembrane domains of proteins is an understudied application. The products of SPPS were analyzed using a Liquid Chromatography Mass Spectroscopy instrument and purified using High Performance Liquid Chromatography. The spin-label was then deprotected and evaluated using Electron Spin Resonance (ESR) Spectroscopy. There are two primary future directions following this research project: first, the generation of viral proteins with spin labels incorporated in different positions to determine the relative depth of each position within the membrane;second, the incorporation of spin labels into SARS-CoV- 2 proteins to develop a model for in vitro evaluation of retro-inverso peptide assembly inhibitors. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.
ABSTRACT
Background: Five variants of concern (VOCs) have dominated COVID-19 disease etiology since 2020-Alpha, Beta, Gamma, Delta, and Omicron-possessing over 150 defining genomic alterations. Here, we used global proteomic and genomic approaches to study the host responses and selective forces driving VOC evolution. Method(s): We infected Calu-3 human lung epithelial cells with 5 VOCs and 2 wave 1 (W1) controls and performed mass spectrometry abundance proteomics, phosphoproteomics, and mRNA sequencing at 10 and 24 hours post infection. We additionally performed affinity purification mass spectrometry (APMS) by individually expressing all VOC mutant viral proteins (52) and corresponding W1 forms in human cells to quantify differential virus-host protein-protein interactions. Data was integrated using network modeling and bioinformatics to pinpoint VOC-specific differences. Four novel mutant viruses were developed using reverse genetics technology to validate the impact of specific genomic alterations. Result(s): We discovered VOCs evolved convergent molecular strategies to remodel the host response by modulating viral RNA and protein levels (most notably of N, Orf9b, and Orf6), altering nucleocapsid phosphorylation, and rewiring virus-host protein complexes. Integrative systems analyses revealed that Alpha, Beta, Gamma, and Delta ultimately converged in the suppression of interferon stimulated genes (ISGs) relative to W1 viruses, but Omicron BA.1 did not, and Delta induced more pro-inflammatory genes compared to other VOCs. Altered regulation of ISGs correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b;for example, Omicron BA.1 depicted a 2-fold decrease in Orf6 expression. We identified mutations that alter expression of Orf9b (N D3L and N -3A del) and the novel VOC protein N* (N R203K/G204R), and confirmed Orf6 innate immune antagonism using recombinant virus technology. Remarkably, Omicron BA.4 and BA.5 regained strengthened innate immune antagonism compared to BA.1, which again correlated with enhanced Orf6 expression, though dampened in BA.4 by a mutation (D61L) that we discovered disrupts the Orf6-nuclear pore interaction. Conclusion(s): Collectively, our findings suggest SARS-CoV-2 convergent evolution overcomes human innate immune barriers, laying the groundwork to understand future coronavirus evolution associated with immune escape and enhanced human-to-human transmission.
ABSTRACT
Background: Owing to the recent scenario on this ongoing Coronavirus pandemic outbreak around the world, the present study has been undertaken. Aim(s): In this study, we adopted two strategies, i.e., via computational method, a search for the novel plant secondary metabolites from the Indian Traditional Medicine to target and combat the enduring novel 2019 CoVs main protease that causes pneumonia, followed by the effect of these selected secondary metabolites on the host's immune system for their immunomodulatory potential on Interleukin-2. Method(s): A detailed literature review has been done to identify the assorted plant secondary metabolites from the natural sources, which have been extensively used traditionally for their immunomodulatory potential. Next, the resulting compounds have processed for the molecular docking study to predict whether the compounds have the potency to fight against 2019-CoVs protein or it could have the ten-dency to battle the cytokines, which are responsible for the immune response of the host, thereby pre-venting the CoVs caused infection in humans. Furthermore, to explore molecular mechanics, the in-silico docking study with COVID-19 Mpro and Interleukin-2 has been performed. Results & Discussion: Among the six secondary metabolites selected, five compounds showed its possible promising potency with COVID-19 and IL-2 proteins, which are compared with the standard drug Remdesivir, one of the anti-viral drugs for treating and managing the present coronavirus condition and an IL-2 inhibitor, which is the native IL-2 ligand protein (i.e., from PDB Id-1PW6) itself. Besides, based on the docking scores, the Curcumin (from Curcuma longa) showed the highest score towards these two targets taken for this study. The identified compounds have a promising binding affinity with the Mpro receptors, in the narrow range of binding energy for the protein PDB Id: 6LU7 and the score range between-10.9102 to-19.8790 kcal/mol: when compared to the standard-21.8600 kcal/mol. Whereas, the binding affinity with the Interleukin-2 receptor, for the protein PDB Id: 1PW6 the range between-11.3899 to-17.1366 kcal/mol: when compared to that of standard-16.9554 kcal/mol. Conclusion(s): Our result findings demonstrate that the integrated Indian traditional herbal treatment might be hopefully used for the viral respiratory infection due to either it may have acted directly on the viral protein or through regulating the immune response, which could lead to the rapid drug discovery of the drug leads with clinical potency towards the novel infectious disease, where there is no drug or vaccines are available.Copyright © 2021 Bentham Science Publishers.
ABSTRACT
A century after the outbreak of the Spanish flu, the world is suffering from another pandemic because of the coronavirus. The virus took a toll on more than millions of lives worldwide and continues to affect the health and socio-economic infrastructure all over the world. This study explores the epidemiology, etiology, and transmission of the virus and its phylogenetic relationship with SARS and MERS coronavirus responsible for the 2002 and 2012 viral outbreak. Furthermore, this review highlights the key features of the viral genome and essential viral proteins responsible for the viral life cycle, evading host immune response, and viral immunopathology with therapeutics from "Recovery" and "Solidarity" trials. The review culminates with a discussion on different classes of prominent vaccines and their efficacy. An overall understanding of essential viral proteins and their role in pathogenesis, repurposed drugs, and vaccine development is the rationale of the present review.Copyright © 2021 Bentham Science Publishers.
ABSTRACT
The severe consequences and high mortality of COVID-19 prompted the development of a wide range of preventive vaccines. The first vaccines to be tested were developed in China and formulated as inactivated SARS-CoV-2 adsorbed on aluminium hydroxide. One of the quality indicators for inactivated adsorbed vaccines is the degree of adsorption, which can be used to control the content not only of non-adsorbed antigen, but also of specific antigen in one dose of a vaccine. The aim of the study was to investigate the possibility of desorbing SARS-CoV-2 antigen from formulated adsorbed vaccines and the possibility of measuring its concentration using the BioScan-SARS-CoV-2 (S) ELISA kit for SARS-CoV-2 S-protein content determination. Material(s) and Method(s): the study used four batches of BBIBP-CorV by CNBG, Sinopharm (China) and three batches of CoronaVac by Sinovac Biotech (China). The authors desorbed SARS-CoV-2 S antigen in accordance with monograph FS.3.3.1.0029.15 of the State Pharmacopoeia of the Russian Federation edition XIV (Ph. Rus.), and quantified it using the BioScan-SARS-CoV-2 (S) ELISA kit by Bioservice Biotechnology Co. Ltd. (Russia). Result(s): mean S-antigen concentrations in the desorbed samples ranged from 61 to 129 ng/mL for BBIBP-CorV and from 461 to 533 ng/mL for CoronaVac. Conclusion(s): the study demonstrated the possibility of specific SARS-CoV-2 antigen desorption from the surface of aluminium hydroxide using the Ph. Rus. method, as well as the possibility of S-antigen quantification in desorbed medicinal products and supernatants using the BioScan-SARS-CoV-2 (S) ELISA kit. The authors observed 3.6- to 8.7-fold difference between the S-antigen concentrations of the desorbed preparations by the two manufacturers.Copyright © 2023 Safety and Risk of Pharmacotherapy. All rights reserved.
ABSTRACT
COVID-19 is a respiratory infection caused by a newer strain of coronavirus known as SARS-CoV-2. The major problem of COVID-19 infections is the ARDS, followed by respiratory failure, organ failure, and even death with multiple organ dysfunction, including cardiovascular collapse. Moreover, it affects the old age population with co-morbid conditions. The deficiency of diet, micronutrients, and vitamins also plays a key role in diminishing the immune power, and increases the rate of viral infectivity. The possible reasons and management methods are discussed in this review. The management methods enhance the host immune system via multi-functional and multi-targeted actions. The global rate of COVID-19 outbreak necessitates the need to develop newer medicines. The drug discovery process is based on the exposure of viral proteins, genome sequence, replication mechanisms, pathophysiological mechanisms, and host cell components (as a target) reactions. This article highlights the overview of coronavirus components, the replications process, and possible targets for the management of coronavirus infections. It may lead to the rapid development of newer medicines for the treatment of coronavirus in-fections.Copyright © 2022 Bentham Science Publishers.
ABSTRACT
Virus pandemics and endemics cause enormous pain and economic, political, and social costs and turmoil. While the Covid19 pandemics induced obvious damages, the "silent" Hepatitis C virus (HCV) infection induced liver damages are the main reason for liver transplantations. HCV-generated virus genome replication factories are housed within virus-induced intracellular structures termed membranous webs (MW) which are derived from the Endoplasmatic Reticulum (ER). Up to now, very advanced experimental data such as highly spatially resolved fuorescence and electron-tomography data often do not enter computational HCV viral RNA (vRNA) cycle models. Based upon difusion-reaction partial differential equation (PDE) models, we are developing fully 3D resolved "in silico microscopes" to mirror in vitro / in vivo experiments of the intracellular vRNA cycle dynamics. Our first models described the major components (vRNA, non-structural viral proteins-NSPs-and a host factor). The next steps incorporated additional parameters: Different aggregate states of vRNA and NSPs, and population dynamics inspired difusion and reaction co-Effcients instead of multilinear ones. Our work in progress framework presently is merging effects restricted to 2D manifold surface grids (e.g. ER surface, NSP difusion) with others occurring in 3D volume meshes (e.g. cytosol, host factor supply). We estimate and incorporate realistic parameters such as NSP difusion constants. The simulations are performed upon experimental data based reconstructed cell geometries and help understanding the relation of form and function of virus replication. In the long run, our framework might help to facilitate the systematic development of Effcient direct antiviral agents and vaccines.