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1.
Cytotherapy ; 25(6 Supplement):S267-S268, 2023.
Article in English | EMBASE | ID: covidwho-20240749

ABSTRACT

Background & Aim: Gene therapies has become recognized for its remarkable clinical benefits in a variety of medical applications, in particular recent approval of an Ad vector-based COVID-19 vaccines have attracted recent global attention. Here, we present key considerations for GMP compliant process development for Coxsackie virus type B3 (CVB3), an oncolytic virus designed for clinical trial in triple-negative breast cancer. Methods, Results & Conclusion(s): CVB3 is a non-enveloped, linear single-strand RNA virus with a size of approximately 27-33 um in diameter. From the initial type using the zonal rotor centrifuge to the advanced type using the tangential flow filtration system and ion chromatograph, we considered the points of the design concept in constructing the manufacturing process. The final design system is constructed as a closed and single-use manufacturing system in which all processes from upstream large-scale cell culture to downstream target purification and concentration steps. In brief, HEK293 cell suspension extended in 3L serum-free medium infected with CVB3, up to 3.6 times 10 to 7 of TCID50 /mL before going to downstream steps, made total 150 mL of final products as 8.43 times 10 to 7 of TCID50/mL concentration. Although further quality control challenges remain that is removal of product-related impurities such as human cellular proteins and residual DNA/RNA to increase virus purity, this concept is effectively applicable even for other types of viruses as GMP manufacturing processes, and would be also important for technology transfer to future commercial production.Copyright © 2023 International Society for Cell & Gene Therapy

2.
Medicina (Kaunas) ; 59(5)2023 May 14.
Article in English | MEDLINE | ID: covidwho-20244340

ABSTRACT

Background and Objectives: COVID-19 infection may influence many physiological processes, including glucose metabolism. Acute hyperglycaemia has been related to a worse prognosis in patients with severe COVID-19 infection. The aim of our study was to find out if moderate COVID-19 infection is associated with hyperglycaemia. Materials and Methods: A total of 235 children were enrolled in the study between October 2021 and October 2022, 112 with confirmed COVID-19 infection and 123 with other RNA viral infection. In all patients, types of symptoms, glycaemia at the time of admission, and basic anthropometric and biochemical parameters were recorded. Results: Average glycaemia was significantly higher in COVID-19 patients compared to other viral infections (5.7 ± 1.12 vs. 5.31 ± 1.4 mmol/L, p = 0.011). This difference was more obvious in subgroups with gastrointestinal manifestations (5.6 ± 1.11 vs. 4.81 ± 1.38 mmol/L, p = 0.0006) and with fever (5.76±1.22 vs. 5.11±1.37 mmol/L, p = 0.002), while no significant difference was found in subgroups with mainly respiratory symptoms. The risk of hyperglycaemia (>5.6 mmol/L) was higher in COVID-19 patients compared to other viral infections (OR = 1.86, 95%CI = 1.10-3.14, p = 0.02). The risk of hyperglycaemia was significantly higher in COVID-19 compared to other viral infections in the subgroups of patients with fever (OR = 3.59, 95% CI 1.755-7.345, p = 0.0005) and with gastrointestinal manifestations (OR = 2.48, 95% CI 1.058-5.791, p = 0.036). Conclusion: According to our results, mild hyperglycaemia was significantly more common in children with moderate COVID-19 infection compared to other RNA virus respiratory and gastrointestinal infections, especially when accompanied by fever or gastrointestinal symptoms.


Subject(s)
COVID-19 , Hyperglycemia , Child , Humans , Hyperglycemia/complications , COVID-19/complications , Child, Hospitalized , Prognosis , Hospitalization
3.
International Journal of Infectious Diseases ; 130(Supplement 2):S144-S145, 2023.
Article in English | EMBASE | ID: covidwho-2324119

ABSTRACT

Intro: Deoxyribozymes (Dz) are short synthetic DNA oligonucleotides that catalyze the cleavage of a phosphodiester bond between nucleotides in the presence of divalent metal ions. The use of DNAzymes in the in vitro diagnostics increases the specificity and versatility of the analysis. Method(s): We took the well-studied Dz 10-23 with high catalytic activity as the basis of our system. The biosensor is divided into two fragments according to the binary probe principle (Dz1 and Dz2), which consist of target RNA binding sites, a fluorescent substrate (Fsub), and half of the Dz 10-23 catalytic center sequence. Assembly of the Dz 10-23 active center with subsequent Fsub cleavage and registration of a fluorescent signal is possible only if the target RNA is present in the sample. Finding(s): To assess the diagnostic potential of the biosensor, we measured FAM fluorescence in a solution containing synthetic RNA 35 nucleotides long (nip35) corresponding to the NiV target sequence, Fsub labeled with the FAM-BHQ1 and Dz_NiV pair. A mixture of Dz_NiV and Fsub was used as a control. The detection limit of the target RNA reached 5 nM, the signal development time was 30 minutes at a temperature of 37 C . Discussion(s): The specificity of Dz_NiV was evaluated in the presence of synthetic RNAs from six other RNA viruses of similar length: Hendra, Machupo, Sabia, Junin, Guanarito, and SARS-CoV. A fluorescent signal was recorded only in the presence of nip35 in the reaction mixture. The efficiency of Dz_NiV on a long fragment was tested using a plasmid with a cloned target sequence. The site is about 700 b.p. was amplified by PCR, followed by transcription. Conclusion(s): It was developed the highly specific biosensor Dz_NiV for the detection of Nipah virus RNA with a sensitivity limit of 5 nM at 37 C .Copyright © 2023

4.
EBioMedicine ; 92: 104608, 2023 Jun.
Article in English | MEDLINE | ID: covidwho-2326835

ABSTRACT

BACKGROUND: SARS-CoV-2 is a single-stranded positive-sense RNA virus. Several negative-sense SARS-CoV-2 RNA species, both full-length genomic and subgenomic, are produced transiently during viral replication. Methodologies for rigorously characterising cell tropism and visualising ongoing viral replication at single-cell resolution in histological sections are needed to assess the virological and pathological phenotypes of future SARS-CoV-2 variants. We aimed to provide a robust methodology for examining the human lung, the major target organ of this RNA virus. METHODS: A prospective cohort study took place at the University Hospitals Leuven in Leuven, Belgium. Lung samples were procured postmortem from 22 patients who died from or with COVID-19. Tissue sections were fluorescently stained with the ultrasensitive single-molecule RNA in situ hybridisation platform of RNAscope combined with immunohistochemistry followed by confocal imaging. FINDINGS: We visualised perinuclear RNAscope signal for negative-sense SARS-CoV-2 RNA species in ciliated cells of the bronchiolar epithelium of a patient who died with COVID-19 in the hyperacute phase of the infection, and in ciliated cells of a primary culture of human airway epithelium that had been infected experimentally with SARS-CoV-2. In patients who died between 5 and 13 days after diagnosis of the infection, we detected RNAscope signal for positive-sense but not for negative-sense SARS-CoV-2 RNA species in pneumocytes, macrophages, and among debris in the alveoli. SARS-CoV-2 RNA levels decreased after a disease course of 2-3 weeks, concomitant with a histopathological change from exudative to fibroproliferative diffuse alveolar damage. Taken together, our confocal images illustrate the complexities stemming from traditional approaches in the literature to characterise cell tropism and visualise ongoing viral replication solely by the surrogate parameters of nucleocapsid-immunoreactive signal or in situ hybridisation for positive-sense SARS-CoV-2 RNA species. INTERPRETATION: Confocal imaging of human lung sections stained fluorescently with commercially available RNAscope probes for negative-sense SARS-CoV-2 RNA species enables the visualisation of viral replication at single-cell resolution during the acute phase of the infection in COVID-19. This methodology will be valuable for research on future SARS-CoV-2 variants and other respiratory viruses. FUNDING: Max Planck Society, Coronafonds UZ/KU Leuven, European Society for Organ Transplantation.


Subject(s)
COVID-19 , Humans , SARS-CoV-2 , RNA, Viral , Prospective Studies , Lung
5.
Microbiol Spectr ; 11(3): e0118623, 2023 Jun 15.
Article in English | MEDLINE | ID: covidwho-2325934

ABSTRACT

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, is a highly contagious positive-sense RNA virus. Its explosive community spread and the emergence of new mutant strains have created palpable anxiety even in vaccinated people. The lack of effective anticoronavirus therapeutics continues to be a major global health concern, especially due to the high evolution rate of SARS-CoV-2. The nucleocapsid protein (N protein) of SARS-CoV-2 is highly conserved and involved in diverse processes of the virus replication cycle. Despite its critical role in coronavirus replication, N protein remains an unexplored target for anticoronavirus drug discovery. Here, we demonstrate that a novel compound, K31, binds to the N protein of SARS-CoV-2 and noncompetitively inhibits its binding to the 5' terminus of the viral genomic RNA. K31 is well tolerated by SARS-CoV-2-permissive Caco2 cells. Our results show that K31 inhibited SARS-CoV-2 replication in Caco2 cells with a selective index of ~58. These observations suggest that SARS-CoV-2 N protein is a druggable target for anticoronavirus drug discovery. K31 holds promise for further development as an anticoronavirus therapeutic. IMPORTANCE The lack of potent antiviral drugs for SARS-CoV-2 is a serious global health concern, especially with the explosive spread of the COVID-19 pandemic worldwide and the constant emergence of new mutant strains with improved human-to-human transmission. Although an effective coronavirus vaccine appears promising, the lengthy vaccine development processes in general and the emergence of new mutant viral strains with a potential to evade the vaccine always remain a serious concern. The antiviral drugs targeted to the highly conserved targets of viral or host origin remain the most viable and timely approach, easily accessible to the general population, in combating any new viral illness. The majority of anticoronavirus drug development efforts have focused on spike protein, envelope protein, 3CLpro, and Mpro. Our results show that virus-encoded N protein is a novel therapeutic target for anticoronavirus drug discovery. Due to its high conservation, the anti-N protein inhibitors will likely have broad-spectrum anticoronavirus activity.


Subject(s)
COVID-19 , Humans , SARS-CoV-2 , COVID-19 Vaccines , Pandemics/prevention & control , Caco-2 Cells , Drug Discovery , Antiviral Agents/therapeutic use , Nucleocapsid Proteins
6.
Biochim Biophys Acta Biomembr ; 1865(6): 184174, 2023 Aug.
Article in English | MEDLINE | ID: covidwho-2324713

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Cell Membrane/genetics , Cell Membrane/metabolism , COVID-19/metabolism , Membrane Proteins/metabolism , SARS-CoV-2/genetics , Viral Regulatory and Accessory Proteins/metabolism
7.
Journal of Biological Chemistry ; 299(3 Supplement):S687, 2023.
Article in English | EMBASE | ID: covidwho-2318717

ABSTRACT

RNA viruses are diverse and abundant pathogens responsible for numerous human ailments, from common colds to AIDS, SARS, Ebola, and other dangerous diseases. RNA viruses possess relatively compact genomes and have therefore evolved multiple mechanisms to maximize their coding capacities, often using overlapping reading frames. In this way, one RNA sequence can encode multiple proteins via mechanisms including alternative splicing and ribosomal frameshifting. Many such processes in gene expression involve the RNA folding into three-dimensional structures that can recruit ribosomes without initiation factors, hijack host proteins, cause ribosomes to frameshift, and expose or occlude regulatory protein binding motifs to ultimately control each key process in the viral life cycle. I will discuss the RNA structure of HIV-1 and SARS-CoV-2 and the importance of alternative conformations assumed by the same RNA sequence in controlling gene expression of viruses and bacteria.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

8.
Journal of Biological Chemistry ; 299(3 Supplement):S154, 2023.
Article in English | EMBASE | ID: covidwho-2317598

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.

9.
Circulation Conference: American Heart Association's ; 144(Supplement 2), 2021.
Article in English | EMBASE | ID: covidwho-2314887

ABSTRACT

Case Presentation: A 19 year old male presented with sudden onset chest pain radiating to back. He was a smoker and denied using cocaine since his last hospitalization for cocaine-induced myocardial infarction 2 years ago. UDS was negative. EKG showed normal sinus rhythm with no ST-T wave changes. Initial troponin was 0.850. Potassium levels were low at 2.9 mmol/L but other labs were normal. Chest CT angiography ruled out aortic dissection. He was started on heparin drip. Stat Echocardiogram showed LVEF of 55-60% with no wall motion abnormalities. Repeat potassium levels normalized after replacement, however, his troponins were trending up from 3.9 and 11.5. He continued to complain of severe chest pain, so underwent cardiac catheterization which showed normal coronary arteries and LVEF 55-60%. Heparin drip was discontinued and NSAIDs and colchicine were started. Cardiac MRI (see Figure) was done that showed patchy mid-wall and epicardial delayed gadolinium enhancement involving the basal inferolateral wall, with mild hyperintense signal on the triple IR sequence, suggestive of myocarditis. On further probing, he reported receiving a second dose of Moderna COVID vaccine 3 days prior to presentation. Discussion(s): In December 2019, a novel RNA virus causing COVID-19 infection was reported, which quickly reached a pandemic level. COVID-19 vaccines were granted emergency use authorization by FDA. With millions of people receiving COVID-19 vaccinations worldwide, rare adverse effects are now being reported. The benefits of vaccination undoubtedly outweigh any minor side effects. However major adverse effects like this are potentially fatal. This case report warrants further investigation into the association of myocarditis with COVID-19 vaccinations and further recommendations regarding vaccination in younger adults.

10.
Topics in Antiviral Medicine ; 31(2):213-214, 2023.
Article in English | EMBASE | ID: covidwho-2313407

ABSTRACT

Background: SARS-CoV-2 evolution has contributed to successive waves of infections and severely compromised the efficacy of available SARS-CoV-2 monoclonal antibodies. Decaying vaccine-induced immunity, vaccine hesitancy, and limited vaccine protection in older and immunocompromised populations further compromises vaccine efficacy at the population level. Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease due to COVID-19. An orally bioavailable RDV analog could facilitate earlier widespread administration to non-hospitalized COVID-19 patients. Method(s): We synthesized monoalkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. We evaluated the in vivo efficacy of our lead compound, 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (V2043), in an oral treatment model of murine SARS-CoV-2 infection. We then synthesized numerous phospholipid analogs of RVn and determined which modifications enhanced in vitro antiviral activity and selectivity. The most effective compounds against SARS-CoV-2 were then evaluated for antiviral activity against other RNA viruses. Result(s): Oral treatment of SARS-CoV-2 infected BALB/c mice with V2043 (60 mg/kg once daily for 5 days, starting 12 hrs after infection) reduced lung viral load by more than 100-fold versus vehicle at day 2 and to below the LOD at day 5. V2043 inhibited previous and contemporary SARS-CoV-2 Variants of concern to a similar degree, as measured by the half maximal effective concentration (EC50) in a human lung epithelial cell line (Calu-3). Evaluation of multiple RVn analogs with hydrophobic esters at the sn-2 of glycerol revealed that in vitro antiviral activity was improved by the introduction of a 3-fluoro-4-methoxysubstituted benzyl or a 3-or 4-cyano-substituted benzyl. These compounds showed a 2-to 6-fold improvement in antiviral activity compared to analogs having an unsubstituted benzyl, such as V2043, and were more active than RDV. These compounds also showed enhanced antiviral activity against multiple contemporary and emerging RNA viruses. Conclusion(s): Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections and as preparation for future outbreaks of pandemic RNA viruses.

11.
Microbiol Spectr ; 11(3): e0099423, 2023 Jun 15.
Article in English | MEDLINE | ID: covidwho-2316423

ABSTRACT

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD. IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines.


Subject(s)
COVID-19 , Chiroptera , Animals , Humans , Antiviral Agents/pharmacology , SARS-CoV-2/metabolism , Nucleocapsid Proteins , Pandemics , Viral Proteins/metabolism , RNA, Double-Stranded
12.
Letters in Applied NanoBioScience ; 11(2):3573-3585, 2022.
Article in English | Scopus | ID: covidwho-2301600

ABSTRACT

Foot-and-mouth disease (FMD) and Coronavirus Disease 2019 (COVID-19) are transboundary diseases caused by single-stranded positive-sense RNA viruses with similarities in genome replication and viral protein synthesis. In FMD, asymptomatic infection leads to carrier status and persistently infected animals that threaten the animals vaccinated with a trivalent inactivated whole virus vaccine. Similar information on COVID-19 is not yet available. As COVID-19 vaccination is introduced in January 2021 (since 16 January 2021 in India), its outcome can be assessed by the year-end;and while doing so, the experiences gained in the control of FMD in livestock worldwide can be applied, including monitoring of vaccination response, duration of immunity, level of herd immunity developed, and antigenic matching of the vaccine virus. Antigenic divergence of the virus is a major issue in FMD, and different geographical regions in the world use different virus strains in vaccine preparations to antigenically match circulating virus strains in respective regions for control of the disease. Non-synonymous mutations in the critical antigenic determinants of SARS-CoV-2 have been observed, and there is likely the existence/development of antigenic variants. Therefore, during the post-COVID-19 vaccination regime, it will be essential to monitor the suitability of the in-use vaccine strain region-wise from time to time, as there could be an eruption of isolated outbreaks in a country arising due to antigenic variation and variants. In the context of the present scenario of COVID-19 around the Globe and multiple ongoing efforts to develop suitable vaccine(s) to control the disease, it is a must to develop NSP-antibody (that differentiate infected from vaccinated) assays to differentiate infected from vaccinated individuals(DIVI;DIVA in veterinary epidemiology). The techniques used and experiences gained in ongoing FMD control programs in the endemic countries can be applied to COVID-19 control in a country;and finally, the Globe. After achieving the control of COVID-19, the aim would be to eradicate the virus, which will be tough even with vaccination, as the disease/infection may become endemic during the time to come. To achieve this, applying the principles of Progressive Control Pathway for Foot-and-Mouth Disease (PCP-FMD;FAO/OIE) to COVID-19 control will be beneficial in its control. The present review discusses the issue of control of COVID-19. © 2021 by the authors.

13.
Wellcome Open Res ; 6: 241, 2021.
Article in English | MEDLINE | ID: covidwho-2293550

ABSTRACT

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

14.
Pathogens ; 12(3)2023 Mar 14.
Article in English | MEDLINE | ID: covidwho-2292284

ABSTRACT

Cells have developed different strategies to cope with viral infections. Key to initiating a defense response against viruses is the ability to distinguish foreign molecules from their own. One central mechanism is the perception of foreign nucleic acids by host proteins which, in turn, initiate an efficient immune response. Nucleic acid sensing pattern recognition receptors have evolved, each targeting specific features to discriminate viral from host RNA. These are complemented by several RNA-binding proteins that assist in sensing of foreign RNAs. There is increasing evidence that the interferon-inducible ADP-ribosyltransferases (ARTs; PARP9-PARP15) contribute to immune defense and attenuation of viruses. However, their activation, subsequent targets, and precise mechanisms of interference with viruses and their propagation are still largely unknown. Best known for its antiviral activities and its role as RNA sensor is PARP13. In addition, PARP9 has been recently described as sensor for viral RNA. Here we will discuss recent findings suggesting that some PARPs function in antiviral innate immunity. We expand on these findings and integrate this information into a concept that outlines how the different PARPs might function as sensors of foreign RNA. We speculate about possible consequences of RNA binding with regard to the catalytic activities of PARPs, substrate specificity and signaling, which together result in antiviral activities.

15.
Viruses ; 15(4)2023 03 23.
Article in English | MEDLINE | ID: covidwho-2299889

ABSTRACT

The virus-host interaction is dynamic and evolutionary. Viruses have to fight with hosts to establish successful infection. Eukaryotic hosts are equipped with multiple defenses against incoming viruses. One of the host antiviral defenses is the nonsense-mediated mRNA decay (NMD), an evolutionarily conserved mechanism for RNA quality control in eukaryotic cells. NMD ensures the accuracy of mRNA translation by removing the abnormal mRNAs harboring pre-matured stop codons. Many RNA viruses have a genome that contains internal stop codon(s) (iTC). Akin to the premature termination codon in aberrant RNA transcripts, the presence of iTC would activate NMD to degrade iTC-containing viral genomes. A couple of viruses have been reported to be sensitive to the NMD-mediated antiviral defense, while some viruses have evolved with specific cis-acting RNA features or trans-acting viral proteins to overcome or escape from NMD. Recently, increasing light has been shed on the NMD-virus interaction. This review summarizes the current scenario of NMD-mediated viral RNA degradation and classifies various molecular means by which viruses compromise the NMD-mediated antiviral defense for better infection in their hosts.


Subject(s)
Nonsense Mediated mRNA Decay , RNA Viruses , RNA Viruses/genetics , Protein Biosynthesis , Codon, Terminator , Antiviral Agents
16.
Investigacion Clinica (Venezuela) ; 64(1):108-122, 2023.
Article in English | EMBASE | ID: covidwho-2254138

ABSTRACT

SARS-CoV-2 is a single-stranded RNA virus that belongs to the group of seven coronaviruses that affect humans, and its infection causes the COVID-19 disease. The association between the COVID-19 condition and risk factors of neurological manifestations is unclear to date. This review aims to update the main neurological manifestations associated with SARS-CoV-2 disease. First, we present the hypothesis of the neuroinvasion mechanisms of SARS-CoV-2. Then, we discuss the possible symptoms related to patients with COVID-19 infection in the central and peripheral nervous systems, followed by the perspectives of diagnosis and treatment of possible neurological manifesta-tions. The hypothesis of the neuroinvasion mechanism includes direct routes, as the virus crosses the blood-brain barrier or the ACE2 receptor pathway role, and indirect pathways, such as malfunctions of the immune system and vascular system dysregulation. Various studies report COVID-19 consequences, such as neuroanatomic alterations and cognitive impairment, besides peripheral condi-tions, such as anosmia, ageusia, and Guillain Barre Syndrome. However, the het-erogeneity of the studies about neurologic damage in patients after COVID-19 infection precludes any generalization of current findings. Finally, new studies are necessary to understand the adequate diagnosis, therapeutic method of early treatment, and risk group of patients for neurological manifestations of COVID-19 post-infection.Copyright © 2023, Instituto de Investigaciones Clinicas. All rights reserved.

17.
Coronaviruses ; 2(8) (no pagination), 2021.
Article in English | EMBASE | ID: covidwho-2251617

ABSTRACT

The emerging new COVID 2019 pandemic, which started in 2019 in China (Wuhan) and is caused by SARS-CoV-2, raises critical concerns due to high morbidity and mortality. As many patients are infected and the numbers still increase, this may suggest that there are different variants of the virus and some of them are more pathogenic. Besides, the virus is suspected to have various evolutionary pathways since SARS-CoV-2 belongs to the RNA viruses' family, which is characterized by a high mutation rate. Additionally, it is crucial to understand the life cycle of the virus to be able to urge antiviral studies. Genotyping studies about viruses are also important in order to understand the transmission and evolution of the virus. The genome of SARS-CoV-2 has a furin-like cleavage site in its S protein that may affect its pathogenicity. It was found that insertions and deletions in S protein have an impact on the transmission and fusion of the virus. The single nucleotide polymorphisms (SNP) genotypes are used to track the relationship of virus isolates. Se-quence alignment revealed the presence of hundreds of inter-host mutations during person-to-per-son transmission. Furthermore, genetic recombination provided a second mechanism for virus evo-lution. In this review, we highlight the life cycle of the virus and methods of virus evolution caused by mutations or recombination of viral genomes.Copyright © 2021 Bentham Science Publishers.

18.
Coronaviruses ; 2(11) (no pagination), 2021.
Article in English | EMBASE | ID: covidwho-2251388

ABSTRACT

Background: The deadly outbreak of COVID-19 disease caused by novel SARS CoV2 has created an unprecedented global health crisis affecting every sectors of human life and enor-mous damage to world's economy. With >16.1 million infections and >650,000 deaths worldwide as of July 27, 2020, there is no treatment for this disease neither is there any available vaccine. Seri-ous research efforts are ongoing on all fronts including treatment, prevention and diagnosis to combat the spread of this infection. A number of targets that include both viral and host proteins have been identified and became part of intense investigation. In this respect the viral surface spike (S) glycoprotein caught the attention most. It is cleaved by multiple host proteases to allow recognition by host receptor human Angiotensin Converting Enzyme2 (hACE2) leading to fusion and viral re-plication. Natural products, small compounds, antioxidants, peptides, proteins, oligonucleotides, antibodies and other compounds are under investigation for development of antiviral agents against COVID-19. Objective(s): Recently cholesterol lowering phytocompounds Quercetin, Swertiamarin and Berberine which promote human Low Density Lipoprotein Receptor (hLDLR) via inhibition of human Pro-protein Convertase Subtilisin Kexin9 (hPCSK9) have been shown to block viral infections caused by ebola, influenza, Respiratory Syncytial Virus (RSV), Hepatitis C virus (HCV) and other RNA type viruses. Since SARS CoV2 is a RNA virus with similar genetic structure and infection machin-ery, it is hypothesised that these phytocompounds may also exhibit antiviral property against COVID-19. Method(s): Our above concept is based on recently published studies as well as our herein presented in silico modeling and computational data which suggested strong interactions of hPCSK9 with above phytocompounds and most importantly with hACE2 following its complexation with receptor binding domain (RBD) of SARS CoV2 S protein. Result(s): These results and a proposed schematic model showing association of hPCSK9 with SARS CoV2 infection are presented in this manuscript. It is proposed that hPCSK9 plays the role of a co-receptor in binding with hACE2:RBD complex and thereby facilitates viral fusion. Conclusion(s): Our studies suggest that PCSK9 inhibitors may provide beneficial effect against COVID-19 infection by retarding viral fusion through displacement of bound hPCSK9 from its complex with ACE2:RBD of SARS CoV2 S protein.Copyright © 2021 Bentham Science Publishers.

19.
Kidney International Reports ; 8(3 Supplement):S457, 2023.
Article in English | EMBASE | ID: covidwho-2250936

ABSTRACT

Introduction: Immunoglobulin (Ig)G antibodies against SARS-CoV-2 are implicated in herd immunity. Humoral response to vaccines in kidney transplant recipients (KTRs) is documented to be sub-optimal. However, the response to non-messenger RNA(mRNA) based vaccines in KTRs is not known Methods: SARS-CoV-2 spike protein IgG antibody response was assessed in KTRs using chemiluminescence immunoassay. Patients were characterized by the number of vaccine doses received and Coronavirus disease 2019 (COVID-19) infection in past. Result(s): Out of 224 KTRs evaluated, 197 (87.94%) had positive S1/S2 IgG anti-SARS-CoV-2 antibodies with a median [IQR] titre of 307.5 AU/ml [91 AU/ml - 400 AU/ml]. High titres (in neutralizing range) were found in 170/224 (75.9%) KTRs. Seropositivity rates after 2 doses of vaccination were significantly higher than unvaccinated KTRs (88.67% vs 66.7%;p = 0.006). After adjusting for cofounders, KTRs with diabetes at the time of vaccination were less likely to develop antibody response (aOR 0.31, 95% confidence interval [CI] - 0.10, 0.90;p = 0.032). Higher eGFR was also an independent predictor of antibody response (aOR 1.04 95% CI - 1.01, 1.08;p = 0.005). KTRs vaccinated with CovishieldTM developed higher antibody response as compared to CovaxinTM (aOR 5.04, 95% CI - 1.56, 16.22;p = 0.007). Conclusion(s): A high rate of seroconversion was seen in KTRs after SARS-CoV-2 vaccination with non mRNA vaccines. The presence of diabetes and decreased eGFR independently predicted lower seroconversion rates. No conflict of interestCopyright © 2023

20.
Coronaviruses ; 2(1):59-72, 2021.
Article in English | EMBASE | ID: covidwho-2250392

ABSTRACT

Background: COVID-19 is an infectious disease caused by SARS-CoV-2. The disease has hit hard around the globe and is now a pandemic. As of April 01, 2020, a total of 875,560 cases have been reported and the figures are increasing day by day. Currently, there is no treatment or vaccine available for curing COVID-19 and pharmaceutical companies are racing toward the common goal of achieving the cure. Method(s): Scientific databases, including Science direct, Pub med, Elsevier, Scopus, and Nature, were explored. Data has also been accessed from case reports, newspaper reports, internet data, World Health Organisation (WHO) reports, and Centre of Disease Control (CDCs) reports. The US National Library of Medicine, Clinicaltrials.gov, were accessed to get information about the ongoing clinical trials. The literature survey started in the first week of February 2020 and was completed in the first week of April 2020. Additional literature survey was done in the second week of June 2020. Result(s): The epicentre of COVID-19 is Wuhan City, Hubei Province, China. Coronavirus belongs to Order Nidovirale and is subdivided into four groups alpha, beta, gamma, and delta. Coronavirus 229E, NL63, HKU1, MERS-CoV and SARS-CoV are known to infect humans. It is an enveloped, non-segmented positive-sense RNA virus of size 30-32 kb with several structural and accessory proteins. The pathogenesis of COVID-19 involves attachment of Spike (S) protein of SARS-CoV-2 to the angio-tensin-converting enzyme 2(ACE2) receptor present on the host cell membrane. Clinical manifestation of COVID-19 include fever, cough, complicated dyspnoea, pneumonia, etc. Real-time-PCR is a sensi-tive test for the detection of SARS-CoV. Remdesivir, Bevacizumab, Darunavir and cobicistat, lopinavir-ritonavir, Oseltamavir, hydroxychloroquine, Sarilumab, mRNA-1273, Ad5-nCoV are some of the drugs under the clinical phase of the trial. People with A-positive blood group, with comorbidities like diabe-tes, hypertension, chronic pulmonary obstructive disease, substance abuse disorders, immunocom-promised individuals, health care workers, and older adults are at high risk of getting infected with SARS-CoV-2 Conclusion(s): This article gives insight into the occurrence of COVID-19, classification and structure of SARS-CoV-2, pathogenesis, pathological findings, clinical manifestation, diagnosis, potential treatment options and prevention, and people at risk of COVID-19.Copyright © 2021 Bentham Science Publishers.

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