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1.
J Virol Methods ; 301: 114463, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1796383

ABSTRACT

PURPOSE: With the rise of the different Variants of Concern (VOC) and Variants of Interest (VOI) in order to control the SARS-CoV-2 pandemic, strategies for accurately tracking these different variants have been developed. While most of these strategies rely heavily on specific PCRs targeting the characteristic mutations of some lineages, several approaches using the alterations at the cycle threshold (Ct) of different commercial PCR diagnostic tests have been described. The objective of this study is to analyse the use of the Ct difference at the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Seegene, Korea) between the Nucleocapside (N) and the Spike (S) or RNA-dependent RNA polymerase (RdRP) genes as a preliminary screening for variant tracking. METHODS: The samples analysed with the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay from 1st of March 2021 to 26th of December 2021 were selected. The Ct values for N, S, RdRP were collected, and the differences between N and S (ΔS) and N and RdRP (ΔRdRP) were calculated. Using ΔS and ΔRdRP a diagnostic test was designed and these results were compared to the routine Variant assessment. RESULTS: The mean ΔS and ΔRdRP were characteristic for Alpha and Delta. This difference was statistically significant. For Every analysed Variant the diagnostic test achieved a higher than 90% sensitivity with a noteworthy performance with the Omicron variant (97% sensitivity and 90% specificity). CONCLUSIONS: The analysis of the Ct alterations at the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay may be a suitable method for an early approach to SARS-CoV-2 variant assessment.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Testing , Humans , RNA-Dependent RNA Polymerase/genetics
2.
Rev Peru Med Exp Salud Publica ; 38(4): 595-600, 2021.
Article in Spanish, English | MEDLINE | ID: covidwho-1780357

ABSTRACT

The present work validated and evaluated a duplex real-time RT-PCR using specific primers and probes for genes RdRp from SARS-CoV-2 and GAPDH from humans; the latter was used as an endogenous control in all reactions. We evaluated the specificity, the sensitivity, the robustness, the reproducibility, the repeatability, the comparability, and the limit of detection. The predictive positive and negative values (PPV and PNV, respectively) and all the parameters evaluated using our duplex real-time RT-PCR was 100%. The detection limit was 100 copies/µL according to the acceptance criteria established for the validation of this protocol. Our duplex real-time RT-PCR demonstrated to be a good alternative for the diagnosis of COVID-19; in addition, this PCR was used adequately in suspicion of COVID-19, allowing it to control the number of false-negatives.


Se validó y evaluó un método de RT-PCR en tiempo real usando cebadores y sondas específicas para los genes RdRP de SARS-CoV-2 y GAPDH de humanos; este último fue usado como control endógeno. Se evaluó la especificidad y sensibilidad; además, se evaluó otros parámetros como la robustez, la repetibilidad, reproducibilidad, comparabilidad y el límite de detección. La sensibilidad, especificidad, los valores predictivos positivo y negativo, la robustez, comparabilidad y la repetibilidad-reproducibilidad de la prueba de RT-PCR en tiempo real dúplex fue de 100%, con un límite de detección de 100 copias/µL, de acuerdo con los criterios de aceptación establecidos para validación del protocolo. Esta prueba estandarizada es una buena alternativa para el diagnóstico de COVID-19; además, la prueba fue aplicada de manera exitosa en personas sospechosas de la enfermedad permitiendo controlar el número de falsos negativos.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , RNA, Viral/genetics , RNA-Dependent RNA Polymerase , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity
3.
Sci Rep ; 12(1): 5771, 2022 Apr 06.
Article in English | MEDLINE | ID: covidwho-1778635

ABSTRACT

SARS-CoV-2 is still a health problem worldwide despite the availability of vaccines. Therefore, there is a need for effective and safe antiviral. SARS-CoV-2 and HCV necessitate RNA-dependent RNA polymerase (RdRp) for replication; therefore, it has been hypothesized that RdRp inhibitors used to treat HCV may be effective treating SARS-CoV-2. Accordingly, we evaluated the effect of the sofosbuvir/velpatasvir (SOF/VEL) combination in early SARS-CoV-2 infection. A multicenter case-control study was conducted, enrolling 120 patients with mild or moderate COVID-19, of whom 30, HCV coinfected or not, received SOF/VEL tablets (400/100 mg) once daily for 9 days within a median of 6 days from the beginning of infection and 90 controls were treated with standard care. The primary endpoint was the effect on viral clearance, and the secondary endpoint was the improvement of clinical outcomes. Nasal swabs for SARS-CoV-2 by PCR were performed every 5-7 days. Between 5-14 days after starting SOF/VEL treatment, SAS-CoV-2 clearance was observed in 83% of patients, while spontaneous clearance in the control was 13% (p < 0.001). An earlier SARS-CoV-2 clearance was observed in the SOF/VEL group than in the control group (median 14 vs 22 days, respectively, p < 0.001) also when the first positivity was considered. None of the patients in the SOF/VEL group showed disease progression, while in the control group, 24% required more intensive treatment (high flow oxygen or noninvasive/invasive ventilation), and one patient died (p < 0.01). No significant side effects were observed in the SOF/VEL group. Early SOF/VEL treatment in mild/moderate COVID-19 seems to be safe and effective for faster elimination of SARS-CoV-2 and to prevent disease progression.


Subject(s)
COVID-19 , Hepatitis C , Antiviral Agents/adverse effects , COVID-19/drug therapy , Carbamates , Case-Control Studies , Disease Progression , Genotype , Hepacivirus/genetics , Hepatitis C/drug therapy , Heterocyclic Compounds, 4 or More Rings/adverse effects , Humans , RNA-Dependent RNA Polymerase , SARS-CoV-2 , Sofosbuvir , Treatment Outcome
4.
Environ Monit Assess ; 194(5): 342, 2022 Apr 07.
Article in English | MEDLINE | ID: covidwho-1777746

ABSTRACT

The present study tracked the city-wide dynamics of severe acute respiratory syndrome-corona virus 2 ribonucleic acids (SARS-CoV-2 RNA) in the wastewater from nine different wastewater treatment plants (WWTPs) in Jaipur during the second wave of COVID-19 out-break in India. A total of 164 samples were collected weekly between February 19th and June 8th, 2021. SARS-CoV-2 was detected in 47.2% (52/110) influent samples and 37% (20/54) effluent samples. The increasing percentage of positive influent samples correlated with the city's increasing active clinical cases during the second wave of COVID-19 in Jaipur. Furthermore, wastewater-based epidemiology (WBE) evidence clearly showed early detection of about 20 days (9/9 samples reported positive on April 20th, 2021) before the maximum cases and maximum deaths reported in the city on May 8th, 2021. The present study further observed the presence of SARS-CoV-2 RNA in treated effluents at the time window of maximum active cases in the city even after tertiary disinfection treatments of ultraviolet (UV) and chlorine (Cl2) disinfection. The average genome concentration in the effluents and removal efficacy of six commonly used treatments, activated sludge process + chlorine disinfection (ASP + Cl2), moving bed biofilm reactor (MBBR) with ultraviolet radiations disinfection (MBBR + UV), MBBR + chlorine (Cl2), sequencing batch reactor (SBR), and SBR + Cl2, were compared with removal efficacy of SBR + Cl2 (81.2%) > MBBR + UV (68.8%) > SBR (57.1%) > ASP (50%) > MBBR + Cl2 (36.4%). The study observed the trends and prevalence of four genes (E, RdRp, N, and ORF1ab gene) based on two different kits and found that prevalence of N > ORF1ab > RdRp > E gene suggested that the effective genome concentration should be calculated based on the presence/absence of multiple genes. Hence, it is imperative to say that using a combination of different detection genes (E, N, RdRp, & ORF1ab genes) increases the sensitivity in WBE.


Subject(s)
COVID-19 , Wastewater-Based Epidemiological Monitoring , Biofilms , Bioreactors , COVID-19/epidemiology , Chlorine , Environmental Monitoring , Humans , RNA, Viral , RNA-Dependent RNA Polymerase , SARS-CoV-2 , Waste Water
5.
Virol J ; 18(1): 177, 2021 08 28.
Article in English | MEDLINE | ID: covidwho-1767103

ABSTRACT

BACKGROUND: The development of an influenza RNA-dependent RNA polymerase (RdRp) inhibitor is required; therefore, a method for evaluating the activity of influenza RdRp needs to be developed. The current method uses an ultracentrifuge to separate viral particles and quantifies RdRp activity with radioisotope-labeled nucleosides, such as 32P-GTP. This method requires special equipment and radioisotope management, so it cannot be implemented in all institutions. We have developed a method to evaluate the mRNA transcription activity of RdRp without using ultracentrifugation and radioisotopes. RESULTS: RdRp was extracted from viral particles that were purified from the culture supernatant using anionic polymer-coated magnetic beads that can concentrate influenza virus particles from the culture supernatant in approximately 30 min. A strand-specific real-time reverse transcription polymerase chain reaction (RT-PCR) method was developed based on reverse transcription using tagged primers. RT primers were designed to bind to a sequence near the 3' end of mRNA containing a poly A tail for specific recognition of the mRNA, with an 18-nucleotide tag attached to the 5' end of the sequence. The RT reaction was performed with this tagged RT primer, and the amount of mRNA was analyzed using real-time qPCR. Real-time qPCR using the tag sequence as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the mRNA of segments 1, 4, and 5. The temperature, reaction time, and Mg2+ concentration were determined to select the optimum conditions for in vitro RNA synthesis by RdRp, and the amount of synthesized mRNAs of segments 1, 4, and 5 was determined with a detection sensitivity of 10 copies/reaction. In addition, mRNA synthesis was inhibited by ribavirin triphosphate, an RdRp inhibitor, thus indicating the usefulness of this evaluation method for screening RdRp inhibitors. CONCLUSION: This method makes it possible to analyze the RdRp activity even in a laboratory where ultracentrifugation and radioisotopes cannot be used. This novel method for measuring influenza virus polymerase activity will further promote research to identify compounds that inhibit viral mRNA transcription activity of RdRp.


Subject(s)
Influenza, Human , Orthomyxoviridae , RNA-Dependent RNA Polymerase , Reverse Transcription , Humans , Orthomyxoviridae/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
6.
Viruses ; 14(3)2022 03 03.
Article in English | MEDLINE | ID: covidwho-1767152

ABSTRACT

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3'-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5'-end of nascent host mRNA.


Subject(s)
Influenza, Human , Orthomyxoviridae , Humans , Orthomyxoviridae/genetics , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , Viral Transcription , Virus Replication
7.
Molecules ; 27(6)2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1742558

ABSTRACT

Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.


Subject(s)
COVID-19 , Encephalitis Viruses, Tick-Borne , Humans , Pandemics , RNA-Dependent RNA Polymerase , SARS-CoV-2
8.
Talanta ; 243: 123393, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1740208

ABSTRACT

We present a fast, reliable and easy to scale-up colorimetric sensor based on gold nanoparticles (AuNPs) to detect the sequences coding for the RdRp, E, and S proteins of SARS-CoV-2. The optimization of the system (so-called "the sensor") includes the evaluation of different sizes of nanoparticles, sequences of oligonucleotides and buffers. It is stable for months without any noticeable decrease in its activity, allowing the detection of SARS-CoV-2 sequences by the naked eye in 15 min. The efficiency and selectivity of detection, in terms of significative colorimetric changes in the solution upon target recognition, are qualitatively (visually) and quantitatively (absorbance measurements) assessed using synthetic samples and samples derived from infected cells and patients. Furthermore, an easy and affordable amplification approach is implemented to increase the system's sensitivity for detecting high and medium viral loads (≥103 - 104 viral RNA copies/µl) in patient samples. The whole process (amplification and detection) takes 2.5 h. Due to the ease of use, stability and minimum equipment requirements, the proposed approach can be a valuable tool for the detection of SARS-CoV-2 at facilities with limited resources.


Subject(s)
COVID-19 , Metal Nanoparticles , COVID-19/diagnosis , Colorimetry , Gold , Humans , RNA, Viral/genetics , RNA-Dependent RNA Polymerase , SARS-CoV-2/genetics
9.
Nature ; 603(7899): 25-27, 2022 03.
Article in English | MEDLINE | ID: covidwho-1730273

Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Clinical Trials as Topic , Drug Repositioning , Host-Pathogen Interactions/drug effects , SARS-CoV-2/drug effects , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/therapeutic use , Administration, Oral , Alanine/administration & dosage , Alanine/analogs & derivatives , Alanine/therapeutic use , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/economics , Antibodies, Neutralizing/therapeutic use , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacology , COVID-19/economics , COVID-19/immunology , COVID-19/mortality , COVID-19/virology , COVID-19 Vaccines , Cytidine/analogs & derivatives , Cytidine/therapeutic use , Depsipeptides/pharmacology , Depsipeptides/therapeutic use , Dexamethasone/administration & dosage , Dexamethasone/therapeutic use , Drug Combinations , Drug Synergism , Esters/pharmacology , Esters/therapeutic use , Guanidines/pharmacology , Guanidines/therapeutic use , Hospitalization , Humans , Hydroxylamines/therapeutic use , Internationality , Lactams/therapeutic use , Leucine/therapeutic use , Mice , National Institutes of Health (U.S.)/organization & administration , Nitriles/therapeutic use , Peptide Elongation Factor 1/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Peptides, Cyclic/therapeutic use , Proline/therapeutic use , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , RNA-Dependent RNA Polymerase/antagonists & inhibitors
10.
Viruses ; 12(6)2020 06 25.
Article in English | MEDLINE | ID: covidwho-1726024

ABSTRACT

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has highlighted the importance of reliable and rapid diagnostic testing to prevent and control virus circulation. Dozens of monoplex in-house RT-qPCR assays are already available; however, the development of dual-target assays is suited to avoid false-negative results caused by polymorphisms or point mutations, that can compromise the accuracy of diagnostic and screening tests. In this study, two mono-target assays recommended by WHO (E-Sarbeco (enveloppe gene, Charite University, Berlin, Germany) and RdRp-IP4 (RdRp, Institut Pasteur, Paris, France)) were selected and combined in a unique robust test; the resulting duo SARS-CoV-2 RT-qPCR assay was compared to the two parental monoplex tests. The duo SARS-CoV-2 assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. We demonstrated that combining two single systems into a dual-target assay (with or without an MS2-based internal control) did not impair performances, providing a potent tool adapted for routine molecular diagnosis in clinical microbiology laboratories.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA-Dependent RNA Polymerase/genetics , Real-Time Polymerase Chain Reaction/methods , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/virology , Coronavirus RNA-Dependent RNA Polymerase , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/analysis , SARS-CoV-2 , Sensitivity and Specificity , World Health Organization
11.
Commun Biol ; 5(1): 154, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1699831

ABSTRACT

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Exonucleases/antagonists & inhibitors , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Sequence , Anilides/pharmacology , Animals , Base Sequence , Benzimidazoles/pharmacology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Drug Synergism , Exonucleases/genetics , Exonucleases/metabolism , Humans , Proline/pharmacology , Pyrrolidines/pharmacology , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Valine/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Virus Replication/genetics
12.
Nat Commun ; 13(1): 621, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1671551

ABSTRACT

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Guanosine Monophosphate/analogs & derivatives , Phosphoramides/chemistry , Phosphoramides/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolism , COVID-19/virology , Cryoelectron Microscopy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Guanosine Monophosphate/chemistry , Guanosine Monophosphate/pharmacology , Humans , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Proteins/genetics
13.
Antiviral Res ; 198: 105254, 2022 02.
Article in English | MEDLINE | ID: covidwho-1654045

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Nucleic Acid Synthesis Inhibitors/pharmacology , SARS-CoV-2/drug effects , Tubercidin/analogs & derivatives , Cell Line , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/pharmacology , Drug Evaluation, Preclinical/methods , HEK293 Cells , Humans , Microbial Sensitivity Tests , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/genetics , Thionucleosides/pharmacology , Tubercidin/pharmacology , Vidarabine/analogs & derivatives , Vidarabine/pharmacology , Vidarabine Phosphate/analogs & derivatives , Vidarabine Phosphate/pharmacology
14.
Antiviral Res ; 198: 105252, 2022 02.
Article in English | MEDLINE | ID: covidwho-1654043

ABSTRACT

We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cytidine/analogs & derivatives , Hydroxylamines/therapeutic use , Lactams/therapeutic use , Leucine/therapeutic use , Nitriles/therapeutic use , Proline/therapeutic use , SARS-CoV-2/drug effects , Adenosine/analogs & derivatives , Adenosine/therapeutic use , Adenosine Monophosphate/therapeutic use , Alanine/therapeutic use , Animals , Cell Line , Chlorocebus aethiops , Coronavirus 3C Proteases/antagonists & inhibitors , Cytidine/therapeutic use , Humans , Microbial Sensitivity Tests , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Vero Cells , Virus Replication/drug effects
15.
J Microbiol ; 60(3): 347-354, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1652455

ABSTRACT

Coronavirus disease (COVID-19) can cause critical conditions that require efficient therapeutics. Several medicines are derived from plants, and researchers are seeking natural compounds to ameliorate the symptoms of COVID-19. Viral enzymes are popular targets of antiviral medicines; the genome of coronaviruses encodes several enzymes, including RNA-dependent RNA polymerase and viral proteases. Various screening systems have been developed to identify potential inhibitors. In this review, we describe the natural compounds that have been shown to exert inhibitory effects on coronavirus enzymes. Although computer-aided molecular structural studies have predicted several antiviral compound candidates, the current review focuses on experimentally proven natural compounds.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 , Enzyme Inhibitors , Phytochemicals/pharmacology , COVID-19/drug therapy , Enzyme Inhibitors/pharmacology , Humans , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
16.
Science ; 375(6577): 161-167, 2022 Jan 14.
Article in English | MEDLINE | ID: covidwho-1648160

ABSTRACT

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus, Human/drug effects , SARS-CoV-2/drug effects , Uracil Nucleotides/pharmacology , Administration, Oral , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/metabolism , COVID-19/virology , Cell Line , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Disease Models, Animal , Female , Ferrets , Humans , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Mononegavirales/drug effects , Mononegavirales/physiology , RNA-Dependent RNA Polymerase/metabolism , Respiratory Mucosa/virology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/physiology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Transcription, Genetic , Uracil Nucleotides/administration & dosage , Uracil Nucleotides/metabolism , Virus Replication/drug effects
17.
Molecules ; 27(3)2022 Jan 26.
Article in English | MEDLINE | ID: covidwho-1648677

ABSTRACT

The human population is still facing appalling conditions due to several outbreaks of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus. The absence of specific drugs, appropriate vaccines for mutants, and knowledge of potential therapeutic agents makes this situation more difficult. Several 1, 2, 4-triazolo [1, 5-a] pyrimidine (TP)-derivative compounds were comprehensively studied for antiviral activities against RNA polymerase of HIV, HCV, and influenza viruses, and showed immense pharmacological interest. Therefore, TP-derivative compounds can be repurposed against the RNA-dependent RNA polymerase (RdRp) protein of SARS-CoV-2. In this study, a meta-analysis was performed to ensure the genomic variability and stability of the SARS-CoV-2 RdRp protein. The molecular docking of natural and synthetic TP compounds to RdRp and molecular dynamic (MD) simulations were performed to analyse the dynamic behaviour of TP compounds at the active site of the RdRp protein. TP compounds were also docked against other non-structural proteins (NSP1, NSP2, NSP3, NSP5, NSP8, NSP13, and NSP15) of SARS-CoV-2. Furthermore, the inhibition potential of TP compounds was compared with Remdesivir and Favipiravir drugs as a positive control. Additionally, TP compounds were analysed for inhibitory activity against SARS-CoV RdRp protein. This study demonstrates that TP analogues (monomethylated triazolopyrimidine and essramycin) represent potential lead molecules for designing an effective inhibitor to control viral replication. Furthermore, in vitro and in vivo studies will strengthen the use of these inhibitors as suitable drug candidates against SARS-CoV-2.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/drug effects , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Pyrimidines/pharmacology , Triazoles/pharmacology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Amides/pharmacology , COVID-19/drug therapy , COVID-19/metabolism , Catalytic Domain/drug effects , Computational Biology/methods , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pyrazines/pharmacology , Pyrimidines/chemistry , RNA, Viral/drug effects , RNA-Dependent RNA Polymerase/drug effects , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Triazoles/chemistry , Virus Replication/drug effects
18.
Antiviral Res ; 198: 105247, 2022 02.
Article in English | MEDLINE | ID: covidwho-1632314

ABSTRACT

Massive usage of antiviral compounds during a pandemic creates an ideal ground for emergence of resistant strains. Remdesivir, a broad-spectrum inhibitor of the viral RNA-dependent RNA polymerase (RdRp), was extensively prescribed under emergency use authorization during the first 18 months of the COVID19 pandemic, before randomized controlled trials showed poor efficacy in hospitalized patients. RdRp mutations conferring resistance to remdesivir are well known from in vitro studies, and the huge SARS-CoV-2 sequencing effort during the ongoing COVID19 pandemic represents an unprecedented opportunity to assess emergence and fitness of antiviral resistance in vivo. We mined the GISAID database to extrapolate the frequency of remdesivir escape mutations. Our analysis reveals very low levels of remdesivir resistance worldwide despite massive usage.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Resistance, Viral/genetics , SARS-CoV-2/genetics , Adenosine Monophosphate/therapeutic use , Alanine/therapeutic use , Drug Repositioning , Genome, Viral/genetics , Humans , Polyproteins/genetics , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Proteins/genetics
19.
J Virol Methods ; 302: 114471, 2022 04.
Article in English | MEDLINE | ID: covidwho-1638654

ABSTRACT

Routine SARS-CoV-2 surveillance in the Western Cape region of South Africa (January-August 2021) found a reduced RT-PCR amplification efficiency of the RdRp-gene target of the Seegene, Allplex 2019-nCoV diagnostic assay from June 2021 when detecting the Delta variant. We investigated whether the reduced amplification efficiency denoted by an increased RT-PCR cycle threshold value (RΔE) can be used as an indirect measure of SARS-CoV-2 Delta variant prevalence. We found a significant increase in the median RΔE for patient samples tested from June 2021, which coincided with the emergence of the SARS-CoV-2 Delta variant within our sample set. Whole genome sequencing on a subset of patient samples identified a highly conserved G15451A, non-synonymous mutation exclusively within the RdRp gene of Delta variants, which may cause reduced RT-PCR amplification efficiency. While whole genome sequencing plays an important in identifying novel SARS-CoV-2 variants, monitoring RΔE value can serve as a useful surrogate for rapid tracking of Delta variant prevalence.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/virology , Diagnostic Tests, Routine , Humans , RNA , RNA-Dependent RNA Polymerase , SARS-CoV-2/genetics
20.
Molecules ; 27(2)2022 Jan 17.
Article in English | MEDLINE | ID: covidwho-1635108

ABSTRACT

The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2' or C3' is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2' was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Carbon/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Nucleotides/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/chemistry , Nucleotides/chemical synthesis , Nucleotides/chemistry , SARS-CoV-2/drug effects , Stereoisomerism
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