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Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α.

ABSTRACT: Inhaled nebulized interferon (IFN)-α and IFN-β have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 μM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.

SUBMITTER: Furutani Y

PROVIDER: S-EPMC8583800 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The extraordinary global demand for reagents and diagnostic instruments needed for timely detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly affected their availability. In order to meet diagnostic needs, it has been necessary to develop new diagnostic procedures. To date, molecular diagnostic tools have represented the gold standard for diagnosis of SARS-CoV-2 infection, and thus an alternative and real-time PCR system was required. To this aim, a molecular rapid test which works with direct real-time RT-PCR may be a relevant aid. In the present work, the accuracy, sensitivity, and specificity of the bKIT Virus Finder COVID-19 rapid molecular test by Hyris Ltd. was evaluated. Moreover, the influence of a different swab storage medium composition was examined relative to that of a routinely used comparator assay. The Hyris Ltd. assay showed an overall agreement of 100% with the comparator based on a panel consisting of 74 retrospective positive nasopharyngeal swabs (NPSs), collected either in universal transport medium (UTM) or using ESwab. No false-positive result was achieved on samples that previously tested negative. Cross-reactivity screening on microorganisms that commonly colonize the human upper respiratory tract was not detected, excluding the risk of false-positive results. Simultaneously, drugs frequently administered to cure respiratory diseases did not interfere with the analytical performance of the assay. Our results showed that the Hyris Ltd. bKIT Virus Finder COVID-19 is a reliable assay for rapid qualitative detection of SARS-CoV-2, providing the advantage of less complex and unambiguous interpretation of results. Indeed, skilled technicians are not required, and thus the Hyris system is suitable as a rapid and easy system for SARS-CoV-2 diagnosis. IMPORTANCE In order to overcome the increased demand for diagnostic tools for the timely detection of SARS-CoV-2 infection, we tested the bKIT Virus Finder COVID-19 molecular rapid test by Hyris Ltd. The new system was confirmed as a reliable assay for rapid SARS-CoV-2 detection, since sensitivity and specificity parameters were fully satisfied. Moreover, the bKIT Virus Finder COVID-19 provides the advantage of easy results interpretation, since skilled technicians are not required, and thus the Hyris system is a valuable SARS-CoV-2 rapid diagnosis system.

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Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α.

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