Dry loop mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens (preprint)

Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries.

The inhaled steroid ciclesonide blocks SARS-CoV-2 RNA replication by targeting viral replication-transcription complex in culture cells (preprint)

We screened steroid compounds to obtain a drug expected to block host inflammatory responses and MERS-CoV replication. Ciclesonide, an inhaled corticosteroid, suppressed replication of MERS-CoV and other coronaviruses, including SARS-CoV-2, the cause of COVID-19, in cultured cells. The effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 M. Ciclesonide inhibited formation of double membrane vesicles, which anchor the viral replication-transcription complex in cells. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in non-structural protein 3 (nsp3) or nsp4. Of note, ciclesonide still suppressed replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients. ImportanceThe outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. To identify the effective antiviral agents to combat the disease is urgently needed. In the present study, we found that an inhaled corticosteroid, ciclesonide suppresses replication of coronaviruses, including beta-coronaviruses (MHV-2, MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alpha-coronavirus (HCoV-229E) in cultured cells. The inhaled ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS, and COVID-19.

Ultra-Rapid Real-Time RT-PCR Method for Detecting Middle East Respiratory Syndrome Coronavirus Using a Mobile PCR Device, PCR1100.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is usually diagnosed through highly sensitive and specific genetic tests such as real-time reverse transcription polymerase chain reaction (RT-PCR). Currently, two real-time RT-PCR assays targeting the upE and ORF1a regions of the MERS-CoV genome are widely used, and these are the standard assays recommended by the World Health Organization (WHO). The MERS outbreaks to date suggest that rapid diagnosis and subsequent isolation of infected patients, particularly superspreaders, are critical for containment. However, conventional real-time RT-PCR assays require large laboratory instruments, and amplification takes approximately 2 h. These disadvantages limit rapid diagnosis. Here, an ultra-rapid real-time RT-PCR test was established comprising a multiplex assay for upE and ORF1a running on a mobile PCR1100 device. As few as five copies of the MERS-CoV RNA can be detected within 20 min using the standard WHO assays in the mobile PCR device, with the sensitivity and specificity being similar to those of a conventional real-time PCR instrument such as the LightCyler, thereby enabling timely intervention to control MERS-CoV infection.

The inhaled corticosteroid ciclesonide blocks coronavirus RNA replication by targeting viral NSP15 (preprint)

Steroid compounds, which are expected to have dual functions in blocking host inflammation and MERS-CoV replication, were screened from a chemical library. Within this library, ciclesonide, an inhaled corticosteroid, suppressed human coronavirus replication in cultured cells, but did not suppress replication of respiratory syncytial virus or influenza virus. The effective concentration of ciclesonide to block SARS-CoV-2 (the cause of COVID-19) replication (EC90) was 6.3 M. After the eleventh consecutive MERS-CoV passage in the presence of ciclesonide, a resistant mutation was generated, which resulted in an amino acid substitution (A25V) in nonstructural protein (NSP) 15, as identified using reverse genetics. A recombinant virus with the mutation was also resistant to ciclesonide suppression of viral replication. These observations suggest that the effect of ciclesonide was specific to coronavirus, suggesting this is a candidate drug for treatment of patients suffering MERS or COVID-19.