Vr 1558

RNA was extracted from 140 μl of nasopharyngeal (NP) specimens using the QIAmp Viral RNA Mini Kit (Qiagen, Inc.,USA) according to the manufacturer’s specifications. Viral RNA was used as a template for real time RT-PCR using HCoV-specific primers as previously described by Kuypers et al. [16 (link)]. For MERS-CoV detection, samples were analysed using NCV-2012rRT-PCR assay according to the CDC protocols. Control RNAs used were obtained from ATCC (VR-1558D for OC43 and VR740D for 229E) as well as NL63 RNA from the KEMRI-Welcome Trust research program. The MERS-CoV control RNA came along with the test kit from CDC. Samples that tested positive by real time RT-PCR were inoculated onto monolayers of LL-CMK2 cells (ATCC strain CCL-7) in culture tubes (Nunc, Denmark) to contain 100 μl of clinical specimen. The culture medium consisted of Dulbecco’s minimum essential medium (DMEM, Life Technologies, NY, USA) supplemented with 0.04 μg/ml gentamycin, 100U/mL of penicillin & 100 μg/mL streptomycin (Sigma-Aldrich Co. MO, USA). These were incubated at 33 °C under 5 % CO₂, and virus growth was monitored for cytopathic effects (CPEs) for up to 10 days. Culture controls were obtained from ATCC and included betacoronavirus 1 (ATCC® VR-1558™) and human coronavirus 229E (ATCC® VR-740™).

Human muscle rhabdomyosarcoma (RD), embryonic kidney (293T), and lung adenocarcinoma epithelial (A549) cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco). Rhesus monkey kidney epithelial cells (LLC-MK2) and African green monkey kidney (Vero-E6) cells were cultured at 37°C or 33°C in minimum essential medium (MEM; Gibco) containing 10% FBS (Gibco). Cells at 80 to 90% confluence were challenged with CV-A6 (2009-96014), CV-A16 (2010-96057), and CV-B3 (obtained from Chang Gung MEMorial Hospital), EV-A71 (Tainan/4643/98), EV-D68 (TW-02795-2014), influenza A virus (A/WSN/1933), Zika virus (PRVABC59), CoV-229E (ATCC VR-740™), CoV-OC43 (ATCC VR1558), or SARS-CoV-2 (CGMH-CGU-01) at varied MOIs. After 1 h of adsorption at 37°C or 33°C in serum-free DMEM or MEM, cells were washed with phosphate-buffered saline (PBS) and incubated with DMEM or MEM containing 2% FBS.

The original Betacoronavirus 1 HCoV-OC43 VR-759 and the HCoV-OC43 rOC/US183-2 double S protein mutant (H183R & Y241H) derived from VR-759 (herein described as VR-759 dm; gifts from Dr. Talbot, INRS, QC, Canada) were passaged twice on HRT-18 cells and harvested as previously described (Favreau et al., 2009 (link)). The HCoV-OC43 variant VR-1558 (ATCC, Manassas, VA, USA) was passaged twice on MRC-5 cells to obtain a working P3 stock. P3 viral stocks were independently prepared from the supernatant and the cell-associated fractions. Hence, cells were scraped and centrifuged at low speed (500×g, 10 min, 4 °C) to separate extracellular and cell-associated viruses. Extracellular virions were next concentrated by ultracentrifugation (60,000×g, 1 h, 4 °C) and resuspended in a minimal volume of DMEM while the intracellular viruses were released by two rounds of freeze-thawing. Both fractions were sonicated 15 times for 1s at a power of 8 of a Sonic Dismembrator Model 100 using a cup-horn setting (Fischer Scientific, Hampton, NH, USA), flash frozen in liquid nitrogen and stored at −80 °C. These stocks were then used to infect cells as described below.

The specificity of the SARS-CoV-2 RT-LAMP assay was evaluated using 17 common respiratory viruses. Two human coronaviruses (hCoVs), HCoV-OC43 (VR-1558) and HCoV-229E, were from standard strains of VR-1558 and VR-740, respectively, which were purchased from the American Type Culture Collection (ATCC). Nucleic acids of another 15 viruses (including: influenza A, B, and C viruses; parainfluenza viruses type 1–3; enterovirus; RSV A and B groups; HCoV-HKU-1; HCoV-NL63; human rhinovirus; human metapneumovirus; adenovirus; and bocavirus) were obtained from positive clinical samples from children with acute respiratory tract infections.