Cell culture and virus growth
Human coronavirus 229E (HcoV-229E) (ATCC, Cat. No. VR-740) was added at a multiplicity of infection (MOI) of 1.0 to an approximately 85% confluent T75 flask of MRC-5 cells (ATCC, Cat. No. CCL-171), 48 h after plating. The flask was maintained with DMEM (Fisher Scientific, Cat. No. 11–965-118) supplemented with 5% heat inactivated fetal bovine serum (Gemini Bioproducts, Cat. No. 100–500) and 1% L-Glutamine (Gemini Bioproducts, Cat. No. 400–106), incubated at 37 °C with 5% CO2 [15]. The cell culture supernatant was harvested at 72 h post infection when 80% cytopathic effect (CPE) was observed.
Viral transport media formulation and production
Viral transport media (VTM) was produced following the US CDC (Atlanta, GA, USA) guidelines, found freely available on their website for formulation of VTM in a laboratory as an alternative to commercial VTM purchases. 500 mL of Hanks Balanced Salt Solution (HBSS) 1X with calcium and magnesium ions (no phenol red) (Fisher Science, Cat. No. SH3058801) was supplemented with 2% heat inactivated fetal bovine serum (Gemini Bioproducts, Cat. No. 100–500). 100 mg of Gentamicin (Gemini Bioproducts, Cat. No. 400-100P) and 500 μg of Amphotericin B (Gemini Bioproducts, Cat. No. 400-104P) was added to the mixture and mixed thoroughly to create a final product of VIRAL TRANSPOT MEDIA, 2% FBS, 100 μg/mL Gentamicin, 0.5 μg/mL Amphotericin B. This viral transport media was used for storage and processing of all swab samples in this manuscript.
Swab viral spike
Sterile cotton swabs (Fisher Science, Cat. No. 22–029-488) were submerged for 5 s in viral solutions ranging from 1.2 × 107 to 1.2 × 101 viral copies/mL [16]. The swabs were exposed in a serial dilution pattern, with three swabs being exposed at each concentration log to evaluate the detection capabilities of this method. The saturated swabs were then placed in a 2 mL screw capped tube (Omni International, Cat. No. 19–648) prefilled with 1 mL of viral transfer buffer [17]. The stem of the swab was then broken off at a level even with the top of the tube to allow for the cap to be screwed on for transporting and processing. The samples were prepared at 23 °C and then incubated for 1 h at 23 °C prior to processing.
Shaker-mill swab processing for viral lysis
To maintain optimal levels of biosafety, the following shaker-mill processing was completed in a biosafety cabinet to protect the user from any potential aerosol production during processing. Twenty-four 2 mL screw cap tubes containing the virally spiked swabs were processed on the Omni Bead Ruptor Elite (Omni International, Cat. No. 19-040E) for 30 s at 4.2 m/s. This processing generated froth within the tube which was allowed to settle prior to removal of 1 μL of lysate for RT-qPCR (Fig. 1).
HcoV-229E RT-qPCR
HcoV-229E nucleocapsid gene (N gene) was selected as a target for RT-PCR from Vabret et al. [1, 18]. The N gene was targeted with forward primer 5′-AGGCGCAAGAATTCAGAACCAGAG-3′ and reverse primer 5′-AGCAGGACTCTGATTACGAGAAAG-3′ [1]. 1 μL of sample lysate was added to create a final reaction volume of 20 μL using the proportions of primers, sample, SYBR, RT, and DEPC-treated H2O as laid out in the New England Biologics Luna RT-qPCR Kit (NEB, Cat. No. E3005S). Amplification of lysate was performed for 44 cycles and the resulting amplicons were loaded into a 2% agarose (Bio-Rad, Cat. No. 1613101) gel for product visualization. Out of abundance of caution, the loading of the PCR plate with viral lysate should be completed in a biosafety cabinet to protect the user from any potentially viable virus particles remaining following shaker-mill homogenization.
Plaque assay protocol for viral quantification
HcoV-229E was quantified with standard plaque assay protocols [19]. HEK-293 cells (ATCC, Cat. No. CRL-1573) were seeded at 2.0 × 105 cells per well in 6 well tissue culture treated plates (Fisher Science, Cat. No. 07–200-601) with 2 mL of DMEM (Fisher Scientific, Cat. No. 11–965-118) infused with 5% heat inactivated fetal bovine serum (Gemini Bioproducts, Cat. No. 100–500). Once the cells achieved 85% confluence, 200 μL of HcoV-229E stock was added to the media of the first well. The media was gently mixed and 200 μL of the infected media was transferred into the adjacent well. This was repeated to create serial dilutions throughout the plate. After 24 h of incubation, the virally infected media was removed from each well and replaced with 2 mL of DMEM infused with 5% heat inactivated FBS and 2% agarose (Bio-Rad, Cat. No. 1613101). The plate was incubated for an additional 5 days at 35 °C with 5% CO2 and plaques were counted to determine viral concentration in plaque forming units/mL (PFU/mL).